The Mechanism Of Mfn2Gene To Promote Cholesterol Efflux In Vascular Smooth Muscle Cell-derived Foam Cells

Part Ⅰ The mechanism of Mfn2gene inhibits foam cell formation1. PurposeTo explore the effect of expression of exogenous Mitofusin2(Mfn2) levels on rats vascluar smooth muscle-derived foam cells and the molecular mechanism of cellular cholesterol efflux that may exist.2. MethodsThe flesh serum of healthy individuals were collected and oxidated by copper ion after seperated LDL by density gradient centrifugation. Rats vascluar smooth muscle cells (rVSMCs) were co-cultured with oxidated LDL in vitro to induct the formation of foam cell, we make the intervention in different concentration of Adv-Mfn2(20pfu/cell,40pfu/cell, and60pfu/cell) in the induction of rVSMCs derived foam cells model. Foam cells were stained with oil red O and observed under a light microscope. The cholesterol level of foam cells were detected in the enzyme-chemical method and the ratio of total cholesterol to free cholesterol were calculated. The Cholesterol transporters adenosine triphosphate-binding cassette subfamily A member l(ABCA1) and adenosine triphosphate-binding cassette subfamily G member1(ABCG1)and the phosphorylation of PPARy protein level were dectect by Western blot.3. ResultsWhile the rVSMCs were transfected with Adv-Mfn2(60pfu/cell) for24h with oxidated ldl(80ug/mL), the lipid droplets in foam cells were reduced significantly. The ratio of intracellular free cholesterol ester compared to total cholesterol levels dropped significantly detected by chemical-enzyme assay. The phosphorylation level of PPARy protein decreased and the expression level of ABCA1and ABCG1increased detected by Western blot.4. ConclusionsAdv-Mfn2could significantly reduce the formation of foam cells after transfected at the appropriate level, and furthermore it could decreases the intracellular cholesterol levels to prompt the expression of cholesterol transport protein ABCA1and ABCG1. Part II The effect of exogenous Mfn2gene on mitogen-activated protein kinase signaling pathway in foam cell formation1. PurposeTo investigate the the mechanism of different expression concentration of mitofusin2gene on the phosphorylation level of MAPKs signaling pathway related to the formation of smooth muscle-derived foam cells.2. MethodsrVSMCs were transfected different titers of adv-Mfn2(20,40and60pfu/cell) for24h, and then it were co-cultured with the ox-LDL and for24hours. The phosphorylation level of ERK1/2, JNK and p38signaling pathway proteins were detected by Western blot. Statistical significances were compared using Tukey-Kramer post hoc test and student t-test by SPSS12.0. Differences were considered significant when P<0.05.3. ResultsThe Western Blot results showed that when foam cells were transfected with60pfu/cell, the phosphorylation levels of ERK1/2of p38proteins of the rVSMC derived foam cells were significantly reduced, regardless of the phosphorylation level of JNK.4. ConclusionsThe moderate level of exogenous Mfn2expression in rVSMC derived foam cells could inhibit the induction of foam cell formation with oxidiated ldl in vitro, which were achieved by reducing the the phosphorylation level of ERK1/2and p38signaling pathway, and independent of the phosphorylation level of JNK signaling pathway.