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NADPH oxidase activity does not affect cellular cholesterol loading in vascular smooth muscle cells

Yuan Zhonghua, Madamanchi N R, Vendrov A E, Niu Xilin, Li Juxiang, Runge M S

Institute of Cardiovascular Disease,Key Laboratory for Arteriosclerology of Hunan Province,University of South China.Hengyang 421001,Hunan；USA

Abstract

Reactive oxygen species generated by NADPH oxidase enhance aortic vascular smooth muscle cell proliferation and migration which play an important role in the pathophysiology of atherosclerosis. We investigated the role of NADPH oxidase in the cellular cholesterol metabolism in vascular smooth muscle cells using p47phox-deficient cells. Wild-type and p47phox knockout vascular smooth muscle cells were loaded with cholesterol for 72 h by using 10 mg/L cholesterol:methyl-#beta#-cyclodextrin complexes and then incubated with or without 0.3 mg/L thrombin for 10 min. Foam cell formation was determined by accumulation of intracellular cholesterol, oil Red O-stained lipid droplets. After cholesterol loading, cellular lipid droplets raised sharply, cellular cholesterol increased from (31.4±2.0) to (61.0±2.1) mg/g protein (P<0.05) in wild-type cells, and from (29.8±2.5) to (51.3±3.1) mg/g protein (P< 0.05) in p47phox deficient cells, but the difference between the two cell types was not significant. Immunostaining showed decreased levels of smooth muscle #alpha#-actin and increased levels of macrophage marker Mac-2 in both wild-type and p47phox deficient vascular smooth muscle cells. One of the macrophage-related inflammation genes, monocyte chemoattractant protein-1 (MCP-1) expression didnot change in both two cell types detected by immunostaining. Although additional incubating with thrombin, another macrophage-related inflammation gene, vascular cell adhesion molecule-1 (VCAM-1) expression was similar in all groups analyzed by real-time RT-PCR. However, the expression of ATP-binding cassette transporter A1 (ABCA1), acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), the key proteins in cellular cholesterol metabolism, were similarly increased (P<0.05) in both two cell types as determined by quantitative real-time RT-PCR and Western blot, and it was not related to the state of oxidative stress. Interestingly, the expression of adipophilin, the lipid droplet related protein, had the similar results with ABCA1 and ACAT1, but, in wild-type cells, its expression also increased merely incubating with thrombin as determined by quantitative real-time RT-PCR. Together, these results suggest that p47phox-dependent NADPH oxidase is not involved in transdifferentitation of vascular smooth muscle cells into macrophage-like state after cholesterol loading. Deleting p47phox gene does not affect the cellular cholesterol metabolism in vascular smooth muscle cells.

Key words: NADPH oxidase;Cholesterol;gene deletion;myocytes, smooth muscle;atherosclerosis

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Citing This Article：

Yuan Zhonghua, Madamanchi N R, Vendrov A E, Niu Xilin, Li Juxiang, Runge M S. NADPH oxidase activity does not affect cellular cholesterol loading in vascular smooth muscle cells. Acta Physiol Sin 2008; 60 (4): 511-519 (in Chinese with English abstract).