Meeting

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


minmin

Rat Heart-derived decellularized ECM


minmin


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)


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



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


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



Both anterior-posterior and Both anterior-posterior and lateral video images lateral video images acquired. 30frames/sacquired. 30frames/s





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










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


LV infarcted segmentLV infarcted segment epicardial epicardial circumference and endocardial circumference and endocardial


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)


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


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


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

Thanx for your listening

Meeting

Health & Medicine

pbs solution pbs solution

c shaker

tissuederived ecm

myocardial tissue

moll nacl2 solution

lv function

week ip

intubated thoracotomy