meeting
TRANSCRIPT
Intramyocardial Injection of Heart Tissue-
Derived Extracellular Matrix Improves
Post-infarction Cardiac Function in Rats
Wangde Dai, MD, Paul Gerczuk, MD Yuanyuan Zhang, MD,Leona Smith, MD, Oleg Kopyov, MD, Gregory L. Kay, MD,Aarne J. Jyrala, MD, and Robert A. Kloner, MD, PhD
Journal of Cardiovascular
Pharmacology and Therapeutics
Introduction
myocyte necrosis
fibrotic scar tissuebreaking down extracellular matrix(ECM)myocyte slippingventricular wall thinning &dilationinfarct expansion
Ischemic injury
Introductionventricular wall thinning &dilation
Laplace’s lawpost-myocardial
infarction
thickness Tension
IntroductionGoal: thicker the
ventriclemethods:•Stem cell•Acellur injectiondecellularized ECM
1.Small intestine2.Pericardium3.Myocardial tissue
IntroductionImplantation of heart-
tissue-derived ECM
•vascularization•c-kit+ cell•paradoxical LV sytolic bulging•LV function
Left Ventricular wall (LV wall)
Material & Method•Rat Heart-derived decellularized
ECM
•Surgical Procedures for coronary ligation &matrix injection
•Assessment of cardiac function by Echocardiography and Left Ventriculography
•Measurement of hemodynamics
•Assessment of postmortem LV volumes & histological parameters
•Immunohistochemical C-kit staining
Rat Heart-derived decellularized ECM
Fisher rat’s heart
cut into 1-mm thick sections
50ml conical tube50ml conical tubeHeart sectionsHeart sections
PBS+1% antibiotic PBS+1% antibiotic antimitiotic solutionantimitiotic solution
““rinserinse””
50ml conical tube50ml conical tubeHeart sectionsHeart sections
PBS+1% antibiotic PBS+1% antibiotic antimitiotic solutionantimitiotic solution
““rinserinse””
T10 basic ULTRA-TURRAX T10 basic ULTRA-TURRAX DisperserDisperser
Heart sctionsHeart sctions3.4 mol/L NaCl2 solution3.4 mol/L NaCl2 solution
““three 30-minute treatmentsthree 30-minute treatments””
T10 basic ULTRA-TURRAX T10 basic ULTRA-TURRAX DisperserDisperser
Heart sctionsHeart sctions3.4 mol/L NaCl2 solution3.4 mol/L NaCl2 solution
““three 30-minute treatmentsthree 30-minute treatments””
4ºC Shaker 1hour4ºC Shaker 1hourHeart tissue sampleHeart tissue samplePBS nuclease solutionPBS nuclease solution
(50 units/mL DNAse + 10mg/mL (50 units/mL DNAse + 10mg/mL RNAse)RNAse)
““DecellularizationDecellularization””
4ºC Shaker 1hour4ºC Shaker 1hourHeart tissue sampleHeart tissue samplePBS nuclease solutionPBS nuclease solution
(50 units/mL DNAse + 10mg/mL (50 units/mL DNAse + 10mg/mL RNAse)RNAse)
““DecellularizationDecellularization””
centrifuged at centrifuged at 3000 rpm for 10 3000 rpm for 10
minmin
centrifuged at centrifuged at 3000 rpm for 10 3000 rpm for 10
minmin
Rat Heart-derived decellularized ECM
centrifuged at centrifuged at 3000 rpm for 10 3000 rpm for 10
minmin
centrifuged at centrifuged at 3000 rpm for 10 3000 rpm for 10
minmin
4ºC Shaker 1hour4ºC Shaker 1hourHeart tissue sampleHeart tissue sample1% Triton X-1001% Triton X-100
4ºC Shaker 1hour4ºC Shaker 1hourHeart tissue sampleHeart tissue sample1% Triton X-1001% Triton X-100
PBS solutionPBS solutionwash 3 times wash 3 times (1 hour per (1 hour per washing)washing)
PBS solutionPBS solutionwash 3 times wash 3 times (1 hour per (1 hour per washing)washing)
Heart ECM pelletsHeart ECM pelletsStored at -80ºCStored at -80ºCHeart ECM pelletsHeart ECM pelletsStored at -80ºCStored at -80ºC
Surgical Procedures for coronary ligation &matrix injection
IP anesthetized with ketamine(75 mg/kg) & IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)xylazine(5 mg/kg)
IP anesthetized with ketamine(75 mg/kg) & IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)xylazine(5 mg/kg)
intubated intubated intubated intubated
Thoracotomy - heart exposedThoracotomy - heart exposedThoracotomy - heart exposedThoracotomy - heart exposed
Left coronary artery ligatedLeft coronary artery ligatedLeft coronary artery ligatedLeft coronary artery ligated
Chest closed (suturing muscle & stapling Chest closed (suturing muscle & stapling skin)skin)
Chest closed (suturing muscle & stapling Chest closed (suturing muscle & stapling skin)skin)
Bupreax (0.001 mg/100 g) sc for Bupreax (0.001 mg/100 g) sc for 2days twice daily2days twice daily
Bupreax (0.001 mg/100 g) sc for Bupreax (0.001 mg/100 g) sc for 2days twice daily2days twice daily
Recover for a weekRecover for a weekRecover for a weekRecover for a week
Surgical Procedures for coronary ligation &matrix injection
Recover for a weekRecover for a weekRecover for a weekRecover for a week
IP anesthetized with ketamine(75 mg/kg) & IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)xylazine(5 mg/kg)
IP anesthetized with ketamine(75 mg/kg) & IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)xylazine(5 mg/kg)
intubated intubated intubated intubated
Thoracotomy - heart exposedThoracotomy - heart exposedThoracotomy - heart exposedThoracotomy - heart exposed
Matrix(75 µL) or saline (75 µL) directly Matrix(75 µL) or saline (75 µL) directly injected in to the LV infarcted areainjected in to the LV infarcted area
Matrix(75 µL) or saline (75 µL) directly Matrix(75 µL) or saline (75 µL) directly injected in to the LV infarcted areainjected in to the LV infarcted area
Recover for 6 weeksRecover for 6 weeksRecover for 6 weeksRecover for 6 weeks
I am ready!I am ready!I am ready!I am ready!
Assessment of cardiac function by Echocardiography
Sono 5500 ultrasound system, 15MHZ Sono 5500 ultrasound system, 15MHZ transducertransducer
Motion-mode(M-mode)Motion-mode(M-mode)
Sono 5500 ultrasound system, 15MHZ Sono 5500 ultrasound system, 15MHZ transducertransducer
Motion-mode(M-mode)Motion-mode(M-mode)
FS (%) = [ (EDD-ESD) / FS (%) = [ (EDD-ESD) / EDD ]*100%EDD ]*100%
FS: Frictional shorteningFS: Frictional shortening
FS (%) = [ (EDD-ESD) / FS (%) = [ (EDD-ESD) / EDD ]*100%EDD ]*100%
FS: Frictional shorteningFS: Frictional shortening
EDD:EDD: The diameter across a The diameter across a ventricle at the end of diastoleventricle at the end of diastole (End-diastolic dimension)(End-diastolic dimension)
ESD: similar to the end-ESD: similar to the end-diastolic dimension, but is diastolic dimension, but is measured at the end of measured at the end of systolesystole (End-systolic dimension)(End-systolic dimension)
EDD:EDD: The diameter across a The diameter across a ventricle at the end of diastoleventricle at the end of diastole (End-diastolic dimension)(End-diastolic dimension)
ESD: similar to the end-ESD: similar to the end-diastolic dimension, but is diastolic dimension, but is measured at the end of measured at the end of systolesystole (End-systolic dimension)(End-systolic dimension)
global LV functionglobal LV functionglobal LV functionglobal LV function
3 consecutive 3 consecutive beats and beats and averagedaveraged
3 consecutive 3 consecutive beats and beats and averagedaveraged
Assessment of cardiac function by Left ventriculography
Xi Scan 1000 C-arm X-ray SystemXi Scan 1000 C-arm X-ray SystemXi Scan 1000 C-arm X-ray SystemXi Scan 1000 C-arm X-ray System
EF(%)=[ (ED_LV_Volumn - ES_LV_Volumn) ] / EF(%)=[ (ED_LV_Volumn - ES_LV_Volumn) ] / ED_LV_VolumnED_LV_Volumn
EF: LV ejection fractionEF: LV ejection fraction
EF(%)=[ (ED_LV_Volumn - ES_LV_Volumn) ] / EF(%)=[ (ED_LV_Volumn - ES_LV_Volumn) ] / ED_LV_VolumnED_LV_Volumn
EF: LV ejection fractionEF: LV ejection fraction
catheter inserted into left catheter inserted into left jugular vein and injected 1 jugular vein and injected 1
mL nonionic contrastmL nonionic contrast
catheter inserted into left catheter inserted into left jugular vein and injected 1 jugular vein and injected 1
mL nonionic contrastmL nonionic contrast
Both anterior-posterior and Both anterior-posterior and lateral video images lateral video images acquired. 30frames/sacquired. 30frames/s
Both anterior-posterior and Both anterior-posterior and lateral video images lateral video images acquired. 30frames/sacquired. 30frames/s
global LV functionglobal LV functionglobal LV functionglobal LV function
3 consecutive 3 consecutive beats and beats and averagedaveraged
3 consecutive 3 consecutive beats and beats and averagedaveraged
Assessment of cardiac function by Left ventriculography
Paradoxical systolic bulging(%) = Paradoxical systolic bulging(%) = ( circumference of the bulging segment / ( circumference of the bulging segment / total LV systolic circumference ) *100%total LV systolic circumference ) *100%
Paradoxical systolic bulging(%) = Paradoxical systolic bulging(%) = ( circumference of the bulging segment / ( circumference of the bulging segment / total LV systolic circumference ) *100%total LV systolic circumference ) *100%
Xi Scan 1000 C-arm X-ray SystemXi Scan 1000 C-arm X-ray SystemXi Scan 1000 C-arm X-ray SystemXi Scan 1000 C-arm X-ray System
catheter inserted into left catheter inserted into left jugular vein and injected 1 jugular vein and injected 1
mL nonionic contrastmL nonionic contrast
catheter inserted into left catheter inserted into left jugular vein and injected 1 jugular vein and injected 1
mL nonionic contrastmL nonionic contrast
Both anterior-posterior and Both anterior-posterior and lateral video images lateral video images acquired. 30frames/sacquired. 30frames/s
Both anterior-posterior and Both anterior-posterior and lateral video images lateral video images acquired. 30frames/sacquired. 30frames/s
global LV functionglobal LV functionglobal LV functionglobal LV function
3 consecutive 3 consecutive beats and beats and averagedaveraged
3 consecutive 3 consecutive beats and beats and averagedaveraged
ES tracing extend out side the ES tracing extend out side the ED tracingED tracingES tracing extend out side the ES tracing extend out side the ED tracingED tracing
Measurement of hemodynamics
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)mg/kg)
IP anesthetized with ketamine(75 mg/kg) & xylazine(5 IP anesthetized with ketamine(75 mg/kg) & xylazine(5 mg/kg)mg/kg)
2F high-fidelity, catheter-tipped micronanometer 2F high-fidelity, catheter-tipped micronanometer inserted into carotid artery inserted into carotid artery
2F high-fidelity, catheter-tipped micronanometer 2F high-fidelity, catheter-tipped micronanometer inserted into carotid artery inserted into carotid artery
record arterial blood pressure and heart raterecord arterial blood pressure and heart raterecord arterial blood pressure and heart raterecord arterial blood pressure and heart rate
Assessment of postmortem LV volumes
Unisperse blue dye 0.6mL 50% stain Unisperse blue dye 0.6mL 50% stain the Blood Vessel the Blood Vessel in the LV wall in the LV wall Unisperse blue dye 0.6mL 50% stain Unisperse blue dye 0.6mL 50% stain the Blood Vessel the Blood Vessel in the LV wall in the LV wall
euthanized with 1mL KCL (149mg/mL)euthanized with 1mL KCL (149mg/mL)euthanized with 1mL KCL (149mg/mL)euthanized with 1mL KCL (149mg/mL)
Harvest the heartHarvest the heartHarvest the heartHarvest the heart
LV Volumes = Water in the cavityLV Volumes = Water in the cavityLV Volumes = Water in the cavityLV Volumes = Water in the cavity
Assessment of Postmortem Histological Parameters
Hearts cut into 3 Hearts cut into 3 transverse slicestransverse slicesHearts cut into 3 Hearts cut into 3 transverse slicestransverse slices
Middle slice for histologyMiddle slice for histologyMiddle slice for histologyMiddle slice for histology
sectioned into 5µm sectioned into 5µm thicknessthickness
sectioned into 5µm sectioned into 5µm thicknessthickness
stained with H&E and stained with H&E and Picrosirius redPicrosirius red
stained with H&E and stained with H&E and Picrosirius redPicrosirius red
Computerized planimetry Computerized planimetry tracingtracing
Computerized planimetry Computerized planimetry tracingtracing
scar thicknessscar thicknessscar thicknessscar thickness
total LVtotal LV epicardial epicardial circumference and endocardial circumference and endocardial
circumferencecircumference
total LVtotal LV epicardial epicardial circumference and endocardial circumference and endocardial
circumferencecircumference
LV infarcted segmentLV infarcted segment epicardial epicardial circumference and endocardial circumference and endocardial
circumferencecircumference
LV infarcted segmentLV infarcted segment epicardial epicardial circumference and endocardial circumference and endocardial
circumferencecircumferenceLV cavity area LV cavity area
and total LV areaand total LV areaLV cavity area LV cavity area
and total LV areaand total LV area
10 microscopic fields at 10 microscopic fields at x400 at infarct area choose x400 at infarct area choose randomly for counting the randomly for counting the
blue particlesblue particles
10 microscopic fields at 10 microscopic fields at x400 at infarct area choose x400 at infarct area choose randomly for counting the randomly for counting the
blue particlesblue particles
Blood vessel Blood vessel densitydensity
Blood vessel Blood vessel densitydensity
Unisperse blue dye 0.6mL 50% Unisperse blue dye 0.6mL 50% stain the Blood Vessel stain the Blood Vessel in the in the
LV wall LV wall
Unisperse blue dye 0.6mL 50% Unisperse blue dye 0.6mL 50% stain the Blood Vessel stain the Blood Vessel in the in the
LV wall LV wall
Immunohistochemical C-kit Stain
Slices stained with Slices stained with antibodies against c-kit (a antibodies against c-kit (a marker for stem cell) marker for stem cell)
Slices stained with Slices stained with antibodies against c-kit (a antibodies against c-kit (a marker for stem cell) marker for stem cell)
Cardioprotective c-kit+ cells Cardioprotective c-kit+ cells are from the bone marrow and are from the bone marrow and
regulate the myocardial balance regulate the myocardial balance of angiogenic cytokines.of angiogenic cytokines.
Cardioprotective c-kit+ cells Cardioprotective c-kit+ cells are from the bone marrow and are from the bone marrow and
regulate the myocardial balance regulate the myocardial balance of angiogenic cytokines.of angiogenic cytokines.
SlicesSlicesSlicesSlices
EDTA PH9.0EDTA PH9.0““epitope epitope retrievalretrieval””
EDTA PH9.0EDTA PH9.0““epitope epitope retrievalretrieval””
0.01% Triton0.01% Triton““permeabilizedpermeabilized””0.01% Triton0.01% Triton““permeabilizedpermeabilized””rabbit anti-c-rabbit anti-c-
kitkitprimary primary
antibodiesantibodies4ºC over night4ºC over night
rabbit anti-c-rabbit anti-c-kitkit
primary primary antibodiesantibodies
4ºC over night4ºC over nightbiotinylated biotinylated horse anti-horse anti-
rabbit-rabbit-secondary secondary antibodiesantibodies
biotinylated biotinylated horse anti-horse anti-
rabbit-rabbit-secondary secondary antibodiesantibodies
Image Pro Plus SoftwareImage Pro Plus Software““QuantificationQuantification””
Image Pro Plus SoftwareImage Pro Plus Software““QuantificationQuantification””
ResultsThe LV FS by
Echocardiography
Saline (n=17) Matrix (n=19)Before coronary occlusion
47.3%±1.3% 46.5%±0.9%
After coronary occlusion1 week
21.4%±1.6% 20.7%±2%
After coronary occlusion6 week
17.8%±1.5% 26.2%±2.2%
ResultsThe LV FS by
Echocardiography
Figure 2. Matrix injection significantly improved the left ventricular(LV) fractional shortening compared to saline at 6 weeks after
treatment(P=0.0034).
ResultsThe LV EF and Paradoxical Systolic Bulging by LV
Ventriculography
Figure 3. A: left ventricular ejection fraction (LVEF) calculated by angiography at 6 weeks after matrix or saline injection directly into the scar area of myocardial infarction in
rats. Matrix significantly increased the LVEF (P=0.043). Panel B: The extent of paradoxical systolic bulging is expressed as (circumference of the bulging segment/total LV systolic circumference) *100%. Matrix implantation significantly prevented paradoxical LV systolic
bulging in the matrix-treated group compared to the saline-treated group (P =0.048).
ResultsHemodynamics
No significant differences were No significant differences were noted in heart rate, systolic noted in heart rate, systolic and diastolic blood pressureand diastolic blood pressure
No significant differences were No significant differences were noted in heart rate, systolic noted in heart rate, systolic and diastolic blood pressureand diastolic blood pressure
ResultsPostmortem LV Volumes, Scar Thickness, Infarct Sizes,
and Expansion Index
Scar thickness Scar thickness significantly increasesignificantly increase
Infarct expansion Infarct expansion significantly decreasesignificantly decrease
Scar thickness Scar thickness significantly increasesignificantly increase
Infarct expansion Infarct expansion significantly decreasesignificantly decrease
ResultsPostmortem LV Volumes, Scar Thickness, Infarct Sizes,
and Expansion Index
Figure 5. Scar thickness, average of the measurements at 5 equidistantpoints of the
infarcted left ventricular wall, is significantly higherin the matrix group compared
to the saline group (P =0.0084).
ResultsBlood Vessel Density and Recruitment of Endogenous Stem
Cells in Infarcts
Saline (n=17)
Matrix (n=19)
BV density in scar area
(vessels/mm2)
189±12 165±23
P=0.077 (>0.05 No significant difference)
ResultsBlood Vessel Density and Recruitment of Endogenous Stem
Cells in Infarcts
Figure 6. Immunohistochemical staining with primary antibody against c-kit of the rat hearts (A) control received
saline and (B) received matrix implantation. The red arrows identify positive c-kit stained cells (brown color stained) within he infarct
area(magnification 400, scale bar . 20 mm).
Saline (n=6)
Matrix (n=6)
The number of c-kit+ cells
105±13 111±11
Similar!!
Discussion
Biomaterial Implantation Has Biomaterial Implantation Has Similar Similar EffectsEffects to That of to That of Cell Therapy for Cell Therapy for
Myocardial InfarctionMyocardial Infarction
Biomaterial Implantation Has Biomaterial Implantation Has Similar Similar EffectsEffects to That of to That of Cell Therapy for Cell Therapy for
Myocardial InfarctionMyocardial Infarction
LV infarct wall LV infarct wall thickenedthickenedLV infarct wall LV infarct wall thickenedthickened
Paradoxical systolic bulging Paradoxical systolic bulging decreaseddecreased
Paradoxical systolic bulging Paradoxical systolic bulging decreaseddecreased
LV EF LV EF enhancedenhancedLV EF LV EF enhancedenhanced
For the FIRST For the FIRST TIME!TIME!
ECM can do ECM can do this!this!
For the FIRST For the FIRST TIME!TIME!
ECM can do ECM can do this!this!
Discussion
New Blood Vessel FormationNew Blood Vessel Formation Within the Within the Implanted BiomaterialsImplanted Biomaterials
New Blood Vessel FormationNew Blood Vessel Formation Within the Within the Implanted BiomaterialsImplanted Biomaterials
In our present study, injected In our present study, injected materials materials did notdid not promote promote
neovascularization.neovascularization.
In our present study, injected In our present study, injected materials materials did notdid not promote promote
neovascularization.neovascularization. Our study suggests that the Our study suggests that the cardiac ECM that we utilized cardiac ECM that we utilized
prevented LV remodeling prevented LV remodeling withoutwithout requiring an increase in requiring an increase in
neovascularizationneovascularization
Our study suggests that the Our study suggests that the cardiac ECM that we utilized cardiac ECM that we utilized
prevented LV remodeling prevented LV remodeling withoutwithout requiring an increase in requiring an increase in
neovascularizationneovascularization
ResultsBlood Vessel Density and Recruitment of Endogenous Stem
Cells in Infarcts
Saline (n=17)
Matrix (n=19)
BV density in scar area
(vessels/mm2)
189±12 165±23
P=0.077 (>0.05 No significant difference)
Discussion
Recruitment of Endogenous Stem Cells by Recruitment of Endogenous Stem Cells by Implanted BiomaterialsImplanted Biomaterials
Recruitment of Endogenous Stem Cells by Recruitment of Endogenous Stem Cells by Implanted BiomaterialsImplanted Biomaterials
Many studies proved that!Many studies proved that!Many studies proved that!Many studies proved that!
There was no evidenceThere was no evidence that ECM that ECM enhanced recruitment of these cells enhanced recruitment of these cells above and beyond infarction alone.above and beyond infarction alone.
There was no evidenceThere was no evidence that ECM that ECM enhanced recruitment of these cells enhanced recruitment of these cells above and beyond infarction alone.above and beyond infarction alone.
Therefore, any benefit of the ECMTherefore, any benefit of the ECM cannot cannot be attributedbe attributed to an increase in c-kit to an increase in c-kit
cells in our studycells in our study
Therefore, any benefit of the ECMTherefore, any benefit of the ECM cannot cannot be attributedbe attributed to an increase in c-kit to an increase in c-kit
cells in our studycells in our study
ResultsBlood Vessel Density and Recruitment of Endogenous Stem
Cells in Infarcts
Figure 6. Immunohistochemical staining with primary antibody against c-kit of the rat hearts (A) control received
saline and (B) received matrix implantation. The red arrows identify positive c-kit stained cells (brown color stained) within he infarct
area(magnification 400, scale bar . 20 mm).
Saline (n=6)
Matrix (n=6)
The number of c-kit+ cells
105±13 111±11
Similar!!
Discussion
Biomaterial Implantation Prevents Post-Biomaterial Implantation Prevents Post-infarctioninfarction Paradoxical LV Systolic BulgingParadoxical LV Systolic BulgingBiomaterial Implantation Prevents Post-Biomaterial Implantation Prevents Post-
infarctioninfarction Paradoxical LV Systolic BulgingParadoxical LV Systolic Bulging
YES,YES,ECM can do ECM can do
this!this!
YES,YES,ECM can do ECM can do
this!this!
ConclusionImplantation of heart-
tissue-derived ECM
•paradoxical LV sytolic bulging•LV EF function
Left Ventricular wall (LV wall)
AngiogenesisAngiogenesisAngiogenesisAngiogenesis
Recruitment of Recruitment of endogenous stem endogenous stem
cellscells
Recruitment of Recruitment of endogenous stem endogenous stem
cellscells