Date post: | 15-Jan-2016 |
Category: |
Documents |
Upload: | jasper-merritt |
View: | 223 times |
Download: | 0 times |
Understanding the Mechanisms and the Potential of Cell Therapy for the Repair of
the Adult Mammalian Heart
Keng Ang
VII International Symposium on Stem Cell Therapy
Madrid, 6-7 May 2010
Intramuscular injection and cytokine mobilisation of bone marrow cells repair infarct myocardium
Orlic D et al Nature 2001;410:701Orlic D et al PNAS 2001;98:10344
Haematopoietic Stem Cells Do Not Transdifferentiate Into Cardiac
Myocytes in Myocardial Infarcts
•Murry CE et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004;428:664
•Balsam LB et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428:668
• 14 patients: one or more MI (>3 months)• Elective CABG surgery• Bone marrow:
– aspirated from sternum
– mixed with serum (1:2 ratio)
– injected into scarred areas at end of surgery
– 250µl/injection 1cm apart into mid-depth
– flow cytometry analysis of nucleated cell count and CD34+/CD117+ cells
1st Phase Study on BMCs
Galiñanes et al. Cell Transplantation 2004;13:7-13
Before Surgery
6 weeks 10 months 2 years
After Surgery
1
2
3
4
LV s
eg
me
nta
l WM
SI
Rest Low Dobutamine Peak Dobutamine
Before Surgery
6 weeks 10 months 2 years
After Surgery
Before Surgery
6 weeks 10 months 2 years
After Surgery
**
**
* *
BM Transplant Alone (n=11 segments)
Bypass Graft Alone (n=13 segments)
BM Transplant + Bypass Graft (n=10 segments)
Galiñanes et al. Cell Transplantation 2004;13:7-13
Dobutamine Stress Echocardiography
2nd Phase Study on BMCs
Objectives
1. To determine whether the transplantation of autologous BMCs into myocardial scar improves systolic function
2. And whether this improvement, if any, depends on the route of administration:
– Intramuscular (IM)
– Intracoronary (IC)
Study Design63 eligible patients
undergoingCABG
Control IM IC
BMCs → mid-depth
of scar(500µl/injection)
BMCs →via graft
Supplying scarNo BMCs
BMCs preparation & administration:
•aspirated from iliac crest at the start of operation;
•Separated by density gradient & diluted in autologous serum
•Administered IM or IC before cross-clamp release
Investigations:
•DSE: Pre-op & 6 month
•MRI: Pre-op & 6 month
% Systolic Fractional Thickening in Scarred Segments
(Systolic segmental thickness – Diastolic segmental thickness)% FT= ------------------------------------------------------------------------------------------------ x 100
Diastolic segmental thickness
LOW DOSE DSE
-20
-15
-10
-5
0
Control IM IC
Treatment group
%F
TPreop Postop
REST
-20
-15
-10
-5
0
Control IM IC
Treatment group
%F
T
Preop Postop
% Infarct Volume
0
10
20
30
40
50
60
70
80
Control IM IC
Treatment group
%In
farc
t v
olu
me
Preop Postop
Infarct volume% Infarct volume = --------------------------------------------------------- x 100% Left ventricular myocardial volume
Left Ventricular Ejection Fraction
0
5
10
15
20
25
30
35
40
45
Control IM IC
Treatment group
%
Preop Postop
Summary
Administration of BMCs (IM or IC) into myocardial scar during CABG is safe, but:
• Do not improve segmental systolic function • Do not reduce infarct size• Do not influence global LV parameters
Martin-Rendon E et al. Eur Heart J 2008;29:1807
If BMCs cannot differentiate into cardiac tissue, is the observed
beneficial effect due to
improvement in cell survival stimulation of cardiac progenitor cells
Cardioprotection by BMCs
• Right atrial appendage from patients undergoing elective cardiac surgery
• Slices 300-500µm thickness, 30-50mg weight• Incubation Krebs solution at 37°C • 90-min simulated ischemia/120-min reoxygenation• End-points:
– CK release during reoxygenation
– Necrosis assessed by PI at the end of reoxygenation
– Apoptosis assessed by TUNEL at the end of reoxygenation
• Bone marrow was aspirated from the iliac crest of the patients and separated by density gradient
Anti-ischemic Effect of BMC
0
0.5
1
1.5
CK
re
lea
se (
IU/m
g w
et
tiss
ue
)
control bmc
*
0
5
10
15
Ce
ll n
ecr
osi
s (%
of
ae
rob
ic)
control bmc
*0
5
10
15
Ce
ll a
po
pto
sis
(%o
f a
ero
bic
)
control bmc
*
Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112
CK
rele
ase
(IU
/mg
wet
tiss
ue)
0
0.5
1
1.5
*
Control- BM +BM - BM +BM
Chelerythrine
The Role of PKC
Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112
Cell
necro
sis
(% o
f aero
bic
)
-10
0
10
20
30
40
*
Control- BM +BM - BM +BM
Chelerythrine
Cell
ap
op
tosi
s (%
of
aero
bic
)
0
10
20
30
40
*
Control- BM +BM - BM +BM
Chelerythrine
The Role of p38MAPK
CK
rele
ase
(IU
/mg
wet
tis
sue)
0
0.5
1
1.5
2
*
Control- BM +BM - BM +BM
SB203580
Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112
Cell
necro
sis
(% o
f aer
ob
ic)
-10
0
10
20
30
40
50
*
Control- BM +BM - BM +BM
SB203580
Cell
ap
op
tosi
s (%
of
aero
bic
)
-10
0
10
20
30
40
*
Control- BM +BM - BM +BM
SB203580
Is Protection Against Ischemic Injury Cell Type Specific?
0
0.5
1
1.5
en
doth
elia
l cells
*
CK
re
lea
se (
IU/m
g w
et
tiss
ue
)
kera
tin
ocyte
s
con
trol
bm
c
Kubal C et al J Thorac Cardiovasc Surg 2006;132:1112
What Is the Most Effective Dose of BMCs-induced Cardioprotection?
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
How Potent is BMCs-induced Cardioprotection?
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
Cardioprotective Efficacy of Allogenic BMCs
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
Does Manipulation of Cells Affect BMCs-induced Cardioprotection?
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
Does the Time of Administration Influence BMCs-induced
Cardioprotection?
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
Do BMCs Precondition the Myocardium?
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
Is the Cardioprotection Induced by BMCs Triggered by Secreted Factor(s)?
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
The Role of IGF-1R in Mediating BMCs-induced Cardioprotection
Lai et al. J Thorac Cardiovasc Surg. 2009; 138:1400
• BMCs possess potent cardioprotective properties • Protection is triggered by a secreted factor(s) • Protection is mediated by IGF-1R and by activation
of the protein kinases PKC and p38MAPK
Summary
44 elective CABG patients: • randomised to control or BMCs group
BMCs group: • BMCs harvested & administered at the end of
each cardioplegia dose as an adjunct(49.6±28.7 x 106 cells/injection).
Primary end point:• plasma cardiac enzymes (troponin I, CK-MB)
during the first 48 hours after CPB.
RCT on the cardioprotective effects of BMCs in patients undergoing CABG
Ang et al. Eur Heart J. 2009;30:2354
Plasma cardiac enzymes
Ang et al. Eur Heart J. 2009;30:2354
Mycardial injury before & after CPB
Pre- CPB 10 mins after starting CPB
Ang et al. Eur Heart J. 2009;30:2354
If BMCs cannot differentiate into cardiac tissue, is the observed
beneficial effect due to
improvement in cell survival stimulation of cardiac progenitor cells
Methodological difficulties in the identification of cardiomyocyte nuclei
Confocal microscopy – advocated, but diagnostic accuracy has not been previously tested.
• MHC-nLAC mice (ß-GAL is expressed in 100% of myocyte)
• membrane marker (WGA)• markers involved in cardiogenesis such as
GATA4
Ang et al. Am J Physiol Cell Physiol (2010, in press)
Troponin – Red; DAPI nucleus – Blue
Troponin – Red; DAPI nucleus – Blue; Membrane – White
Troponin – Red; DAPI nucleus – Blue; Membrane – White; Myocyte nucleus - Green
Sensitivity and specificity of myocyte nuclei identification in presence & absence of WGA
Orientation ObserverSensitivity (%) [CI] Specificity (%) [CI]
without WGA with WGA without WGA with WGA
Transverse
1 45.1 [37.7 - 52.8] 64.6 [57.1 - 71.5] 90.1 [87.6 - 92.1] 98.4 [97.1 - 99.1]
2 48.8 [41.2 - 56.4] 68.9 [61.5 - 75.5] 89.5 [86.9 - 91.6] 99.0 [97.9 - 99.5]
3 54.3 [46.6 - 61.7] 72.6 [65.3 - 78.8] 94.4 [92.4 - 95.9] 98.4 [97.1 - 99.1]
Longitudinal
1 28.9 [21.8 - 37.3] 59.4 [50.7 - 67.5] 83.4 [80.0 - 86.3] 94.0 [91.7 - 95.7]
2 34.4 [26.7 - 43.0] 56.3 [47.6 - 64.5] 84.5 [81.2 - 87.4] 95.5 [93.4 - 97.0]
3 39.8 [31.8 - 48.5] 61.7 [53.1 - 69.7] 88.5 [85.5 - 90.9] 95.3 [93.2 - 96.8]
Overall
1 38.0 [32.6 - 43.7] 62.3 [56.6 - 67.7] 87.1 [85.1 - 88.9] 96.5 [95.3 - 97.4]
2 42.5 [36.9 - 48.2] 63.4 [57.7 - 68.7] 87.3 [85.3 - 89.0] 97.4 [96.4 - 98.2]
3 48.0 [42.3 - 53.7] 67.8 [62.2 - 72.9] 91.7 [90.1 - 93.2] 97.0 [95.9 - 97.8]
Abbreviation: WGA – wheat germ agglutinin;CI – Confidence interval
Diagnostic accuracy of myocyte nuclei identification in the presence and absence of WGA
Abbreviation: WGA – wheat germ agglutinin; CI – Confidence interval
Orientation Observer Diagnostic accuracy without WGA (%) [CI] Diagnostic accuracy with WGA (%) [CI]
Transverse
1 81.4 [78.6 - 83.8] 91.8 [89.7 - 93.4]
2 81.5 [78.7 – 84.0] 93.1 [91.2 - 94.6]
3 86.5 [84.0 - 88.7] 93.3 [91.4 - 94.8]
Longitudinal
1 72.9 [69.4 - 76.2] 87.4 [84.6 - 89.7]
2 74.9 [71.5 – 78.0] 88.0 [85.3 - 90.2]
3 79.1 [75.8 – 82.0] 88.9 [86.3 - 91.0]
Overall
1 77.6 [75.4 - 79.6] 89.8 [88.2 - 91.2]
2 78.6 [76.4 - 80.6] 90.8 [89.3 - 92.2]
3 83.2 [81.3 – 85.0] 91.4 [89.8 - 92.7]
Diagnostic performance of GATA4 immune-reactivity
Sensitivity of 91% & specificity of 88%Positive predictive power of 72% & Negative predictive power of 97% Diagnostic accuracy for myocyte nuclei (89.5%)
Summary:• concerns about the diagnostic accuracy of confocal
approaches for the correct identification of cardiomyocyte nuclei events
• transgenic models (MHC-nLAC) can be of help for a more accurate identification of cardiomyocyte nuclei
CONCLUSIONS
• BMCs induce survival of the myocardium but its potential to stimulate the proliferation of cardiac resident stem cells needs to be elucidated
• There is a need to improve & refine the accuracy of the methodological tools used so far for the identification of cardiomyocytes’ nuclei
Acknowledgements
University of Leicester• Keng Ang• Vien K Lai• Lincoln Shenje• José Linares-Palomino• Catrin Pritchard• Derek Chin
Goodhope H (Birmingham)• Francisco Leyva• Paul Foley
University of Indiana• Loren Field• Michael Rubart• Mark Soonpa
Bernado Nadal-Ginard (JMLU)
Funding:• Bristol-Myer Squibbs • British Heart Foundation• Vietnamese Government