Cellular Repressor Of E1a-stimulated Genes Overexpression In Bone Mesenchymal Stem Cells Protect Against Rat Myocardial Infarction

Background:Myocardial infarction(MI) is one of the most common human diseases. Even if the patient can survive through the acute phase, cardiomyocytes will lose irreversibly and be replaced by fibrous scar tissue due to the poor regeneration, leading to heart failure finally.It’s reported that bone marrow mesenchymal stem cells(BMSCs) transplantation can be used to treat myocardial infarction. They can effectively prevent pathological ventricular remodeling, enhance cardiac contractile function, and promote the recovery of heart function. BMSCs induce angiogenesis and improve infarct zone microenvironment by the paracrine of various growth factors after transplanting into the marginal zone of infarcted myocardium. Therefore, increasing the secretion of angiogenic factors after BMSCs transplantation for MI is the hot spot of cardiovascular, tissue engineering and regenerative research. Cellular repressor of E1A-stimulated genes(CREG) widely existsin adult tissues and cells, but hardly be found in primitive stem cells. It is reported that CREG promote the vascular endothelial growth factor(VEGF) secretion in vascular endothelial cells. In addition, over-expressing CREG in embryonic stem cells not only can promote differentiation into endothelial cell under directional revulsant but also not affect the angiogenesis function of endothelial cells. We have also found that previous studies in vitro overexpression of CREG in BMSCs can enhance its capability against apoptosis under pathological conditions. However, the effect of BMSCs overexpressing CREG for the MI animal treatment is unclear and the mechanism is also confusing. We designed to study the cardioprotective effects of CREG overexpression in BMSCs(CREGBMSCs) after transplantation into infarcted heart of rats in order to provide new tools and theories for preventing myocardial infarction.Objectives:1. To establish rats MI model and to observe the effect ofCREGBMSCs treating for MI rats.2. To determine thatCREGBMSCs reduce injury of MI rats through increasing the secretion of angiogenic factors.3. To investigate whether CREG participates in the process of HIF-1α promoting VEGF paracrine, and to elucidate the relating mechanism.Methods:1. To produce the BMSCs overexpressing CREG: Adenoviral GFP(Ad-GFP) and GFP-CREG(Ad-GFP-CREG) transiently transfect BMSCs respectively. The effect is d determine by fluorescence microscope. When the transfection efficiency beyond 95%,they can be used for experiment.2. Rat MI model: Male Sprague-Dawley rats were anesthetized with pentobarbital sodium(50 mg/kg, i.p.). Myocardial infarction was induced by performing a left thoracotomy at the fourth rib, exposing the heart, placing a 4-0 silk around the left anterior descending coronary artery(LAD), near its origin from the left coronary artery, and making a knot to permanently occlude it. 5 place around the infarct zone were been chosen for injection.3. Three and fourteen days after BMSCs transplantation for MI.(1) Doppler echocardiography was used to detect the cardiac function;(2) Masson staining was used to measure the size of heart fibrosis;(3) Western blot was used to detect the expression of Cleavece-caspase 3 in heart tissue;(4) ELISA was used to detect the expression of VEGF;(5) Confocal microscope was used to observe CD31 staining in heart.4. We observe whether CREG affect the growth factor paracrine of BMSCs under hypoxic conditions in vitro by ELISA and the angiogenesis with matrigel.5. We explore the mechanism of CREG regulating BMSCs paracrine under hypoxic conditions in vitro by western blot and real-time PCR.Results:1. In vivo(1) Comparing with other groups, the LVEF and LVFS ofCREGBMSCs are significantly improved after transplantation for MI at 3 day and 14 day. It showed thatCREGBMSCs could improve BMSC’s treatment effectiveness and cardiac function for MI after transplantation further.(2) Comparing with other groups, the fibrosis size of heart is the minimum inCREGBMSCs transplantation for MI at 3 day and 14 day. It showed thatCREGBMSCs could attenuate fibrosis and prevent ventricular remodeling further.(3) The expression of Cl-caspase 3 is lowest inCREGBMSCs transplantation for MI at 3 day and 14 day compared with other groups. It showed thatCREGBMSCs could inhibit the myocardial apoptosis further.(4) The expression of CD31 is highest inCREGBMSCs transplantation for MI at 3 day and14 day compared with other groups. It showed thatCREGBMSCs could promote the myocardial angiogenesis and improve the microenvironment of the infarct zone further.(5) CREG can delay the BMSCs apoptosis after transplantation for MI and extend VEGF paracrine of it.2. In vitro(1) Under hypoxia in vitro, BMSCs overexpressing CREG can increase the secretion of VEGF and its supernatant can promote vascular endothelial cells generate networks.(2) CREG increases HIF-1α expression and nuclear translocation resulting in the increaseof VEGF in BMSCs under hypoxia.(3) CREG increases VEGF expression and promotes angiogensis through HIF-1α has been validated by the Si RNA of HIF-1α in BMSCs.(4) CREG attenuates the synthesis of VHL m RNA which regulates the degradation of HIF-1α resulting in the increase of HIF-1α.Conclusion:1. Transplantation of BMSCs overexpressing CREG for MI can improve myocardial function, inhibit fibrosis and attenuate apoptosis further.2. BMSCs overexpressing CREG can prolong its VEGF’s secretion and promote angiogenesis after transplantation for MI.3. CREG attenuates the synthesis of VHL m RNA which regulates the degradation of HIF-1α, resulting in secretion of VEGF and angiogenesis.