The Effects Of EPC And MSC Derived From The Rat Bone Marrow On The Repair Of Atherosclerosis Vascular Endothelium

Atherosclerosis (AS) is responsible for a significant part of morbidity and mortality all over the world. The clinical manifestation of AS includes myocardial infarction, stroke, aortic aneurysm, aortic dissection as well as intermittent lameness. Not only the pathogenic factors leading to atherosclerosis are highly heterogeneous, but the causative risk factors include hyperlipidemia, hypertension, diabetes mellitus and smoking can also damage endothelial function.Endothelial dysfunction is an early hallmark of atherosclerotic disease and may correlate to ischemic events even in the absence of arterial obstruction. Thus, repair of the injured endothelial cells is an emphasis on AS therapy.In the context of regeneration, published data from animal studies have unveiled that endothelial progenitor cells(EPC) effectively contribute in restoring endothelial function.The mechanisms involved in regeneration of damaged endothelial cells have been suggested in some studies, showing that regeneration of endothelial cells were promoted when EPC were injected into rats of vascular injury. Animal studies have demonstrated that tissue ischemia upregulates many growth factors and cytokines, such as VEGF and SDF-1, which stimulated the release of EPC through eNOS and MMP-dependent pathways in the bone marrow. In addition, specific homing of EPC at the damaged site is directed by the interaction between chemokines produced locally and their specific receptors.Mesenchymal stem cells(MSC) first identified in the marrow stroma are a group of multipotent progenitor cells which can differentiate along multiple lineages giving rise to cartilage, bone, fat, muscle and vascular tissue. Some studies indicate that MSC play important roles in the progress of atherosclerosis. Specifically,MSC can differentiate into ECs that regenerate the damaged endothelial layer. Moreover some cytokine could be secreted by MSC,which provides a proper microenvironment for repairing the vascular injury and eovascularization.In this study, we establish an experimental animal model of atherosclerosis using rats by high-fat diet, and investigate the therapeutic effect of bone marrow-derived EPC and MSC on AS.PartⅠIsolation and culture of rat bone marrow endothelial progenitor cells and mesenchymal sten cellsobjectiveIn this study, we established the method of isolation and culture bone marrow-derived endothelial progenitor cells and mesenchymal stem cells, and investigated their biological characteristics and multipotential capacity.Methods1.Rat bone marrow-derived MSC were isolated by density gradient centrifugation, and cultured in MSC culture medium. Biological characteristics of MSC were detected by immunofluorescence staining, flow cytometry and growth curve, the differentiation potential of cells were tested using standard differentiation conditions for osteoblasts and adipocytes.2.Rat bone marrow-derived EPC were isolated by density gradient centrifugation, and cultured in EPC culture medium. immunophenotypic characterizations were analyzed using cytoimmunostaining, the ability of uptaking of acetylated low-density lipoprotein (acetylated LDL) and binding to UEA-1 was detected, and the growth characteristics and clonogenic potential were observed.Results1.MSC revealed a surface antigen profile that is coincidence with that reported for BM-MSC, that is, CD29+,CD90+, and do not express surface markers of hematopoetic cells and endothelial cells, namely CD34-,CD45-, CD31-,vWF-. Moreover, these cells could differentiate into osteoblasts and adipocytes under specified culture condition. The growth curve of the third passage MSC showed that the cells grew slowly in the first two days and begin to speed up from the third day after plating. The doubling time was 49.13±0.7 hours. 2.The endothelial specific markers vWF,CD31 and CD 144 were expressed by the cultured cells and these cells were capable of incorporating Dil-Ac-LDL and binding to lectin UEA-1. EPC showed different proliferative potential in different passages. The proliferative potency of the second passage cells was higher than fifth passage, their doubling time was 82.70±4.28 and 90.88±5.30 hours, respectively. The formation of endothelial cell colonies was found when the cells were plated at a low density. There was a linear relationship between the number of plated cell and the number of coloniesConclusionEPC and MSC can be isolated and cultured succesfully from the rat bone marrow.Part IIThe establishment of AS rat model and analysis of endothelial senescence after high-fat diet challengeObjectiveThe aim of this study was to establish an experimental rat model of atherosclerosis, and investigate the changes of rat arterial endothelial senescence.Methods1. Twenty eight male healthy SD rats were randomly divided into four groups. In the control group (A) rats were fed with basic food, while in other groups rats were fed with high fat-diet. After three months the levels of plasma total cholesterol (TC), triglyceride (TG) and high density lipoprotein-cholesterol (HDL-C) were measured with biochemical techniques. The pathological changes of arterial vessels were examined by HE staining.2. The endothelial cells from arterial vessels of high-fat and normal diet rat were isolated and identified by immunocytochemistry after a 12-wk high-fat dietary treatment, Senescence-associated (3-galactosidase (SA-β-gal) activity was examind using the method of cytochemistry.Results1. Adult SD male rat were fed with high-fat diet to induce to heperlipemia. After 12 weeks of feeding, the levels of serum triacetylglycerid, total cholesterol and high density lipoprotein cholesterol were elevated in high-fat fed mice compared with normal diet fed mice.2. No pathological changes were observed in the aorta in control group by HE staining, but the arterial vessels showed pathology changes of atherosclerosis.3. High-fat diet increased the prevalence of endothelial senescence, SA-β-gal acitivies increased significantly in postdietary challenge.Conclusion1. The AS rat model was established succesfully.2. High-fat diet damaged arterail vessels and caused arterial endothelial cell senescence, and increased senescence-associated SA-β-gal activities in rat.PartⅢThe repairing effect of bone marrow-derived EPC and MSC on ASObjectiveIn this study, the repairing effect of bone marrow-derived EPC and MSC on AS was investigated.Methods1. rAAV2-IRES-GFP was packed and purified, and was used to transduce EPC and MSC.1,packing and purifing rAAV2-IRES-GFP,and EPC and MSC were transferred by rAAV2-IRES-GFP2. Group A (normal control) was injected with saline; Group B (untreated model control) was injected with saline; Group C EPC transduced by rAAV2-IRES-GFP were transplanted into model rat; Group D MSC transduced by rAAV2-IRES-GFP were transplanted into model rat. After 2 months, the level of blood lipid was tested. The change of aorta histology was observed by HE staining and GFP-labeled cells by frozen section.3.Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression level of eNOS, ICAM-1 and APOE in artery vessel. ResultsThe level of blood lipid in all treated rats decreased substantialy compared with the model group. The EPC and MSC transferred with rAAV2-IRES-GFP were found in the endothelial monolayer of artery vessel of EPC-treated group and MSC-treated group, respectively. The result of HE staining suggested that, the lipid deposits in aortic endothelium in both cell transferred groups were less than those in the untreated group.RT-PCR suggested that eNOS, apoE expression level of arterial vascular wall in MSC treatment group and EPC treatment group was both significantly higher than untreated control group, but still lower than the normal diet controls. The expression levels of ICAM-1 in treatment group was significantly lower than the pathological control group (p<0.05).These results showed that the therapeutical effect of EPC on AS was more obvious than that of MSC.ConclusionBoth bone marrow-derived EPC and MSC had the repairing effect on arterial endothelium of AS, but the therapeutical effect of EPC was more obvious than that of MSC on the damaged arterial endothelium.