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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2016 Mar 24;36(6):1197–1208. doi: 10.1161/ATVBAHA.116.307421

Figure 1.

Figure 1

LPA-mediated sustained CD36 transcriptional repression may be associated with histone deacetylase activity in endothelial cells. A, LPA exposure suppresses CD36 promoter activity. Primary HMVECs were transfected with reporter constructs containing the CD36 proximal promoter fused to the pGL3-basic (pCD36-luc) for 24 hrs with co-transfection of pRL Renilla luciferase for normalization. The cells were then exposed to LPA for 24 hrs before measurement of luciferase activity with Dual-Luciferase® Reporter Assay System. Promoter activity was expressed as the fold-change in relative luciferase activity compared with pGL3.2 basic control (mean ± S.E. of three independent experiments). B, LPA mediates sustained CD36 mRNA downregulation. Primary HMVECs were exposed to LPA (10 μM) for 24 hrs, and then LPA was removed and replaced with complete media for 48 hrs. The mRNA level was assayed with quantitative real time RT-PCR. Triplicate experiments were performed (p <0.01). C, LPA signaling significantly reduces CD36 mRNA expression in cardiac microvascular endothelial cells (HMVEC-C). Primary HMVEC-Cs (passage 3) were exposed to LPA (10 μM) for 24 hrs or pretreated with LPA receptor1,3 antagonist Ki16425 (2 μM) for 30 min followed by LPA (10 μM) for 24 hrs. Total RNA was obtained for quantitative real time RT-PCR. D, ChIP analyses targeting the CD36 proximal promoter in LPA-treated (10 μM, 24 hrs) HMVECs using antibodies against AcH3 (K9). Input shows DNA isolated from the lysate before immunoprecipitation (10% of the total chromatin was used for the PCR reactions). Lane labeled “IgG” is ChIP performed with a non-immune IgG. E, CD36 expression in primary HMVECs treated with LPA and histone deacetylase inhibitors suberanilohydroxamic acid (SAHA) or trichostatin (TSA). The HMVECs were pre-exposed to 500 nM of SAHA or 500 ng/ml of TSA for 30 min followed with 10 μM of LPA for 24 hrs. Cell lysates were subjected to Western blots for CD36 protein expression (right panel). Densitometry was performed and relative expression levels calculated for triplicate experiments (left panel).