The Expression Of Steroidogenic Acute Regulatory Protein And Its Significance In Vascular Endothelial Cells And Mice Liver

The steroidogenic acute regulatory protein (StAR) was first found in steroidogenic tissues. It could transport cholesterol from the outer mitochondrial membrane into the inner, which play an important role in the rate-limiting step in steroidogenesis. Cholesterol can be oxidized to the more polar oxysterols by the enzyme in the inner mitochondrial membrane. StAR can contribute to the cholesterol efflux and may be involved in the reverse cholesterol transport.With the studies developing, it was reported that StAR also express in the non steroidogenesis tissue such as liver with similar function. Cholesterol delivery to the inner mitochondrial membrane was the predominant rate-determining step for bile acid synthesis via the alternative pathway. Interestingly, it could produce high levels of potent regulatory oxysterols in this process. By binding and activating nuclear sterol receptors, the generated regulatory oxysterols can regulate the expression of key genes involved in maintenance of cholesterol homeostasis.It is known that hypercholesterolemia is one of the important risk factors of atherosclerosis. In vessels, endothelial cells (ECs) are the first line of defense against constant insults from circulating risk factors for atherosclerosis and other cardiovascular diseases. Homeostasis of cholesterol is an effective prevention manner for atherosclerosis. We hypothesized that endothelial cells may play an important role in cholesterol efflux from cells by transformating it to oxysterols, which can prevent the lipid aggradation into the tissue under endothelial cells. The role of StAR in lipid metablism was investagted in the cultured endothelial cells in vitro and the expression of StAR and its significance were also studied in the animal model of atherosclerosis established by apoE-knockout mice in vivo.This paper is composed of four parts as following.Part I .To establish a method of primary culturing the rat myocardial microvascular endothelial cells (RMMVEC). The morphology of RMMVECs was studied by light and electronic microscopy. To study the cytological and genetic characteristics of primary RMMVECs by microarray, the RMMVECs were cultured by the method of planting myocardial tissue based on the previous work of our lab. Its molecular markers were observed by immunocytochemistry. Cell proliferation kinetic was analyzed by cell number counting. The gene expression of the RMMVECs was studied by endothelial cell biology gene microarray. The results suggested that primary RMMVECs have morphological characteristics of microvascular endothelial cells (MVEC), growing in a cobblestone pattern, forming tube-like structure (TLS) or capillary network and having microvilli on cell surface. The RMMVECs showed positive staining for CD34, CD31, CD 105, vWF and Tie-2. The relatively high levels expression of VEGFR, ICAM-1, VCAM-1, angiopoietinl, PECAM1 (CD31) and other genes closely related to microvascular endothelial functions in primary and 2^nd passage RMMVECs were detected by gene microarray analysis. In summary, the RMMVECs cultured by this method possess typical characteristics of MVEC. The cytological characteristics were steady in the cultured cells of primary and 2^nd passage. It can be utilized to study the mechanisms of some cardiovascular diseases. However, the expression of characteristic genes in 5^th passage cells disappeared and it can not be used as the microvascular endothelial cells.Part II. To study the expression of StAR mRNA and protein and its regulation by lipids in endothelial cells. The expression and location of StAR protein in various endothelial cells were studied by immunochemistry. The regulation of StAR mRNA and protein expression in bEnd.3 cells by cholesterol, LDL and 25-hydroxycholesterol were investigated by Real-time RT-PCR and Western blot. The present study shows for the first time that StAR also expressed in the endothelial cells, including the primary cultured rat myocardial microvascular endothelial cells (RMMVECs), microvascular endothelial cell line-bEnd.3 cells, intima of aorta and mini-vein of human connective tissue, which was mainly located in cytoplasm. All of three lipid compounds could increase StAR expression in a time-dependent and dose-independent manner. The results of Real-time RT-PCR showed that after 4 h incubated with 10μg/ml CHO,. the expression of StAR mRNA reached the peak (P<0.05 vs. control), then returns to basal levels after 24h. The same result was achieved when incubated with 15μg/ml LDL (P<0.05 vs. control) while treated with 10μg/ml 25-hydroxycholesterol, the maximal time is at 12 h (P<0.01 vs. control). In the dose course experiment, the most effective concentration of CHO, LDL and 25-OH were 10μg/ml (P<0.05 vs. control), 15μg/ml (P<0.01 vs. control) and 10μg/ml (P<0.05 vs. control) respectively. The results of Western blot indicated that after 8 h incubation with 10μg/ml CHO, the expression of StAR protein arrived to the maximum (P<0.05 vs. control), and then returns to basal levels. When incubated with 15μg/ml LDL the maximum time is 24h (P<0.01 vs. control), while treated with 10μg/ml 25-hydroxycholesterol, the maximal time is at 4 h (P<0.05 vs. control). In the dose course experiment, the most effective concentration of CHO, LDL and 25-OH were 10μg/ml (.P<0.05 vs. control), 15μg/ml (,P<0.01 vs. control) and 10μg/ml (P<0.01 vs. control) respectively. The results indicated that StAR also express in RMMVECs, bEnd.3 cells, intima of aorta of human and mini-vein of human connective tissue, which was mainly located in cytoplasm. CHO, LDL and 25-OH could up-regulate the StAR expression in bEnd.3 cells in a time- and dose-dependent minner.Part III. To study the role of StAR on the cholesterol reverse transporters in bEnd.3 cells by overexpression StAR using adenovirus system. Real-time RT-PCR and Western blot were used to detect the overexpression of StAR and the expression of cholesterol reverse transporters ABCA1/ABCG1 in bEnd.3 cells. We found that the efficiency of the adenovirus system was over 96%, which suggested that it was an effective model to infect bEnd.3 cells. After overexpression StAR by adenoviral-mediate for 48 hours, ABCA1 and ABCG1 mRNA in bEnd.3 cells increased by 50% (P<0.05 vs. control and null adenovirus) and 80% (P<0.01 vs. control and null adenovirus) respectively. And ABCA1 and ABCG1 protein increased by 2 folds (P<0.01 vs. control and P<0.01 vs.null adenovirus) and 0.9 fold (P<0.01 vs. control and null adenovirus) respectively. Our results indicated that overexpression StAR in endothelial cells could increase the expression of the cholesterol reverse transporter ABCA1 and ABCG1. As a result, it may promote the efflux cholesterol from cells, reduced the accumulation of cholesterol in endothelial cells.Part IV .To study the expression of StAR in the apoE-knockout mice atherosclerosis model compared to the C57BL/6J mice which have the same genetic background, and the effects of lipids and age on StAR expression. The serum lipids levels were'detected by kits. The expression of StAR in mice liver of different age and sex were detected by Real-time RT-PCR and Western blot. We found that StAR mRNA and protein expression decreased with aging in C57BL/6J mice, reached its peak at one month and reduced since then with little change of lipids. The lipids increased with aging, and LDL-C was the most distinct one in the four lipids in apoE-knockout mice (3 month and 5 month vs. 1 month P<0.01, 5 month vs. 3 month P<0.01). StAR mRNA and protein expression increased at first and then decreased, 1 month old mice were higher than neonatale mice (P<0.05), 3 month old mice have the highest expression (P<0.01 vs. neonatal) and 5 month old mice have lower expression compared to 3 month old mice (P<0.01 vs. 3 month mice), there is no significance between neonatale mice and 5 month old mice. The levels of lipids in apoE-knockout mice were higher (P<0.01) and the StAR mRNA expression in mice liver were also higher than the corresponding C57BL/6J mice (P<0.01 in neonatal mice and 1 month old mice, P<0.05 in 3 and 5 month old mice). In the other hand, there was no significance in the StAR protein expression between the two type mice. Our results suggested that the StAR expression in mice liver was associated with age and serum lipids levels.In summary, the present study demonstrated that the cultured primary and 2^nd passage RMMVECs possessed typical characteristics of MVEC. StAR expressed in RMMVECs, bEnd.3 cells, intima of aorta of human and mini-vein of human connective tissue, which was mainly located in cytoplasm. CHO, LDL and 25-OH could up-regulate the StAR expression in bEnd.3 cells in a time-dependent and a dose-dependent manner. StAR could increase the expression of ABCA1 and ABCG1 mRNA and protein in endothelial cells and then promote the efflux cholesterol from cells, as a result, reduce the accumulation of cholesterol in endothelial cells. StAR expression in vivo was associated with the age and serum lipids levels.