CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs
Wei Li, … , Masayuki Yamamoto, Roy L. Silverstein
Wei Li, … , Masayuki Yamamoto, Roy L. Silverstein
Published October 11, 2010
Citation Information: J Clin Invest. 2010;120(11):3996-4006. https://doi.org/10.1172/JCI42823.
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Research Article Cardiology

CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs

Abstract

CD36 is a membrane glycoprotein expressed on platelets, monocytes, macrophages, and several other cell types that was recently demonstrated to be involved in platelet activation in response to oxidized phospholipids, including oxidized LDL. Although the role of CD36 in other vascular cells has not been well defined, previous studies have demonstrated that cd36-knockout (cd36–/–) mice have prolonged thrombosis times after vascular injury, which can be protective in the state of hyperlipidemia. Here, we found significantly less ROS in the vessel walls of cd36–/– mice compared with WT after chemically induced arterial injury, suggesting that CD36 may contribute to ROS generation in the VSMCs themselves. Gene expression analysis revealed that the antioxidant enzymes peroxiredoxin-2 (Prdx2) and heme oxygenase-1 were upregulated in cd36–/– VSMCs. Molecular dissection of the pathway in isolated mouse VSMCs revealed CD36 ligand-dependent induction of Fyn phosphorylation, with subsequent phosphorylation and degradation of the redox-sensitive transcription factor Nrf2. Chromatin immunoprecipitation experiments further showed that Nrf2 directly occupied the Prdx2 promoter. The importance of this pathway was evidenced by increased ROS generation in prdx2–/– mice and decreased thrombosis times in both prdx2–/– and nrf2–/– mice after vascular injury. These data suggest that CD36-mediated downregulation of antioxidant systems in VSMCs may contribute to its prothrombotic, proinflammatory, and atherogenic effects.

Authors

Wei Li, Maria Febbraio, Sekhar P. Reddy, Dae-Yeul Yu, Masayuki Yamamoto, Roy L. Silverstein

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Figure 7

CD36 inhibits VSMC response to oxidative stress in vitro, and Prdx2 deficiency increases ROS generation and promotes thrombosis in vivo.

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CD36 inhibits VSMC response to oxidative stress in vitro, and Prdx2 defi...
(A) Western blot shows that cd36–/– cells have increased basal expression HO-1 compared with WT (lanes 6 and 7 versus lanes 1 and 2). After exposure to H2O2 (250 μM) for 3–6 hours, more HO-1 expression increased in the cd36–/– cells, but not in the WT. Stripped membranes were reblotted for actin as loading control. (B) Quantification of ROS in VSMCs by HPLC detection of EOH. WT, cd36–/–, and human Prdx2 cDNA–transfected WT VSMCs or mouse Nrf2 shRNA–transfected cd36–/– VSMCs were treated with FeCl3 for 30 minutes and incubated with DHE for another 30 minutes; cellular extracts were then examined by HPLC to detect and quantify EOH. Data are shown as fold changes of absence of FeCl3 per se; n = 3. *P < 0.001 versus others. (C) Topical application of FeCl3 induces increased ROS in carotid arteries of prdx2–/– mice. prdx2–/– and WT mice were treated as in Figure 1, and FeCl3-induced ROS generation was detected using DHE as a fluorescence probe. *P < 0.05, prdx2–/– versus WT. (D) Prdx2 and Nrf2 deficiency are prothrombotic. Vessel occlusion time after FeCl3-induced carotid artery injury was measured as in Figure 1 in prdx2–/–, nrf2–/–, and WT mice (n = 8 in each group). *P < 0.01, WT versus prdx2–/– or nrf2–/–. One-way ANOVA (Bonferroni/Dunn) was used to determine the differences among groups. Data are represented as mean ± SEM.