Inflammatory stress increases receptor for lysophosphatidylcholine in human microvascular endothelial cells

Abstract

The atherogenic serum lysophosphatidylcholine (LPC) is known to mediate vascular endothelial responses ranging from upregulation of adhesion molecules and growth factors to secretion of chemokines and superoxide anion. We investigated whether endothelial cells express receptors for LPC, which may account for their actions. Human brain microvascular (HBMEC) and dermal microvascular endothelial cells (HMEC) were prepared for RT-PCR analysis for possible expression of the G protein-coupled receptors, GPR4 and G2A, which are believed to be specific LPC receptors. Results indicated that HBMEC expressed low basal GPR4 mRNA, but stimulation with tumor necrosis factor-alpha (TNF-alpha) (100 U/ml) or H2O2 (50 micromol/l) for 2 h or overnight upregulated expression severalfold. In contrast, HMEC expressed high basal GPR4 mRNA, which was not further increased by either TNF-alpha or H2O2 stimulation. Another LPC receptor, G2A, was not detected in either endothelial cell type. Competition binding studies were made to evaluate specific binding of [3H]LPC to the intact endothelial cell monolayer. Basal specific [3H]LPC binding in HBMEC was approximately eight times lower than in HMEC; however, TNF-alpha or H2O2 stimulation increased [3H]LPC binding on HMBEC but not HMEC. The results indicated that GPR4 expression was consistent with specific [3H]LPC binding. Overall, we report that endothelial cells selectively expressed GPR4, a specific LPC receptor. Furthermore, GPR4 expression by HBMEC, but not HMEC, was increased by inflammatory stresses. We conclude that endogenous GPR4 in endothelial cells may be a potential G protein-coupled receptor by which LPC signals proinflammatory activities.

Loss of G2A promotes macrophage accumulation in atherosclerotic lesions of low density lipoprotein receptor-deficient mice

“…A recent study also reported GPR4 expression in endothelial cells and further suggested that GPR4 expression is transcriptionally induced by inflammatory stress in human vascular endothelial cells (24). However, this study did not detect G2A expression, which contradicts our observations.…”

“…Although this may reflect the use of human versus murine cells, it is also possible that differences in their preparation, or immortalization in the case of cell lines, may influence endothelial cell receptor expression, as G2A is subject to transcriptional regulation by multiple stress stimuli (25). Furthermore, it is possible that endothelial cells from different vascular sites exhibit varying patterns of receptor expression (24). Nevertheless, our data show that G2A but not GPR4 is expressed in inflammatory cells, whereas GPR4 is the predominant receptor in aortic endothelial cells that also express lower levels of G2A.…”

Reactive oxygen species mediate high glucose-induced heparanase-1 production and heparan sulphate proteoglycan degradation in human and rat endothelial cells: a potential role in the pathogenesis of atherosclerosis

“…PI-88 was kindly provided by Progen Pharmaceuticals (Toowong, QLD, Australia). Human brain microvascular endothelial cells (HBMEC) and human dermal microendothelial cells (HDMEC) were used in our previous study [21]. HBMEC were grown in RPMI 1640 containing 10% FBS, MEM vitamins, non-essential amino acid, sodium pyruvate (Invitrogen, Carlsbad, CA, USA), penicillin (100 U/ml), streptomycin (100 μg/ml) and 2 mmol/l L-glutamine.…”

Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A

“…34 While a recent report argues against SPC and LPC as ligands for GPR4, 35 another line of evidence from competition binding assays shows that LPC binds to microvascular endothelial cells where GPR4 but not G2A is expressed. 36 Given the distinct expression patterns of G2A and GPR4, one may speculate that these 2 receptors are responsible for LPC-induced chemotaxis differentially in various cell contexts. In this respect, it is interesting to note that although expression of GPR4 is lower than G2A in mouse primary and J774A.1 macrophages by RT-PCR (L.V.Y.…”