Original ArticleLysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines
Introduction
Vascular endothelium maintains the non-thrombogenic and selective permeability properties of blood vessels. Endothelium also plays a role in inflammation when it is activated by multiple adverse conditions, including infection, hypoxia, altered blood flow or vascular injury [1]. Activated endothelial cells (EC) express adhesion molecules and secrete chemokines, which facilitate the recruitment of leukocytes to the sites of inflammation [1], [2], [3]. If unregulated, EC chemokines can promote pathogenic inflammation [2], [3].
We have focused on the role of lysophosphatidylcholine (LPC), a bioactive product of phospholipase A2 (PLA2) activity [4] and a component of oxidized low-density lipoprotein (LDL) [5], [6], in the induction of chemokines in EC. LPC elicits multiple responses in EC. It has been shown to induce the elaboration of growth factors [7], [8], adhesion molecules [9], [10] and the chemokine MCP-1 [11], [12], [13], but the mechanisms underlying these effects are not fully understood. LPC has been reported to be chemotactic for human leukocytes [14], [15], [16], and PLA2 inhibitors can block chemotaxis regulated by PLA2 [16]. PLA2 has also been implicated in inflammation [17]. These findings suggest a role for LPC in inflammation.
In this study, we have examined tissue-specific induction of chemokines by LPC using human EC isolated from three large vessels (pulmonary artery, aorta and umbilical vein) and microvessels, each representing a distinct area of the vasculature with differing vessel sizes and hemodynamics [18]. Of eight chemokines examined using a multi-probe ribonuclease protection assay (RPA), LPC markedly regulated the expression of only RANTES, MCP-1 and IL-8, which are chemotactic factors to T cells, monocytes and neutrophils, respectively. We show that LPC differentially regulates the expression of chemokines in small vs. large vessel EC. We investigated the signaling mechanisms for LPC induction of chemokines in microvascular EC (MVEC) because of the preferential expression of RANTES. We report that LPC induction of chemokines in MVEC is Gi-protein dependent, and the activities of phosphoinositide 3 kinase (PI3 kinase) and p38 MAP kinase are required for optimal induction of RANTES and IL-8 by LPC.
Section snippets
Cell culture
EC were isolated by collagenase digestion from sterile segments of human non-atherosclerotic thoracic aorta (HAEC) and pulmonary artery (HPAEC) after obtaining institutional review board approved family consent [19]. Human umbilical vein EC (HUVEC) were isolated as described earlier [20], and human dermal and lung MVEC were obtained commercially (Clonetics, MD). All EC were subject to two or more passages on fibronectin (1 μg/cm2) coated dishes under the same culture conditions prior to
Differential induction of chemokines by LPC in small and large vessel EC
Three EC preparations from large vessels (aorta, pulmonary artery and umbilical vein) and one from tissue microvessels were examined for the expression of a panel of eight chemokines in response to 100 μmol/l LPC. LPC regulated the expression of only three chemokines, viz. RANTES, MCP-1 and IL-8, which attract different subsets of leukocytes. Autoradiographs from an RPA are shown in Fig. 2. LPC induced the expression of RANTES in MVEC, but not in large vessel EC (Fig. 2, panels A and D). IL-8
Discussion
This is the first report of LPC induction of the T-cell chemoattractant RANTES [27] in EC. In this report, we demonstrate that LPC can differentially regulate the expression of chemokines in small vs. large vessel EC. We also show that LPC induction of chemokines in MVEC involves differential mechanisms.
Acknowledgements
This work was supported by a beginning grant-in aid from the Ohio Valley Affiliate of the American Heart Association (No. 0060321B to G.M.), and NIH grants (2R01 NS 32151 to R.M.R. and HL 29582 to G.M.C.). The authors thank the Perinatal Clinical Research Center (NIH GCRC RR-00080) of the Cleveland MetroHealth Hospital and Dr. Paul DiCorleto of the Cleveland Clinic Foundation for HUVEC.
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