Effects Of Pioglitazone On Bone Marrow-derived Endothelial Progenitor Cells Of Spontaneously Hypertensive Rats

Background and Objective: Along with rapid development of material civilization, people's income increases and their living standard advances.But accompanied by a higher standard of living,they intake high cholesterol and calorie diet. In addition, high degree of mechanization makes people inactivity and more stresses which lead to the high incidences of hypertention and coronary heart disease nowdays. As one of the most important risk factors of coronary heart disease, high blood pressure will lead to small artery endothelial cells damage and dysfunction, participate in the process of atherosclerosis. Endothelial progenitor cell (EPC) is one kind of the most important stem cells, which participate in the vasculogenesis in the early stage of embryo development. With the active proliferation and differentiation capacity and vasculogenesis ability independent of vascular endothelial cells,the EPCs can be used as an ideal cell donor for stem cell therapy. But for limited sources of EPCs,the EPCs transplantation is an individualized treatment. There are a relative small number of cells even culturing cells in vitro. Rencent researches show that the microenvironment is more important than stem cells themselves in determining their fate. The changes of the microenvironment may play a pivotal role in the directed differentiation of stem cells, but may have a side effect on their proliferation and functions. Unfortunately researchers have not paid much attention to the double effects. People are seeking effective intervension to improve mobilization and differentiation of stem cells.PPARγnot only participates the process of serum glucose and lipid regulation, has the capacity of anti-inflammatory and antioxidation, but also improves functions of endothelial cells. In a word, PPARγcould protect the cardiovascular sysytem. Researches abroad pointed that EPCs express PPARγ. Activated PPARγcan up-regulate EPCs numbers, improve function of EPCs and inhibit apoptosis in health individual. But there is few research about the relationship between PPARγand EPCs in hypertension individual. The present study wants to: (1) investigate the effects of hypertension on bone marrow-derived EPCs of rats; (2) investigate the effects of pioglitazone (PPARγagonist) on EPCs of spontaneously hypertensive rats (SHR).This study is helpful for further study of biological characteristics and regulation mechanism of EPCs ,and provides evidences for clinical application of pioglitazone.Methods and Results: In the first part of the study, we used SHR and WKY of different ages. Age-matched SHR and WKY were used as one pair in the proportion of 1:1. 4, 8, 16, 24-week rats were defined as A, B, C D groups. Mononuclear cells of rats were isolated from rat bone marrows by Ficoll density gradient centrifugation. The cells were cultured with the EPCs specific medium with recombinant VEGF and bFGF in it. Adhering cells were harvested, and identified to EPCs by Dil-ac-LDL and FITC-UEA-I double staining. Number of EPCs were counted, proliferative capacity,NO secretion and rate of cell apoptosis were evaluated. Results: 1. In A group, there is no difference of BP in SHR and paired age-match WKY. But BP of SHRs were getting higher than paired age-matched WKY since the 8th week.(B, C, D group), while BP of different ages WKY were uniformity.2. The Ficoll density gradient centrifugation method is an easy way with lowcost, as well as a 100% success rate for culturing EPCs. The purity of EPC is about 70%~90% by identification with immuno-fluorescence dyes and biomarkers. There is no significant differents in number of bone-marrow derived EPCs between SHR and WKY in A group. But in B, C, D groups, numbers of bone-marrow derived EPCs of WKY were more than age-match SHR.3. There are no significant differences in proliferative capacity,NO secretion and rate of apoptosis of bone-marrow derived EPCs between SHR and WKY in A group. But in B,C,D groups,there are significant differences. The proliferative capacity and NO secretion of bone-marrow derived EPCs from SHR were lower. The rate of cell apoptosis were higher..In the second part, 12-week SHR and WKY rats were randomized to receive pioglitazone (PIO) (20mg?kg-1?d-1) or normal saline for 14 days. Body weight (BW), heart rate (HR) and blood pressure (BP) were monitored during the pretreatment. Arterial blood were collected at the first and the last day of the pretreatment in order to investigate the fasting blood glucose (FBG), fasting insulin (FIN) level. Ratio of FBG/FIN was calculated. Bone marrow-derived EPCs of rats were isolated and cultured with the mothed used in part 1. Number of EPCs was counted, proliferative capacity, NO secretion and rate of cell apoptosis were evaluated. Results:There were no significant differences of BP, BW, HR, FBG, FIN and the ratio of FBG/FIN in WKY after pretreatment, as well as no differences of EPCs proliferation, NO secretion and rate of apoptosis between pretreatment group and control group in WKY. However, PIO significantly decreased level of BP, FBG,FIN in SHR while no influences on BW and HR. PIO markedly increased bone marrow-derived EPC number in SHR, and improved proliferation and NO secretion. PIO decreases BP, FBG and FIN in SHR, improves insulin sensitivity, up-regulates bone marrow-derived EPC number, improves EPC dysfunction and inhibits EPCs apoptosis in SHR. The result suggested that PIO could protect EPCs derived from bone-marrow of SHR.In the third part, isolated EPCs from SHR were divided into PIO group (with 10μmol /mL PIO in the culture medium) and blank group. Proliferative capacity,NO secretion and rate of cell apoptosis were evaluated. Results: EPCs proliferative capacity,NO secretion in PIO group were significantly better than that in blank group, rate of cell apoptosis was lower. The result suggested that PIO has a direct protect effect on SHR bone-marrow derived EPCs. Conclusion:The result we obtained showed that: 1.high blood pressure is harmful for bone-marrow derived EPCs; 2. PIO pretreatment (20mg?kg-1?d-1) can lower BP, FBG, and improve insulin sensitivity in SHR, as well as can up-regulates bone marrow-derived EPC number, improves EPC dysfunction and inhibit EPCs apoptosis in SHR; 3. Ex vivo study had also suggested that PIO has a direct protect effect on SHR bone-marrow derived EPCs.