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179 chlamydophila pneumoniae

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Silvana Vielma,MD Gregor Krings,BS Maria Lopes-Virella, MD, PhD Medical University of South Carolina and Ralph Johnson VA Medical Center, Charleston, SC Chlamydophila pneumoniae Chlamydophila pneumoniae Induces Induces ICAM-1 Expression In Human Aortic ICAM-1 Expression In Human Aortic Endothelial Cells Endothelial Cells Via Protein Kinase C - Dependent Via Protein Kinase C - Dependent Activation of Nuclear Factor- Activation of Nuclear Factor- B B
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Page 1: 179 chlamydophila pneumoniae

Silvana Vielma,MD Gregor Krings,BS

Maria Lopes-Virella, MD, PhD

Medical University of South Carolina and Ralph Johnson VA Medical Center, Charleston, SC

Chlamydophila pneumoniae Chlamydophila pneumoniae Induces Induces ICAM-1 Expression In Human AorticICAM-1 Expression In Human Aortic

Endothelial Cells Endothelial Cells Via Protein Kinase C - Dependent Via Protein Kinase C - Dependent

Activation of Nuclear Factor-Activation of Nuclear Factor-BB

Page 2: 179 chlamydophila pneumoniae

ENDOTHELIAL CELL DYSFUNCTIONENDOTHELIAL CELL DYSFUNCTION

Cardiovascular Disease StartsStarts and EndsEnds with Endothelial Cell Dysfunction

Page 3: 179 chlamydophila pneumoniae

Factors Leading to Endothelial Factors Leading to Endothelial DysfunctionDysfunction

• ConventionalConventional: Dyslipidemia, Hyperglycemia, Smoking• Non-ConventionalNon-Conventional:

– Increased Homocysteine, Angiotensin II, Pro-thrombotic Factors, Pro-Inflammatory Factors, Oxidative Stress

– Infectious Processes: Cytomegalovirus and other viral infections , C. pneumoniae

Page 4: 179 chlamydophila pneumoniae

C. pneumoniae and Arteriosclerosis• C. pneumoniae has an epidemiological link with

arteriosclerosis and acute cardiovascular events

• C. pneumoniae has been detected in carotid, abdominal aorta, coronary , femoral, pulmonary and popliteal arteries

• C. pneumoniae is able to replicate in macrophages, endothelial and smooth muscle cells

Page 5: 179 chlamydophila pneumoniae

C. pneumoniae Infects and Activates Endothelial Cells

MonocyteV-CAM-1E-SelectinICAM-1

Adhesion molecules

Induction of ChemokinesIL-8, MCP-1

C. pneumoniaeElementary bodies

Endothelial cells

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ADHESION MOLECULESADHESION MOLECULES

• Activation of Endothelial Cells by C. pneumoniae leads to increased expression of adhesion molecules and, as a consequence, increased adherence of monocytes to the endothelium, an early hallmark of atherogenesis

• Adhesion molecules:– serve as mediators of cell-cell and cell-matrix

interactions– participate in cell migration and signaling functions

Page 7: 179 chlamydophila pneumoniae

Sequential steps of leukocyte adhesion

Capture/Tethering Rolling Firm Adhesion Transmigration

L-selectin

P-selectin

E-selectin

Integrins, ICAM,VCAM

ICAMPECAM

Endothelial cells

Price et. al. 1999

Page 8: 179 chlamydophila pneumoniae

RATIONALE and GOALS It is known that C. pneumoniae activates p42/p44 (ERK1/2) and NF-B in endothelial cells. Nothing is known, however, about regulation of ICAM-1 expression in chlamydia-infected HAEC. Thus the GOALGOAL of this study is:

To determine which signaling transduction To determine which signaling transduction pathways are involved in the regulation of ICAM-1 pathways are involved in the regulation of ICAM-1 by by C. pneumoniaeC. pneumoniae in human aortic endothelial cells in human aortic endothelial cells

Page 9: 179 chlamydophila pneumoniae

Structure and regulation of ICAM-1 promoter

ARE

ICAM-1ICAM-1

TATAIRETATA

H2O2IL-1TNF

AP-1AP-1/ETS

AP-1/ETS

NF-kB

C/EBP-AP-3

TFIID AP

-1C/EBP

NF-kB Ets-1 STAT

Sp1AP-2

TFIID

IFN-

• Major intracellular signal transduction pathways– NF-B pathway – Mitogen-Activated Protein (MAP) kinase (ERK,

JNK, and p38) pathway– Protein kinase C (PKC) pathway

Page 10: 179 chlamydophila pneumoniae

Protocol to infect HAEC with C. pneumoniae AR39 (ATCC)

Cycloheximide Tx Hep-2 cells

Chamber-slide system

Mechanical dysruption and sonication

IF staining

Rocker platform x 2h at 37CIncubation, 37°C, 1h

MOI: 5-10 EB/cell

HAEC

Differential centrifugation

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Time Course Expression of ICAM-1 in C. pneumoniae-Infected

Human Aortic Endothelial Cells

NI: Non-infectedTNF: TNF-treated cellsCp: C.pneumoniae infected cellsUV: Cells infected with UV-treated CpH: Cells infected with Heat-inactivated CpM: Mock cells

Page 12: 179 chlamydophila pneumoniae

Time dependent activation of MAPK pathway in C. pneumoniae-infected HAEC

Page 13: 179 chlamydophila pneumoniae

ICAM-1 expression by C.pneumoniae- infected HAEC is not mediated by MAPK

Activation

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NF-B activation mediates C. pneumoniae-induced ICAM-1 expression

NI: Non-infected cellsTNF: TNF-treated cellsCAPE: Caffeic acid phenethyl ester

Page 15: 179 chlamydophila pneumoniae

ICAM-1 expression induced by C. pneumoniae is PKC-dependent

Cy:CytosolM: Membrane

NI:Non-infected cellsCal C: Calphostin CBis I: Bisindolyl- maleimide I

Page 16: 179 chlamydophila pneumoniae

PKC isozymes depletion in C. pneumoniae-infected HAEC

Cy: CytosolM: Membrane NI: Non-infected cellsPMA: PMA-treated cells

Page 17: 179 chlamydophila pneumoniae

ICAM-1 up-regulation in C. pneumoniae-infected cells is PKC and NF-B dependent

NI:Non-infected cellsTNF:TNF-treated cellsCal C: Calphostin CBay: Bay 117085

Page 18: 179 chlamydophila pneumoniae

Summary of ResultsSummary of Results• The up-regulation of ICAM-1 expression in C. pneumoniae-infected

HAEC is time-dependent. Heat and UV inactivation of C. pneumoniae elementary bodies completely abolished the upregulation of ICAM-1

• Up-regulation of ICAM-1 expression in C. pneumoniae-infected HAEC is not mediated by MAPK activation

• Inhibition of NF-B activation completely abolishes C. pneumoniae-induced ICAM-1 expression by HAEC

• ICAM-1 upregulation in C. pneumoniae-stimulated HAEC is PKC dependent

• Activation of PKC leads to NF-B activation and that, in turn, leads to increased transcription of the ICAM-1 gene

Page 19: 179 chlamydophila pneumoniae

C. pneumoniae’s EB

HAEC Toll-like receptorsLeucine-like receptors?

ICAM-1

NIKIKK

IKKIKK

IkB- p and ubiquination

NF-kB translocation

P65(RelA)

cSrc

PKCCalphostinC

CAPE

BAY117085

U0126PD98059

c-Fos, Ets-1, Sap1, STAT

c-Raf-1MEK1/2ERK1/2

c-Jun

JNKSAPK

MEKK1MKK4 IkB

NF-kB

Signal transduction pathways that mediate ICAM-1 Signal transduction pathways that mediate ICAM-1 up-regulation in up-regulation in C. pneumoniae-C. pneumoniae-infected HAECinfected HAEC

Page 20: 179 chlamydophila pneumoniae

CONCLUSIONSCONCLUSIONS1. We have shown for the first time that PKC-mediated

activation of NF-kB by C. pneumoniae leads to a specific up-regulation of ICAM-1 in human aortic endothelial cells

2. Up-regulation of ICAM-1 by C. pneumoniae contributes to the chronic inflammatory events associated with atherosclerosis

Page 21: 179 chlamydophila pneumoniae

Questions?Questions?

Vielma SA, Kreggs G, Lopes-Virella MF: Vielma SA, Kreggs G, Lopes-Virella MF: Circulation ResearchCirculation Research 92: 1130-7, 2003 92: 1130-7, 2003


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