The Related Mechanism Of Free Cholesterol Induced Cell Injury In Smooth Muscle Cells

Atherosclerosis is one of the most common cardiovascular diseases in the modern society. Several important environmental and genetic risk factors have been associated with atherosclerosis, including hypertension, high blood cholesterol, diabetes, obesity, gender and unhealthy lifestyles. The appearance of cholesterol and cholesteryl esters-laden foam cells in advanced atherosclerotic lesions is a hallmark of atherosclerosis. In the advanced plaques, excessive death of smooth muscle cells (SMCs) may compromise plaque integrity, weaken the stability of fibrous cap, lead to plaque rupture and finally trigger thrombosis and vessel occlusion.The original of foam cell was identified both from macrophages and SMCs. Most research works of the foam cell was focused on macrophage-original cell models. Recent studies have found that excess intracellular free cholesterol (FC) is the reason of macrophage-derived foam cell death. FC-overloading activated both endoplasmic reticulum-(ER) and mitochondrial-dependent apoptosis pathway and induced macrophage apoptosis. However, little is known about the relationship between excessive cytoplasmic cholesterol and SMCs death.In present study, rat smooth muscle cells were loaded with cholesterol:methyl-β-cyclodextrin (CHOL:MPCD) complexes accompanied with the ACAT inhibitor Sandoz58035. The potential impairment of the cellular calcium concentration, mitochondrial calcium concentration, ROS concentration and the dynamic change of the mitochondrial morphology and mitochondrial membrane potential were analyzed. We also estimated the cytotoxicity of excess FC and detected the activation of related mitochondrial-and ER-dependent cell apoptosis proteins. In order to evaluate the activation of autophagy in FC-overloading SMCs, we observed the conversion of microtubule-associated protein-1 light chain 3-I (LC3-I) to a phosphatidylethanolamine-conjugated form (LC3-II) and the formation of autophagic vacuoles (AVs). Combined with pharmacology experiments, we further explained the cellular and molecular mechanism of the activation of autophagy in SMCs. In conclusion, our experiments simulated the physiological and pathological changes of the SMCs under the FC-overloading condition and demonstrated that FC-overloading SMCs underwent a complex mode of cell death, including cell apoptosis and autophagy.The major results of our study were:1. Using CHOL:MβCD to establish the SMC-derived foam cell model. The FC-overloading cell model was then established with CHOL:MPCD accompanied with the ACAT inhibitor Sandoz58035.2. Compared with foam cell model, we found that the intracellular calcium concentration and mitochondrial calcium concentration were down-regulated while the ROS concentration was up-regulated in the FC-overloading cell model.3. After incubated with CHOL.MβCD plus 58035 for 4-8 h, the normal tubular mitochondrial network was completely converted to a punctual conformation.4. Our experiments herein revealed a decrease of mitochondrial membrane potential in FC-overloading SMCs. After a widespread mitochondrial dysfunction, FC-overloading caused an increase of cellular Bax accompanied with a decrease of Bcl-2 and finally caused the release of cytochrome C. Our experiments also showed that treatment with excess FC induced ER stress and caused UPR-mediated SMCs death, as demonstrated by upregulation of KDEL and inducing of CHOP, respectively.5. The excess intracellular FC led to a large-scale cellular organelles damage which further activated formation of AVs and LC3 processing. Enhanced autophagy played as a prosurvival mechanism that prevented cell death in FC-overloading SMCs.In this paper, the usage of water-soluble cholesterol and ACAT inhibitor to create SMC-original foam cell model and FC-overloading cell model were more stable and repeatable. Our experiments explained the cellular mechanism of FC-overloading induced SMCs damage through multiple intracellular signaling pathways. And the concept of autophagy was introduced into these cell models for the first time which is also an innovative point in this study.