The Therapeutic Effect On Ischemia Heart Disease By Hypoxic Preconditioning Of Endothelial Progenitor Cells Transplantation

Objective: We aimed at hypoxic preconditioning endothelial progenitor cells(HEPCs), investigating the therapeutic effectiveness of transplantation HEPCs into acute infarcted myocardium and potential molecular biological mechanism.Content:In present study, we observe the effectiveness of hypoxic preconditioning on endothelial progenitor cells; study the potential molecular biological mechanism mediated by SDF-1α/CXCR4,PI3K/Akt signal pathway; evaluate the therapeutic effect of transplantation HEPCs into infarcted myocardium on ventricular remodeling, cardiac function and haemodynamics.Method: Bone marrow endothelial progenitor cells(EPCs) were islolated from syngeneic adult male Wistar rats, as demonstrated by the double affinity for Di I-ac LDL and UEA-1, as well as the expressions of CD34 and CD133. The BM-EPCs were then subjected to 72 h of normoxia followed by either another 72 h normoxia or increasing periods of hypoxia(1%O2+5%CO2+94%N2) for 24,48 and 72 h. BM-EPCs were showed to be sensitive to hypoxia, and changed morphologically and functionally in response to hypoxic preconditioning. Using Annexin V/PI to evaluate anti-apoptosis ability of the cells. Boyden Chamber assay was used to compare migration ability between groups towards 100ng/ml bone marrow derived factor-1α(SDF-1α). Matrigel assay was used to screen angiogenisis of BM-EPCs. Using RT-PCR to quantity relative gene expression, such as CXCR4, PI3 K, Akt, NF-κB. Using western blot assay to detect protein expression of CXCR4, PI3 K and Akt. Then, 34 syngeneic adult male Wistar rats, 8-week old, 250g~280g, were randomized into 3 groups: Control(n=12),EPCs(n=10) and HEPCs(n=12),set up acute myocardium infarction animal model by ligation left anterior descending coronary artery. At infarction, the rats received 5-points peri-infarct intramyocardial injections of PBS 200 ul, 2×106 EPCs and 2×106 hypoxia 24 h EPCs. After 4 weeks, the rats underwent echocardiography to evaluate morphological changes of the heart, using closed chest pressure-volume conductance catheter to analyze improvement of cardiac function by haemodynamics parameters.Result: After cultrue bone marrow endothelial progenitor cells in 1%O2+5%CO2+94%N2, the cells are still alive. After changing culture media for 1 day, attachment and cluster formation were developed. As time went on, more and more spindle shaped attaching cells appeared. Cord-like structures were observed after changing media for 3 days. Cells were demonstrated by the double affinity for Di I-ac LDL and UEA-1, as well as the expressions of CD34 and CD133.The time course exposure to hypoxia for 24 h caused significant enhancements in the formation of tube like structure and motility of BM-EPCs(p<0.05), while the tube formation ability and motility activity of BM-EPCs were reduced after prolonged hypoxic preconditioning for 48 and 72 h, as compared with the BM-EPCs under normoxic condition(p<0.05). However, hypoxic preconditioning of BM-EPCs for 24 h in our study did not resulted in increased apoptosis resistance, and apoptosis of BM-EPCs was even enhanced by hypoxic preconditioning for 48 and 72 h. The m RNA expressions of CXC chemokine receptors 4(CXCR4), as well as pro-survival factors PI3 K, AKT, and NF-κB were induced by 24 h hypoxic preconditioning when compared with cells grew under normoxic condition, and the data were reversed to some extent by prolonged hypoxia. Despite of these, our in vivo transplantation experiments demonstrated the beneficial effect of hypoxic preconditioning on the left ventricular(LV) functions after acute myocardial infarction in rat.Conclusion: To sum up, our present study showed evidence suggested that hypoxic preconditioning did exert further beneficial effects of BM-EPCs on preservation of LV function after acute myocardial ischemia. The short term exposure to hypoxia for about 24 h provided better condition for BM-EPCs survival and differentiation, and further study is still needed to optimize the hypoxic pattern of BM-EPCs preconditioning so as to better protect heart from myocardial ischemic injury.