The Protective Role Of Steroidogenic Acute Regulatory Protein(StAR) In Endothelial Dysfunction Induced By Palmitic Acid

Steroidogenic acute regulatory protein (StAR) is one of the cholesterol transporters, which is mainly involved in cholesterol metabolism and the synthesis of steroid hormones. Our previous studies showed that StAR expressed in liver, vascular endothelial cells and macrophages with the function that transporting cholesterol from outer mitochondrial membrane to inner mitochondrial membrane and regulating the lipid metabolism.As the fundamental element of endothelial structure and function, endothelial cells not only keep vascular wall integrity, but also play an important role in vascular homeostasis. First, the endothelium regulates vascular tone via producing and relwasing nitric oxide (NO), bradykinin, endothelin, prostacyclin. Secondly, the endothelium inhibits leucocyto or platelet aggregation and adhesion, smooth muscle cells proliferation and migration. Some endogenous and exogenous factors, such as psychogenous or physiologic stress, atherosclerosis, hypertension, aging, inflammation and diabetes, would induce to endothelial dysfunction. Endothelial dysfunction is generally defined as disequilibrium between vasodilation and vasoconstriction, thrombogenesis and fibrinolysis, inhibition and promotion of proliferation, which causes by impairment of endothelium-dependent vasodilation, inflammation and thrombogenesis. Endothelial dysfunction is considered an early marker for atherosclerosis and other cardiovascular diseases, and induced by prolonged exposure to the risk factors of cardiovascular diseases. The most important risk factor is lipid disorder, which is characterized by high level of cholesterol, especially high level of free fatty acid (FFA) in circulating plasma. Excess FFA has been shown to impair endothelial nitric oxide synthase activity, inhibit endothelium-dependent vascular vasodilation, stimulate inflammatory factors release and induce apoptosis. Therefore, the investigations of the mechanisms of FFA-mediated endothelial dysfunction might have a great significance in protection of endothelial dysfunction, and the further prevention and treatment of atherosclerosis. In the present study, we investigate whether StAR plays a protective role in palmitic acid induced endothelial dysfunction via StAR overexpression in the primary cultured rat aortal endothelial cells (RAECs).The present study is composed of two main parts as following. Part Ⅰ. The effect of StAR on lipid metabolism in rat aortic endothelial cells.We used the RAECs as the research objects, which were successfully cultured using the method of planting aorta rings. The morphology of RAECs was studied by inverted microscopy, its molecular makers were observed by immunocytochemistry. RAECs were transfected with adenovirus encoding StAR gene. Then real-time quantitative PCR and western blot were employed to demonstrate StAR overexpression in RAECs and genes expression related to lipid metabolism. The results showed that primary RAECs migrated from the aorta rings after72hours planting without movements. These cells have morphological characteristics of vascular endothelial cells, that are cobblestone pattern and tube-like structure (TLS). At the same time, RAECs showed positive staining of CD31and vWF. After infection with adenovirus at different multiplicity of infection (MOI=10,20,50,100) for48hours, mRNA and protein levels of StAR in RAECs increased followed with MOI increment, which indicated successful overexpression of StAR. Besides, the mRNA levels of FAS, ACC-1, HMGR, LDLR and SREBP-2mRNA decreased significantly after48hours transfection (P<0.05vs. EGFP). In some experiments, intracellular cholesterol and free fatty acid were extracted by chloroform/isopropanol/NP-40and chloroform/Triton X-100respectively, and analyzed by cholesterol detection kit and free fatty acid hypersensitivity assay kit. The results indicated that StAR overexpression decreased the intracellular total cholesterol and free fatty acid(P<0.05, vs. EGFP).Part Ⅱ. The protective role of StAR in endothelial dysfunction induced by palmitic acid (PA).We used the RAECs as the research objects, and overexpress StAR in RAECs by infection with adenovirus encoding StAR gene. After infection for48hours, RAECs were incubated in serum-and growth factor-free media in the presence of PA bound to BSA. Total RNA was extracted at different times for studying IL-1β, TNF-α, IL-6and VCAM-1mRNA levels by real-time quantitative PCR. ELISA was used to test the contents of inflammatory factors in cultural medium. Meanwhile, the cytoplasmic and nuclear protein of cells were extracted for detecting NFκB expression in cytoplasm and nuclear respectively by western blot. The results showed that StAR overexpression inhibited PA-mediated elevated gene expressions and release of inflammatory factors. The blocking of NFκB nuclear translocation and finally inhibiting the transcription of NFκB downstream inflammatory factors by StAR may involved in the mechanisms. After48hours infection, RAECs were incubated in serum- and growth factor-free media containing PA and BSA at different time. Further, total proteins were extracted for western blot analysis of protein expression related to p-IRS-1/p-Akt/p-eNOS signal pathway, and the culture supernatant was collected for NO detection at the same time. The results indicated that StAR overexpression could redeem the inhibition effect of PA on p-IRS-1/p-Akt/p-eNOS/NO pathway, and NO production and secretion, and finally maintaining the balance between vasodilation and vasoconstriction.In conclusion, RAECs were successfully cultured using the method of planting aorta rings and authenticated by morphology and cell surface markers. StAR was successfully overexpressed in RAECs by infection with adenovirus encoding StAR gene. Overexpression of StAR in RAECs inhibits the expression of key enzymes involved in synthesis of cholesterol and fatty acid, and also inhibits PA-mediated inflammation and decrease of NO production via regulating lipid metabolism and reduction of fatty acid synthesis. These findings may represent a new theoretical and experimental basis for prevention and treatment of atherosclerosis or diabetes.