Endothelial Progenitor Cells and Development of Collateral Formation in Patients with Chronic Total Coronary Artery Occlusion and Transplantation of EPCs in an Experimental Model Presented by Doaa Mostafa Gharib, M.D

I have no real or apparent conflicts of interest to report. Doaa Mostafa Gharib, MD I have no real or apparent conflicts of interest to report.

EPCs in Chronic total occlusion Chronic Total Occlusion (CTO) remains one of the most difficult challenges for the coronary arteries intervention. Endothelial progenitor cells (EPCs ) are bone marrow derived stem cells that were experimentally shown to incorporate into sites of neovascularization and home to sites of endothelial denudation (Caroline et al., 2005). Therefore, EPCs might be useful for cell therapy to potentially promote both neovascularization and cardiac regeneration in patients with ischemic heart disease as CTO patients.  

THIS WORK AIMS TO: Aim of The Work Study whether or not the number and function of EPCs are associated with the development of collateral formation in patients with single-vessel coronary artery disease of CTO. We aimed also to examine the ability of EPCs to form new blood vessels and to differentiate into cardiomyocytes in an experimental model (canines) with acute myocardial infarctions (AMI).

The design of this work was divided into two major parts: I-Clinical study: Twenty patients who had CTO in one major coronary artery diagnosed by coronary angiography (CA) were divided according to Rentrop collateral grading (Rentrop et al., 1985) into 2 groups : Group 1: 10 patients with good coronary collaterals. Group 2: 10 patients with poor coronary collaterals.

I-Clinical study: EPCs were isolated from peripheral blood samples, cultured and their phenotypes were confirmed by uptake of acetylated LDL and binding of fluorescein isothiocyanate (FITC) - labeled Ulex europaeus agglutinin 1 (UEA-1) lectin. EPCs were enumerated and their endothelial function and activity was determined by Quantitative RT- PCR for : Human Vascular Endothelial Growth Factor Receptor-2 (hVEGFR-2). Human endothelial nitric oxide synthase (eNOS). Number and function of EPCs were correlated with the development of collateral formation in these patients.

II-Experimental study: Isolated EPCs from CTO patients with good coronary collaterals were transplanted intramyocardially in an experimental model (canines) with acute myocardial infarctions (AMI).  Experimental animals were divided into 2 groups: Group 1: EPCs-AMI treated canines (n=6). Group 2: AMI canines (n=6) were subjected to intramyocardial saline injection as control placebo group. In all canines, MI was induced by ligation of the left anterior descending coronary artery (LAD) distal to the first diagonal branch.

Results of the Clinical Study

1-Patients’ characterization: parameter All subjects (No=20) Good collateral (No=10) Poor collateral (No=10) P value Age 56.4± 8.25 55.3 ± 9 57.5 ± 7.66 NS Sex M/F (M/F %) 16/4(80/20) 9/1(90/10) 7/3(70/30) Smoking, no. (%) 11(55) 6(60) 5(50) Hypertension, no. (%) 13(65) 7(70) Diabetes, no. (%) 8(40) 4(40) Previous MI 12(60) Statin 16(20) 9(90) CTO grading 20 2,3 (10) 0,1 (10) Plasma cholesterol mg/dl 271.8±92.24 301.8 ± 104.39 241.8±71.17 Plasma TG mg/dl 146.45±62.07 161.6 ±80 131.3±34.8 Total CK U/L 122±66.92 145.3±70.1 98.70±57.63 CK-MB U/L 58.85±27.93 69.3±26.34 48.4±26.65

A B 2-EPCs characterization and identification: a- EPCs-Colony Forming Unit (CFU) characterization in culture: A B EPC-CFU as a central core of rounded cells surrounded by elongated spindled-shaped cells cultured on fibronectin plate. A: at 24 hours culture (200X magnification) B: at 7 days of culture (400 X magnification)

b- Analysis of hEPCs based on cell surface marker expression : This figure represents FACS analysis for CD34+ of cultured EPCs as an identification surface marker of EPCs.

A 3- EPCs characterization by specific fluorescent stains: B: Characterization of viability of cultured EPCs by positive-DAPI blue cytoplasm counterstaining (400X magnification) A: Merged picture for Specific DiLDL-UAE-1 double staining of EPCs in culture for characterization (400X magnification)

VEGFR-2 gene expression 4.98±1.32 5.63 ± 0.7 4.33 ± 1.50 0.024* 4-Relation of EPCs counting & function with the development of collateral formation in good and poor collaterals of CTO patients: parameter All subjects (No=20) Good collateral (No=10) Poor collateral (No=10) P value EPCsX106 count/mL 1.88 ±1.08 2.39 ± 0.63 1.37±1.23 0.033* eNOS gene expression 6.46±1.38 7.08 ± 0.69 5.84± 1.63 0.041* VEGFR-2 gene expression 4.98±1.32 5.63 ± 0.7 4.33 ± 1.50 0.024* P value <0.05 was significant *

Results of the Experimental Study

a) CK-MB &Troponin I levels in group1 EPCs-AMI treated canines: 1- Biochemical analysis of troponin I and CK-MB: a) CK-MB &Troponin I levels in group1 EPCs-AMI treated canines: CK-MB U/L Troponin I ng/mL Mean ± SD after ligation (24h) 59.50 ± 6.156 0.4117±0.05076 after EPCs injection (1 week after ligation) 39.17 ± 2.041 0.1833±0.01966 at scarification (26±4 days after EPCs injection) 30.67 ± 1.366 0.1400±0.01095

b) CK-MB &Troponin I levels in group2 AMI control canines: CK-MB U/L Troponin I ng/mL Mean ± SD after ligation (24h) 62.00±2.449 0.3733 ±.04082 after saline injection (1 week after ligation) 41.83±0.983 0.1717 ±0.01472 at scarification ( at variable times) 31.67±0.516 0.1300±.01789

2-Electrocardiogram tracings at the indicated time intervals before and after intramyocardial infusion of the hEPC: A: After 2 hours of AMI, Starting ST segment elevation. B: After 24 hours of AMI, More ST segment elevation. C: After one week of AMI, Resolution of ST segment elevation. D: After one month of AMI, Resolution of ST segment to the normal basal level.

3-Localization of injected labelled EPCs under fluorescent microscope to assess homing and differentiation: A B (A) homing and localization of injected Dil-labeled hEPCs within cardiac striated muscles of canine (100X magnification). (B) positive-DAPI staining of injected EPCs to document their viability after transplantation (100X magnification).

A B C 4- Analysis of canine heart histopathology: a) Normal striated cardiac musculature: A B C b) Infracted muscle:

c- Histopathological cardiac tissue analysis of infarction injected with human EPCs: B (A): indicates collection of large vessels & many budding capillaries within fibrosed infarction HE (magnification 200X). (B): Dense fibrosis with many newly formed capillaries surrounding necrotic muscle MT(magnification 200X).

A C B (A): neovascularization & branched congested blood vesselswithin fibrosed infarction, HE (magnification 10X). (B):increased intramuscular vessels, MT(magnification 100X). (C): Dil-labeled human EPCs within cardiac tissue of canine was branching into newly formed blood vessel.

5-Immunohistochemistry: Transdifferentiation of injected hEPCs into cardiomyocyte-like cells and their green staining for troponin I as a cardiac marker (magnification 100X)

6- QRT-PCR gene expression of human VEGFR-2 and eNOS to assess hEPCs homing in canine heart tissues: a) eNOS & VEGFR-2 gene expression: Mean Std. Deviation Sig. eNOS gene expression on adjacent tissues 0.114 0.0096 0.283 on infarcted tissues 0.175 0.0116 VEGFR-2 gene expression 0.189 0.0069 0.347 0.260 0.0139

Conclusion: Our findings suggested that EPC-mediated angiogenesis might be associated with coronary collateral formation in humans. EPCs are able to form new blood vessels and to differentiate into cardiomyoctes in an experimental model (canines) with acute myocardial infarction.

THANK YOU