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.
Highlights
Several homologs of the gp91phox (Nox2) catalytic moiety of multisubunit NADPH oxidase (Nox): Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2, have been recently identified [1]
Our results demonstrate that NADPH oxidase plays a critical role in the regulation of both CD44 and hyaluronic acid (HA) expression in vascular smooth muscle cells (VSMC) treated with thrombin
Thrombin Induces Cd44 Expression in VSMC through NADPH Oxidase-dependent Activation of AP-1—We previously reported NADPH oxidase-dependent regulation of CD44 expression in wild-type VSMC treated with thrombin for 8 h [4]
Summary
Chemicals and Reagents—If not stated otherwise, all chemicals were obtained from Sigma. 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) was purchased from Calbiochem. Rac Activation Assay—Cell lysates from thrombin- and LMW-HA-treated VSMC were analyzed for Rac activation using the PAK-1 PBD pull-down method (Rac Activation Assay Kit, Millipore). This assay uses the Rac downstream effector, p21-activated protein kinase (PAK), to isolate the active GTP-bound form of Rac from the sample. Cells were incubated with 2 units/ml of thrombin, rat anti-mouse CD44 monoclonal antibody (Millipore), or normal mouse IgG (Santa Cruz Biotechnology) at 37 °C for 30 min, and 2 ϫ 104 cells were seeded on the top of Transwell filters. The Human CD44 ELISA kit (Cell Sciences) was used to measure soluble CD44 in mouse plasma and CD44 in protein lysates from mouse aortas.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
