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NADPH oxidases regulate CD44 and hyaluronic acid expression in thrombin-treated vascular smooth muscle cells and in atherosclerosis.

ABSTRACT: The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE(-/-) mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47(phox-/-) VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE(-/-) compared with apoE(-/-)/p47(phox-/-) mice. HA-regulated pro-inflammatory gene expression was higher in apoE(-/-) than apoE(-/-)/p47(phox-/-) mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.

SUBMITTER: Vendrov AE

PROVIDER: S-EPMC2924092 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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NADPH oxidases regulate CD44 and hyaluronic acid expression in thrombin-treated vascular smooth muscle cells and in atherosclerosis.

Vendrov Aleksandr E AE Vendrov Aleksandr E AE Madamanchi Nageswara R NR Niu Xi-Lin XL Molnar Kimberly C KC Runge Mason M Szyndralewiez Cédric C Page Patrick P Runge Marschall S MS

The Journal of biological chemistry 20100617 34

The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE(-/-) mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ...[more]

PMID: 20558727

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Project description:Hyaluronic acid (HA) is an extracellular matrix (ECM) component that has been shown to play a significant role in regulating muscle cell behavior during repair and regeneration. For instance, ECM remodeling after muscle injury involves an upregulation in HA expression that is coupled with skeletal muscle precursor cell recruitment. However, little is known about the role of HA during skeletal muscle development. To gain insight into the way in which HA mediates embryonic myogenesis, we first determined the spatial distribution and gene expression of CD44, RHAMM and other HA related proteins in embryonic day (E)10.5 to E12.5 murine forelimbs. While HA and CD44 expression remained high, RHAMM decreased at both the protein (via immunohistochemistry) and RNA (via qPCR) levels. Next, we determined that 4-methylumbelliferone-mediated knockdown of HA synthesis inhibited the migration and proliferation of E11.5/E12.5 forelimb-derived cells. Then, the influence of CD44 and RHAMM on myoblast and connective tissue cell behavior was investigated using antibodies against these receptors. Anti-RHAMM, but not anti-CD44, significantly decreased the total distance myogenic progenitors migrated over 24?h, whereas both inhibited connective tissue cell migration. In contrast, anti-CD44 inhibited the proliferation of connective tissue cells and muscle progenitors, but anti-RHAMM had no effect. However, when myoblasts and connective tissue cells were depleted of CD44 and RHAMM by shRNA, motility and proliferation were significantly inhibited in both cells indicating that blocking cell surface-localized CD44 and RHAMM does not have as pronounced effect as global shRNA-mediated depletion of these receptors. These results show, for the first time, the distribution and activity of RHAMM in the context of skeletal muscle. Furthermore, our data indicate that HA, through interactions with CD44 and RHAMM, promotes myogenic progenitor migration and proliferation. Confirmation of the role of HA and its receptors in directing myogenesis will be useful for the design of regenerative therapies that aim to promote the restoration of damaged or diseased muscle.

Project description:Asymmetrical dimethylarginine inhibits nitric oxide synthase, cationic amino acid transport, and endothelial function. Patients with cardiovascular risk factors often have endothelial dysfunction associated with increased plasma asymmetrical dimethylarginine and markers of reactive oxygen species. We tested the hypothesis that reactive oxygen species, generated by nicotinamide adenine dinucleotide phosphate oxidase, enhance cellular asymmetrical dimethylarginine. Incubation of rat preglomerular vascular smooth muscle cells with angiotensin II doubled the activity of nicotinamide adenine dinucleotide phosphate oxidase but decreased the activities of dimethylarginine dimethylaminohydrolase by 35% and of cationic amino acid transport by 20% and doubled cellular (but not medium) asymmetrical dimethylarginine concentrations (P<0.01). This was blocked by tempol or candesartan. Cells stably transfected with p22(phox) had a 50% decreased protein expression and activity of dimethylarginine dimethylaminohydrolase despite increased promoter activity and mRNA. The decreased DDAH protein expression and the increased asymmetrical dimethylarginine concentration in p22(phox)-transfected cells were prevented by proteosomal inhibition. These cells had enhanced protein arginine methylation, a 2-fold increased expression of protein arginine methyltransferase-3 (P<0.05) and a 30% reduction in cationic amino acid transport activity (P<0.05). Asymmetrical dimethylarginine was increased from 6+/-1 to 16+/-3 micromol/L (P<0.005) in p22(phox)-transfected cells. Thus, angiotensin II increased cellular asymmetrical dimethylarginine via type 1 receptors and reactive oxygen species. Nicotinamide adenine dinucleotide phosphate oxidase increased cellular asymmetrical dimethylarginine by increasing enzymes that generate it, enhancing the degradation of enzymes that metabolize it, and reducing its cellular transport. This could underlie increases in cellular asymmetrical dimethylarginine during oxidative stress.

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NADPH oxidases regulate CD44 and hyaluronic acid expression in thrombin-treated vascular smooth muscle cells and in atherosclerosis.

Publications

NADPH oxidases regulate CD44 and hyaluronic acid expression in thrombin-treated vascular smooth muscle cells and in atherosclerosis.

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