Angiopoietin-1 and -2 in Infectious Diseases Associated with Endothelial Cell Dysfunction
by
Andrea Vaughn Page, B.Sc(H), MD, FRCPC
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science University of Toronto
© Copyright by Andrea Vaughn Page 2012
ii
Angiopoietin-1 and -2 in Infectious Diseases Associated with
Endothelial Cell Dysfunction
Andrea Vaughn Page, B.Sc(H), MD, FRCPC
Master of Science
Institute of Medical Science
University of Toronto
2012
Abstract
Normal endothelial cell function is controlled in part by a tightly regulated balance between
angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and
increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and
prothrombotic. Since an activated endothelial phenotype is seen in invasive group A
streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis,
we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and
to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased
Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical
outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by
combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore
represent a shared final common pathway to endothelial activation as well as a clinically useful
prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.
iii
Acknowledgments
The author would like to acknowledge Dr. W. Conrad Liles (supervisor) for his guidance and
mentorship, Drs. Kevin C. Kain, James W. Scholey, and Susan E. Quaggin (members of the
Program Advisory Committee) for their advice and direction, Drs. Malak Kotb, Allison McGeer,
Donald E. Low, Phillip I. Tarr, Shevin T. Jacob, Christopher C. Moore, and W. Michael Scheld
for their collaboration and for their provision of banked patient specimens, Ms. Nimerta Rajwans
for her technical expertise and her generous and unfailing support throughout the work, and Dr.
Laura Erdman, Dr. Andrea Conroy, and the other members of the Kain-Liles laboratory for their
encouragement. This work was funded in part by the University of Toronto Department of
Medicine Clinician-Scientist Training Program (A.V.P.), the McLaughlin Centre for Molecular
Medicine (W.C.L.), the Canadian Institutes of Health Research (CIHR) Canada Research Chair
program (W.C.L., K.C.K.), CIHR Team Grant in Malaria (K.C.K.), and CIHR MOP-13721
(K.C.K.), Genome Canada through the Ontario Genomics Institute (K.C.K.), and the Defense
Advanced Research Projects Agency (K.C.K.).
iv
Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Publications 2008-2011 ..................................................................................................... vii
List of Abbreviations ................................................................................................................... viii
List of Tables ............................................................................................................................... xiv
List of Figures ............................................................................................................................... xv
Chapter 1 Literature Review ......................................................................................................... 1
1.1 The Vascular Endothelium ................................................................................................. 1
1.1.1 Streptococcal Toxic Shock Syndrome .................................................................... 3
1.1.2 Hemolytic-Uremic Syndrome ................................................................................. 3
1.1.3 Sepsis ...................................................................................................................... 4
1.2 Angiogenesis ....................................................................................................................... 6
1.3 Molecular Structure and Function of the Angiopoietins and their Receptor(s) .................. 7
1.3.1 Regulation of Angiopoietin Expression and Release .............................................. 7
1.3.2 Angiopoietin Receptor(s) and Signalling ................................................................ 8
1.3.3 Downstream effects of Angiopoietin-1 and -2 ...................................................... 10
1.4 Angiopoietin-1 and -2 in Development ............................................................................ 13
1.5 Angiopoietin-1 and -2 in Mature Endothelial Cells .......................................................... 15
1.6 Angiopoietin-3 and -4, and Angiopoietin-like proteins .................................................... 16
1.7 Dysregulation of Angiopoietin-1 and -2 in Human Disease ............................................. 17
1.7.1 Angiopoietins in Critical Illness ........................................................................... 17
1.7.2 Angiopoietins in Malaria ...................................................................................... 18
1.7.3 Angiopoietins in Cardiovascular Disease ............................................................. 19
1.7.4 Angiopoietins in Chronic Kidney Disease ............................................................ 20
v
1.7.5 Angiopoietins in Pregnancy and Preeclampsia ..................................................... 21
1.7.6 Angiopoietins in Malignancy ................................................................................ 22
1.7.7 Angiopoietins in Respiratory Diseases ................................................................. 23
1.7.8 Angiopoietins in Autoimmune Connective Tissue Diseases ................................ 24
1.7.9 Angiopoietins in Miscellaneous Diseases and Syndromes ................................... 25
1.8 Angiopoietin-based Therapies .......................................................................................... 26
1.9 Biomarkers of Endothelial Dysfunction in Sepsis ............................................................ 27
1.9.1 Procalcitonin ......................................................................................................... 27
1.9.2 C-reactive protein .................................................................................................. 36
1.9.3 sTREM-1 ............................................................................................................... 36
1.9.4 Chi3L1 (YKL-40) ................................................................................................. 37
1.9.5 IP-10 ...................................................................................................................... 38
1.9.6 PF4 ........................................................................................................................ 38
1.9.7 vWF ....................................................................................................................... 39
1.9.8 sICAM-1 ............................................................................................................... 39
1.9.9 VEGF .................................................................................................................... 40
1.9.10 sFlt-1 ..................................................................................................................... 40
1.9.11 Miscellaneous Markers of Endothelial Dysfunction in Sepsis ............................. 41
1.9.12 Conclusion ............................................................................................................ 42
Chapter 2 Research Aims and Hypotheses .................................................................................. 43
2.1 Research Aims .................................................................................................................. 43
2.2 Hypotheses ........................................................................................................................ 43
Chapter 3 Angiopoietin-1 and -2 in Invasive Group A Streptococcal Infection ......................... 44
3.1 Introduction ....................................................................................................................... 44
3.2 Methods ............................................................................................................................. 44
3.3 Results ............................................................................................................................... 46
vi
3.4 Discussion/Conclusion ...................................................................................................... 50
Chapter 4 Angiopoietin-1 and -2 in the Hemolytic-Uremic Syndrome ...................................... 53
4.1 Introduction ....................................................................................................................... 53
4.2 Methods ............................................................................................................................. 53
4.3 Results ............................................................................................................................... 55
4.4 Discussion/Conclusion ...................................................................................................... 59
Chapter 5 Prognostic Biomarkers for Sepsis in a Predominantly HIV-infected Population in
a Resource-limited Setting ....................................................................................................... 62
5.1 Introduction ....................................................................................................................... 62
5.2 Methods ............................................................................................................................. 62
5.3 Results ............................................................................................................................... 65
5.4 Discussion/Conclusion ...................................................................................................... 79
Chapter 6 Discussion ................................................................................................................... 84
Chapter 7 Conclusion .................................................................................................................. 89
Chapter 8 Future Directions ........................................................................................................ 93
8.1 Effect of Ventilatory Strategy on Angiopoietin levels in ARDS ...................................... 94
8.2 Angiopoietins in HIV ........................................................................................................ 96
8.3 Angiopoietins in Obstructive Sleep Apnea ....................................................................... 97
8.4 Procalcitonin in patients with Hematologic Malignancy .................................................. 98
8.5 Systematic review of procalcitonin for the prediction of mortality in sepsis and severe
bacterial infection ............................................................................................................ 100
References ................................................................................................................................... 101
vii
List of Publications 2008-2011
1. Petruzziello TN, Yuen DA, Page AV, et al. Functional contribution of the
CXCR4/CXCR7/SDF-1 pathway to E. coli O157:H7 pathogenesis. J Clin Invest. 2011; In
press.
2. Page AV, Liles WC. Colony-stimulating factors (CSFs) in the prevention and management of
infectious diseases. Infect Dis Clin North Am. 2011; 25: 803-817.
3. Page AV, Kotb M, McGeer A, et al. Systemic dysregulation of angiopoietin-1/2 in
streptococcal toxic shock syndrome. Clin Infect Dis. 2011; 52: e157-e161.
4. Makhani N, Morris S, Page AV, et al. A Twist on Lyme: The challenge of diagnosing
European Lyme Neuroborelliosis. J Clin Micro. 2011; 49: 455-457.
5. Boggild AK, Page AV, Keystone JS, et al. Delay in diagnosis: malaria in a returning
traveller. CMAJ. 2009; 180: 1129-1131.
6. Page AV, Liles WC. Immunomodulators (Chapter 42). Principles and Practices of Infectious
Diseases. Ed. GL Mandell et al. 7th
ed. 2009.
7. Page AV, Liles WC. Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-
Macrophage Colony-Stimulating Factor (GM-CSF), and other immunomodulatory therapies
for the treatment of infectious diseases in solid organ transplant recipients. Curr Opin Organ
Transplant. 2008; 13: 575-580.
viii
List of Abbreviations
ABC Abacavir
ACCP American College of Chest Physicians
ACS Acute Coronary Syndrome
ALI Acute Lung Injury
AMI Acute Myocardial Infarction
Ang-1 Angiopoietin-1
Ang-2 Angiopoietin-2
Ang-3 Angiopoietin-3
Ang-4 Angiopoietin-4
APACHE Acute Physiology and Chronic Health Evaluation
ARDS Acute Respiratory Distress Syndrome
ART Antiretroviral Therapy
AUC Area under the Curve
CAP Community-acquired Pneumonia
CaRT Classification and Regression Tree
CHF Congestive Heart Failure
Chi3L1 Chitinase-3-like protein-1
CI Confidence Interval
CKD Chronic Kidney Disease
ix
CLL Chronic Lymphocytic Leukemia
COPD Chronic Obstructive Pulmonary Disease
CPAP Continuous Positive Airway Pressure
CRP C-reactive Protein
DIC Disseminated Intravascular Coagulation
Dll4 Delta-like ligand 4
DMARDs Disease-modifying Anti-rheumatic Drugs
ED Emergency Department
EGF Epidermal Growth Factor
EGR-1 Early Growth Response-1
ELISA Enzyme-linked Immunosorbent Assay
ESR Erythrocyte Sedimentation Rate
FDP Fibrinogen Degradation Product
FGF-2 Fibroblast Growth Factor-2
GAS Group A Streptococcus (Streptococcus pyogenes)
Gb3 Globotriaosylceramide
HAP Hospital-acquired Pneumonia
Hb Hemoglobin
HbA1c Hemoglobin A1c
HFO High Frequency Oscillation
x
HIF-2α Hypoxia-inducible Factor-2α
HIV Human Immunodeficiency Virus
HMG-CoA 3-hydroxy-3-methyl-glutaryl-CoA (reductase inhibitor)
HUS Hemolytic-uremic Syndrome
ICAM-1 Intercellular Adhesion Molecule-1
ICH Intracranial Hemorrhage
ICU Intensive Care Unit
IGRA Interferon-γ Release Assay
IL Interleukin
IP-10 Interferon-γ-inducible Protein - 10 kDa
IQR Interquartile Range
LPS Lipopolysaccharide
MAPK Mitogen-Activated Protein Kinase
MCP-1 Monocyte Chemoattractant Protein-1
MEDS Mortality in Emergency Department Sepsis (score)
MHC Major Histocompatibility Complex
MMP Matrix Metalloproteinase
mRNA messenger Ribonucleic Acid
NF-κB Nuclear Factor-κB
NIHSS National Institutes of Health Stroke Scale
xi
NO Nitric Oxide
NS Non-significant
OR Odds Ratio
OSA Obstructive Sleep Apnea
PaCO2 Partial pressure of Carbon Dioxide (arterial)
PAH Pulmonary Arterial Hypertension
PAI-1 Plasminogen Activator Inhibitor-1
PaO2 Partial pressure of Oxygen (arterial)
PCR Polymerase Chain Reaction
PCT Procalcitonin
PF4 Platelet Factor 4
PI3k Phosphatidylinositol-3-kinase
POCT Point-of-care Test
PROWESS recombinant human activated Protein C Worldwide Evaluation in Severe Sepsis
PSI Pneumonia Severity Index
RA Rheumatoid Arthritis
ROC Receiver Operating Characteristic (curve)
RR Relative Risk
S1P Sphingosine-1-phosphate
SAPS Simplified Acute Physiology Score
xii
SCCM Society of Critical Care Medicine
sFlk-1 soluble Fms-like tyrosine kinase receptor-1 (VEGFR2 - Vascular Endothelial
Growth Factor Receptor 2)
sFlt-1 soluble Fms-like tyrosine kinase receptor-1 (VEGFR1 - Vascular Endothelial
Growth Factor Receptor 1)
Shh Sonic hedgehog
SIRS Systemic Inflammatory Response Syndrome
siRNA small interfering Ribonucleic Acid
SLE Systemic Lupus Erythematosus
SN Sensitivity
SOFA Sequential Organ Failure Assessment
SP Specificity
STEC Shiga Toxin-producing Escherichia coli (E. coli)
sTie-2 soluble Tyrosine kinase with immunoglobulin and epidermal growth factor
homology domain-2
sTREM-1 soluble Triggering Receptor Expressed on Myeloid cells-1
STSS Streptococcal Toxic Shock Syndrome
TB Tuberculosis
Th1 T helper cell, subset 1
Tie-1 Tyrosine kinase with immunoglobulin and epidermal growth factor homology
domain-1
xiii
Tie-2 Tyrosine kinase with immunoglobulin and epidermal growth factor homology
domain-2
TLR4 Toll-like Receptor 4
TNF Tumour Necrosis Factor
tPA tissue Plasminogen Activator
UC Ulcerative Colitis
VAP Ventilator-associated Pneumonia
VCAM-1 Vascular Cell Adhesion Molecule-1
VE-cadherin Vascular Endothelial-cadherin
VEGF Vascular Endothelial Growth Factor
VILI Ventilator-induced Lung Injury
vWF von Willebrand Factor
WPB Weibel-Palade Body
xiv
List of Tables
Table 1. Selected studies evaluating the use of procalcitonin to predict mortality in 30
adult patients with sepsis or severe bacterial infection
Table 2. Significant predictors of mycobacterial infection in Ugandan patients with 77
sepsis
xv
List of Figures
Figure 1. Angiopoietin-1 and -2 (Ang-1 and Ang-2) in patients with invasive Group A 47
streptococcal disease with and without streptococcal toxic shock syndrome (STSS)
Figure 2. Angiopoietin-1 and -2 (Ang-1 and Ang-2) concentrations, and the ratio 49
between the two (Ang-2:Ang-1), in matched acute and convalescent plasma samples
from patients with STSS
Figure 3. Timeline of blood sampling and division into study groups of participants 54
at various stages of E. coli O157:H7 infection
Figure 4. Serum angiopoietin concentration in E. coli O157:H7 infection 56
Figure 5. Serum angiopoietin concentration in individual patients with 58
E. coli O157:H7 infection before and after the diagnosis of HUS
Figure 6. Plasma biomarkers associated with in-hospital mortality in Ugandan 67
patients with sepsis
Figure 7. Plasma biomarkers associated with 28-day mortality in Ugandan patients 70
with sepsis
Figure 8. Classification and Regression Tree (CaRT) analysis of biomarker 72
combinations to predict mortality following an episode of sepsis
Figure 9. Plasma biomarkers associated with positive aerobic blood cultures 74
Figure 10. CaRT analysis for the prediction of positive mycobacterial cultures 78
1
Chapter 1 Literature Review
1.1 The Vascular Endothelium
The vascular endothelium, once conceptualized as little more than the inert and passive lining of
blood vessels, is now known to be a complex organ responsible for interacting with, and
responding to, its environment.1 As reviewed by Aird, bloodborne mediators (including
cytokines, pro-inflammatory molecules, and growth factors), surrounding and supporting cells,
mechanical shear stress, and physico-chemical factors (including temperature and pH) can all act
on endothelial cells to influence their function. This influence is revealed in the endothelial cell
phenotype, typically dichotomized as either activated or resting/quiescent, although intermediary
states are also possible. Quiescent endothelial cells are anticoagulant, anti-adhesive, and
vasodilatory, while activated endothelial cells are pro-coagulant, adhesive, and contractile. These
properties are reflected in cell surface marker expression, secreted molecules, and the integrity of
the endothelial cell barrier.
As reviewed by Levi and van der Poll, as well as Schouten et al, resting endothelial cells express
thrombomodulin and the endothelial protein C receptor on their surface, both of which facilitate
the generation of the crucial anticoagulant molecule, activated protein C.2, 3
Downregulation of
thrombomodulin expression, as occurs in activated endothelial cells, leads to reduced protein C
generation and subsequently, increased thrombin generation. Likewise, tissue factor pathway
inhibitor, tissue-type plasminogen activator, heparan sulfates, prostacyclin, and antithrombin are
either expressed or released by the anticoagulant, resting endothelium, while tissue factor,
plasminogen-activator inhibitor (PAI)-1 and von Willebrand Factor (vWF) are upregulated in the
procoagulant, activated endothelium. Differential cell surface molecule expression between
quiescent and activated endothelial cells influences not only the relative balance between pro-
and anti- coagulant activity, but also the degree of adhesion of circulating hematologic cells. E-
selectin is expressed on activated endothelial cells, where, in combination with P-selectin, it
facilitates rolling of leukocytes along the endothelial layer as a prelude to leukocyte adhesion
(facilitated by the upregulation of ICAM-1 (intercellular adhesion molecule-1) and VCAM-1
(vascular cell adhesion molecule-1) to activated endothelial cells and subsequent transmigration
across the endothelial barrier to a site of injury or inflammation.4 It should be noted that although
2
this is the classical paradigm of endothelial activation and leukocyte response, there is some
organ-specific variability as described in a second review by Aird.4
In addition to cell surface marker expression, activation of the endothelium also influences its
secretory function. As reviewed by Lowenstein et al, endothelial cells contain Weibel-Palade
bodies (WPBs), intracellular structures that store a variety of molecules for rapid release or
externalization upon exposure of the cell to inflammatory or noxious stimuli, including
mechanical factors (hypoxia, trauma, or shear stress), lipids (sphingosine-1-phosphate, ceramide,
and oxidized low-density lipoprotein), inflammatory mediators (thrombin, fibrin, terminal
complement components, and leukotrienes), and angiogenic factors (VEGF).5 Nitric oxide (NO)
impairs or inhibits WPB release.6 WPB exocytosis occurs within minutes, in contrast to the
transcriptional response which requires hours, and liberates the following: vWF, P-selectin,
tissue plasminogen activator, Interleukin (IL)-8, endothelin-1, and angiopoietin-2 (Ang-2),
among others. Consistent with the opposing actions of certain of these molecules, differential
regulation of WPB contents and exocytosis is known to exist.7 Nonetheless, the majority of the
molecules contained in WPBs are prothrombotic, inflammatory, or vasoconstrictive,
characteristics associated with an activated endothelium.
Finally, endothelial cell activation may manifest as abnormal barrier function. The resting
endothelium relies on stable endothelial cell morphology and intact inter-endothelial cell
adherens junctions (comprised largely of the protein vascular endothelial (VE)-cadherin) and
tight junctions (composed of the occludin, claudin, and junctional adhesion molecule proteins) to
limit vascular leak.8 Junctional protein stability is maintained in part by close association with
actin bundles. Under the various conditions which activate the endothelium (described in the
preceding paragraphs), junctional proteins may be downregulated, mis-located, or
phosphorylated, all leading to loss of junctional integrity. Furthermore, many vaso-active agents
activate the Rho GTPases (RhoA or Rac1), which in turn activate Rho kinase, leading to myosin
light chain phosphorylation, stress fibre (as opposed to the usual actin fibre) formation, and
increased cell contractility. Ultimately, this sequence of events produces inter-endothelial cell
gaps, and consequently, vascular leak.9
Under normal circumstances, all of the above responses are appropriately adaptive in the setting
of tissue inflammation or injury. However, these responses can be detrimental when they are
3
exaggerated or excessive, as is the case in certain infectious disease syndromes associated with
prominent endothelial cell activation.
1.1.1 Streptococcal Toxic Shock Syndrome
According to the Working Group on Severe Streptococcal Infections, Group A Streptococcal
(GAS; Streptococcus pyogenes) toxic shock (STSS) is defined by the isolation of Group A
streptococcus from a normally sterile site and hypotension (systolic blood pressure ≤ 90 mmHg),
as well as at least 2 of the following 6 signs of major organ dysfunction: acute kidney injury,
coagulopathy, necrotizing fasciitis, erythematous rash that may desquamate, elevated liver
enzymes, or acute respiratory distress syndrome (ARDS).10
STSS results largely from the action
of a bacterial-derived exotoxin that functions as a superantigen to activate a polyclonal subset of
T cells far in excess of that recruited during a normal immune response.11
Massive pro-
inflammatory cytokine release follows. Ultimately, endothelial activation leads to profound
hypotension and vascular leak, however, the mechanism by which this occurs has not yet been
fully explored.12
Antibiotics are administered in STSS but have little effect on pre-formed toxin. Therefore,
patients require aggressive supportive care, commonly in the intensive care unit (ICU).13
In
addition, mortality from STSS remains high, reported at 43% in the most recent epidemiologic
study.14
As a result, detailed exploration of all potential mediators of endothelial activation in
STSS will be crucial to the discovery of the new therapeutic targets necessary to improve patient
outcome.
1.1.2 Hemolytic-Uremic Syndrome
Shiga (or vero-) toxin-producing Escherichia coli (STEC) are a potentially serious cause of
foodborne infectious diarrhea and were responsible for a recent and well-publicized European
epidemic centered in Germany in 2011. During that outbreak, at least 4075 patients were
infected, and 908 subsequently developed the hemolytic uremic syndrome (HUS).15
Thirty-four
of those patients died.
HUS is defined by the triad of non-immune hemolytic anemia, thrombocytopenia, and acute
renal failure. Clinical signs and symptoms result from circulating bacterial-derived Shiga toxin,
since STEC infections are rarely associated with frank bacteremia.16
Shiga toxin binds to its
4
receptor, globotriaosylceramide (Gb3), which is concentrated on renal glomerular endothelial
cells, and induces the characteristic pathophysiologic changes associated with HUS, namely
endothelial cell swelling and detachment from the basement membrane.17
Endothelial
dysfunction, characterized by increased procoagulant activity, vWF release, and leukocyte
adhesion, is present even earlier in the disease process, before the clinical signs and symptoms of
HUS are manifest.18-20
Whether this occurs directly as a result of toxin interaction with the
endothelial cells or secondarily as a result of elevated pro-inflammatory cytokines is not clear, as
both mechanisms have been documented.21-23
Furthermore, although the inciting factors (Shiga
toxin and/or cytokines) and sequelae (procoagulant and adhesive endothelium) are well-
described, the intervening mediators of endothelial dysfunction have not been determined.
1.1.3 Sepsis
Prior to 1992, proposed definitions of sepsis were often vague, contradictory, and fluid. With the
publication of consensus definitions by the American College of Chest Physicians (ACCP) and
Society of Critical Care Medicine (SCCM), early patient diagnosis and treatment, as well as
clinical trial enrolment or inclusion criteria, could be standardized.24
The Systemic Inflammatory
Response Syndrome (SIRS) was defined as the existence of two or more of: temperature >38oC
or < 36oC, heart rate > 90 beats per minutes, respiratory rate > 20 breaths per minute or PaCO2
(partial pressure of carbon dioxide) < 32 mmHg, and leukocyte count > 12 x 109 cells/L, < 4000
x 109 cells/L, or > 10% immature (band) forms. SIRS may result from a variety of both
infectious and non-infectious conditions, with pancreatitis, ischemia, trauma, and burns being
among the more common causes in the latter group. Sepsis was then defined as SIRS occurring
as a result of infection. As elegantly illustrated in a Venn diagram from the original manuscript,
not all patients with infection have SIRS, and not all patients with SIRS have infection, however,
all patients with sepsis have both (suspected infection and SIRS). Bacteremia was recognized as
the most common, but not sole, cause of sepsis. Severe sepsis was defined as sepsis associated
with organ dysfunction, hypoperfusion, or hypotension, while septic shock was defined as
sepsis-induced hypotension, hypoperfusion, or need for inotropic or vasopressor support despite
adequate fluid resuscitation. Upon re-evaluation in 2001, the basic concepts behind each
definition were maintained, and more specific guidance provided regarding vital sign,
inflammatory, hemodynamic, organ dysfunction, and perfusion parameters that might be used to
5
make the diagnosis of sepsis.25
Despite concerns about specificity, the original definitions of
SIRS and sepsis remain in common use.
In 2009, the Canadian Institute for Health Information released updated statistics on the
epidemiology of sepsis in Canada (excluding Quebec).26
In 2008-2009, there were more than
30 000 sepsis-related hospitalizations in Canada, an increase of more than 4000 in 4 years, with
an associated mortality rate of 30.5%. Severe sepsis accounted for 39% of those admissions, and
45% of the deaths. Patients with sepsis remained in hospital for a median of 9 days longer than
did patients hospitalized for other indications, and 45% of patients with sepsis required ICU care,
further adding to the healthcare cost. This problem is mirrored in other high-income countries in
which sepsis remains a leading cause of healthcare expenditure and patient mortality.27-30
As recently reviewed by Lee and Liles, the clinical manifestations of sepsis ultimately derive
from diffuse and profound endothelial activation, with all of its associated characteristics:
hypercoagulability, inflammation, and vascular leak.31
In a mouse model of sepsis, disruption of
endothelial-specific NF-κB reduced thrombin-antithrombin complex formation (a marker of
coagulation), diminished neutrophil infiltration, attenuated pulmonary edema and major organ
dysfunction, and reversed hypotension, all without enhancing bacterial clearance, suggesting that
the documented benefits were a result of improved endothelial function.32
In vivo studies of
sepsis have documented impaired endothelium-dependent microvascular reactivity and elevated
levels of circulating markers of endothelial activation (including E-selectin, soluble ICAM-1,
soluble VCAM-1, and PAI-1), directly proportional to disease severity.33, 34
The stimuli for
endothelial activation in sepsis are bacterial products, such as lipopolysaccharide (LPS), detected
by pattern recognition molecules, such as toll-like receptor 4 (TLR4) on endothelial cells and
monocytes and macrophages of the innate immune system. Endothelial activation via an
LPS/TLR4 signalling pathway has been shown to augment neutrophil recruitment and
extravasation, endothelial contractility and vascular leak, and upregulation of procoagulant
genes, while activation of the innate immune system leads to production of pro-inflammatory
cytokines and subsequent activation of endothelial cells.35-38
Typically, such responses are
appropriate, adaptive, and localized, and the inciting infection is cleared. However, when these
responses are exaggerated, excessive, or prolonged, sepsis and critical illness result.
6
1.2 Angiogenesis
Angiogenesis, the development and maturation of new blood vessels or their repair following
injury, is a critical function of the healthy endothelium and is mediated by both positive and
negative regulatory factors. Given the complexity of the process (reviewed in detail by Carmeliet
and Jain) and the intent of this review to focus specifically on the angiopoietins, only a limited
discussion of angiogenesis will be included here.
The quiescent endothelium is maintained in a resting state by cell survival signals produced by
the pericytes that underlie and support the endothelial cell monolayer.39
These signals include
VEGF, which is anti-apoptotic via the phosphatidylinositol-3-kinase (PI3k)/Akt pathway, as well
as Dll4/Notch, and angiopoietin-1 (Ang-1). Ang-1 has been shown to upregulate Delta-like
ligand 4 (Dll4) in endothelial cells to facilitate basal signalling through the Notch receptor and
promote continued endothelial quiescence.40
In response to hypoxic (hypoxia-inducible factor
(HIF)-2α), inflammatory, or mechanical stimuli, angiopoietin-2 (Ang-2) is released from
endothelial cells, and mediates endothelial cell detachment from the basement membrane via
matrix metalloproteinases.41-43
Endothelial permeability is further increased by VEGF, which
allows plasma proteins to establish a subendothelial extracellular matrix. The sprouting
angiogenesis that follows requires the migration of Dll4-expressing “tip” endothelial cells in
response to a concentration gradient of VEGF on the extracellular matrix, followed by the
proliferation of Notch1-expressing “stalk” endothelial cells in response to high levels of the
Notch1 ligand Jagged 1. Akin to its function in resting endothelial cells, Dll4 on the tip cell is
thought to interact with Notch on the stalk cells to prevent concurrent sprouting or branching of
the stalk cells, thereby facilitating vessel growth in a single direction.44
Following migration, the
endothelial cells form tubules and return to quiescence to complete maturation of the new vessel,
a process guided in part by Ang-1.45
7
1.3 Molecular Structure and Function of the Angiopoietins and their Receptor(s)
Ang-1 and Ang-2 are glycoproteins with a similar size of 70 kDa and coiled-coil domains at the
amino termini that facilitate oligomerization.46
Ang-1 must, at minimum, form a tetramer in
order to activate its receptor, but is in fact usually found in superclusters of oligomers.47
Ang-2,
in contrast, typically exists in dimeric form in vivo. The carboxy terminus of both molecules
consists of a fibrinogen-related domain that regulates binding to their shared receptor, Tie-2.
Traditionally, Ang-1 has been considered an agonist, and Ang-2 an antagonist, at the Tie-2
receptor, inducing opposite effects on endothelial cell function.
1.3.1 Regulation of Angiopoietin Expression and Release
Ang-1 is produced primarily in the pericytes and smooth muscle cells that surround the
endothelial cell monolayer, but can also derive from platelets.48, 49
More recent evidence suggests
that Ang-1 is also expressed in neutrophils in vitro, but the in vivo significance of this finding is
not clear.50
Ang-1 production by pericytes is constitutive, but can be upregulated by Sonic
hedgehog (Shh) and downregulated by Fibroblast Growth Factor-2 (FGF-2).51, 52
In contrast,
FGF-2 upregulates Ang-2 expression in endothelial cells and VEGF expression in stromal cells,
illustrating the positive/negative regulatory factor interplay common in vascular physiology.
Ang-2 is produced by endothelial cells and stored in the Weibel-Palade bodies for rapid release
upon exposure to various physico-chemical, mechanical, and inflammatory stimuli (discussed in
Chapter 1.1). Of particular relevance to the angiogenic system, WPB exocytosis can be induced
by VEGF and sphingosine-1-phosphate (S1P).53, 54
S1P is a phospholipid signalling molecule
that is secreted from platelets, activated monocytes and mast cells, and vascular endothelial
cells,55
providing evidence of an additional degree of interaction between the inflammatory and
angiogenic pathways. The net effect of S1P on the vasculature may be context-dependent, as S1P
not only has a role in Ang-2 release via WPB exocytosis, but is also implicated as a mediator of
Ang-1 signalling. Ang-1 has been found to increase intra-endothelial cell concentrations of S1P
and to be at least partly dependent on S1P for its effects on vascular permeability.56
8
1.3.2 Angiopoietin Receptor(s) and Signalling
The primary angiopoietin receptor, Tie-2 (tyrosine kinase with immunoglobulin and epidermal
growth factor homology domain-2), as well as Tie-1 (tyrosine kinase with immunoglobulin and
epidermal growth factor homology domain-1), both belong to a family of vascular tyrosine
kinase receptors expressed primarily, but not exclusively, in endothelial cells.57
The extracellular
domain of Tie-2 contains three immunoglobulin-like domains and three epidermal growth factor
(EGF)-like domains.58
Binding of the Tie-2 agonist Ang-1 leads to phosphorylation of the receptor and downstream
signalling. Ang-2 binds at the same site, but its effects may be agonistic or antagonistic
depending on the microenvironment.59
In cultured endothelial cells, Ang-2, in the absence of
Ang-1, can act as a partial agonist at Tie-2, leading to phosphorylation of the receptor and to the
downstream effects characteristic of Ang-1/Tie-2 signalling.60
However, Yuan et al propose that
since Ang-1 binds the Tie-2 receptor with 20 times the affinity of Ang-2 (perhaps due to the
multimerization of Ang-1 which would support clustering and activation of multiple Tie-2
molecules on the cell surface), Ang-2 acts as a relative antagonist in the presence of Ang-1,
leading to relatively weaker signalling when it (Ang-2) binds to Tie-2. Consistent with this, Ang-
2 induces a lesser degree of signalling through Tie-2 than does Ang-1, with reduced downstream
phosphorylation of Akt. Interestingly, release kinetics prior to receptor internalization also differ
between the two molecules, with Ang-2 being released more rapidly than Ang-1.61
Taken
together, the above studies indicate that the degree of activation of Tie-2 is determined by the
relative balance between Ang-1 and Ang-2.
Like other members of the tyrosine kinase receptor family, Tie-2 exists on the cell surface in pre-
formed clusters of oligomers.62
Since tetrameric Ang-1 is the smallest oligomer that can activate
Tie-2, receptor grouping likely facilitates ligand binding. Tie-2 is located on both the apical and
basolateral membranes, thereby allowing it to be bound by circulating Ang-1/Ang-2, as well as
by Ang-1/Ang-2 secreted by pericytes and endothelial cells, respectively.63
Upon activation by
Ang-1, Tie-2 is rapidly internalized through clathrin-coated pits, ubiquitylated, and degraded,
likely to regulate the duration of signalling, as is common within the receptor family.62, 64
As
noted above, ligands appear to be released prior to internalization.61
In keeping with its largely
antagonist activity and its rapid rate of release after binding, Ang-2 is only a weak stimulus for
9
the internalization and degradation of Tie-2. Tie-2 is also regulated by constitutive and VEGF-
inducible ectodomain cleavage, leading to a soluble Tie-2 (sTie-2) fragment that can bind Ang-1
(and Ang-2) and inhibit signalling through intact, transmembrane Tie-2 receptors.65
The function of the alternate angiopoietin receptor, Tie-1, has only recently been determined.
Ang-1 can induce Tie-1 phosphorylation in a Tie-2-dependent manner, however, Ang-1-induced
activation of Tie-1 downregulates the Akt and 42/44 MAPK (mitogen-activated protein kinase)
pathways, resulting in inhibition of Tie-2-mediated effects.66
Tie-1 has been shown to form
heterodimers with Tie-2 on the endothelial cell surface, and experiments using inactive forms of
Tie-1 or Tie-2 tyrosine kinases suggest that it is Tie-2 tyrosine kinase activity that results in Tie-
1 phosphorylation.67
In keeping with their opposing actions, Ang-1 has been found to
preferentially bind non-Tie-1-associated Tie-2.68
In addition to acting as a regulator of Ang-
1/Tie-2 signalling, Tie-1 has also been proposed as a second mechanism by which Ang-2 may
exert its context-dependent agonist/antagonist effects. In the presence of Tie-1, Ang-2 functions
as an antagonist, binding but not signalling at Tie-2, and in the absence of Tie-1, Ang-2 functions
as an agonist, binding and signalling through Tie-2.69
Tie-1 also mediates, in part, the cross-talk
between the angiopoietins and VEGF. VEGF indirectly phosphorylates Tie-2 by facilitating
proteolytic cleavage of the extracellular domain of Tie-1, thereby enhancing access of Ang-1 to
the binding site of Tie-2.70, 71
In addition to VEGF, inflammatory stimuli and shear stress can
also rapidly induce cleavage of the Tie-1 ectodomain.72, 73
Taken together, these findings suggest
that endothelial function can be precisely regulated in response to changes in the
microenvironment, and that this regulation can occur by a variety of means, including changes in
receptor cell surface expression, cleavage of the Tie-1 extracellular domain, and the relative
agonist/antagonist concentrations.
The function of Tie-1 independent of Tie-2 remains controversial. Disruption of the Tie-1 gene is
embryonic lethal at day E13.5 due to edema, hemorrhage, and microvascular rupture.74
Prior to
death, endothelial cells in these animals demonstrate increased filopodia and resulting enhanced
capillary density, as well as abnormal lymphangiogenesis.75
Double Tie-1 and Tie-2 knockout
(-/-, null) mice display a phenotype more severe than that seen with knockout of either gene
alone.76
Beyond the developmental stage, however, the Tie-2-independent function of Tie-1 is
less clear. In a mouse model of atherosclerosis, Tie-1 was overexpressed at regions of shear
stress, while Tie-1 knockdown attenuated atherosclerotic changes.77
In vitro studies have
10
suggested that Tie-1 may have its own function in the endothelial response to inflammation.
Overexpression of Tie-1 in endothelial cells lead to auto-phosphorylation and subsequent
upregulation of cell surface adhesion molecules, including E-selectin, VCAM-1, and ICAM-1,
while knockdown of Tie-1 by siRNA (small interfering RNA) lead to downregulation of certain
pro-inflammatory molecules, including IL-1β.78, 79
However, these findings have yet to be
duplicated in higher order models. Furthermore, other authors have not detected an effect of
siRNA knockdown of Tie-1 on Ang-1 function.80
In summary, the net signalling that occurs through Tie-2 is regulated by the balance in local
concentrations of Ang-1 and Ang-2, as well as by the degree of co-localization and ectodomain
cleavage of Tie-1. Through the latter mechanism, VEGF and inflammatory stimuli can modulate
the magnitude of Ang-1 and Ang-2 signalling (or lack thereof) through Tie-2, and influence
endothelial cell function.
1.3.3 Downstream effects of Angiopoietin-1 and -2
Consistent with their classic roles as competitive antagonists of the same receptor, Ang-1 and
Ang-2 typically exert opposite effects on signalling through Tie-2. Via Tie-2, Ang-1 activates
both the Erk and Akt pathways. Erk is thought to be important for cell migration and
proliferation, while Akt modulates cell survival and inhibits apoptosis.81
Multimeric Ang-1
results in relocation of Tie-2 to areas of cell-to-cell contact, subsequently leading to Ang-1
bridging and trans-activation of Tie-2, preferentially activating Akt, which subsequently
activates endothelial Nitric Oxide Synthase (eNOS) and phosphorylates the forkhead
transcription factor Foxo1.82
This results in Foxo1 exclusion from the nucleus, and reduced
transcription of its dependent genes. The net result is vascular quiescence in the setting of cell-to-
cell contact. Sensing of neighbouring endothelial cells may be facilitated through Tie-2 receptors
clustered in lipid rafts that also contain VE-cadherin, and appears to hinge on the expression of
the transcription factor Krüppel-like factor 2.83, 84
In the absence of cell-to-cell contact, Ang-1
can be bound to fibronectin, collagen, vitronectin, and other components of the extracellular
matrix to anchor Tie-2 to the cell surface in contact with the substratum. In this situation,
signalling through Tie-2 results in preferential activation of the Erk pathway, resulting in
increased cell mobility and enhanced likelihood of forming a functional endothelial cell
monolayer.82
Also necessary for cell migration is Tie-2-dependent caveolin-1 polarization, and
11
Ang-1-induced Early Growth Response-1 (Egr-1).67
Egr-1 is a transcription factor that
upregulates multiple growth factors, cytokines, adhesion molecules, and pro-angiogenic factors.
In siRNA knockdown studies, loss of Egr-1 abolished Ang-1-induced endothelial cell
migration.85
Gene expression profiling of Ang-1-activated endothelial cells reveals upregulation
of genes involved in endothelial cell proliferation, differentiation, migration, and survival, and
downregulation of those involved in apoptosis and inhibition of transcription.86
Besides its anti-apoptotic effect, Ang-1 exerts an anti-permeability effect on the endothelium, at
least partly through increasing the depth and glycosaminoglycan content of the endothelial
glycocalyx.87
Ang-1 also activates Rac1 and phosphorylates RhoGAP, while at the same time
inhibiting RhoA, which is responsible for Ang-2 mediated cytoskeletal rearrangements and
vascular permeability (see below).88
Ang-1 also has anti-thrombotic properties: downregulation
of Ang-1 leads to enhanced VEGF- and tumour necrosis factor (TNF)- induced tissue factor
expression through loss of Ang-1 negative regulation.89, 90
Finally, Ang-1 is anti-inflammatory. It
has been shown to downregulate endothelial cell production of the pro-inflammatory molecule
endothelin-1, as well as LPS- and VEGF- induced cell surface expression of the leukocyte
adhesion markers E-selectin, ICAM-1, and VCAM-1.91-93
Consistent with its role as a Tie-2 antagonist, Ang-2 typically opposes the effects of Ang-1 and
therefore induces pro-apoptotic, pro-permeability, and pro-adhesive changes in the endothelium.
Blockade of signalling through Tie-2 leads to endothelial cell apoptosis via inhibition of the Akt
pathway, as well as increased expression of the antiangiogenic molecule thrombospondin.94
Ang-
2 increases thrombin-induced vascular permeability by impairing VE-cadherin organization and
increasing intercellular gap formation, possibly through actions on Rho kinase and myosin light
chain phosphorylation, or by inducing αvβ3 integrin internalization and degradation.9, 95, 96
Tie-2
knockdown mimics the effect of Ang-2 on endothelial barrier function. Treatment with
exogenous Ang-2 has been shown to result in endothelial detachment from the basement
membrane within 4 hours.97
Finally, in a transgenic mouse model, inducible endothelial cell-
specific Ang-2 expression enhanced myeloid cell recruitment, adhesion to ICAM-1 on the
endothelial cell surface, and tissue infiltration.98
12
A simplified summary of these effects casts Ang-1 as an effector of vascular quiescence, and
Ang-2 as an effector of vascular activation, although there are certainly context-dependent
nuances to this interpretation of the function of both molecules.
13
1.4 Angiopoietin-1 and -2 in Development
Disruption of the Ang-1 gene in mice is embryonic lethal at day E12.5.99
Prominent
developmental defects include a simplified endocardium detached from the myocardial wall, loss
of myocardial trabeculations (suggestive of diminished cross-talk between myocardial and
endocardial cells), and generalized dilatation and loss of complexity (diminished branching) of
vascular networks. Ultrastructural examination of these abnormal vessels reveals rounded
endothelial cells separated from the underlying supportive cells. Disruption of Tie-2 is also
embryonic lethal in mice, resulting in death by day E10.5.74
The Tie-2 null phenotype is one of
large, unbranched blood vessels, and a simplified vascular network, similar to that seen with
Ang-1 knockout.
Overexpression of Ang-2 in the vascular endothelium (transgenic mice created using Tie-2
transcriptional regulatory elements) is embryonic lethal at day E9.5-10.5.100
Pathology studied at
day E9 revealed a disorganized and discontinuous vascular network, and confirmed the basic
concept that Ang-2 antagonizes the effect of Ang-1; detachment of the endocardium from the
underlying myocardium with overexpression of Ang-2 was identical to that seen in Tie-2 and
Ang-1 knockout mice. Similarly, detachment of the endothelial layer and rounding of individual
endothelial cells was seen in the vessel walls and was equivalent in Ang-2 overexpression and
Ang-1 or Tie-2 underexpression.
In contrast to all of the above phenotypes, knockout of Ang-2 does not result in embryonic
lethality. Instead of being a key component of vascular development, Ang-2 appears to be crucial
for vascular remodelling. Using the retinal vasculature as a representative vascular bed, Ang-2 -/-
mice were found to have grossly normal vessels at birth.101
However, they failed to show
evidence of angiogenesis in response to hypoxia-induced VEGF, and similarly failed to
demonstrate the usual and necessary hyaloid vascular regression. Similarly, in a rat pup model of
angiogenesis, Ang-2 mediated new vessel sprouting in the presence of VEGF, and vessel
regression and endothelial apoptosis in its absence.102
That Ang-2 is involved in the coordinated
vascular sprouting and regression necessary to produce a mature vascular network is consistent
with its known actions as a context-dependent antagonist/agonist at Tie-2. Of note, Ang-2 -/-
mice typically die within two weeks of birth with profound chylous ascites ± pleural effusions.101
These mice were found to have a leaky and disorganized lymphatic network thought to result
14
from the lack of Ang-2 agonist activity at Tie-2 in the presence of VEGF. This is consistent with
more recent in vitro work that confirms a prominent agonist effect of Ang-2 on lymphatic
endothelial cells.103
Finally, Ang-2 -/- mice are unable to mount an appropriate inflammatory
response to either chemical or bacterial insult, partly as a result of the loss of Ang-2-induced
sensitization of endothelial cells to the effects of TNF, in particular to TNF-induced expression
of cell adhesion molecules.104
15
1.5 Angiopoietin-1 and -2 in Mature Endothelial Cells
In healthy adult human tissue, Ang-1 is widely expressed in most organs while Ang-2 mRNA is
detectable primarily in those anatomic sites with significant angiogenesis, namely the ovary
(which must create a densely vascularized corpus luteum after follicular rupture), uterus, and
placenta.100
It was early studies involving the rat ovary that characterized the influence of VEGF
on the vascular action of Ang-2: in the presence of VEGF, as is found in the evolving corpus
luteum, excess Ang-2 promoted vessel sprouting by blocking the stabilizing effect of Ang-1,
while in the absence of VEGF, as is found in follicular atresia, excess Ang-2 promoted vessel
regression.100
Reflecting its role in destabilizing the mature endothelium (whether physiologic, in
the promotion of angiogenesis, or pathologic, in the initiation of endothelial dysfunction),
administration of exogenous Ang-2 leads to severe vascular leak and pulmonary edema in mice.9
In contrast, transgenic mice that overexpress Ang-1 were found to have leak-resistant vessels,
even in the presence of inflammatory mediators that typically increase vascular permeability.105
Furthermore, while isolated VEGF overexpression enhances angiogenesis but produces
pathologically permeable vessels, co-expression of VEGF with Ang-1 leads to the development
of new, functional, appropriately permeable vessels. In addition, while adenoviral-delivered
VEGF lead to diffuse edema and death in mice, this was blocked by the pre-administration of
adenoviral-delivered Ang-1.106
Further parsing out the role of Ang-1 in the adult vasculature, a
murine Cre-Lox model of conditional Ang-1 gene knockout demonstrated no obvious phenotype
during vascular quiescence with Ang-1 gene disruption initiated after E13.5, but significantly
altered vascular remodelling in response to injury.107
Taken together, the studies of the angiopoietins in embryonic development and in the mature
endothelium indicate that Ang-1 signalling through the Tie-2 receptor is indispensible for the
formation of new blood vessels whether during embryogenesis or during repair of the adult
vasculature. Although Ang-1 plays a role in the maintenance of vascular quiescence, it is not an
absolute pre-requisite for the resting endothelial state. In contrast, Ang-2 is not absolutely
required during embryogenesis, but is necessary for vascular remodelling thereafter, during
which its function is context-dependent as per the presence or absence of VEGF.
16
1.6 Angiopoietin-3 and -4, and Angiopoietin-like proteins
While Ang-1 and Ang-2 are the best characterized, they are not the only members of the
angiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) are Tie-2 receptor agonists,
capable of inducing Tie-2 phosphorylation and subsequent angiogenesis.108
Both are species-
specific, with Ang-3 limited to mice and Ang-4 to humans, and represent mouse-human
orthologues based on equivalent chromosomal loci.109
The functional role of Ang-4 remains
unclear: some in vitro studies have found that Ang-4 inhibits endothelial cell migration and
supports endothelial barrier function,110
while others have described increased angiogenesis and
tumour progression in glioblastoma multiforme,111
suggesting that the effect of Ang-4 may be
context-dependent.
The physiologic role of the angiopoietin-like proteins is currently under active study.
Angiopoietin-like proteins 1-7 all have a coiled-coil domain at the N-terminus and a fibrinogen-
like domain at the C-terminus, akin to the angiopoietins, but none bind Tie-2 or Tie-1, and most
appear to have important in vivo functions unrelated to angiogenesis.112
As an example of the
pleiotropic effects of this family of molecules, Angiopoietin-like protein-4 participates both in
increased endothelial permeability through direct interactions with integrin α5β1 (and subsequent
Rac signalling), VE-cadherin, and claudin-5, and in hypertriglyceridemia in response to
glucocorticoids.113, 114
Angiopoietin-like protein-2 is highly expressed in adipose tissue, increases
in response to chronic hypoxia and inflammation, and is correlated with insulin resistance in
obese patients.115
Angiopoietin-like protein-3 increases plasma levels of cholesterol and
triglycerides, and mutations in its gene have recently been implicated in familial combined
hypolipidemia.116
Angiopoietin-like protein-6 has also been implicated in obesity, although the
literature on the subject is conflicting.117
17
1.7 Dysregulation of Angiopoietin-1 and -2 in Human Disease
Because of the central role of angiogenesis in the development, proper function, defence, and
subsequent repair of every organ system, it is not surprising that the angiopoietins, important
regulators of angiogenesis, have been widely studied and implicated in a variety of human
diseases and syndromes. Although the occasional study has attempted to explore angiopoietin
levels in parenchymal tissue or non-hematogenous bodily fluids, the vast majority of the studies
presented below have examined serum, plasma, or whole blood concentrations of Ang-1 and
Ang-2, in keeping with their vascular origins and targets. Because of the breadth and depth of the
literature in this field, the following review will focus primarily on clinical studies, with
reference to in vitro or animal models where required to provide supporting evidence or
rationale. It should be noted that, in healthy individuals, the serum/plasma concentration of Ang-
1 is greater than that of Ang-2, and that angiopoietin dysregulation refers to a perturbation of this
typical ratio (decreased Ang-1, increased Ang-2, and an increased Ang-2:Ang-1 ratio).118
1.7.1 Angiopoietins in Critical Illness
The majority of work in this area has focused on the association of the angiopoietins with sepsis
and its diagnosis, treatment, complications, and outcome. Angiopoietin dysregulation, and in
particular a net excess of Ang-2, would be predicted to create a pro-inflammatory, pro-coagulant,
and pro-adhesive endothelium that would in turn manifest as tissue inflammation, microvascular
thrombosis, and hypotension, all frequently observed in septic patients. In healthy volunteers
who received an infusion of LPS, serum Ang-2 levels began to rise 2 hours after the infusion and
peaked at 4.5 hours, while neither sTie-2 nor Ang-1 was appreciably different from baseline.119
The increase in Ang-2 occurs coincident with that of IL-6, 30 minutes after that of TNF, and 30
minutes before that of IL-8. Of note, Ang-2 levels declined more slowly than did those of the
cytokines, yet faster than both E-selectin and ICAM-1. An accompanying study of 21 septic
patients found higher Ang-2 levels in non-survivors than survivors (28-day mortality), both on
admission and at 72 hours. Likewise, in a study of 50 critically ill patients, plasma Ang-2 levels
taken within 12 hours of the diagnosis of sepsis were predictive of ICU and 28-day mortality.120
Ang-2 levels on the day of enrolment and throughout the ICU admission correlated with fluid
balance, with standard indices of pulmonary dysfunction, and with mortality, suggesting a
potential role for serial measurements of Ang-2. vWF was not as uniformly associated with
18
illness severity, prompting the authors to speculate that, despite co-storage in the Weibel-Palade
bodies, differential regulation might be relevant.7 Other studies have found a similar relationship
between Ang-2 concentration and the severity of illness in sepsis: Orfanos et al described a
correlation between serum Ang-2, severe sepsis, and TNF,121
Siner et al, Davis et al, and
Kümpers et al between serum Ang-2, Sequential Organ Failure Assessment (SOFA) and Acute
Physiology And Chronic Health Evaluation (APACHE) II scores, and mortality,122-124
and Parikh
et al between serum Ang-2 (but not Ang-1) and impaired pulmonary gas exchange, but not with
survival or APACHE II score.9 Ang-2 has been found to be similarly predictive of mortality in
certain subsets of critically ill patients, including those requiring renal replacement therapy and
in children with septic shock.125, 126
In contrast, decreased plasma Ang-1 at ICU admission predicted 28-day mortality in 70 patients
with severe sepsis, while decreased Ang-1 levels were also associated with mortality in a study
of Malawian children with severe bacterial infection.127, 128
Furthermore, activated protein C,
which until recently was approved for the treatment of severe sepsis, has been found to
upregulate both Ang-1 and Tie-2, thereby improving endothelial barrier function in a manner
which may have been responsible for some of the therapeutic benefit documented in early
clinical trials.129
Importantly, the clinical utility of Ang-1 as a prognostic biomarker may be
limited by the frequent fluctuations in circulating levels seen in individual patients with hourly
sampling over a 24-hour time period.118
The Ang-2:Ang-1 ratio, reflecting perturbations in one
or both of the angiopoietins, has been found to be predictive of the eventual development of
septic shock at the time of fever onset in neutropenic patients.130
Finally, although the soluble
Tie-2 receptor does increase in sepsis, it has no apparent effect on Ang-1/2 function or
circulating levels, and was not found to be a significant indicator of pulmonary edema nor a
significant predictor of mortality in a multivariate analysis.131
1.7.2 Angiopoietins in Malaria
In malaria, endothelial activation leads to adherence of parasitized erythrocytes, microvascular
obstruction, and tissue ischemia. Ang-2, but not VEGF, was found to be elevated in severe
malaria (and a better predictor of outcome than lactate) in association with reduced NO
bioavailability.132
Subsequent studies, however, have all identified Ang-1 as a more consistent
biomarker in malaria, discriminating between cerebral malaria and uncomplicated malaria in
19
Thai adults and Ugandan children, between cerebral, severe, and uncomplicated malaria in Thai
adults, and between cerebral malaria with retinopathy and febrile, non-malarial decreased level
of consciousness in Malawian children.133-135
Furthermore, decreased Ang-1 was associated with
Plasmodium falciparum malaria in pregnancy, while an increased Ang-2:Ang-1 ratio was
associated with both placental malaria and low birth weight infants.136
1.7.3 Angiopoietins in Cardiovascular Disease
Given the prominence of the angiopoietin system in vascular remodelling, Ang-1 and Ang-2
have been proposed as both mediators and potential therapeutic targets for a variety of primary
cardiovascular diseases. Higher levels of Ang-2 were associated with systolic blood pressure,
diabetes, and the metabolic syndrome in 3778 third-generation cohort participants of the
Framingham Heart Study.137
Likewise, David et al found a higher level of plasma Ang-2 in
hypertensive patients as compared to healthy controls, and in those patients with atherosclerotic
disease as compared to those without.138
Despite purported anti-inflammatory effects separate
from their lipid-lowering properties, a six-week course of therapy with a 3-hydroxy-3-methyl-
glutaryl (HMG)-CoA reductase inhibitor (“statin”) did not lower Ang-2 levels in this population.
It should be noted that other studies have documented an elevation in plasma angiogenic factors,
including VEGF, Ang-1, Ang-2, and sTie-2, in treated hypertensive patients with high
Framingham risk scores, attributed to the relative hypoxia resulting from rarefaction or loss of
the microcirculation that occurs in hypertension.139
As noted in the Framingham study, angiopoietin dysregulation has also been associated with
disorders of glucose and lipid metabolism. In vitro studies involving human cardiac
microvascular endothelial cells and human macrovascular endothelial cells have demonstrated
that exposure to high concentrations of glucose leads to specific inhibition of Ang-1/Tie-2-
induced Akt phosphorylation, resulting in a predicted loss of cell survival signalling.140
The Ang-
1/Tie-2-induced phosphorylation of Erk 1/2 was not affected, leading to a predicted net signal
favouring endothelial cell activation and migration. In clinical studies, Ang-2 is higher in
patients with diabetes than in healthy control subjects, and elevated Ang-2 is independently
associated with an elevated hemoglobin A1c (HbA1c), a marker of longterm glycemic control, as
well as with diabetic retinopathy and a composite marker of macrovascular disease that included
cardiovascular and cerebrovascular disease as well as neuropathy.141-143
20
Given its association with multiple cardiovascular risk factors, it is not surprising that elevated
levels of Ang-2 have been associated with coronary artery disease. Plasma levels of Ang-2 at
presentation are significantly higher in patients with acute myocardial infarction (AMI) than in
patients with unstable or stable angina, in whom Ang-2 levels are significantly higher than in
healthy controls.144
Microarray analysis of myocardial biopsies from patients undergoing urgent
coronary artery bypass grafting for AMI confirms significant upregulation of the Ang-2 gene.145
Ang-2 levels are also elevated in acute, versus chronic, congestive heart failure (CHF),146
and at
a baseline routine health examination in patients who experienced AMI within the next ten
years.147
The latter association was independent of the traditional cardiovascular risk factors.
Since vascular stabilization, repair, and regeneration are important prerequisites for functional
recovery following stroke, high post-event Ang-1 levels in the cerebral vasculature would be
predicted to have a beneficial effect. Indeed, in experimental animal models and in one
prospective clinical trial involving 95 patients, elevated Ang-1 levels were associated with
improved outcome after intracerebral hemorrhage (ICH).148
A serum Ang-1 level of ≥ 35 ng/mL
at 72 hours post-ICH yielded an odds ratio (OR) of 14.7 for the occurrence of a good outcome at
three months, defined as an improvement in the NIHSS (National Institutes of Health Stroke
Scale) of ≥ 50%.149
Consistent with these observations, patients with subarachnoid hemorrhage
who exhibited post-event cerebral vasospasm on transcranial Doppler ultrasound were found to
have more prolonged and severe suppression of serum Ang-1 levels when compared with both
healthy controls and patients with SAH but without cerebral vasospasm.150
Higher Ang-1 levels
likewise correlate with more rapid middle cerebral artery recanalization after tPA (tissue
plasminogen activator) therapy, while higher Ang-2 levels have been associated with risk of
recurrence following lacunar infarct.151, 152
Elevated levels of Ang-2 have also been implicated in
post-stroke breakdown of the blood-brain barrier and subsequent cerebral edema.153
1.7.4 Angiopoietins in Chronic Kidney Disease
A role for the angiopoietins in chronic kidney disease (CKD) was first suggested by animal
studies. Podocyte-specific overexpression of Ang-2 lead to albuminuria and apoptosis of the
glomerular endothelial cells, and to decreased expression of VEGF,154
an important local
regulator of the antagonist/agonist effects of Ang-2. In contrast, exogenous Ang-1 has been
found to attenuate renal dysfunction in a variety of models of acute and chronic kidney
21
injury.93,
155, 156
Angiopoietin dysregulation appears to be greatest in patients on hemodialysis
(although the comparison with those on peritoneal dialysis trended towards but did not reach
statistical significance), and resolves with renal transplantation.157
Neither time of sampling (pre-
or post- dialysis) nor transition to nocturnal hemodialysis for a period of 6 months altered
angiopoietin dysregulation. Furthermore, in patients with CKD on dialysis (either hemo- or
peritoneal), serum Ang-2 levels correlated with the presence of coronary heart disease and
peripheral arterial disease. The same authors described a progressive increase in serum Ang-2
with each successive stage of chronic kidney disease, and attributed angiopoietin dysregulation
in this setting to the reduction in nitric oxide, a known inhibitor of Weibel-Palade body
exocytosis, seen in CKD.158
Given the relatively high molecular weight of the angiopoietins, and
their existence as multimers, they are not candidates for glomerular filtration, nor are they
detectable in urine (Page AV, unpublished observations). Therefore, elevated serum levels of
Ang-2 in CKD are likely related to increased release, rather than decreased renal clearance.
Further work by Kümpers et al determined that elevated Ang-2 was an independent predictor of
mortality in CKD.159
1.7.5 Angiopoietins in Pregnancy and Preeclampsia
The angiopoietins are frequently implicated in the vascular complications of pregnancy, but
published reports differ with regard to the direction of change of Ang-1, Ang-2, and soluble Tie-
2. Serum Ang-2 levels are higher in healthy pregnant women than in non-pregnant or post-
partum women.160
Circulating Ang-2 levels were initially reported to be lower, and Ang-1 levels
higher, in women with preeclampsia than in those with normal pregnancies.161
Further study
revealed that this difference might be present in the third trimester only: the two groups of
patients are indistinguishable in the first trimester, and those patients who will subsequently
develop preeclampsia typically have higher serum Ang-2 concentrations in the second
trimester.162, 163
More recently, several authors reported a high Ang-2:Ang-1 ratio, primarily as a
result of lower Ang-1, in women in the second trimester of pregnancy who would later develop
preeclampsia, and in those with established preeclampsia.164, 165
Low soluble Tie-2 levels have
also been found in women with preeclampsia.166
In summary, while angiopoietin dysregulation
as a marker or contributor to preeclampsia and other hypertensive disorders in pregnancy is
certainly plausible, additional research is needed to clarify the relevant timing, direction, and
magnitude of change of Ang-1, Ang-2, and soluble Tie-2.
22
Of note, Ang-2 has also been detected in the amniotic fluid of healthy women in the second and
third trimesters of pregnancy, and is elevated in those with an intrauterine infection, consistent
with its known links to inflammation.167
1.7.6 Angiopoietins in Malignancy
Consistent with what is known about the role of Ang-2 in development, tumours from transgenic
mice that overexpress Ang-2 have increased microvessel density, an immature vascular
phenotype with a deficit of pericytes, and an enhanced rate of endothelial cell apoptosis.168
Various malignant cells, including chronic lymphocytic leukemia (CLL) cells, can secrete Ang-
2.169
In addition, gastrointestinal stromal tumours, leiomyomas, schwannomas, colorectal
adenocarcinomas, and to a lesser extent Kaposi‟s sarcomas and cutaneous angiosarcomas, have
all been shown to harbour Ang-1, Ang-2, Ang-4, Tie-1, and Tie-2 in the tumour cell
cytoplasm.170-172
Although the clinical significance of this finding is not known, the presence of
both the ligand and receptor in the same cell suggests that the tumour cells may be able to
respond to autocrine or paracrine signalling through the angiopoietin/Tie system.
Further evidence of the interaction between the angiopoietin/Tie system and various
malignancies is derived from cohort studies. Elevated circulating Ang-2 levels have been
associated with reduced time to first treatment and higher mortality in CLL,169
increased
mortality in acute myeloid leukemia (14.7% three-year survival in patients with Ang-2 levels ≥
1495.6 pg/mL versus 64.7% in those with Ang-2 levels < 1495.6 pg/mL),173
stage of disease and
bone involvement in multiple myeloma,174
baseline disease and cardiac involvement in primary
light chain (AL) amyloidosis,175
baseline disease and lymph node metastases in early gastric
cancer,176
baseline disease in castration-resistant prostate cancer,177
reduced survival in
pancreatic ductal adenocarcinoma,178
baseline disease, residual disease after debulking surgery,
and poor overall and disease-free survival in ovarian carcinoma,179
increased mortality at the
time of diagnosis of colorectal carcinoma,180
baseline disease, tumour burden, and prognosis in
neuroendocrine tumours,181, 182
disease stage, metastases, and overall survival in non-small cell
lung cancer,183
survival and disease progression in metastatic malignant melanoma,184
baseline
disease in liver cirrhosis and hepatocellular carcinoma,185
increased tumour size in soft tissue
sarcoma,186
baseline disease in breast cancer,187
and inversely with tumour burden in response to
treatment in metastatic renal cell carcinoma.188
Interestingly, high bone marrow concentrations
23
of Ang-1 (studied following reports of increased bone marrow microvessel density) have
recently been reported to be independently associated with a higher rate of transformation to
acute leukemia and with overall mortality in myelodysplastic syndromes.189
Of note, the angiopoietin/Tie system has also been implicated in adverse effects associated with
cancer therapy. Vascular leak syndrome occurs in more than half of the patients treated with
high-dose IL-2 for metastatic renal cell carcinoma or melanoma, and in these patients, Ang-2
levels rose with each consecutive day of therapy and declined with discontinuation.190
Furthermore, when applied to endothelial cells in culture, patient serum from the final day of IL-
2 therapy resulted in endothelial gaps and increased actin stress fibres, pathophysiologic
correlates of the vascular leak syndrome in vivo. In patients receiving the anti-VEGF antibody
bevacizumab, upregulation of Ang-1 as a result of VEGF inhibition limits tumour hypoxia and
protects the tumour vasculature from regression, thereby potentially contributing to tumour
recurrence despite ongoing VEGF blockade.191
1.7.7 Angiopoietins in Respiratory Diseases
The rationale for the study of the angiopoietins in obstructive airway diseases derives from
clinicopathologic studies that have documented an increase in the number and permeability of
blood vessels in the airway walls in chronic obstruction as recently reviewed by Zanini et al, and
from the growing need for novel, non-steroid-based therapies.192
Expression of Tie-2 is
upregulated in airway epithelial cells and selected macrophages in the lungs of ovalbumin
-sensitized and -challenged mice, a recognized animal model for airway hyperresponsiveness,
and is correlated with the degree of airway remodeling.193
Ang-2 expression was increased, and
Ang-1 expression decreased, in the same cells. In clinical studies, asthmatics who smoked had
significantly higher levels of Ang-2 in induced sputa than did asthmatics who did not smoke, and
these levels were positively correlated with the airway vascular permeability index.194
In patients
with chronic obstructive pulmonary disease (COPD), serum Ang-2 levels were significantly
higher in those with stable disease as compared to healthy controls, and higher still in those with
acute exacerbations.195
Ang-2 levels in this instance were inversely correlated with PaO2.
Furthermore, both former and current smokers had higher serum levels of Ang-2 than did those
patients who had never smoked. Since VEGF is also increased in obstructive airway diseases,196
and since Ang-2 is known to promote new vessel sprouting with endothelial migration and
24
proliferation in the presence of VEGF, the predicted impact of the net increase in Ang-2 is
increased angiogenesis, thereby providing a plausible explanation and potential therapeutic target
for the airway vascular proliferation characteristic of chronic obstructive lung diseases.
Chronic pulmonary vascular remodelling also occurs in pulmonary hypertension. In
experimental models of pulmonary arterial hypertension (PAH), Ang-1 has been found to be
protective against vascular remodelling, while Ang-2 is elevated in hypoxia-induced PAH,
consistent with the ability of hypoxia to trigger Weibel-Palade body release from endothelial
cells.197, 198
In patients with PAH, elevated plasma Ang-2 has been found to be inversely
correlated with the response to treatment, and to be an independent predictor of mortality.199, 200
1.7.8 Angiopoietins in Autoimmune Connective Tissue Diseases
Angiopoietin dysregulation has been associated with a variety of autoimmune disorders. When
compared to healthy controls, patients with newly diagnosed rheumatoid arthritis (RA) had
elevated levels of serum Ang-2, and these levels correlated with the severity of disease and
measures of disease activity.201
Interestingly, patients who later developed cardiovascular disease
(at a mean interval of 12.5 years after the onset of RA) had higher levels of Ang-2 at arthritis
diagnosis than did those patients who never developed cardiovascular disease over the same
length of follow-up. Elevated serum Ang-2 levels (and the Ang-2:Ang-1 ratio) have also been
found to correlate with disease activity and proteinuria in patients with Systemic Lupus
Erythematosus (SLE), and with disease activity and/or renal involvement in patients with various
vasculitides.202-204
Furthermore, Ang-2 expression was upregulated in the glomeruli of patients
with SLE-associated renal disease. Additional data suggesting that the angiopoietin system may
contribute to the pathogenesis of autoimmune diseases is derived from patients with autoimmune
thyroid disease, in whom monocyte overexpression of the Tie-2 receptor appears to enhance the
chemotactic response to Ang-1 and Ang-2 overproduction by thyroid follicular cells, leading to
an inflammatory cell infiltrate in the thyroid parenchyma.205
Serum Ang-1 levels in patients with early RA (at the time of first clinic visit) were found to be
elevated when compared with those in patients with other collagen vascular diseases, a
discriminatory effect not seen with serum C-reactive protein or rheumatoid factor.206
Serum
Ang-1 levels are likewise elevated in patients with Behçet‟s disease as compared to healthy
controls,207
suggesting that angiopoietin dysregulation is not a uniform process across
25
rheumatologic disorders, and therefore, that its clinical utility should be carefully evaluated with
particular attention to the type, stage, and degree of complications in each autoimmune disease.
1.7.9 Angiopoietins in Miscellaneous Diseases and Syndromes
Plasma levels of Ang-2 have been shown to rise within 30 minutes of major trauma, and to
correlate with the severity of injury, the degree of tissue hypoperfusion, and in-hospital mortality
in trauma victims.208
Circulating Ang-2 is elevated in asymptomatic patients with sickle cell
disease as compared to healthy controls (HbSS > HbSC > HbAA), and is further elevated in both
groups during painful crises.209, 210
Since vascular remodelling is prominent in the
pathophysiology of inflammatory bowel disease, serum Ang-2 was investigated and found to be
elevated in patients with Crohn‟s disease and ulcerative colitis (UC).211
Epithelial cells from
crypt abscesses in biopsy specimens from UC patients were found to overexpress Ang-1, Ang-2,
and Tie-2, while the neutrophils in those abscesses overexpressed Tie-2, suggesting the ability to
respond to Ang-1 or Ang-2 as chemotactic factors.212
These findings are strikingly similar to
those of autoimmune thyroid disease (discussed above). In patients with acute pancreatitis,
admission serum Ang-2 levels were found to be predictive of later organ failure that persisted for
more than 48 hours.213
Finally, serum Ang-2 levels have been found to be increased in patients
with hepatitis C-induced liver disease as compared to healthy controls, and to decline in patients
with a sustained virologic response to therapy.214
Consistent with these findings, in vitro
experiments have shown that Ang-2 is upregulated in hepatocytes infected with either hepatitis C
or hepatitis B.215
26
1.8 Angiopoietin-based Therapies
Treatment strategies targeting the angiopoietin/Tie-2 system have been proposed for a wide
variety of disorders and diseases, although most have not yet progressed beyond testing in
experimental models. In vitro or animals models using Ang-1 mimetic or Tie-2 stimulatory
therapies have been successful in the treatment of: acute lung injury (ALI),216
pulmonary
hypertension,217
diabetic wounds,218
fractures, ischemic necrosis, and destructive bone diseases
(through enhancement of osteoblast differentiation and to promote angiogenesis in ex vivo bone
tissue engineering),219-221
cardiac, cerebral, and limb ischemia,222-224
ischemia-reperfusion-
induced acute kidney injury,155
radiation-induced apoptosis of the microcapillary endothelial
cells of the intestinal villi,225
and spinal cord injury,226
while those using Ang-2 inhibitory
therapies have demonstrated positive results in the treatment of hematologic and solid tumour
malignancies.227, 228
Not all studies have been positive, however. Despite multiple animal and
human studies documenting a link between angiopoietin dysregulation and pulmonary edema,
treatment with Ang-1 did not attenuate ventilator-induced lung injury in mice.229
The sole
clinical study of angiopoietin therapy involved AMG 386, a peptide-Fc fusion protein that blocks
the Tie-2 receptor. In a Phase 1 study of 22 patients with various advanced solid tumour
malignancies, AMG 386 was well-tolerated and appeared to exert some anti-tumour effect,
although all patients were receiving concomitant full-dose standard chemotherapy.230
While the
angiopoietin/Tie-2 system is an attractive therapeutic target, further studies are clearly needed to
determine when, how, and in what patient population anti-angiogenic therapy is best deployed.
27
1.9 Biomarkers of Endothelial Dysfunction in Sepsis
The manifestations of sepsis are notoriously protean (the 2001 International Definitions
Conference limned 24 criteria, some or all of which may be used to diagnose sepsis in
association with documented or suspected infection), yet early diagnosis and treatment is crucial,
with delay directly associated with mortality.25, 231, 232
The results of confirmatory diagnostic
tests, and in particular microbiologic cultures, can take hours to days. Furthermore, clinical
scores and traditional markers of inflammation (leukocyte count) and tissue hypoxia (lactate) are
non-specific or reflect the end result of sepsis rather than the underlying pathophysiology.
Therefore, the ideal biomarker for use in sepsis would derive from one of the two major
dysregulated systems (either the inflammatory or endothelial/vascular system), would
discriminate between septic and non-septic patients with SIRS, would allow prognostication at
the time of first contact with the healthcare system, and would be reproducible across various
patient populations. What follows is a discussion of the evidence for and against several
proposed diagnostic and/or prognostic biomarkers, representative of either the inflammatory or
vascular pathway, and their use in sepsis.
1.9.1 Procalcitonin
A prodigious literature has been generated describing the rationale for and clinical utility of
procalcitonin (PCT) as a diagnostic and prognostic biomarker in a variety of infectious diseases,
and has been most recently reviewed by Gilbert.233
Procalcitonin is the 12.6 kDa prohormone of
calcitonin, itself produced by the medullary C cells of the thyroid gland in response to
hypercalcemia. Plasma levels in healthy adults are low, but procalcitonin levels rise within 2-4
hours of bacteremia or endotoxemia, earlier than other markers of inflammation such as C-
reactive protein (CRP) or the erythrocyte sedimentation rate (ESR). PCT appears to be relatively
specific for infection, and does not increase in response to non-infectious inflammatory states
such as autoimmune connective tissue disease or inflammatory bowel disease.234, 235
Interestingly, PCT production by endothelial-adherent monocytes is transient, and has only been
documented in vitro.236
PCT in patients with infection appears to arise from monocyte-stimulated
adipocytes, and has been documented to act as an in vitro monocyte chemoattractant.237, 238
Human neutrophils increase production of pro-inflammatory cytokines in response to PCT in
vitro, but demonstrate decreased migration.239
Hence, the pathophysiologic role of PCT in the in
28
vivo response to infection is not clear. Further obscuring its true in vivo effect are studies
demonstrating that exogenous procalcitonin produces no effect when administered to healthy
animals, but increases mortality when administered during sepsis.240
Despite its questionable role in the immune response, much interest has been generated in the
potential utility of PCT to differentiate bacterial from non-bacterial infection. A 2004 meta-
analysis by Simon et al included 12 prospective studies that compared PCT and CRP, and
calculated sensitivities of 85% and 82%, and specificities of 83% and 88%, for the use of PCT to
differentiate between bacterial infection and either non-infectious inflammatory states or viral
infections, respectively.241
In both cases, PCT outperformed CRP. A second meta-analysis
focused on PCT in the Emergency Department or at admission for the diagnosis of bacteremia,
and included 17 studies (none included in the 2004 analysis) with a total of 2008 patients.242
PCT
was found to be moderately predictive of bacteremia, with a negative likelihood ratio of 0.3
helping to rule out the diagnosis. PCT levels have also been found to discriminate between Gram
positive and Gram negative bacteremia in septic patients in the ICU.243
A cut-off value of 16
ng/mL yielded a positive predictive value of 83% and a negative predictive value of 74% for the
diagnosis of Gram negative bacteremia in this population. Neither CRP, nor leukocyte count, nor
the SOFA score were discriminative in this setting. Notably, however, mortality was also higher
in those patients with Gram negative bacteremia, and the positive likelihood ratio of 4.2
indicated only moderate clinical utility. When followed routinely in the ICU, a decrease in PCT
levels between days 2 and 3 is predictive of appropriate empiric antibiotic therapy,244
suggesting
that PCT may have a role in monitoring therapeutic response. PCT was able to distinguish
between patients with community-acquired pneumonia and those with non-infectious
exacerbations of asthma or COPD, however, CRP was slightly more predictive than PCT in this
setting.245
PCT at a cut-off value of 0.8 ng/mL had a sensitivity of 91% and specificity of 68%
for the diagnosis of secondary bacterial infection during the 2009 H1N1 pandemic influenza
season.246
It should be noted, however, that not all studies have confirmed the specificity of PCT
for bacterial infection.247
PCT has been evaluated in at least two meta-analyses, one including 33 studies and the other 18,
for its utility in distinguishing sepsis from non-infectious inflammatory causes of SIRS in
critically ill patients. However, the two studies reported widely divergent results. The first, by
Uzzan et al, evaluated 3943 patients and concluded that PCT had good predictive value and
29
outperformed CRP for the differentiation of sepsis versus SIRS in critically ill patients after
surgery or trauma, while the second, by Tang et al, evaluated 2097 patients and concluded that
PCT was insufficiently sensitive (71%) for the differentiation of sepsis versus SIRS in a mixed
population (medical and surgical) of critically ill adults.248, 249
The diagnostic odds ratio for PCT
reported by Tang et al was less than half that reported by Uzzan et al. In at least one study
reported since, of 539 consecutive adult patients presenting to the Emergency Department with
suspected infection, PCT could distinguish between severe sepsis and other infectious/non-
infectious presentations, but not amongst non-severe sepsis, non-septic bacterial infection, and
non-infectious presentations, the clinical situations in which it would be most helpful.250
Based
on these results, the utility of PCT seems to vary significantly according to the clinical setting
and patient population, making generalization difficult and reinforcing the need for specific
study in each population in which its use is intended.
Importantly, procalcitonin levels are not affected by advanced human immunodeficiency virus
(HIV) disease, and can distinguish between respiratory infection with Pneumocystis jirovecii,
Mycobacteria tuberculosis, and community-acquired bacteria in this population, as well as
between bacteremia and disseminated viral, mycobacterial, and fungal infections, localized
bacterial infections, and cerebral toxoplasmosis.251-253
In addition to its potential role in the diagnosis of bacterial infection, procalcitonin has also been
proposed as a prognostic marker in sepsis and severe bacterial infection. Table 1 presents a
thorough, though not exhaustive, compilation of positive and negative studies examining
procalcitonin for the prediction of mortality at Emergency Department presentation, hospital or
ICU admission, onset of illness, or up to 1 week into the course of illness. Some studies have
also advocated the use of the PCT trajectory over time as a means to increase predictive value.
Regardless, as is illustrated by Table 1, the most appropriate use of PCT (in terms of timing,
duration of sampling, and target patient population) for the prediction of mortality in sepsis
remains controversial.
30
Author Study Population Significant PCT
Measure
Predicted
Outcome
Notes
Bloos F et
al 254
175 patients requiring
mechanical ventilation for CAP,
HAP, and (new) VAP
PCT on days 1-14,
starting within 48 hours
of diagnosis
28-day mortality Daily PCT levels higher in non-
survivors from days 1-14; Initial cut-off
value 1.1 ng/mL, peak cut-off value 7.8
ng/mL
Predictive value equal to APACHE II
Boeck L et
al 255
101 patients with VAP Serum PCT at diagnosis 28-day mortality PCT levels failed to decline over time
in non-survivors
Boussekey
N et al 256
110 patients requiring ICU
admission for CAP
Serum PCT within 48
hours of ICU admission
ICU mortality CRP not significant
Charles PE
et al 244
180 patients with sepsis in the
ICU
PCT on day 3 ICU mortality PCT levels on day 1 not predictive
Clec‟h C et
al 257
75 consecutive ICU patients
with shock
Serum PCT on day of
diagnosis
ICU mortality SN 87.5%, SP 54%, 6 ng/mL cut-off
31
Author Study Population Significant PCT
Measure
Predicted
Outcome
Notes
Dahaba AA
et al 258
69 patients requiring ICU
admission for severe sepsis
within 24 hours of surgery
Plasma PCT at day 6 28-day mortality
due to sepsis
No difference in PCT between
survivors and non-survivors on days 1-
4; SN 85%, SP 89%, 3.2 ng/mL cut-off
day 6
Giamarellos-
Bourboulis
EJ et al 259
1156 hospitalized patients with
sepsis (234 in the ICU)
PCT with 24 hours of
diagnosis of sepsis
In-hospital
mortality
Cut-off values 0.12 ng/mL in non-ICU
patients and 0.85 ng/mL in ICU
patients
Gibot S et
al 260
63 critically ill patients with
sepsis
None ICU mortality Only sTREM-1 remained predictive of
mortality in a multivariate analysis
Plasma PCT within 12 hours of ICU
admission was measured
Guan J et
al 261
37 patients with septic shock and
initial PCT levels > 10 ng/mL
Δ PCT from days 1-5 28-day mortality
Hillas G et
al 262
45 consecutive patients with
VAP
PCT and ΔPCT on days
1 and 7
28-day mortality OR 7.23 for ΔPCT1-7
32
Author Study Population Significant PCT
Measure
Predicted
Outcome
Notes
Huang DT et
al 263
1651 patients presenting to the
ED with CAP
PCT at presentation 30-day mortality PCT identifies low-risk patients from
amongst those identified as high risk by
PSI and CURB-65
Karlsson S
et al 264
242 critically ill patients with
sepsis
None In-hospital
mortality
Serum PCT at day 0 measured
Lee CC et
al 265
525 consecutive patients
presenting to the ED with sepsis
Serum PCT on
admission
Early (≤ 5 d) and
late (6-30 d)
mortality
MEDS score > PCT > CRP for
predicting either outcome
Menendez R
et al 266
453 patients hospitalized for
CAP
None 30-day mortality Only CRP remained a significant
predictor of mortality in a multivariate
analysis
PCT on admission measured
Meng FS et
al 267
86 patients with severe sepsis
requiring admission to the ICU
Serum PCT on the day
of ICU admission
28-day mortality Outperformed CRP and APACHE II
for prediction of mortality
33
Author Study Population Significant PCT
Measure
Predicted
Outcome
Notes
Novotny A
et al 268
160 patients with sepsis
following major visceral surgery
Serum PCT on the day
of diagnosis of sepsis
Sepsis-related
mortality
SN 71%, SP 77% for PCT + APACHE
II at diagnosis
Oberholzer
A et al 269
124 patients with severe sepsis None 28-day mortality Neither baseline plasma PCT nor
change over days 1-4 were predictive
of mortality
Phua J et
al 270
72 consecutive patients with
septic shock within 24 hours of
admission to the ICU
Increase in plasma
lactate + serum PCT
between days 1 and 2
28-day mortality APACHE II was the only significant
variable at admission; IL-1β, IL-6, IL-
10, TNF not significant
Rau BM et
al 271
82 patients with secondary
peritonitis, within 96 hours of
symptom onset
PCT ≥ 1 ng/mL after
the first week
In-hospital
mortality
Outperformed CRP
Ruiz-
Alvarez MJ
et al 272
103 consecutive patients
requiring ICU admission for
suspected sepsis
None ICU mortality Neither serum PCT nor CRP within 24
hours of admission were predictive of
mortality
34
Author Study Population Significant PCT
Measure
Predicted
Outcome
Notes
van
Langevelde
P et al 273
464 febrile patients presenting to
the Medicine ED
PCT at presentation In-hospital
mortality
RR 5.0 (95% CI 1.7 - 15) for PCT level
≥ 0.5 ng/mL
Viallon A et
al 274
131 consecutive patients
presenting to the ED with sepsis
PCT at presentation 30-day mortality SAPS II and lactate significant; serum
IL-6, IL-8, TNF, and CRP not
significant
Wunder C et
al 275
33 consecutive ICU patients
with severe sepsis
Plasma PCT 28-day mortality APACHE III score also significant, but
not IL-6 or IL-10
Table 1. Selected studies evaluating the use of procalcitonin to predict mortality in adult patients with sepsis or severe bacterial
infection. Sepsis defined as per ACCP/SCCM standard definitions unless otherwise indicated.24
Community-acquired pneumonia (CAP),
hospital-acquired pneumonia (HAP), ventilator-associated pneumonia (VAP), Procalcitonin (PCT), Acute Physiology And Chronic
Health Evaluation (APACHE), Intensive Care Unit (ICU), C-reactive protein (CRP), Sensitivity (SN), Specificity (SP), soluble Triggering
Receptor Expressed on Myeloid cells-1 (sTREM-1), Emergency Department (ED), Pneumonia Severity Index (PSI), CURB-65 (alternate
pneumonia severity score), Mortality in Emergency Department Sepsis (MEDS) score, Odds Ratio (OR), Tumour Necrosis Factor (TNF),
Relative Risk (RR), Simplified Acute Physiology Score (SAPS), Change in (Δ).
35
Procalcitonin has also been proposed for use as a prognostic indicator in non-septic patients. In a
study of 472 consecutive patients admitted to the ICU of a tertiary care hospital in Denmark,
routine daily procalcitonin measurements revealed that a maximum procalcitonin level of ≥ 1
ng/mL and a rising procalcitonin level were both independent predictors of 90-day all-cause
mortality.276
It should be noted that leukocyte count and C-reactive protein (CRP) were also
measured but were not similarly predictive. Schneider et al studied 220 unselected patients who
required post-operative admission to an intensive care unit for therapy or monitoring and found
that, in a multivariate analysis, admission procalcitonin values were independently predictive of
in-hospital mortality (sensitivity 81% and specificity 80% for a cut-off value of 1.44 ng/mL) and
were superior in this regard to the APACHE II score and to IL-6.277
In children, procalcitonin levels have been associated with severity of illness in
meningococcemia and sepsis post-bone marrow transplant, and performed better than CRP in
both populations.278, 279
In the latter group, procalcitonin was also associated with mortality.
Similarly, in a study of 75 children with septic shock, ICU admission levels of procalcitonin,
TNF, and IL-10 were all associated with mortality, however, none performed as well as a
pediatric clinical risk score unless followed serially.280
In a recent meta-analysis, procalcitonin
proved to have good discriminative capacity for the differentiation of sepsis from other critical
illnesses in neonates.281
Carrol et al evaluated 377 Malawian children with meningitis or
pneumonia, of whom 279 had a confirmed bacterial etiology.282
Plasma procalcitonin was higher
in severe bacterial infection regardless of HIV status (as was CRP), and predicted mortality. Of
note, however, the area under the ROC curve for procalcitonin in the prediction of mortality was
only 0.61 (95% CI 0.50 - 0.71). Furthermore, the ability of both PCT and CRP to distinguish
between viral and bacterial pneumonia in Mozambican children was abolished in the presence of
detectable Plasmodium falciparum (malaria) parasitemia,283
indicating the need for caution in
both the use and study of PCT in malaria-endemic areas.
In summary, PCT may be useful in the diagnosis of bacterial infections, and may be a more
specific predictor of outcome than cytokines and other non-specific markers of inflammation
such as CRP. However, it may need to be measured over time, and it is not clear that it is a better
predictor of outcome in all cases than are standard clinical prediction scores. Furthermore, PCT
36
shows considerable variability in its utility in different populations, particularly amongst those
with malarial co-infection, indicating the need for further study.
1.9.2 C-reactive protein
C-reactive protein (CRP) is a 21 kDa acute phase reactant protein synthesized by the liver in a
process upregulated by inflammation.241
When evaluated for use as a biomarker, CRP has often
yielded contradictory results in patients with sepsis. As illustrated in Table 1, CRP, when
compared to PCT, has variously been found to be more, less, or equally predictive of mortality in
sepsis. CRP levels in ALI/ARDS have been reported in at least one study to be higher in
survivors than non-survivors, even when adjusted for potential confounding factors such as
corticosteroid use and hepatic failure.284
Therefore, despite the fact that CRP assays are widely
available for clinical use, the appropriate interpretation of the results in any given clinical context
will require further study.
1.9.3 sTREM-1
Triggering Receptor Expressed on Myeloid cells (TREM)-1 is found on monocytes,
macrophages, and neutrophils, and consistent with its distribution on cells of the innate immune
system, is upregulated by TLR signalling.285
TREM-1 is thought to amplify the immune response
to pathogens, but whether it is beneficial, detrimental, or potentially both (in a context-dependent
manner) is in question. In a mouse model of pneumococcal pneumonia, TREM-1 signalling
enhances cytokine production, bacterial clearance, and survival.286
Consistent with this
protective effect, soluble TREM-1 (sTREM-1) levels on the day of ICU admission in 63 patients
with sepsis were lower in non-survivors than survivors, and remained predictive of outcome in a
multivariate analysis (in contrast to procalcitonin and CRP).260
In contrast, blockade of TREM-1
has been variously reported to increase both survival and mortality in murine models of
polymicrobial sepsis induced by cecal ligation and puncture, but has also been shown to improve
survival in a murine model of endotoxemia.287, 288
Consistent with the latter finding, sTREM-1
has been shown to correlate with severity of illness in a study of 52 critically ill patients with
sepsis, while PCT and CRP did not.289
sTREM-1 levels were found to be higher in the non-
survivors than survivors at days 10 and 14 of admission. Other authors have also documented an
association between elevated levels of sTREM-1 and severity of illness in sepsis.290
Further
complicating the interpretation of sTREM-1 levels in sepsis are the findings from a study of 65
37
septic patients in a surgical ICU, in whom plasma sTREM-1 measured within 24 hours of
diagnosis could not distinguish between SIRS and sepsis, nor between survivors and non-
survivors (28-day mortality).291
sTREM-1 has also been proposed as a means to distinguish infectious from non-infectious
inflammatory states.292
In a small study of patients with febrile neutropenia, both sTREM-1 and
PCT were associated with bloodstream infections and mortality, with levels of both markers
higher in non-survivors, while in a larger study of patients with febrile neutropenia, sTREM-1
performed better for the diagnosis of microbiologically documented infection than did PCT, with
sensitivities of 88% and 48%, respectively.293, 294
sTREM-1 has been reported to discriminate
between infectious and non-infectious pulmonary infiltrates in hospitalized, non-critically ill
patients with acute respiratory illness, yielding a sensitivity of 83% and specificity of 63%.295
Furthermore, sTREM-1 performed better than usual clinical markers in predicting bacteremia in
patients with community-acquired pneumonia, with low admission sTREM-1 levels
demonstrating a negative predictive value of 99%.296
However, at least one study has reported
that sTREM-1 performs less well than CRP in distinguishing septic versus non-septic patients
with SIRS,297
underscoring the variability found in clinical studies of sTREM-1.
1.9.4 Chi3L1 (YKL-40)
Chitinase-3-like protein 1 (Chi3L1) is a 40 kDa protein secreted by activated neutrophils and
macrophages in response to IL-6. Levels of Chi3L1 peak 6 to 24 hours following IL-6
infusion.298
The function of Chi3L1 is not known, and although it is a secreted protein, no cell
surface or soluble receptors have yet been identified.
In hospitalized patients with bacterial community-acquired pneumonia, serum Chi3L1 levels are
elevated as compared to healthy controls, peak on day 1, and decline to baseline levels by day
3.299
Interestingly, patients with pneumococcal pneumonia demonstrated the greatest elevation in
Chi3L1, while those with pneumonia due to H. influenzae or the “atypical” organisms had
Chi3L1 levels only modestly above baseline. In keeping with this finding, serum Chi3L1 levels
are elevated in patients with pneumococcal bacteremia as compared to healthy controls, and
relate to the severity of infection (defined as need for hemodialysis, vasopressor medications, or
mechanical ventilation) and in-hospital mortality in these patients.300
In a multivariate analysis,
Chi3L1 was found to be an independent predictor of mortality in the same population. Elevated
38
Chi3L1 levels have also been documented in association with Gram negative sepsis. In
experimental human Escherichia coli (E. coli) endotoxemia, Chi3L1 levels rose significantly
above baseline at 2 hours and peaked at 24 hours post-infusion.301
The Chi3L1 response trailed
that of TNF and IL-6, whose levels peaked at 1.5 and 2 hours post-infusion, respectively. In
addition, proteomic analysis identified Chi3L1 as one of three proteins uniquely upregulated in
septic, as compared to non-septic, critically ill patients.302
Serum levels at the time of ICU
admission were significantly higher in septic versus non-septic patients, and correlated with
severity of illness and circulating levels of IL-6.
1.9.5 IP-10
Interferon-γ-inducible protein-10 kDa (IP-10 or CXCL10) is a pro-inflammatory chemokine that
is involved in the chemotaxis of monocytes and T cells to sites of inflammation. IP-10 was found
to correlate with severity of illness and mortality in a univariate analysis of a small study of 16
patients with sepsis, and in addition was found to have excellent sensitivity and specificity in the
diagnosis of bacterial sepsis in very low birth weight neonates.303, 304
IP-10 has been shown to
effectively discriminate between survivors and non-survivors of severe malaria in a study
conducted in Ugandan children,305
a finding in keeping with evidence from animal models in
which IP-10 gene knockout reduced peripheral parasitemia and mortality.306
IP-10 is known to be elevated in both latent tuberculosis (TB) and active TB, regardless of
immunosuppression due to corticosteroids and disease-modifying anti-rheumatic drugs
(DMARDs).307
IP-10 levels in the pleural fluid are significantly different between patients with
tuberculous pleural effusions and those with pleural effusions of other etiologies.308, 309
Furthermore, IP-10 has been studied and found to be effective as a novel measurable end-product
of a modified Interferon-γ Release Assay (IGRA) in which patient plasma is incubated ex vivo
with purified specific M. tuberculosis proteins.310
1.9.6 PF4
Platelet Factor 4 (PF4) is a marker of platelet activation after release from platelet alpha
granules, and is upregulated in septic shock.311
By binding to both protein C and
thrombomodulin, PF4 can augment activated protein C generation in vivo, and improve survival
in animal models of sepsis.312, 313
In a small study of ten patients with sepsis, PF4 levels were
39
elevated in the acute phase of illness and declined significantly with effective therapy,314
but the
utility of PF4 as a biomarker in sepsis has not been further investigated.
1.9.7 vWF
von Willebrand Factor (vWF) is stored in the Weibel-Palade bodies of endothelial cells for
release upon endothelial cell activation.5 It acts to stabilize the adhesion of platelets at sites of
vascular injury, and its release has been associated with microvascular thrombosis in animal
models of experimental endotoxemia, although not in cecal ligation and puncture-induced
polymicrobial sepsis.315, 316
In a small study of 14 patients with sepsis, bacteremia, and
disseminated intravascular coagulation (DIC), plasma levels of vWF were significantly elevated
when compared to non-septic hospitalized patients, and correlated with the plasma level of
fibrinogen degradation products (FDPs), a marker of the severity of DIC and therefore indirectly
of sepsis.317
There was no correlation with lactate or platelet count. Other studies have also
documented an association between vWF and severity of illness in sepsis.318
A study of 50
mechanically ventilated patients in the ICU, enrolled within 12 hours of the diagnosis of sepsis,
found that the vWF concentration on the day of enrolment, but not thereafter, was predictive of
mortality during the ICU admission.120
However, not all studies have confirmed the association
between vWF levels and mortality in sepsis.319
In addition, there is conflicting literature as to
whether vWF is predictive of the development of ARDS in at-risk patients,320-323
although
elevated vWF levels have been associated with mortality in patients with ALI.324
1.9.8 sICAM-1
ICAM-1 plays a crucial role in neutrophil recruitment to sites of inflammation and injury.
Expressed constitutively at low levels on the endothelial cell surface but substantially
upregulated during endothelial cell activation, ICAM-1 interacts with β2-integrin on the surface
of the neutrophil to mediate firm adhesion, a necessary prerequisite to eventual neutrophil
extravasation.325
A soluble form of ICAM-1 can be detected in plasma and increases in concert
with the cell surface form during inflammation. Hence, soluble ICAM-1 (sICAM-1) has been
studied as a marker of inflammation.
In a small study of 25 critically ill patients with sepsis, plasma levels of sICAM-1 at the time of
diagnosis/study entry were significantly higher than those in non-septic hospitalized patients and
40
in eventual non-survivors versus survivors.326
In a multivariate analysis, sICAM-1 remained
predictive of survival. Similar results were seen in other studies, including a large trial of 221
patients with sepsis, however, sICAM-1 performed less well in this setting than did other
markers of endothelial dysfunction such as sFlt-1 (see below).34, 327
1.9.9 VEGF
VEGF plays a pivotal role in angiogenesis as outlined in Chapter 1.2, and is also linked to the
inflammatory pathway.328
In a mouse model of sepsis, circulating VEGF increases in a time-
dependent manner.329
Furthermore, reducing VEGF signalling via a soluble form of its receptor
attenuated the clinical signs of sepsis in the same model. Despite such promising results, the
findings in human studies have been contradictory. While most authors agree that circulating
levels of VEGF are elevated in sepsis,330
studies have yielded conflicting results as to whether
the degree of elevation correlates with disease severity.123, 331-333
Although VEGF has been
associated with the development of ALI/ARDS in septic patients, it is less predictive of this
outcome than are the angiopoietins.320
Also unexpected given the results of the animal model
was a large study of 293 Malawian children with severe bacterial infection, in whom plasma
VEGF concentrations were higher in survivors than in non-survivors.128
However, this difference
did not remain significant in a multivariate analysis. Other studies of sepsis have also failed to
detect an association between VEGF level and outcome.334
1.9.10 sFlt-1
Fms-like tyrosine kinase receptor 1 (sFlt-1) is the first of two VEGF receptors (VEGFR1). Both
the transmembrane and soluble form of Flt-1 may act to trap VEGF and prevent binding to Fms-
like tyrosine kinase receptor (Flk-1; VEGFR2), which is capable of transducing a much stronger
signal.39
This role is consistent with the interactions of sFlt-1 and the Dll4/Notch system. During
sprouting angiogenesis, VEGFR1 is upregulated, and VEGFR2 downregulated, by Notch
signalling in stalk cells, thereby limiting their responsiveness to the surrounding high
concentrations of VEGF required to guide the tip cells, and promoting organized, unidirectional
angiogenesis.335
In this way, sFlt-1 may function to fine-tune the actions of VEGF.
In 83 patients with suspected infection, sFlt-1 levels upon presentation to the Emergency
Department were higher in those with than without shock, and correlated with the APACHE II
41
and SOFA scores.331
A similar study, this time including 221 patients with sepsis of varying
severity, confirmed those findings.34
In 10 patients with febrile neutropenia, sFlt-1 measured 48
hours after fever onset was significantly higher amongst those patients who would develop septic
shock than amongst those who would continue to manifest only uncomplicated sepsis.332
Nonetheless, neither the pathophysiologic role nor the source of sFlt-1 in sepsis is clear.
Of note, sFlt-1 has also been implicated in malaria, and plasma levels were predictive of
mortality in a large study of 156 Ugandan children with malaria.305
1.9.11 Miscellaneous Markers of Endothelial Dysfunction in Sepsis
As might be expected, many other circulating factors have been explored as potential prognostic
indicators in sepsis. A recent review documented 3370 references detailing studies on 178
different biomarkers.336
Few of those that pertain to endothelial dysfunction have as well-defined
a pathophysiologic role and as reproducible a clinical prognostic utility as the angiopoietins. For
instance, although pro-inflammatory cytokines would seem an obvious choice for prognostic
indicators in sepsis, their utility has not been confirmed in large studies. In a study of 60 patients
with severe sepsis, plasma levels of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, and
TNF, were higher at diagnosis in patients with shock than in those without.337
Plasma levels of
IL-8 and monocyte chemoattractant protein (MCP)-1 correlated best with the SOFA score, and
also predicted 48-hour and 28-day mortality, however, only MCP-1 was associated with outcome
in a multivariate analysis. One of the largest studies, involving 1690 patients at enrolment in the
PROWESS (recombinant human activated Protein C Worldwide Evaluation in Severe Sepsis)
study, measured IL-1β, TNF, IL-6, and IL-8, the pro-inflammatory cytokines most commonly
associated with sepsis.338
While IL-8 and TNF correlated with APACHE II score, few patients
had detectable IL-1β, TNF, or IL-10 (an anti-inflammatory cytokine) levels at study entry,
reflecting their early and transient release during sepsis. Furthermore, serum IL-6 levels at the
time of enrolment did not predict 28-day mortality in the 840 patients randomized to the study‟s
placebo arm. In addition, anti-cytokine therapies have not proven useful in the treatment of septic
patients.339, 340
Many other candidate biomarkers have also been studied in sepsis. E-selectin has been associated
with severity in sepsis and mortality in critically ill patients.34, 342
While some studies have
42
documented an association with outcome in severe infection, others have not.326, 342-344
Endothelial progenitor cells are known to play a role in vascular repair, are elevated in sepsis,
and have been found to correlate with the outcome of sepsis in at least one small study.345-347
Circulating endothelial microparticles have also been evaluated as markers of severity in sepsis,
however, controversy still exists as to whether they exert a pro- or anti- inflammatory effect.348,
349 Finally, serum levels of various matrix metalloproteinases (MMPs) were found to be
abnormal at the time of diagnosis of sepsis, although the direction of change was dependent on
the particular MMP studied. MMP-10 was higher in septic than non-septic patients, while MMP-
9 was lower in non-survivors than survivors of sepsis.350
Since conflicting data regarding MMPs
in sepsis exist, further study is needed.351
1.9.12 Conclusion
In summary, although multiple candidate biomarkers have been proposed for use in sepsis, none
have been uniformly and consistently shown to be clinically useful as either diagnostic or
prognostic indicators in sepsis. Nor have any met the prespecified criteria delineated in Chapter
1.9 for the ideal biomarker: one that would derive from one of the two major dysregulated
systems, discriminate between septic and non-septic patients with SIRS, allow prognostication at
the time of first contact with the healthcare system, and be reproducible across various patient
populations. Thus there is the need for further study in this area, and importantly, for the direct
comparison of performance characteristics of candidate molecules in a single patient population.
43
Chapter 2 Research Aims and Hypotheses
2.1 Research Aims
The specific aims of the project are:
1) To determine whether angiopoietin-1 and -2 are dysregulated in human diseases
characterized by diffuse endothelial cell activation,
2) To define the clinical consequences (morbidity and mortality) associated with
abnormal angiopoietin levels, and the prognostic or predictive value of these levels,
3) To explore the clinical utility of biomarker combinations to predict mortality in septic
patients in a resource-limited setting.
2.2 Hypotheses
1) Since the angiopoietins are prominent regulators of endothelial cell function,
dysregulation of the angiopoietins (decreased Ang-1 and increased Ang-2) will
correlate with disease severity and outcome in infectious diseases associated with
significant endothelial cell dysfunction, including STSS, HUS, and sepsis, and will be
predictive of disease progression (reflecting the severity of endothelial dysfunction)
when measured early in illness.
2) As outlined in Chapter 1, multiple pathways mediate reciprocal interactions between
the immune system and the endothelium. Since sepsis is characterized by both
immune and endothelial cell activation, combining biomarkers from the inflammatory
and vascular pathways will more closely approximate the underlying pathophysiology
and therefore will more accurately predict mortality in sepsis.
44
Chapter 3 Angiopoietin-1 and -2 in Invasive Group A Streptococcal Infection
Originally published in Clinical Infectious Diseases, 2011 352
3.1 Introduction
Streptococcal toxic shock syndrome (STSS) remains a serious disease associated with poor
clinical outcomes despite prompt recognition, timely surgical debridement, and rapid initiation of
appropriate antimicrobial therapy.13
Much of the morbidity and mortality results from the release
of toxins by invading group A streptococcal bacteria. GAS pyrogenic exotoxins function as
superantigens to activate T cells in a Major Histocompatibility Complex (MHC) class II
unrestricted manner, initiating a series of inflammatory reactions that result in diffuse endothelial
cell activation, vascular leak, and the clinical characteristics of STSS: hypotension, major organ
dysfunction, and coagulopathy.13
While massive pro-inflammatory cytokine release has been
implicated in the pathogenesis of STSS, we hypothesized that angiopoietin-1 and angiopoietin-2,
critical regulators of endothelial cell activation, might also play central roles in the development
of STSS.
3.2 Methods
Beginning in 1992, prospective, population-based surveillance for invasive group A
streptococcal disease has been undertaken in Ontario, Canada via mandatory laboratory reporting
of S. pyogenes isolates cultured from normally sterile sites.353
Thirty-seven patients, enrolled
between 1999 and 2009, were included in the current study. Informed consent was obtained to
collect bacterial isolates and plasma samples, as well as selected clinical data from interviews
with the attending physicians and patient chart review. Patients with invasive disease were
considered to have streptococcal toxic shock if they met the consensus definition: hypotension in
combination with at least two of coagulopathy, acute kidney injury, elevated serum
aminotransferases, ARDS, rash, or necrotizing fasciitis.13
Of the 37 patients included in this study, 16 had invasive streptococcal infection and toxic shock
(STSS), while 21 had invasive streptococcal infection alone (no STSS). The median age was not
significantly different between these groups (41.5 years with STSS versus 38 years without;
45
range from 10 to 74 years; P = NS). The patients were predominantly female (56% in the group
with STSS and 67% in the group without, P = NS), and otherwise healthy, with only one-third of
the patients in either group describing a pre-existing illness. Only 3 patients (2 with STSS and 1
with invasive disease alone) reported pre-existing illnesses potentially associated with
angiopoietin dysregulation. The underlying source of infection was similar in each group, with
the majority of patients in both groups having skin and soft tissue infections (7 patients (44%)
with STSS and 12 patients (57%) with invasive streptococcal infection alone, P = NS). Amongst
the patients with skin and soft tissue infections, 5 (71%) with STSS and 4 (33%) with invasive
infection alone had necrotizing fasciitis, P = NS. Presenting group A streptococcal infections in
the remaining patients included respiratory tract infections, bacteremia without an identified
source, post-partum infections, and peritonitis, and did not differ significantly between the
groups. The two groups were significantly different only in the diagnostic criteria for STSS;
hypotension was present in 100% of patients with STSS and 33% of patients without (P <
0.0001). Five patients with STSS died as compared to one patient with invasive infection alone
(31% versus 5%, P = 0.06).
Acute phase plasma samples were collected in heparinised tubes upon study enrolment and
stored at -70oC until use. Convalescent plasma samples were collected at least one month post-
enrolment and once all signs and symptoms of infection had resolved. Plasma concentrations of
angiopoietin-1 and -2 were measured by enzyme-linked immunosorbent assay (ELISA; R&D
Systems, Minneapolis MN) according to the manufacturer‟s instructions. Each sample was tested
in duplicate and all samples were assayed simultaneously. The upper and lower limits of
detection for the assays were 10 000 pg/mL and 9.77 pg/mL for Ang-1 and 2520 pg/mL and 2.46
pg/mL for Ang-2, respectively. Samples were diluted in assay diluent (1:20 for Ang-1 and 1:4
for Ang-2) to fall within the range of the standard curves. Laboratory testing was performed by
an investigator blinded to all clinical data.
Statistical analysis was performed using GraphPad Prism v4.03 and Instat v3.06 (GraphPad
Software, San Diego, CA). To describe the study population, categorical variables were analyzed
by the Fisher‟s exact test and continuous variables with the Student‟s t-test. Differences in
median serum angiopoietin levels were assessed by the Kruskal-Wallis test followed by Dunn‟s
test for multiple comparisons. Matched acute and convalescent samples were compared using the
Wilcoxon matched pairs test. Receiver operating characteristic (ROC) curves were generated
46
using patients with STSS as “cases” and those with invasive streptococcal disease alone as
“controls”, with the null hypothesis that the area under the curve (AUC) equals 0.5.
3.3 Results
Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) was associated with disease
severity in patients with invasive group A streptococcal infection (Figure 1A). The median
plasma concentration of Ang-1 was lower during the acute phase of illness in patients with
invasive infection and STSS than in those with invasive streptococcal infection alone (13 915
pg/mL, [interquartile range (IQR): 5295 - 23 876 pg/mL] vs. 29 084 pg/mL [IQR: 11 855 -
54 803 pg/mL]), while the median plasma concentration of Ang-2 was higher (5752 pg/mL,
[IQR: 1250 - 7695 pg/mL] vs. 1337 pg/mL [IQR: 539 - 2371 pg/mL]). As a result, the normally
low Ang-2:Ang-1 ratio was significantly higher amongst patients with invasive infection and
STSS as compared to those with invasive streptococcal infection alone (0.437 [IQR: 0.122 -
0.775] versus 0.048 [IQR: 0.014 - 0.327], P < 0.05).
Ang-1/2 dysregulation declined with convalescence in both groups of patients (Figure 1A). In the
cohort of patients with STSS, the median plasma concentration of Ang-1 rose from 13,915
pg/mL to 21 115 pg/mL (IQR: 3517 - 41 373 pg/mL), the median plasma concentration of Ang-2
decreased from 5752 pg/mL to 378 pg/mL (IQR: 225 - 1097 pg/mL), P < 0.01, and the median
Ang-2:Ang-1 ratio fell from 0.437 to 0.019 (IQR: 0.011 - 0.139), P < 0.05.
Receiver operating characteristic (ROC) curves were generated for Ang-1, Ang-2, and the Ang-
2:Ang-1 ratio, and the area under the ROC curves indicated that the degree of magnitude of Ang-
1/2 dysregulation accurately differentiated between those individuals with STSS and those
without STSS (Figure 1B). The ROC curves for plasma Ang-2 (AUC: 0.759; 95% confidence
interval (CI): 0.596 - 0.921; P = 0.009) and for the Ang-2:Ang-1 ratio (AUC: 0.791; 95% CI:
0.643 - 0.938; P = 0.003) discriminated between patients with STSS and those with invasive
streptococcal infection alone (no STSS). The ROC curve for plasma Ang-1 concentration
trended towards, but did not reach, statistical difference (AUC: 0.683; 95% CI: 0.506 - 0.860; P
= 0.07).
47
Figure 1. Angiopoietin -1 and -2 (Ang-1 and Ang-2) in patients with invasive Group A streptococcal disease with and without
streptococcal toxic shock syndrome (STSS). A. Absolute and median concentrations of Ang-1 and Ang-2, as well as Ang-2:Ang-1
expressed as log base 10, in acute and convalescent plasma from patients with or without STSS. * P < 0.05; ** P < 0.01. = survived; =
died. B. Receiver operating characteristic curves for each marker: Ang-1 (AUC: 0.683; 95% confidence interval (CI): 0.506 - 0.860; P =
0.07), Ang-2 (AUC: 0.759; 95% CI: 0.596 - 0.921; P = 0.009), and Ang-2:Ang-1 (AUC: 0.791; 95% CI: 0.643 - 0.938; P = 0.003).
48
In individual patients with STSS, the matched acute and convalescent plasma Ang-2
concentrations and the Ang-2:Ang-1 ratios also differed significantly (Figure 2). Although the
same pattern was observed in the cohort of patients with invasive streptococcal disease without
STSS, the changes in Ang-1/2 concentrations were more modest. The median plasma
concentration of Ang-1 in this group increased only from 29 084 pg/mL to 31 743 pg/mL (IQR:
8443 - 54 645 pg/mL), while the Ang-2 concentration declined from 1337 pg/mL to 535 pg/mL
(IQR: 203 - 1702 pg/mL), and the Ang-2:Ang-1 ratio decreased slightly from 0.048 to 0.027
(IQR: 0.008 - 0.091), P = NS for all comparisons.
49
Figure 2. Angiopoietin-1 and -2 (Ang-1 and Ang-2) concentrations, and the ratio between the two (Ang-2:Ang-1), in matched acute and
convalescent plasma samples from patients with STSS. P = 0.01 for Ang-2 in acute vs. convalescent plasma, and P = 0.03 for Ang-2:Ang-
1 in acute vs. convalescent plasma by the Wilcoxon matched pairs test.
50
3.4 Discussion/Conclusion
This study represents the first report of an association between angiopoietin dysregulation and
disease severity in invasive streptococcal infection. Ang-1/2 dysregulation, as reflected by an
increased Ang-2:Ang-1 ratio, was significantly greater during the acute phase of illness in
individuals with STSS than in those without (Figure 1). This finding confirms that angiopoietin
dysregulation in invasive streptococcal disease is specifically related to the degree of endothelial
dysfunction present, and not simply associated with the underlying infection. Further, the
observation is consistent with our current understanding of angiopoietin dysregulation in other
infectious disease syndromes associated with prominent endothelial cell activation. Ang-2 and/or
the Ang-2:Ang-1 ratio have been found to be markers of disease severity in sepsis, ALI/ARDS,
febrile neutropenia, and malaria.9, 130, 133, 134, 320
In our study, ROC curves suggested that both the
absolute Ang-2 concentration and the Ang-2:Ang-1 ratio could effectively discriminate between
patients with and without STSS (Figure 1).
The sequence of events leading to angiopoietin dysregulation during STSS is not yet known. S.
pyogenes produces a variety of factors, including the M proteins, that have been shown to
mediate endothelial cell function.354
Streptococcal superantigens may also be involved, whether
through direct action on the vascular endothelium or through intermediary molecules.
Streptococcal superantigens have long been known to mediate the release of pro-inflammatory
cytokines, including TNF, which might function as a link to the angiopoietin system. Circulating
and tissue-infiltrating mononuclear cells from patients with STSS have been shown to produce
significantly higher levels of TNF and other inflammatory cytokines as compared to cells from
patients with invasive streptococcal infection without shock, and TNF itself has been associated
with the upregulation of Ang-2 expression in human endothelial cells in vitro, although not
definitively with Ang-2 release.7, 11, 355
In turn, Ang-2 is known to sensitize endothelial cells to
the pro-inflammatory effects of TNF,104
reflecting the type of complex interplay between the
cytokine and angiopoietin systems that may occur during STSS. Ultimately, increased Ang-2
release in STSS leads to increased Ang-2 binding to the endothelial Tie-2 receptor, with
subsequent induction of the activated, pro-thrombotic endothelial cell phenotype that contributes
to fluid extravasation, shock, organ dysfunction, and coagulopathy.356
A similar mechanism has
been proposed to explain angiopoietin dysregulation in sepsis.356
Together, our findings as well
51
as those of others are consistent with the possibility of a shared final common pathway of
endothelial activation and diffuse vascular leak in the pathogenesis of sepsis, ALI/ARDS, and
STSS.120, 320, 357
Although the current study cannot prove causation, it suggests that angiopoietin
dysregulation may contribute to disease severity, and provides a rationale for future translational
studies in this area.
Consistent with this hypothesis, convalescence and clinical recovery were associated with
progressive normalization of the balance between Ang-1 and Ang-2, most dramatically in
survivors of STSS (Figure 2). Similar results have been reported in sepsis, in which Ang-2 levels
were also found to decline with convalescence.9 In agreement with the observation that
endothelial cell function (activation versus quiescence) reflects the balance between local
concentrations of both Ang-1 and Ang-2, we observed that the Ang-2:Ang-1 ratio was a
consistent marker of both STSS and convalescence.
There are some notable limitations to this study. The small sample size restricted our ability to
detect potentially important differences, particularly in Ang-1 levels that displayed a trend
towards statistical significance. In addition, the overall mortality rate was low and not
significantly different between those with STSS and those without, which limited the potential
for conclusions regarding the prognostic value of plasma concentrations of Ang-1 and/or Ang-2
for clinical outcome. Finally, since serial samples were not available during the acute phase of
illness in the current study, it is not possible to comment upon the predictive value of early
angiopoietin dysregulation for disease progression and the development of STSS in invasive
streptococcal infection. Elevation of the Ang-2:Ang-1 ratio and serum Ang-2 concentration have
recently been reported to be predictive of the development of septic shock in patients with febrile
neutropenia when measured at or within 48 hours of fever onset, respectively.130
It is therefore
possible that close monitoring of the kinetics of angiopoietin dysregulation in the plasma of
patients with invasive streptococcal disease could provide a reliable prognostic biomarker for
disease progression and severity, and an indicator of response to various therapeutic
interventions.
Despite the above limitations, our study adds to the growing body of evidence implicating
endothelial cell dysfunction and angiopoietin dysregulation in life-threatening infectious disease
syndromes, and suggests that angiopoietin dysregulation may be both a contributor to, and a
52
clinically informative biomarker of, disease severity in invasive streptococcal infection. On this
basis, further studies exploring the potential role of angiopoietin dysregulation in the
pathogenesis of STSS and the predictive value of angiopoietin dysregulation for both the
development of STSS and mortality in invasive streptococcal infection are warranted.
53
Chapter 4 Angiopoietin-1 and -2 in the Hemolytic-Uremic Syndrome
Submitted for Publication, 2012
4.1 Introduction
The haemolytic-uremic syndrome (HUS) is defined by the triad of non-immune hemolytic
anemia, thrombocytopenia, and renal failure.16
Once thought to be a consequence of toxin-
induced glomerular endothelial cell ribosomal arrest and apoptosis, recent evidence suggests a
more complex pathogenesis in HUS, involving Shiga toxin-induced microvascular endothelial
cell activation.23
Although studies have shown that Shiga toxin can induce a prothrombotic and
adhesive endothelial cell phenotype,358
the precise mechanism for endothelial cell
activation/dysfunction in HUS has yet to be definitively determined.
We hypothesized that angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) might
be present and contribute to the pathogenesis of HUS. The bacterial serotype most commonly
implicated in typical HUS is E. coli O157:H7, and those most at risk of developing HUS as a
consequence of STEC hemorrhagic colitis are children.359
Therefore, we studied serum
angiopoietin concentrations at various time points throughout illness in children with E. coli
O157:H7 infection, with and without HUS.
4.2 Methods
Study population and specimen collection
Beginning in 1997, population-based surveillance for E. coli O157:H7 infection in children less
than 10 years of age was undertaken in Washington, Oregon, Idaho, and Wyoming through
mandatory laboratory reporting of positive stool cultures as previously described.10
Children
from whom a positive culture was obtained within the first seven days after the onset of diarrhea
were eligible for enrolment. Seventy-eight children, enrolled between 1998 and 2005, were
included in the current study. Written informed consent from the parents, and assent from the
child where appropriate, was obtained at study entry for the collection of epidemiologic and
clinical data and for phlebotomy both at enrolment and as clinically indicated thereafter. HUS
was diagnosed in the setting of hemolytic anemia (a hematocrit < 30% with evidence of
54
schistocytes on peripheral blood film), thrombocytopenia (platelet count < 150 000 cells/mm3),
and renal insufficiency (serum creatinine above the age-adjusted upper limit of normal);
participants who had not met these criteria by day 14 following the onset of diarrhea were
considered to have had uncomplicated infection.18
In total, 84 serum samples were tested (Figure
3): 26 from patients on the day of diagnosis of HUS, 8 from patients who would subsequently be
diagnosed with HUS but had not yet met diagnostic criteria (pre-HUS), and 50 from patients
with uncomplicated infection. Six patients had samples taken both prior to (pre-HUS) and on the
day of, HUS diagnosis.
Figure 3. Timeline of blood sampling and division into study groups (uncomplicated infection,
pre-HUS, HUS) of participants at various stages of E. coli O157:H7 infection.
Measurement of serum Ang-1 and Ang-2
Serum samples were stored in aliquots at -80oC until use. Serum concentrations of Ang-1 and
Ang-2 were measured by ELISA (R&D Systems, Minneapolis MN) as per the manufacturer‟s
instructions. Each sample was tested in duplicate and all samples were assayed simultaneously.
The technical upper limits of detection for the assay were 10 000 pg/mL for Ang-1 and 2520
Uncomplicated Infection
Pre-HUS and HUS
55
pg/mL for Ang-2, yielding effective upper limits of detection of 200 000 pg/mL and 10 080
pg/mL, respectively, for the dilutions employed in the analysis. Lower limits of detection for the
assay were 9.77 pg/mL for Ang-1 and 2.46 pg/mL for Ang-2.
Statistical analyses
Statistical analysis was performed using GraphPad Prism v.4.03 (San Diego, CA). Median serum
angiopoietin levels were compared using the Kruskal-Wallis test followed by Dunn‟s test for
multiple comparisons. Matched pre-HUS and HUS samples were compared using the Wilcoxon
matched pairs test. Receiver operating characteristic (ROC) curves were generated using pre-
HUS patients as “cases” and those with uncomplicated infection as “controls”, with the null
hypothesis that the area under the curve (AUC) equals 0.5.
4.3 Results
In this study of 78 children with E. coli O157:H7 infection, angiopoietin dysregulation
(decreased Ang-1 and increased Ang-2) was associated with illness severity (Figure 4A). The
median serum Ang-1 concentration in those with uncomplicated infection was significantly
higher than in those with HUS (77 357 pg/mL [interquartile range (IQR): 53 437 - 114 889
pg/mL] versus 10 622 pg/mL [IQR: 3464 - 43 523 pg/mL]), P < 0.001. Conversely, the median
serum Ang-2 concentration was significantly lower in those with uncomplicated infection than in
those with HUS (1140 pg/mL [IQR: 845 - 1492 pg/mL] in patients with uncomplicated infection
versus 1959 pg/mL [IQR: 1057 - 2855 pg/mL] in those with HUS), P < 0.05. Finally, the Ang-
2:Ang-1 ratio was 0.014 (IQR: 0.011 - 0.023) in patients with uncomplicated infection, and more
than 10-fold higher, at 0.18 (IQR: 0.066 - 0.51) in those with HUS, P < 0.001.
56
Figure 4. Serum Angiopoietin Concentration in E. coli O157:H7 Infection. A. Angiopoietin-1
(Ang-1), Angiopoietin-2 (Ang-2), and the Ang-2:Ang-1 ratio expressed as log base 10 in
children with uncomplicated E. coli O157:H7 infection (infected), children prior to the diagnosis
of HUS (pre-HUS), and children at the time of diagnosis of HUS (HUS). *P<0.05; ** P<0.001,
o = outlier (1.5 x interquartile range [IQR]); = extreme outlier (3 x IQR). B. Receiver
Operating Characteristic (ROC) curves comparing Ang-1, Ang-2, and the Ang-1:Ang-2 ratio
amongst children with uncomplicated infection and those in the pre-HUS phase of illness, with
the null hypothesis that the area under the curve is 0.5. P = 0.01 for Ang-1.
57
Furthermore, serum Ang-1 concentration at the time of presentation to hospital effectively
discriminated between two populations of clinically indistinguishable children: 1) those with
uncomplicated hemorrhagic colitis and 2) those with hemorrhagic colitis who would eventually
develop HUS (Area under the ROC curve [AUC]: 0.785, 95% confidence interval (CI): 0.641 -
0.923; P = 0.01) (Figure 4B). A serum Ang-1 cut-off value of less than 48 000 pg/mL yields a
sensitivity of 75% and a specificity of 80% for the diagnosis of the pre-HUS phase of illness.
The ROC curves for the serum Ang-2 concentration and the Ang-2:Ang-1 ratio trended towards,
but did not reach, statistical difference (AUC: 0.600, 95% CI: 0.357 - 0.843; P = 0.37) and AUC:
0.685, 95% CI: 0.499 - 0.872; P = 0.09, respectively).
Data from individual patients closely mirrored the aggregate data presented above. Paired
samples from patients on the day of diagnosis of infection (day of positive stool culture, or pre-
HUS) and on the day of diagnosis of HUS, revealed the same trend: serum concentrations of
Ang-1 fell, while the Ang-2:Ang-1 ratio rose (Figure 5).
58
Figure 5. Serum Angiopoietin Concentration in Individual Patients with E. coli O157:H7
Infection before and after the diagnosis of HUS. Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-
2), and the Ang-2:Ang-1 ratio in matched serum samples from individual patients at the time of
diagnosis of E. coli O157:H7 infection (pre-HUS) and at the time of diagnosis of HUS (HUS).
59
4.4 Discussion/Conclusion
The finding of angiopoietin dysregulation in HUS is consistent with the known biologic effects
of Ang-1 and Ang-2, and with their predicted impact on endothelial cell function during the
course of E. coli O157:H7 infection. Ang-1 and Ang-2 compete for binding to the shared Tie-2
receptor.356
In healthy individuals, constitutive release and binding of Ang-1 results in
phosphorylation of the Tie-2 receptor, inhibition of the NF-κB pathway, and endothelial cell
quiescence.356
In patients with HUS, inflammation-induced release and binding of Ang-2 is
predicted to block the action of Ang-1, release the NF-κB pathway from inhibition, and activate
the endothelium.356
The serum Ang-1 and Ang-2 concentrations reported here for children with
uncomplicated infection are comparable to those found in the serum of healthy children and
adults included as control subjects in other studies, and are in keeping with the clinical
observation that there is little if any endothelial dysfunction present in these patients.124, 133
In
contrast, the relative deficit of Ang-1 and excess of Ang-2 in children with HUS would be
expected to produce significant endothelial cell activation. As a consequence, constitutive Ang-1
inhibition of the actions of TNF and VEGF, upregulated by Shiga toxin or in HUS
respectively,360, 361
would be diminished, leading to increased expression of tissue factor,
VCAM-1, ICAM-1, and E-selectin on the endothelial cell surface.81, 90
Furthermore, Ang-2 itself
has been shown to sensitize endothelial cells to the effects of TNF.104
The endothelial phenotype
that is predicted to result from an increased Ang-2:Ang-1 ratio is therefore prothrombotic and
adhesive, and in keeping with the characteristic thrombotic microangiopathy of HUS. Our
observations are similar to those made in other disorders of endothelial cell function, including
sepsis, cerebral malaria, and streptococcal toxic shock.81, 90, 352
In each of these infectious
syndromes, an association has been described between the degree of angiopoietin dysregulation
and the severity of illness, and likely reflects a progressive degree of endothelial dysfunction.
Taken together, these findings suggest that angiopoietin dysregulation may represent a final
common pathway through which various infectious insults act to promote endothelial cell
activation.
In HUS, angiopoietin dysregulation likely occurs either directly or indirectly through the action
of the Shiga toxin. Angiopoietin-2 is stored in the Weibel-Palade bodies of endothelial cells for
rapid release upon detection of an inflammatory stimulus.7 Binding of the Shiga toxin to its
receptor, globotriaosylceramide (Gb3), concentrated in the renal microvasculature, may directly
60
stimulate Weibel-Palade body exocytosis, a hypothesis in keeping with the finding that other
constituents of Weibel-Palade bodies, such as von Willebrand Factor (vWF) are released after
Shiga toxin exposure in vitro.19
Alternatively, Shiga toxin may act indirectly through a mediator
such as VEGF, known to stimulate Weibel-Palade body release.362
In contrast, the decline in serum Ang-1 levels may reflect Shiga toxin-induced injury to the
pericytes, or frank thrombocytopenia in the case of children with HUS. However, the Ang-1
concentration declines in the pre-HUS phase of illness, before the onset of thrombocytopenia,
making the latter an unlikely mechanism.
Previous work by Chandler et al has shown that children in the pre-HUS phase of illness have
normal blood cell counts, yet show evidence of early vascular injury with levels of prothrombin
fragment 1+2, tissue plasminogen activator (tPA) antigen, tPA-plasminogen-activator inhibitor
type 1 complex, and D-dimer, that are significantly higher than those in children with
uncomplicated infection.18
Our work supports the concept that subclinical endothelial
dysfunction precedes the development of overt HUS and can be detected before the classically
recognized markers of disease.
Serum or plasma levels of Ang-1/2 have also been proposed as clinically informative
biomarkers in several other important clinical syndromes, including: sepsis, cerebral malaria,
severe bacterial infection in children.127, 129, 130, 305
Although previous studies have investigated
the role of markers of inflammation, such as procalcitonin and interleukin-6, in established HUS,
our study is unique in that it demonstrates the potential utility of a biomarker to detect impending
HUS in the setting of E. coli O157:H7 infection, a stage of illness for which no diagnostic tests
currently exist.363, 364
Although the degree of overlap between the groups precludes the use of
Ang-1 as a definitive single predictor of HUS, one or both of the angiopoietins might be
combined with other early markers of endothelial cell dysfunction, such as those described by
Chandler et al, to derive useful discriminatory or predictive classification rules. Our study
therefore provides a rationale for prospective validation of circulating angiopoietins as outcome-
specific biomarkers for children with E. coli O157:H7 infection. If the utility of one or more
biomarkers were to be confirmed clinically, it might provide the opportunity to initiate early
aggressive intervention, particularly fluid resuscitation, which has been shown to limit morbidity
in children with E. coli O157:H7 infection who ultimately progress to HUS.365
61
There are some important limitations to our study. We chose to investigate E. coli O157:H7-
associated HUS because it remains the most common serotype, and prior to the European
outbreak, was associated with the most severe disease and sequelae.359
Although the clinical
syndrome of HUS is largely the same regardless of serotype, we cannot conclusively state that
our findings apply to illness caused by non-O157 serotypes. In addition, we cannot exclude the
impact of renal failure itself on angiopoietin dysregulation in HUS. However, renal function was
intact in children in the pre-HUS phase of illness, and therefore, this limitation should not detract
from the finding that vascular injury precedes the overt clinical syndrome of HUS, nor from the
utility of Ang-1 to discriminate between children with uncomplicated infection and those in the
pre-HUS phase of illness. Finally, although we cannot exclude a contribution of platelet-derived
Ang-1, due to ex vivo activation of platelets after venipuncture, to the high serum Ang-1
concentrations found in individuals with uncomplicated infection, we would expect the degree of
ex vivo activation to be the same between the children with uncomplicated infection and those in
the pre-HUS phase of illness. Therefore, it is unlikely that this particular limitation influenced
the relative difference in serum Ang-1 concentration between these two groups.
In conclusion, this study is the first to demonstrate angiopoietin dysregulation in E. coli O157:H7
infection, in which the degree of dysregulation is associated with disease severity and predictive
of disease progression upon presentation with bloody diarrhea. If these observations are
confirmed in prospective clinical studies, peripheral blood levels of Ang-1/2 may be clinically
useful to identify children with E. coli O157:H7 infection at high risk of progression to HUS.
62
Chapter 5 Prognostic Biomarkers for Sepsis in a Predominantly HIV-infected
Population in a Resource-limited Setting
5.1 Introduction
Sepsis is a significant cause of infection-related mortality in high-income countries, and,
although data are limited, an emerging and increasingly recognized issue in low- to middle-
income countries.366
In both settings, careful and directed triage of limited resources and
personnel is required, yet at present there is no well-defined, rapid, and specific test to identify
those patients at highest risk of mortality, and therefore most in need of aggressive therapeutic
intervention.
The clinical manifestations of sepsis occur because of an exaggerated response - both
immunologic and vascular - to an infection, typically bacterial. Angiopoietin-1 and -2, sTie-2,
procalcitonin, C-reactive protein, sTREM-1, Chi3L1, IP-10, PF4, vWF, ICAM-1, VEGF, and
sFlt-1 have each been proposed as individual prognostic biomarkers in sepsis. However, as
described in Chapter 1, no single biomarker has been demonstrated to have adequate sensitivity
and specificity in all populations tested, and controversy persists in the literature surrounding
each candidate molecule. There are few studies in which the prognostic utility of these markers
has been explored in combination, and even fewer in resource-limited settings.129, 305
Therefore, the aim of this study is twofold: 1) to determine whether a combination of
biomarkers, representing activation of both the immunologic and vascular pathways, can predict
mortality from an episode of sepsis in a predominantly HIV-infected population in a resource-
limited setting, and 2) to explore the utility of each of these markers, or a combination thereof, to
predict the etiology of an episode of sepsis in the same setting.
5.2 Methods
Study population and enrolment criteria
Beginning in 2009, consecutive patients who presented on weekdays to the Emergency
Departments of two large referral hospitals in Uganda (Mulago Hospital in Kampala and Masaka
Regional Referral Hospital in Masaka) were evaluated for trial enrolment. Inclusion criteria were
63
defined as 2 of 4 criteria for SIRS or thermal dysregulation alone, in combination with systolic
blood pressure ≤ 100 mmHg, suspected infection as per the admitting physician, and an elevated
plasma lactate level (>2.5 mmol/L) or low Karnofsky Performance score (≤ 40). Use of the
Karnofsky Performance Score, a well-described and commonly cited morbidity assessment tool,
for this indication had been investigated previously and found to correlate well with mortality in
Ugandan patients with sepsis.367
Exclusion criteria included: age < 18 years, pregnancy,
gynecologic or surgical presenting illness, acute stroke, and gastrointestinal hemorrhage.
Informed consent was obtained from the patient and her/his relative(s), and demographic,
clinical, biochemical, microbiologic, and outcome data were collected. Resources available in
each hospital for the diagnosis and treatment of infection were previously outlined by
Jacob et al. 367
In total, 336 patients were included in this study, of whom 170 (50.6%) were female. The median
age at enrolment was 34 years (range: 18 - 73 years). 282 patients (84%) had HIV disease, and
amongst those, the median CD4+ T cell count was 45 cells/μL (range 1 - 746 cells/μL). Previous
studies in this population indicated that only 12% of the patients infected with HIV were actively
receiving antiretroviral therapy.367
Fourteen percent of patients had positive malaria smears. At
enrolment, the median lactate level was 3.9 mmol/L (range 1 - 16.6 mmol/L) and the median
Karnofsky Performance score was 40 (range 10 - 80). The median white blood cell count was 4.8
x 109 cells/L (range 0.1 - 82 x 10
9 cells/L).
In-hospital mortality data was available for 332 of 336 patients, of whom 89 of 332 (27%) died.
Twenty-eight day mortality data was available for 306 of 336 patients, of whom 119 (39%) died.
In a univariate analysis of positive microbiologic results (any mycobacterial culture, aerobic
blood culture, or malaria smear), only a positive mycobacterial culture was significantly
associated with a risk of death (data not shown).
The candidate was not involved in this aspect of the study.
Biomarker Assays
Plasma was obtained at study enrolment, divided into aliquots, and stored at -70oC until use.
Care was taken to maintain the frozen samples during prolonged transportation and shipping.
Plasma concentrations of all biomarkers were measured by ELISA (R&D Systems, Minneapolis
64
MN except where indicated) as per the manufacturer‟s instructions. The technical upper limits of
detection for the assays were 250 ng/mL for vWF, 20 000 pg/mL for Ang-1, sTie-2, and sFlt-1,
7000 pg/mL for Ang-2, 5000 pg/mL for PF4, and 4000 pg/mL for C-reactive protein, sTREM-1,
Chi3L1, IP-10, ICAM-1, and VEGF, yielding effective upper limits of detection of 500 μg/mL
for vWF, 80 000 pg/mL for Ang-1, 400 000 pg/mL for sTie-2, 40 000 pg/mL for sFlt-1, 28 000
pg/mL for Ang-2, 50 ng/mL for PF4, 400 g/mL for CRP, 4000 pg/mL for sTREM-1, 2 ng/mL
for Chi3L1, 8000 pg/mL for IP-10, 4 ng/mL for ICAM-1, and 8000 pg/mL for VEGF, for the
dilutions employed in the analysis. Lower limits of detection were as follows: 244 pg/mL for
vWF, 19.5 pg/mL for Ang-1, sTie-2, and sFlt-1, 6.84 pg/mL for Ang-2, 4.88 pg/mL for PF4, and
3.91 pg/mL for CRP, sTREM-1, Chi3L1, IP-10, ICAM-1, and VEGF. Plasma concentrations of
procalcitonin were measured by ELISA (RayBiotech Inc, Norcross GA) as per the
manufacturer‟s instructions, with a technical upper limit of detection of 60 000 pg/mL, an
effective upper limit of detection of 300 000 pg/mL, and a lower limit of detection of 58.6
pg/mL.
All assays were performed by an investigator blinded to clinical and microbiologic data, as well
as outcome, and each sample was tested in duplicate.
Statistical Analyses
Statistical analysis was performed using GraphPad Prism v.4.03 (San Diego CA) and SPSS
v.16.0 (IBM, Chicago IL). Median plasma levels of all biomarkers upon enrolment in survivors
and non-survivors were compared using the Mann-Whitney test. Receiver operating
characteristic (ROC) curves were generated for each biomarker for the prediction of in-hospital
and 28-day mortality, as well as positive mycobacterial or aerobic bacterial cultures, with the
null hypothesis that the area under the curve (AUC) equals 0.5. Classification and Regression
Tree (CaRT) analysis was performed to predict the outcome (survival versus death) of an episode
of sepsis or the likelihood of a laboratory-confirmed microbiologic diagnosis by a combination
of clinical factors (Karnofsky Performance score) and host biomarkers measured upon first
presentation to hospital. CaRT analysis has been used extensively in biomarker studies in cancer,
cardiology, and infectious diseases, among other disciplines, and uses recursive partitioning to
derive cut-points for the independent variables that are then combined to create an algorithm that
can be used to classify patients according to the dependent variable.305, 368, 369
All biomarkers that
65
discriminated between survivors and non-survivors for each of the primary (in-hospital) and
secondary (28-day mortality, positive aerobic culture, and positive mycobacterial culture)
endpoints on univariate analysis were entered into each combinatorial analysis. The cost of
misclassifying an eventual death or positive culture was considered to be 5 times that of
misclassifying survival or a negative culture. CaRT analysis was chosen for this study because it
yields a clinically relevant and easily followed algorithm, and because it makes no assumptions
about the distribution or completeness of the data.370
Because over-fitting, the creation of an
excessively complex tree that does not generalize well, is a potential limitation of CaRT analysis,
the following criteria for tree construction were specified: minimum 10 cases for a parent node
and 5 for a child node, customized prior probabilities based on a prospective observational study
undertaken at the same sites,367
maximum depth of 3 nodes, and pruning to reduce overfitting.
The cut-points as selected by the analysis for each biomarker are indicated between parent and
child nodes, while the predicted classification of patients in each terminal node (no further
branching) is highlighted.
5.3 Results
In this large cohort of Ugandan patients presenting to the Emergency Department with sepsis,
multiple biomarkers were significantly associated with in-hospital mortality (Figure 6). In
particular, angiopoietin dysregulation (an increase in Ang-2 and the Ang-2:Ang-1 ratio) was
more severe in those patients who later died during hospital admission. The median plasma Ang-
2 level was 1927 pg/mL (IQR: 1073 - 5494 pg/mL) in non-survivors and significantly lower at
1366 pg/mL (IQR: 748 - 3122 pg/mL) in survivors, P < 0.01, while the median Ang-2:Ang-1
ratio was 0.40 (IQR: 0.17 - 1.9) in non-survivors and only 0.23 (IQR: 0.074 - 0.84) in survivors,
P < 0.01. In addition, biomarkers representing both the inflammatory (procalcitonin, sTREM-1,
Chi3L1, and IP-10) and vascular (vWF, ICAM-1, and sFlt-1) pathways were each significantly
different in those patients who survived to hospital discharge versus those who did not. The
median plasma procalcitonin level was significantly elevated in non-survivors (11 104 pg/mL,
IQR: 2666 - 34 475 pg/mL) as compared to survivors (6425 pg/mL, IQR: 815 - 23 411 pg/mL),
P < 0.05, as were the median concentrations of sTREM-1 (247 pg/mL, [IQR: 107 - 446 pg/mL]
versus 124 pg/mL, [IQR: 83 - 223 pg/mL], P < 0.001), Chi3L1 (813 ng/mL, [IQR: 253 - 1589
ng/mL] versus 359 ng/mL, [IQR: 178 - 827 ng/mL], P < 0.001), and IP-10 (771 pg/mL, [IQR:
309 - 1273 pg/mL] versus 540 pg/mL, [IQR: 256 - 942 pg/mL], P < 0.01), all in non-survivors
66
versus survivors, respectively. Amongst markers of endothelial or vascular activation, the
median plasma concentration of vWF was significantly higher in patients who died prior to
hospital discharge than in those who survived (265 ng/mL, [IQR: 188 - 394 ng/mL] versus 186
ng/mL, [IQR: 98 - 297 ng/mL], P < 0.001), as was also true of the median plasma concentrations
of ICAM-1 (564 ng/mL, [IQR: 357 - 929 ng/mL] versus 439 ng/mL, [IQR: 303 - 608 ng/mL], P
< 0.001), and sFlt-1 (1133 pg/mL, [IQR: 39 - 3051 pg/mL] versus 319 pg/mL, [IQR: 39 - 1596
pg/mL], P < 0.01).
ROC curves constructed for each of these biomarkers indicated that all nine indices were
predictive of in-hospital morality, albeit to differing degrees: Ang-2 (AUC: 0.597, 95% CI: 0.529
- 0.665; P < 0.01), the Ang-2:Ang-1 ratio (AUC: 0.611, 95% CI: 0.544 - 0.677; P < 0.01),
procalcitonin (AUC: 0.571, 95% CI: 0.503 - 0.639, P < 0.05), sTREM-1 (AUC: 0.668, 95% CI:
0.599 - 0.738, P < 0.001), Chi3L1 (AUC: 0.634, 95% CI: 0.564 - 0.705, P < 0.001), IP-10
(AUC: 0.610, 95% CI: 0.541 - 0.680, P < 0.01), vWF (AUC: 0.645, 95% CI: 0.580 - 0.710, P <
0.001), sICAM-1 (AUC: 0.634, 95% CI: 0.564 - 0.705, P < 0.001), and sFlt-1 (AUC: 0.600, 95%
CI: 0.529 - 0.671, P < 0.01) (data not shown). ROC curves for the other biomarkers tested did
not meet statistical significance (data not shown). Although the ROC curve for sTREM-1
demonstrated the greatest AUC, a plasma sTREM-1 cut-off value of 164.7 pg/mL yielded only
62% sensitivity and specificity for the prediction of in-hospital mortality at the onset of an
episode of sepsis in this population.
Both plasma lactate and the clinical Karnofsky Performance score were significantly different
between those patients who died prior to hospital discharge and those who survived: 4.4 mmol/L
(IQR: 3.3 - 5.7 mmol/L) versus 3.8 mmol/L (IQR: 3.0 - 4.6 mmol/L), P < 0.001, and 30 (IQR: 30
- 40) versus 40 (IQR: 30 - 50), P < 0.001, respectively (data not shown). ROC curves for both
were significantly predictive of in-hospital mortality, however, plasma lactate performed slightly
worse (AUC: 0.609, 95% CI: 0.543 - 0.676, P < 0.001) than many of the newer biomarkers
discussed above, and Karnofsky Performance score (AUC: 0.668, 95% CI: 0.609 - 0.728, P <
0.001) approximately equally (data not shown).
68
Figure 6. Plasma Biomarkers Associated with In-hospital Mortality in Ugandan patients with
sepsis. Absolute and median concentrations of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2),
Ang-2:Ang-1 ratio, soluble Tie-2 receptor (sTie-2), C-reactive protein (CRP), Procalcitonin
(PCT), soluble Triggering Receptor Expressed on Myeloid cells-1 (sTREM-1), Chitinase-3-like
protein 1 (Chi3L1), Interferon-γ-inducible Protein-10 kDa (IP-10), Platelet Factor 4 (PF4), von
Willebrand Factor (vWF), soluble Intercellular Adhesion Molecule-1 (sICAM-1), Vascular
Endothelial Growth Factor (VEGF), and soluble Fms-like tyrosine kinase-1 (sFlt-1) at the time
of hospital admission in patients with sepsis, divided according to in-hospital mortality . * P <
0.05; ** P < 0.01; *** P < 0.001.
Multiple biomarkers were also significantly associated with 28-day mortality (Figure 7). With
one exception, these markers were identical to the subset that predicted in-hospital mortality.
Again, angiopoietin dysregulation at hospital admission was significantly greater in those
patients who died in the subsequent 28 days than in those who survived. The median plasma
concentration of Ang-2 was 1927 pg/mL (IQR: 871 - 5427 pg/mL) in non-survivors and
significantly lower at 1381 pg/mL (IQR: 758 - 3077 pg/mL) in survivors, P < 0.05, while the
median Ang-2:Ang-1 ratio was 0.49 (IQR: 0.17 - 1.8) in non-survivors and 0.21 (IQR: 0.072 -
0.68) in survivors, P < 0.001. Again mirroring the results found with in-hospital mortality,
biomarkers representing both the inflammatory (sTREM-1, Chi3L1, and IP-10) and vascular
(PF4, vWF, sICAM-1, and sFlt-1) pathways were each significantly different in patients who
survived for at least 28 days after hospital admission versus those who did not. The median
plasma concentrations of sTREM-1 (178 pg/mL, [IQR: 98 - 385 pg/mL] versus 127 pg/mL,
[IQR: 85 - 225 pg/mL], P < 0.01), Chi3L1 (749 ng/mL, [IQR: 228 - 1309 ng/mL] versus 352
ng/mL, [IQR: 154 - 723 ng/mL], P < 0.001), and IP-10 (771 pg/mL, [IQR: 300 - 1319 pg/mL]
versus 504 pg/mL, [IQR: 259 - 870 pg/mL], P < 0.001), all differed significantly amongst non-
survivors versus survivors at 28 days, respectively. Similarly, the median plasma concentrations
of vWF (260 ng/mL, [IQR: 175 - 387 ng/mL] versus 172 ng/mL, [IQR: 89 - 281 ng/mL], P <
0.001), ICAM-1 (536 ng/mL, [IQR: 357 - 880 ng/mL] versus 425 ng/mL, [IQR: 305 - 601
ng/mL], P < 0.001), and sFlt-1 (1159 pg/mL, [IQR: 39 - 3051 pg/mL] versus 125 pg/mL, [IQR:
39 - 1224 pg/mL], P < 0.001) were all significantly higher at hospital admission in those patients
69
who died within 28 days than in those who survived (non-survivor versus survivor, respectively,
for each biomarker). In contrast, the median plasma concentration of PF4 at hospital admission
demonstrated the opposite pattern and was significantly lower in non-survivors (944 ng/mL,
IQR: 342 - 2150 ng/mL) than survivors (1244 ng/mL, IQR: 942 - 3535 ng/mL), P < 0.05.
Notably, the median plasma procalcitonin level upon presentation to hospital did not differ
significantly amongst survivors and non-survivors at 28 days as it did amongst survivors and
non-survivors at hospital discharge, while the opposite was true of the median plasma PF4
concentration.
Each of these nine biomarkers discriminated between survivors and non-survivors at 28 days
according to the area under the ROC curve: Ang-2 (AUC: 0.579, 95% CI: 0.513 - 0.644; P <
0.05), the Ang-2:Ang-1 ratio (AUC: 0.619, 95% CI: 0.554 - 0.683; P < 0.001), sTREM-1 (AUC:
0.607, 95% CI: 0.540 - 0.675, P < 0.01), Chi3L1 (AUC: 0.639, 95% CI: 0.574 - 0.703, P <
0.001), IP-10 (AUC: 0.619, 95% CI: 0.554 - 0.685, P < 0.001), PF4 (AUC: 0.587, 95% CI: 0.521
- 0.652, P < 0.05), vWF (AUC: 0.663, 95% CI: 0.602 - 0.724, P < 0.001), sICAM-1 (AUC:
0.627, 95% CI: 0.562 - 0.693, P < 0.001), and sFlt-1 (AUC: 0.631, 95% CI: 0.564 - 0.698, P <
0.001) (data not shown). However, while statistically significant, none of these indices were
sufficiently sensitive and specific to be clinically useful as the definitive and sole prognostic
indicator in patients presenting to the Emergency Department with sepsis.
Again, both lactate (4.1 mmol/L [IQR: 3.3 - 5.6 mmol/L] versus 3.8 mmol/L [IQR: 3.0 - 4.6
mmol/L], P < 0.01 in non-survivors and survivors, respectively) and Karnofsky Performance
score (30 [IQR: 30 - 40] versus 40 [IQR: 30 - 50], P < 0.001 in non-survivors and survivors,
respectively) differed according to 28-day mortality. ROC curves indicated that both were
predictive of 28-day mortality - lactate AUC: 0.578, 95% CI: 0.518 - 0.639, P < 0.01, and
Karnofsky Performance score AUC: 0.656, 95% CI: 0.601 - 0.712, P < 0.001 - but neither was
sufficiently predictive for use as the sole predictive marker in a clinical setting.
71
Figure 7. Plasma Biomarkers Associated with 28-day Mortality in Ugandan patients with sepsis.
Absolute and median concentrations of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Ang-
2:Ang-1 ratio, soluble Tie-2 receptor (sTie-2), C-reactive protein (CRP), Procalcitonin (PCT),
soluble Triggering Receptor Expressed on Myeloid cells-1 (sTREM-1), Chitinase-3-like protein
1 (Chi3L1), Interferon-γ-inducible Protein-10 kDa (IP-10), Platelet Factor 4 (PF4), von
Willebrand Factor (vWF), soluble Intercellular Adhesion Molecule-1 (sICAM-1), Vascular
Endothelial Growth Factor (VEGF), and soluble Fms-like tyrosine kinase-1 (sFlt-1) at the time
of hospital admission in patients with sepsis, divided according to 28-day mortality . * P < 0.05;
** P < 0.01; *** P < 0.001.
CaRT analysis was undertaken to determine whether a combination of biomarkers might be more
clinically useful for the prediction of mortality at the time of presentation to the Emergency
Department. With in-hospital mortality defined as the dependent variable and those biomarkers
that had effectively discriminated between survivors and non-survivors in univariate analyses
defined as independent variables, CaRT analysis identified a model incorporating sTREM-1 (cut-
off value > 246 pg/mL), the Karnofsky Performance score (cut-off value ≤ 30), and Ang-2 (cut-
off value > 1404 pg/mL) that predicted in-hospital mortality with a sensitivity of 97% (Figure
8A). Sensitivity was not appreciably increased when the model was expanded to incorporate
additional biomarkers, nor when either of the standard predictive measures (Karnofsky
Performance score or lactate) were specified a priori as the first variable in the model (data not
shown). Furthermore, a model incorporating only the Karnofsky Performance score and lactate
yielded a higher misclassification rate (data not shown). For the prediction of 28-day mortality,
CaRT analysis yielded a model that incorporated vWF (cut-off value 194 ng/mL), the Ang-
2:Ang-1 ratio (cut-off value > 0.23), and Chi3L1 (cut-off value > 84 ng/mL), and identified non-
survivors with 93% sensitivity (Figure 8B).
72
Figure 8. Classification and Regression Tree (CaRT) analysis of biomarker combinations to predict mortality following an episode of
sepsis in a Ugandan population. A. sTREM-1, Ang-2, and the Karnofsky Performance score predict in-hospital mortality: sensitivity
97%, specificity 38%, misclassification rate 26%, standard error 2.4%. B. vWF, Ang-2:Ang-1, and Chi3L1 predict 28-day mortality:
sensitivity 93%, specificity 38%, misclassification rate 20%, standard error 2.3%.
A. B.
73
In addition to prognostic indicators, we sought to identify those biomarkers that might be
associated with specific microbial etiologies. Of 226 patients who had blood cultures drawn, 37
(16%) were positive. This is in keeping with the incidence of positive cultures in previous studies
in this population.367
The median plasma concentrations for 5 of 12 biomarkers tested were
significantly different at hospital admission in those patients with positive aerobic blood cultures
than in those patients whose blood cultures remained negative: CRP (178 μg/mL, [IQR: 98 - 385
μg/mL] versus 127 μg/mL, [IQR: 85 - 225 μg/mL], P < 0.01), PCT (749 pg/mL, [IQR: 228 -
1309 pg/mL] versus 352 pg/mL, [IQR: 154 - 723 pg/mL], P < 0.001), sTREM-1 (771 pg/mL,
[IQR: 300 - 1319 pg/mL] versus 504 pg/mL, [IQR: 259 - 870 pg/mL], P < 0.001), Chi3L1 (851
ng/mL, [IQR: 441 - 1992 ng/mL] versus 295 ng/mL, [IQR: 151 - 827 ng/mL], P < 0.001), and
sICAM-1 (500 ng/mL, [IQR: 386 ng/mL - 695 ng/mL], versus 407 ng/mL, [IQR: 287 - 603
ng/mL], P < 0.05), in patients with positive and negative aerobic blood cultures, respectively
(Figure 9A).
Each of these five biomarkers discriminated between those patients with positive blood cultures
and those without as per the area under the ROC curves for each (Figure 9B): CRP (AUC: 0.614,
95% CI: 0.514 - 0.714, P < 0.05), PCT (AUC: 0.757, 95% CI: 0.667 - 0.847, P < 0.001),
sTREM-1 (AUC: 0.623, 95% CI: 0.523 - 0.723, P < 0.05), Chi3L1 (AUC: 0.730, 95% CI: 0.650
- 0.809, P < 0.001), and sICAM-1 (AUC: 0.609, 95% CI: 0.518 - 0.700, P < 0.05). PCT and
Chi3L1 were the best predictors of positive aerobic blood cultures from amongst the five
significant biomarkers, and this was confirmed in a CaRT analysis, in which these two markers,
with the addition of sTREM-1, predicted positive blood cultures with a specificity of 86%
(Figure 9C).
Neither lactate nor Karnofsky performance score differed amongst patients with and without
positive aerobic blood cultures (data not shown).
75
Figure 9. Plasma biomarkers associated with positive aerobic blood cultures. A. Individual and
median plasma biomarker concentrations in patients with positive and negative aerobic blood
cultures. * P < 0.05; ** P < 0.01; *** P < 0.001. C-reactive protein (CRP), Procalcitonin (PCT),
soluble Triggering Receptor Expressed on Myeloid cells-1 (sTREM-1), Chitinase-3-like protein
1 (Chi3L1), and soluble Intercellular Adhesion Molecule-1 (sICAM-1). B. ROC curves for each
biomarker that significantly predicted a positive aerobic blood culture. C. CaRT analysis of a
biomarker combination to predict positive aerobic blood cultures at the time of hospital
admission.
A similar analysis was performed to examine biomarkers predictive of mycobacterial infection.
Results were positive in 71 of 243 patients (29%) from whom mycobacterial cultures were taken,
consistent with the findings of previous studies in the same population.367
In patients presenting
to the Emergency Department with sepsis, all biomarkers tested were significantly different
between those with and without mycobacterial infection except for sTie-2, sFlt-1, and VEGF
(Table 2). Furthermore, ROC curves confirmed that each of these markers were predictive of
positive mycobacterial culture results, albeit to varying degrees. sTREM-1 and IP-10 had the
highest and second highest AUCs by ROC curve analysis, respectively, and CaRT analysis
included both in a model that predicted positive mycobacterial culture results with 79%
sensitivity and 76% specificity (Figure 10). Although plasma lactate was significantly different
between those with and without mycobacterial infection, the associated AUC from the ROC
curve analysis was lower than that of most of the other biomarkers tested (data not shown).
Karnofsky performance score was not significantly different between patients with and without
mycobacterial infection (data not shown).
Interestingly, the most commonly used indicator of infection of any type, leukocyte count, was
not significantly different at hospital admission in those patients with and without either
bacteremia or mycobacterial infection (data not shown). Furthermore, although the white blood
cell count was slightly lower in non-survivors than survivors, and although this difference did
reach statistical significance (P < 0.05), admission white blood cell count was very poorly
76
discriminative of both in-hospital and 28-day mortality, and was outperformed by all other
statistically significant biomarkers (data not shown).
77
Table 2. Significant predictors of mycobacterial infection in Ugandan adults with sepsis. Angiopoietin-1 (Ang-1), Angiopoietin-2
(Ang-2), Ang-2:Ang-1 ratio, C-reactive protein (CRP), Procalcitonin (PCT), soluble Triggering Receptor Expressed on Myeloid cells-1
(sTREM-1), Chitinase-3-like protein 1 (Chi3L1), Interferon-γ-inducible Protein-10 kDa (IP-10), Platelet Factor 4 (PF4), von Willebrand
Factor (vWF), and soluble Intercellular Adhesion Molecule-1 (sICAM-1).
Median Plasma Concentration (IQR) Area under the ROC curve (95% CI)
Biomarker Positive
Mycobacterial culture
Negative
Mycobacterial culture
P P
Ang-1 (pg/mL) 3835 (1425 - 11 032) 5598 (2529 - 13 549) < 0.05 0.588 (0.522 - 0.665) < 0.05
Ang-2 (pg/mL) 2043 (1099 - 5078) 1382 (778 - 3113) < 0.01 0.606 (0.533 - 0.680) < 0.01
Ang-2:Ang-1 0.660 (0.152 - 1.92) 0.225 (0.078 - 0.770) < 0.001 0.641 (0.569 - 0.713) < 0.001
CRP (μg/mL) 134 (78.3 - 221) 84.9 (35.2 - 169) < 0.001 0.631 (0.561 - 0.701) < 0.001
PCT (pg/mL) 13 709 (4858 - 44 738) 5605 (440 - 20 800) < 0.001 0.635 (0.563 - 0.707) < 0.001
sTREM-1 (pg/mL) 294 (160 - 458) 117 (82 - 223) < 0.001 0.777 (0.720 - 0.834) < 0.001
Chi3L1 (ng/mL) 726 (343 - 1309) 348 (172 - 932) < 0.001 0.657 (0.588 - 0.725) < 0.001
IP-10 (pg/mL) 901 (670 - 1342) 444 (216 - 884) < 0.001 0.754 (0.697 - 0.811) < 0.001
PF4 (ng/mL) 794 (317 - 2139) 1180 (509 - 3271) < 0.05 0.587 (0.512 - 0.662) < 0.05
vWF (μg/mL) 249 (177 - 367) 192 (91 - 302) < 0.01 0.625 (0.559 - 0.691) < 0.01
sICAM-1 (ng/mL) 592 (386 - 815) 432 (294 - 631) < 0.001 0.647 (0.577 - 0.716) < 0.001
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Figure 10. Classification and Regression Tree (CaRT) analysis for the prediction of positive
mycobacterial cultures in Ugandan adults with sepsis. Interferon-γ-inducible protein-10 kDa (IP-
10), soluble Triggering Receptor Expressed on Myeloid cells-1 (sTREM-1). Sensitivity 79%,
specificity 76%.
79
5.4 Discussion/Conclusion
The current study of 336 Ugandan patients with sepsis is, to our knowledge, the largest study
published to date that has investigated the role of prognostic biomarkers in a predominantly HIV-
infected population in a resource-limited setting. As a result, a number of unique conclusions can
be drawn.
First, angiopoietin dysregulation is a prominent feature of sepsis in this population, and both the
plasma Ang-2 concentration and the Ang-2:Ang-1 ratio at the time of hospital admission predict
in-hospital mortality and mortality at 28 days. Angiopoietin dysregulation, manifested primarily
by increased Ang-2 resulting in an increased Ang-2:Ang-1 ratio, has been found in septic
patients in high-income countries, and in these settings has been found to correlate with severity
of illness, pulmonary edema, ALI/ARDS, multi-organ dysfunction, and mortality.9, 120-123, 127
The
increase in Ang-2 in sepsis may reflect Weibel-Palade body exocytosis in response to reduced
endothelial nitric oxide bioavailability, increased sphingosine-1-phosphate, or perturbation of
another as yet unidentified factor.5, 53, 123
Since many proposed biomarkers of infection have shown wide variation in performance
characteristics when studied in different patient populations, our study is significant in that it
confirms that the association between angiopoietin dysregulation and mortality in sepsis is
present in a predominantly HIV-infected, significantly immunosuppressed population in which
the median CD4+ T cell count was 45 cells/L. Mankhambo et al conducted the only other
investigation of angiopoietin dysregulation in a similar population: a study of 293 Malawian
children with meningitis or pneumonia of whom 53% were HIV-infected and in whom a low
Ang-1 concentration was associated with mortality in a multivariate analysis.129
Our study is also unique in that, in addition to Ang-2 and the Ang-2:Ang-1 ratio, sTREM-1,
Chi3L1, IP-10, vWF, sICAM-1, and sFlt-1 were all predictive both of in-hospital and 28-day
mortality in this septic population, while PCT and PF4 were predictive of in-hospital and 28-day
mortality, respectively. This is the first study to document an association between mortality in
sepsis and Chi3L1 or PF4, and the second to document the same association with IP-10. Chi3L1
has only been examined once before in sepsis, in a small study of 45 patients in which it was
associated with illness severity but not mortality,302
although elevated Chi3L1 has been
associated with mortality following pneumococcal bacteremia.300
PF4 had previously been
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studied in 10 patients with sepsis, but no attempt was made to correlate increased circulating
levels of PF4 with mortality.314
Finally, IP-10 had previously been studied only in 16 patients
with sepsis, but despite the small sample size elevated levels were nonetheless found to correlate
with mortality.303
Interestingly, all of the biomarkers studied here were higher in non-survivors
than survivors, with the exception of PF4, which demonstrated the opposite pattern. PF4 is
known to facilitate generation of the anticoagulant molecule activated protein C, and, in keeping
with our findings, increased levels of PF4 have been shown to correlate with survival in animal
models of sepsis.312, 313
While the literature is generally in agreement that sTREM-1 can predict prognosis in sepsis,
there is some controversy regard the direction of change. Our results coincide with those of
Zhang et al and Dimopoulou et al, both of whom described higher levels of sTREM-1 in non-
survivors, but conflict with those of Gibot et al, who describe lower sTREM-1 in non-
survivors.260, 289, 290
It is possible that the discrepancy reflects the inherent paradox of sepsis,
namely that the inflammatory response can be both helpful and harmful. sTREM-1 amplifies the
innate immune response induced by pathogens detected by pattern recognition factors such as
TLR4.371
If this response is controlled, carefully regulated, and directed, the end result is
pathogen clearance and minimal host morbidity. If, however, this response is exaggerated,
excessive, and indiscriminate, the end result is sepsis and potentially significant morbidity.
Therefore, the discrepancy may simply reflect the fact that an intermediate level of sTREM-1 is
required for an appropriate response, and levels that are either too high or too low are
detrimental.
PCT, vWF, sICAM-1, and sFlt-1 have all been associated with mortality and/or disease severity
in other studies of septic patients, and in this regard our results confirm previous findings.34, 120,
254-263, 267, 268, 273-275, 318, 326, 331
Of note, we did not detect an association between Ang-1, sTie-2, VEGF, or CRP and mortality in
this population of adult patients with sepsis. Ang-1 has not been identified as a significant
predictor of mortality in sepsis as consistently as have Ang-2 or the Ang-2:Ang-1 ratio.127, 128
Furthermore, Ang-1 has been shown to fluctuate widely in individual patients over a 24-hour
period,118
and therefore may have limited clinical reproducibility and utility. With respect to the
other candidate biomarkers, our results are in keeping with the published literature in which
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neither sTie-2, nor VEGF, nor CRP have been consistently and reliably associated with mortality
in sepsis.128, 131, 266, 272, 334
Despite the number of biomarkers that were positively associated with mortality in this cohort,
none demonstrated sufficient sensitivity or specificity to be employed clinically as the sole
indicator of prognosis. Therefore, we exploited Classification and Regression Tree (CaRT)
analysis to determine whether combinations of biomarkers were more predictive of mortality in
this population than any single marker alone. Combining biomarkers has previously been shown
to enhance their prognostic utility in both sepsis and malaria,127, 305
and likewise the
combinations illustrated in Figures 6 and 7 predict death with better sensitivity than any
individual marker alone. The predictive models for both in-hospital and 28-day mortality are
representative of the underlying pathophysiology of sepsis and incorporate markers from both
the inflammatory and vascular pathways. To maximize clinical utility and feasibility in our
patient population and resource-limited setting, this analysis was performed with the prespecified
goals of prioritizing sensitivity over specificity and limiting the number of markers included in
each model to three. By identifying patients at highest risk of mortality, this approach may be
useful for triage and directed allocation of limited resources. A recent pan-African survey of
anesthesiologists found that < 2% of African hospitals regularly had access to the resources,
including medications, disposable and permanent equipment, and monitoring and diagnostic
tools, required to implement the Surviving Sepsis Campaign guidelines, now considered the
standard of care for patients with sepsis.372, 373
A convenient, inexpensive, point-of-care test that
could be used at the time of hospital admission to identify those patients at highest risk of
mortality and therefore most likely to benefit from intensive therapy would be of value, and
should be studied prospectively. Rapid tests are currently available for the diagnosis of HIV,
syphilis, and malaria, and new microfluidics-based technology allows all of the steps of a
traditional ELISA to be mimicked on a point-of-care chip, making implementation of biomarker
analysis feasible even in resource-limited settings.374, 375
Rapid identification of the specific microbial etiology responsible for an episode of sepsis would
likewise be a key component of any algorithm designed to maximize appropriate resource
allocation. To that end, we confirmed that procalcitonin has reasonable specificity for bacterial
infection despite the high prevalence of co-infection with HIV, malaria, and mycobacteria in this
population. Interestingly, Chi3L1 had equivalent specificity for bacterial infection in this
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population, and has not previously been studied for its discriminative ability in this regard.
Although the known association of Chi3L1 with the specific granules of activated neutrophils is
in keeping with the finding of elevated Chi3L1 in bacterial infection, elevated levels of Chi3L1
have also been reported in association with HIV encephalitis and hepatitis C-induced liver
fibrosis.299, 376, 377
Therefore, further study of Chi3L1 is warranted to confirm its specificity for
bacterial infection in septic patients.
In contrast, IP-10 has been well-studied in association with tuberculosis and was also most
predictive of mycobacterial infection in our cohort of Ugandan patients with sepsis. IP-10 acts to
amplify the adaptive immune response: it can be produced by monocytes, macrophages, and
vascular endothelial cells and act as a specific chemotactic signal to draw activated Th1 cells that
express its cognate receptor, CXCR3, to a site of infection, and in turn, expression of IP-10 is
further upregulated by interferon-γ produced by the infiltrating Th1 cells.378
Consistent with our
findings and its role in the Th1 inflammatory response, elevated levels of IP-10 have been
demonstrated in areas of granulomatous inflammation and pneumonitis in the lungs of patients
with tuberculosis.379
However, it should be noted that elevated IP-10 levels are not
pathognomonic of mycobacterial infection. IP-10 has also been shown to be elevated in, and
crucial to host control of, Toxoplasma gondii and influenza infection.380, 381
If combined in a point-of-care test panel, these biomarkers might facilitate the initiation of rapid,
appropriate, empiric antimicrobial therapy in the Emergency Department, without the time delay
required for standard diagnostic methods. A recent study compared blood culture, bacterial
polymerase chain reaction (PCR), and procalcitonin for the diagnosis of bacteremia, and found
that the average time from testing to the receipt of results was 33 hours for blood cultures, 10
hours for PCR, and 45 minutes for procalcitonin, a significant difference that is not
inconsequential given the well-documented relationship between delay of appropriate antibiotics
and mortality in sepsis.231, 382
Procalcitonin is not a substitute for blood culture since the latter
has a clear advantage in terms of antimicrobial identification and susceptibility testing. However,
procalcitonin and similar markers may be particularly useful in resource-limited settings to
prioritize those patients most likely to benefit from broad-spectrum antibiotics, or, in the case of
IP-10, to identify and appropriately treat those patients likely to have a non-bacterial cause of
sepsis.
83
In summary, our study has demonstrated that a variety of biomarkers from the inflammatory and
vascular pathways are predictive of both in-hospital and 28-day mortality when measured at the
time of hospital presentation in predominantly HIV-infected Ugandan patients with sepsis.
Biomarker combinations warrant further study with the goal of developing rapid, point-of-care
tests to assist Emergency Department physicians in the appropriate allocation of limited
resources, particularly as sepsis becomes increasingly recognized as an important contributor to
morbidity and mortality in low- and middle- income countries.
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Chapter 6 Discussion
Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) is present in streptococcal
toxic shock syndrome, E. coli O157:H7-induced hemolytic-uremic syndrome, and sepsis. This
work is the first to identify angiopoietin dysregulation in both invasive group A streptococcal
infection and E. coli O157:H7 infection, and adds both syndromes to the published literature on
infectious diseases, including sepsis, severe bacterial infection, and malaria, in which
angiopoietin dysregulation has been described.119, 128, 133
No association between angiopoietin
dysregulation and exotoxin-mediated diseases has previously been reported, and therefore, these
findings may provide the rationale for future experimental work in this area, particularly as
regards the direct effects of exotoxins on the endothelium in general and on Weibel-Palade body
exocytosis in particular. The impact of streptococcal exotoxins on the release of WPBs or their
contents has not been studied in vitro, and while Shiga toxins have been linked indirectly to
WPB exocytosis (via rapid toxin-mediated stimulation of vWF release), the mechanism for
decreased Ang-1, as found in our study, is not known.383
In patients with sepsis, our findings of angiopoietin dysregulation, and in particular of increased
plasma Ang-2 and an elevated Ang-2:Ang-1 ratio, were in accordance with those reported in
other cohorts of septic patients.9, 120-123, 127
Furthermore, angiopoietin dysregulation performed
better than lactate, a standard marker of tissue hypoperfusion, for the prediction of mortality in
sepsis, confirming results reported by others.124
Our study, however, was unique in that it was
conducted in a resource-limited setting amongst adult patients with sepsis, of whom 84% were
HIV-infected and 29% had mycobacteremia, and as such, is the only study of its kind to
document the association between angiopoietins and sepsis in this patient population. The study
of biomarkers for sepsis in resource-limited settings is crucial prior to clinical implementation, as
patient populations in these settings differ markedly from those enrolled in usual clinical trials.
Mortality rates are often higher than in settings in which protocolized sepsis bundles are
considered standard of care.367
As demonstrated in the cohort under study here, patients in
resource-limited areas of Africa who present with sepsis have a high rate of co-infections,
including HIV, malaria, and mycobacteremia, not seen in the usual sepsis studies conducted in
North America, Australia, and Europe. Co-infections that influence basal levels of circulating
85
inflammatory and vascular mediators are particularly significant as they may accentuate or mask
between-group differences in the biomarker under study, and as a consequence, render those
biomarkers either more or less useful than they appeared in studies conducted in different target
populations. For example, healthy patients from malaria endemic areas are known to exhibit a
different basal cytokine expression profile than that found in patients from resource-limited but
malaria non-endemic areas, 384, 385
and active malaria infection has been shown to abrogate the
clinical utility of both procalcitonin and C-reactive protein to discriminate between bacterial and
viral pneumonia.283
Similarly, HIV infection is known to induce the production of pro-
inflammatory cytokines that with treatment decline towards levels found in healthy
individuals.386
Our study is therefore significant in that it demonstrates the utility of angiopoietin
dysregulation for the prediction of mortality in a patient population with a high rate of untreated,
severe HIV disease in which the median CD4+ T cell count was 45 cells/μL.
In addition, angiopoietin dysregulation was present despite a high rate of mycobacterial infection
in this Ugandan population. Although plasma levels of the angiopoietins have not been studied
previously in patients with mycobacterial disease, pleural fluid Ang-2 levels were found to be
lower in tuberculous pleural effusions than in other types of exudative effusions.387
Furthermore,
angiopoietin dysregulation has not been found in animal models of pulmonary tuberculosis. In
fact, caseating granuloma formation in these animals has been attributed in part to impaired
neovascularization and subsequent hypoxia as a result of relatively low angiopoietin levels.388
Given these findings, it was important that angiopoietin dysregulation be confirmed specifically
in septic patients with a high rate of mycobacterial disease prior to clinical use or further study in
this population.
If added to the initial panel of diagnostic tests in patients with suspected severe bacterial
infection, the angiopoietins might be useful adjuncts to the clinical assessment of prognosis. The
presence of angiopoietin dysregulation can differentiate between those patients with invasive
streptococcal infection and those with streptococcal toxic shock, between those patients with E.
coli O157:H7 infection who will have an uncomplicated course and those who will develop the
hemolytic-uremic syndrome, and between those who will and will not survive after an episode of
sepsis at the time of presentation to the Emergency Department. By providing information about
the functional status of the endothelium, angiopoietin dysregulation acts as a marker of the extent
of the pathophysiologic abnormalities induced by the disease process, and, at least in the case of
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E. coli O157:H7 infection, is a more sensitive indicator of impending complications than are
traditional laboratory investigations alone. In patients who present to the Emergency Department
with sepsis, angiopoietin dysregulation is associated with an increased risk of both early and late
mortality. The identification of those patients with the greatest degree of angiopoietin
dysregulation, and presumably endothelial dysfunction, may be an important signal for the need
for more aggressive therapeutic intervention. Early, appropriate, supportive therapy is known to
improve outcome in both HUS and sepsis, and the ability to accurately triage or prioritize
patients for the receipt of these interventions would enhance any Emergency Department critical
illness protocol and would be especially crucial in resource-limited settings.231, 232, 365, 366
The
clinical utility of the angiopoietins in multiple different infectious syndromes is a major
advantage over biomarkers with limited or specific indications and argues for their use in the
undifferentiated patient with suspected infection.
In sepsis, the prognostic utility of the angiopoietins, representative of vascular and endothelial
activation, can be enhanced by their use together with other biomarkers representative of
inflammation and immunologic activation. For the prediction of in-hospital mortality, Ang-2 can
be combined with the Karnofsky Performance score (representing clinical assessment) and
sTREM-1 (representing the inflammatory pathway), while for the prediction of 28-day mortality,
the Ang-2:Ang-1 ratio can be combined with vWF and Chi3L1 (the former representing the
vascular pathway and the latter the inflammatory pathway), to create in both cases clinical
prediction pathways capable of identifying those patients at greatest risk of mortality with
sensitivities in excess of 90%. The resulting algorithms are composed of biomarkers from the
two primary pathways perturbed during sepsis and therefore may more accurately represent the
underlying pathophysiology of the sepsis syndrome than might angiopoietin dysregulation alone.
Biomarker combinations have likewise been proposed for use in risk assessment and outcome
prediction during acute coronary syndromes (ACS). As reviewed by Morrow and Braunwald,
ACS results from the maladaptive activation of multiple systems (in a manner strikingly similar
to sepsis), including the inflammatory, thrombotic, and vascular systems, and therefore,
prognostication is best done using an algorithm that incorporates information from each of the
potentially abnormal pathways.389
Testing this theory in the Emergency Department, Birkhahn et
al found that a point-of-care test (POCT) incorporating multiple cardiac biomarkers was equally
accurate for the diagnosis of non-ST elevation myocardial infarction as standard laboratory-
87
based single marker testing, but was significantly faster.390
With regards to infectious syndromes,
biomarker combinations have been proposed for the prediction of mortality amongst Ugandan
children with severe malaria and amongst patients with sepsis in a high-income country,127, 305
and represent the most likely form in which these prognostic biomarkers will be introduced into
clinical settings.
The finding of angiopoietin dysregulation in three seemingly disparate infectious disease
syndromes also suggests the possibility of a shared underlying final common pathway of
endothelial injury or activation. As such, the angiopoietin system becomes an attractive target for
therapeutic intervention, particularly in patients who present with non-specific hypotension and
major organ (such as renal) dysfunction of unclear etiology. In such cases, empiric antibiotic
therapy requires time to take effect, may be initially inappropriate for the causative organism
(particularly in the era of increasingly common multi-drug resistant organisms), or may be
ineffective in the case of toxin-mediated disease. This is perhaps best illustrated by recent
outcome studies in streptococcal toxic shock, in which even with prompt antimicrobial therapy
and surgical debridement where indicated for necrotizing fasciitis, mortality rates remain in
excess of 40%.14
Furthermore, even with protocolized sepsis management guidelines mandating
early goal-directed therapy in the Emergency Department under optimal clinical trial conditions,
in-hospital mortality for patients presenting with sepsis was 30%.391
Amongst those who did not
receive early goal-directed therapy, mortality was 46.5%. Ten years after the advent of early
goal-directed therapy and the subsequent widespread implementation of severe sepsis
resuscitation bundles and the Surviving Sepsis Campaign recommendations, mortality rates from
sepsis remain at 30% and efforts to further improve outcomes have stalled.370, 392, 393
Angiopoietin-based therapies might be useful adjuncts to the current best-practice interventions
to support such patients until the etiology of the presentation is determined and therapy
appropriately targeted.
There are limitations to the body of work presented here. These studies document an association
between angiopoietin dysregulation and each of the syndromes under investigation, and therefore
suggest, but do not prove, that the angiopoietins are involved in the underlying pathogenesis.
Furthermore, the impact of angiopoietin testing on physician decision-making and patient
outcome should be explored in a prospective manner before firm conclusions can be drawn
regarding their true clinical utility. Regardless, the three studies presented here add to the
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growing literature on angiopoietin dysregulation by a) identifying two syndromes (STSS and
HUS) not previously known to be associated with angiopoietin dysregulation and demonstrating
that dysregulation correlates with disease severity in each and is predictive of clinical outcome in
E. coli O157:H7 infection, and b) confirming that angiopoietin dysregulation is associated with
mortality in sepsis in a predominantly HIV-infected patient population and illustrating that a
combination of biomarkers, including the angiopoietins as markers of vascular dysfunction and
either sTREM-1 or Chi3L1 as markers of inflammation, predicts mortality better than any
individual marker alone.
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Chapter 7 Conclusion
Angiopoietin dysregulation is present in invasive group A streptococcal infection, E. coli
O157:H7 infection, and sepsis. It is associated with disease severity in streptococcal toxic shock
and is a prognostic biomarker in undifferentiated E. coli O157:H7 infection and sepsis,
suggesting that measurement of the angiopoietins upon patient presentation with a suspected
severe bacterial infection may offer clinically useful information beyond that which can be
obtained from clinical judgement and traditional diagnostic tests.
However, in recent years many biomarkers have been proposed, studied, and ultimately
associated with a variety of different diseases and syndromes, yet have failed to make the
transition to clinical practice. This has prompted some authors to raise concerns regarding the
true clinical utility of biomarkers identified in association studies such as those presented here.394
Vasan has elegantly outlined the ideal characteristics for biomarkers, referring specifically to
biomarkers of cardiovascular disease but with principles equally applicable to biomarkers of
infectious diseases.395
To be useful in clinical practice, biomarkers should be specific,
reproducible, representative in a single measure but correlated with disease over time, applicable
to and studied in multiple different populations, able to provide information not available from
clinical assessment, and amenable to accurate laboratory testing that can be automated to achieve
high throughput and short turnaround time. Based on these criteria, the angiopoietins appear
well-positioned to evolve from experimental to clinical use, and will likely do so first in sepsis,
in which they have been most often studied.
When compared to other biomarkers recently proposed as prognostic indicators in patients with
sepsis, the angiopoietins have several advantages. First, unlike procalcitonin and C-reactive
protein, the angiopoietins have been consistently associated with disease severity in sepsis in
multiple different studies and wide-ranging patient populations, from undifferentiated critically
ill patients in the ICU to immunocompromised patients with febrile neutropenia to
predominantly HIV-infected patients as studied here. Our study, in particular, is one of the
largest undertaken, with an enrolment of 336 patients, and confirms the results found in earlier,
smaller studies, a major criterion for clinical use not always fulfilled by other proposed
biomarkers.391
While a meta-analysis of studies examining the association between angiopoietin
90
dysregulation and disease severity in sepsis might be useful to obtain a more accurate estimate of
effect size, such an analysis is unlikely to overturn the basic finding of the association itself
given the almost uniformly positive results in studies published to date.
Second, in their use as prognostic biomarkers in sepsis, the angiopoietins have the added benefit
of directly reflecting a component of the underlying pathophysiology, namely endothelial cell
activation. Unlike procalcitonin or Chi3L1, whose precise roles in sepsis and inflammation are
still unclear, angiopoietin dysregulation has a very well-defined and well-studied effect on the
vascular endothelium,9 and therefore makes physiologic sense as a marker of both endothelial
dysfunction and the resultant mortality in sepsis.
Third, the magnitude of angiopoietin dysregulation approximates disease severity both early and
late in sepsis. Angiopoietin dysregulation has been shown to correlate with the evolution of
endothelial activation and its clinical manifestations, including hypotension and ARDS, over
time, such that angiopoietin concentrations remain abnormal during ongoing illness and
normalize with convalescence.9, 127
The persistence of angiopoietin dysregulation during
prolonged illness is a distinct advantage over other proposed biomarkers of sepsis, such as the
pro-inflammatory cytokines IL-6 and TNF, which may be elevated only transiently and therefore
missed during one-time sampling and simply not useful for serial assessments over the course of
a patient‟s hospital or ICU admission.119
Although angiopoietin dysregulation can herald
ongoing endothelial cell activation in prolonged critical illness, it has been studied most often for
its prognostic value upon patient presentation, and has been found to be an equally effective
marker of disease severity in this setting. Ang-2 concentrations rise within two hours of
experimental human endotoxemia, indicating that angiopoietin dysregulation occurs, and
presumably can be detected, in the earliest stages of severe systemic bacterial infection.119
The
phenomenon of early angiopoietin dysregulation is supported by our findings in E. coli O157:H7
infection, in which angiopoietin dysregulation preceded development of the overt clinical
syndrome of HUS, and could distinguish between two clinically indistinguishable patient
populations: those who would experience an uncomplicated course of illness, and those who
would develop HUS.
Finally, angiopoietin dysregulation is widely applicable to a variety of infectious diseases and
syndromes associated with endothelial cell activation or dysfunction. In contrast to a biomarker
91
such as IP-10, which is relatively specific for mycobacterial infection, angiopoietin dysregulation
is sufficiently generalizable to be of potential use in the estimation of disease severity in
undifferentiated patients with suspected severe bacterial infection in the Emergency Department.
At the same time, angiopoietin dysregulation is specific enough to distinguish septic patients
from those with non-infectious critical illness.121, 122
Nonetheless, there are some aspects of the use of angiopoietin dysregulation as a prognostic
biomarker in sepsis that need to be clarified or resolved prior to implementation in clinical
practice. First, the ideal cut-off value for Ang-1, Ang-2, or the Ang-2:Ang-1 ratio needs to be
established. Since this may differ according to whether disease severity or mortality is being
predicted, both values will have to be made available in a format easily interpretable for
clinicians. Furthermore, any biomarker proposed for clinical use must be sufficiently sensitive
and specific to correctly classify patients. To achieve these performance characteristics, it is
likely that angiopoietin dysregulation would need to be combined with other biomarkers in a
prognostic algorithm or clinical decision pathway, and once again, the format of such a pathway
would need to facilitate ease of clinical use. Second, a rapid test, whether laboratory-based or
point-of-care, will need to be established and validated, as the ELISA method used in the studies
presented above will not yield a sufficiently rapid turnaround time to enable use of angiopoietin
dysregulation in clinical decision-making. This test will also need to be relatively inexpensive, or
be associated with a demonstrable net cost savings, in order to justify its use in preference to the
standard (lactate) and free (Karnofsky Performance score) means of prognostication traditionally
used in sepsis. Finally, the ability of angiopoietin monitoring to influence physician management
and patient outcome will need to be proven in prospective studies prior to widespread clinical
implementation. Although there are clearly aspects on which additional research is required,
angiopoietin dysregulation as a prognostic indicator in sepsis fulfills many of Vasan‟s criteria for
the ideal biomarker, and therefore, deserves further study.
Even if the angiopoietins are never employed clinically as biomarkers of infection, the studies
presented here have still identified the angiopoietins as potential mediators of the endothelial
dysfunction present in streptococcal toxic shock syndrome, E. coli O157:H7-induced hemolytic-
uremic syndrome, and sepsis. In that regard, these studies have provided a rationale for further
investigation into the pathophysiology of each syndrome, with a focus on the regulation of
angiopoietin production, release, and impact on the endothelium in each case. Furthermore, these
92
studies provide the basis for pre-clinical and clinical studies to explore the angiopoietins as
potential therapeutic targets in undifferentiated patients with severe bacterial infections. In this
way, studies such as these that document an association between angiopoietin dysregulation and
various infectious diseases characterized by prominent endothelial cell dysfunction can serve to
advance research in the field and potentially impact clinical outcome by means other than simply
through the identification of potential prognostic biomarkers.
93
Chapter 8 Future Directions
The three studies presented above provide the necessary background for conceptualizing future
research on the angiopoietin system. By suggesting that the angiopoietins play a role in the
pathogenesis of toxin-mediated diseases such as streptococcal toxic shock syndrome and the
hemolytic-uremic syndrome, these studies provide a rationale for in vitro experimental work
investigating the interaction between exotoxins and the endothelium. In STSS, we reported
elevated levels of Ang-2 in proportion to disease severity, however, our association study was
not able to determine whether the increased circulating Ang-2 was due to a direct effect of the
streptococcal exotoxin on the endothelium, or whether an intermediary molecule was required.
To determine whether streptococcal exotoxins could induce WPB exocytosis, cultures of human
umbilical vein endothelial cells and human dermal microvascular endothelial cells could be
exposed to streptococcal exotoxins prior to live-cell imaging for the detection of WPB
exocytosis and subsequent measurement of the angiopoietin-2 concentration in the cell culture
supernatant by ELISA. Abrogation of the effect of the streptococcal exotoxin in an experiment
repeated after treatment of the cultured cells with a specific inhibitor of WPB exocytosis would
confirm the mechanism of Ang-2 release. In contrast, in E. coli O157:H7 infection, we reported
decreased circulating Ang-1 levels as the predominant contributor to angiopoietin dysregulation.
Although Ang-1 is produced in part by platelets, decreased Ang-1 levels were present in children
in the pre-HUS phase of illness, prior to the onset of thrombocytopenia, and therefore cannot be
attributed to a decline in platelet numbers. Since pericytes are the primary source of Ang-1, a co-
culture of pericytes and endothelial cells could be exposed to Shiga toxin, with measurement of
Ang-1 levels in the cell culture supernatant both before and after toxin treatment. Cell cultures of
pericytes alone could also be observed and assayed for apoptosis and necrosis after direct
exposure to Shiga toxin. In this way, in vitro studies could explain the observed clinical results.
By suggesting that the angiopoietins may be useful prognostic indicators in E. coli O157:H7
infection and sepsis, these studies provide the rationale for the prospective exploration of the
impact of an angiopoietin-based algorithm on clinical decision-making in the management of
patients with suspected severe bacterial infection. However, as outlined in Chapter 7, appropriate
94
cut-off values need to be defined, and a rapid and perhaps point-of-care test developed before
such studies could be planned or implemented.
Finally, as a continuation of the studies presented above, angiopoietin dysregulation could be
investigated in a variety of clinical conditions with known endothelial dysfunction in which a
demonstrated association may shed light on the underlying, and heretofore unknown,
pathogenesis. Additional work could explore the potential utility of the angiopoietins and other
biomarkers to guide clinical decision-making in areas in which standard diagnostic tools are
either absent or lacking sufficient sensitivity and/or specificity.
8.1 Effect of Ventilatory Strategy on Angiopoietin levels in ARDS
ARDS is characterized by the diffuse leak of intravascular fluid into the alveolar space, resulting
from disruption of the alveolar-capillary membrane barrier. Pulmonary microvascular endothelial
cell dysfunction is a necessary pre-requisite. Consistent with the known effects of the
angiopoietins - endothelial cell quiescence induced by Ang-1 and endothelial cell activation
induced by Ang-2 - a relative excess of Ang-2 has been shown to play a role in the pathogenesis
of ARDS. In vitro, Ang-2 sensitizes human pulmonary microvascular endothelial cells to
thrombin-induced contractility and inter-cellular gap formation.95
In a murine model of LPS-
induced acute lung injury, an increase in alveolar Ang-2 relative to Ang-1 was associated with an
increase in the neutrophil and protein content of bronchoalveolar lavage fluid, suggestive of
pulmonary capillary leak.396
In the same model, overexpression or administration of exogenous
Ang-1 produced improvements in all indices of endothelial function, lung injury, and
inflammation.216, 397, 398
In humans, polymorphisms of the Ang-2 gene have been associated with
increased susceptibility to ALI and ARDS.399, 400
Circulating plasma Ang-2 levels can
differentiate between critically ill patients with ALI and those without.401
Serum from critically
ill patients has been shown to contain high levels of Ang-2 in proportion to the degree of
hypoxemia, and to be capable of disrupting normal barrier function when added to human
microvascular endothelial cells in vitro.9 This effect was reversed by Ang-1 and resolved as
circulating Ang-2 levels declined with convalescence. High Ang2 levels also predicted mortality
in these patients, as has since been confirmed in several other studies of ALI/ARDS in critically
ill patient populations.320, 357, 402
Taken together, these data indicate that angiopoietin
dysregulation is present in, and likely contributes to, the pathogenesis of ALI/ARDS.
95
Mechanical ventilation is the mainstay of supportive therapy for patients with ARDS, however,
ventilator-associated lung injury (also known as „ventilator-induced lung injury‟ [VILI]) remains
a major contributor to morbidity and mortality. High frequency oscillation (HFO) is a means of
delivering small tidal volume ventilation that may limit VILI and the resulting inflammatory
response. When compared to lung-protective conventional ventilation providing similar mean
airway pressures, HFO has been shown to reduce inflammatory cell infiltrate and pro-
inflammatory cytokine production in the lungs in large animal models.403, 404
Human data come
primarily from studies in neonates, which have demonstrated a reduction in the circulating levels
of some, but not all, pro-inflammatory cytokines with the use of HFO.405
Given these findings,
we hypothesize that angiopoietin dysregulation will be present in patients with ARDS receiving
conventional low tidal volume ventilation and will be attenuated in those patients transitioned to
HFO. The only currently existing data on the effect of ventilatory strategy on levels of
circulating angiopoietins are contradictory and derive from two murine models. In the first,
hyperoxia-induced downregulation of Ang-1 was reversed in the setting of permissive
hypercapnia, a model of low tidal volume ventilation.406
In the second, the administration of
exogenous Ang-1 failed to prevent VILI in the setting of high tidal volume ventilation.229
There
are no studies exploring the effect of HFO on circulating levels of the angiopoietins.
There, we propose to measure serum or plasma Ang-1 and Ang-2 levels at study enrolment and
serially thereafter in patients with ALI/ARDS receiving either conventional, lung-protective, low
tidal volume ventilation or high frequency oscillatory ventilation, to test the following
hypotheses:
1. Circulating levels of Ang-1 and Ang-2 will correlate with the severity of illness and will
predict clinical outcome in ventilated patients with ARDS
2. Lower levels of circulating Ang-2 and/or higher levels of circulating Ang-1 (and/or more
rapid correction of angiopoietin dysregulation) will be found in patients receiving HFO
versus those receiving conventional mechanical ventilation
Ultimately, the angiopoietins may be useful biomarkers to guide clinical management decisions
concerning the choice of ventilatory strategies in patients with ARDS.
96
8.2 Angiopoietins in HIV
Untreated HIV disease is associated with endothelial activation and dysfunction, attenuated in
part by the initiation of antiretroviral therapy (ART).407, 408
IL-6, sICAM-1, sVCAM-1, P-
selectin, and vWF are all elevated in patients with HIV disease as compared to healthy
controls.409
In patients receiving ART, both CRP and IL-6 were found to be predictive of the
development of an opportunistic disease.410
Taken together, these results indicate that there is a
baseline level of inflammation and endothelial dysfunction that occurs in chronic HIV infection
and which subsequently diminishes (but does not normalize) with treatment and increases again
in the setting of intercurrent illness or disease.
Although the advent of truly effective ART has been associated with clear survival benefits in
HIV-infected individuals, it has also exposed patients to the consequences and adverse effects of
longterm ART use. Both endothelial activation and an increase in carotid intima-media thickness
(a marker of subclinical atherosclerosis) are well-documented amongst treated patients with HIV
infection.411
Furthermore, certain antiretroviral medications appear to be associated with a
particular increased risk of cardiovascular disease, namely abacavir (ABC) and certain protease
inhibitors (indinavir or lopinavir-ritonavir).412
This risk is not fully accounted for by changes in
lipid profile or other metabolic disorders. Data conflict on the degree of endothelial activation
specifically associated with ABC. VEGF, vWF, IL-6, CRP, and other markers of thrombosis,
inflammation, and endothelial activation have been studied but not found to be different amongst
patients receiving ABC and those receiving non-ABC-containing regimens.413
In contrast, in
vitro studies demonstrated ICAM-1 upregulation and a resultant increase in rolling and adhesion
of neutrophils and peripheral blood mononuclear cells along an endothelial monolayer in the
presence of ABC.414
At this time, the pathogenesis of ABC-induced cardiovascular risk remains
unknown.
Given the known association between angiopoietin dysregulation, cardiovascular disease, and the
traditional risk factors for cardiovascular disease (reviewed in Chapter 1.7.3) in the general
population, I hypothesize that angiopoietin dysregulation might also play a role in ABC-induced
cardiovascular disease in HIV. This hypothesis is further supported by in vitro data that describe
the attenuation of ABC-induced endothelial dysfunction by specific inhibitors of the Erk
97
signalling pathway, one of the major downstream regulators of Tie-2 activation and associated
with endothelial cell proliferation.415
Although the majority of patients in the sepsis study reported here (Chapter 5) were HIV-
infected, most were not receiving ART, and all had angiopoietin levels measured in the setting of
acute illness. To my knowledge, there are no studies that explore the angiopoietins in treated
HIV disease, nor specifically in association with ABC-induced cardiovascular disease.
Therefore, I propose to investigate Ang-1 and Ang-2 levels in a cohort of patients receiving
ABC, and to compare these levels to that of a control group of patients (matched for age,
duration and stage of HIV infection, and cardiovascular risk factors and disease) receiving non-
ABC-containing ARV regimens. I hypothesize that angiopoietin dysregulation (decreased Ang-1
and/or increased Ang-2) will be greater in those patients receiving ABC-containing ARV
regimens than in the control group. I further hypothesize that amongst those patients receiving
non-ABC-containing regimens, angiopoietin dysregulation will be an independent predictor of
cardiovascular disease.
8.3 Angiopoietins in Obstructive Sleep Apnea
Obstructive sleep apnea (OSA) is defined by frequent and recurrent complete or partial collapse
of the upper airway during sleep, resulting in apneas or hypopneas, intermittent asphyxia, and
periodic awakenings leading to daytime somnolence.416
OSA is common, and depending on the
precise disease definition used, has been documented in as many as 24% of middle-aged men.417
OSA is also serious; it is an independent predictor of future drug-resistant hypertension,
myocardial infarction, stroke, and death from cardiovascular disease.416
As recently reviewed by
Drager et al, multiple mechanisms have been proposed to link atherosclerosis and OSA,
including dyslipidemia, the generation of reactive oxygen species and subsequent lipid
peroxidation, and inflammation.416
Each of these risk factors for atherosclerosis can be induced
in a mouse model by intermittent hypoxia, a defining characteristic of OSA in humans.416
As the site of atherosclerosis, the endothelium is clearly implicated as a mediator of many of the
adverse consequences of OSA. However, impaired endothelial function occurs even in the
absence of overt cardiovascular disease, as evidenced by diminished endothelium-dependent
vasodilatation, enhanced expression of cell-surface adhesion molecules, and increased
circulating levels of tissue factor and vWF.418-422
98
Since hypoxia is a known regulator of Weibel-Palade body exocytosis, and since a
vasoconstrictive, adhesive, prothrombotic endothelial cell phenotype such as that seen in OSA is
the predicted result of a net increase in circulating Ang-2, I hypothesize that angiopoietin
dysregulation might occur in obstructive sleep apnea and might provide an additional link
between intermittent hypoxia and the endothelium. Although angiopoietin-like protein 4 has
been associated with chronic intermittent hypoxia through its role in lipid metabolism
(specifically as an inhibitor of the enzyme lipoprotein lipase needed to clear triglyceride-rich
lipoproteins from the circulation), Angiopoietin-1 and -2 have not been studied in OSA.423
To investigate this hypothesis, I propose a study comparing plasma levels of Ang-1 and Ang-2 in
three groups of patients: a) untreated patients with newly diagnosed OSA, b) patients with
treated OSA adherent to the prescribed regimen of nasal continuous positive airway pressure
(CPAP), and c) untreated healthy controls without OSA. Based on the above data, I expect to
find angiopoietin dysregulation, independent of other cardiovascular disease risk factors, present
in untreated patients with OSA. Furthermore, treated patients with OSA who are adherent to
CPAP should manifest an angiopoietin profile akin to that of healthy controls without sleep-
disordered breathing, provided that the prescribed CPAP therapy has been documented with a
titration sleep study to reduce or eliminate apneic/hypopneic episodes. As a substudy, I propose
to follow the newly diagnosed patients with serial measurements of plasma Ang-1 and Ang-2
both before and after the initiation of CPAP with the intent of documenting reduced angiopoietin
dysregulation in individual patients in whom CPAP is successful in eliminating or reducing
intermittent nocturnal hypoxia.
8.4 Procalcitonin in patients with Hematologic Malignancy
The duration of appropriate broad-spectrum antibiotic therapy in patients with prolonged febrile
neutropenia without obvious source is a common and contentious issue.424
Early discontinuation
in the setting of unrecognized infection could lead to morbidity or mortality, while delayed
discontinuation in the absence of infection exposes patients to unnecessary antibiotics and the
inherent risk therein, and may increase antimicrobial resistance. Since microbiologic cultures and
other diagnostic tests are often negative in this patient population, an alternative marker of
infection with sufficient negative predictive value to guide the safe discontinuation of antibiotics
(although not necessarily antifungals) would be of benefit.
99
Procalcitonin has been proposed as a marker to guide antibiotic therapy, and in this role was the
subject of a recent systematic review by Schuetz et al.425
Fourteen randomized controlled trials,
enrolling a total of 4467 patients with respiratory tract infections or sepsis in primary care,
emergency department or ICU settings, were included in the analysis. Use of a procalcitonin cut-
off value of < 0.25 ng/mL in primary care or the ED, or < 0.25 - 1 ng/mL in the ICU to
determine initiation or duration of antibiotics resulted in a significant decrease in antibiotic use
and duration, without a change in mortality. A second systematic review by Agarwal and
Schwartz focused on sepsis in the ICU and confirmed that procalcitonin-based algorithms for
antibiotic discontinuation lead to reduced antibiotic exposure without compromising survival.426
Importantly, this finding was sufficiently robust to withstand relatively high rates of physician
non-adherence to the algorithm (failure to discontinue antibiotics despite a low or declining
procalcitonin level). A third systematic review focused on only those studies exploring PCT-
guided antibiotic therapy in patients with respiratory tract infections, and found results identical
to those of Schuetz and Agarwal and Schwartz.427
However, since the publication of these meta-analyses, a large study of 1200 critically ill patients
randomized to either standard or PCT-guided antibiotic therapy found that PCT-guided therapy
did not improve survival, and in fact prolonged ICU stay and increased use of broad-spectrum
antimicrobial agents.428
In this study, a PCT measurement of ≥ 1 ng/mL triggered escalation and
expansion of the spectrum of the patient‟s current antimicrobial regimen. Nonetheless, PCT may
still be useful to guide antimicrobial discontinuation, as opposed to escalation.
Before such a study is undertaken, the sensitivity and specificity of PCT for the diagnosis of
bacterial infection in this population must be clarified. In a mixed population of
immunocompromised patients (HIV or hematologic/solid tumour malignancies), procalcitonin
levels greater than 0.5 ng/mL displayed excellent sensitivity for the diagnosis of bacterial
infection, and remained an independent predictor of such in a multivariate analysis.429
However,
a separate study found that while all patients with bacteremia in the setting of febrile neutropenia
ultimately manifested a procalcitonin level above 0.5 ng/mL (the lower limit of detection for first
generation assays), a substantial portion of patients with febrile neutropenia without source also
manifested PCT values above this limit.430
While some studies have found that PCT can
effectively identify those patients with bacteremia from amongst the total population of patients
with febrile neutropenia, others have found reduced sensitivity and specificity of PCT in febrile
100
neutropenic as compared to febrile non-neutropenic patients for the diagnosis of bacterial
infection.431, 432
In a study of 90 patients undergoing chemotherapy for a hematologic malignancy
and who presented with febrile neutropenia, admission plasma procalcitonin levels, using a
sufficiently sensitive assay, were unable to discriminate between those with microbiologically or
clinically documented infections and those with fever without source.433
However, if the peak
procalcitonin level occurred more than three days after the onset of febrile neutropenia, it was
predictive of an invasive fungal infection, perhaps indicating that elevated procalcitonin levels
have a different significance or should be interpreted differently in immunocompromised
patients, and certainly indicating that the utility of PCT in patients with febrile neutropenia
requires further exploration.
Consequently, I would propose two sequential studies:
1. Cohort study of the performance characteristics of PCT to discriminate
microbiologically-confirmed bacterial from non-bacterial infection in non-neutropenic
patients with hematologic malignancy and in neutropenic patients with fever of short
duration (< 5 days).
2. Randomized controlled trial of PCT-guided antibiotic discontinuation in prolonged
febrile neutropenia (> 14 days) without source, provided the findings from study 1
confirm a sufficient negative predictive value for PCT in this setting.
8.5 Systematic review of procalcitonin for the prediction of mortality in sepsis and severe bacterial infection
As can be seen from the literature review in Chapter 1.9.1 and from Table 1, there are a
substantial number of publications, many with conflicting results, pertaining to the use of PCT to
predict outcome in sepsis or severe bacterial infection. Distilling and organizing this information
in a systematic fashion may yield a better understanding of the potential benefit, or lack thereof,
to the use of PCT for this indication. To my knowledge, no such review is ongoing, and searches
of both the Cochrane Database of Systematic Reviews and the newly launched Prospero
International Registry of Systematic Reviews yielded no registered reviews.
101
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