The P2Y2 Nucleotide Receptor Mediates UTP-induced Vascular Cell Adhesion Molecule-1 Expression in Coronary Artery Endothelial Cells

Abstract

P2Y2 receptor up-regulation and activation induces intimal hyperplasia and monocyte/macrophage infiltration in the collared rabbit carotid artery model of vascular injury, suggesting a potential role for P2Y2 receptors in monocyte recruitment by vascular endothelium. In this study, we addressed the hypothesis that activation of P2Y2 receptors by extracellular nucleotides modulates the expression of adhesion molecules on vascular endothelial cells that are important for monocyte recruitment. Results indicated that the equipotent P2Y2 receptor agonists UTP or ATP (1–100 μm) stimulated the expression of vascular cell adhesion molecule-1 (VCAM-1) in human coronary artery endothelial cells (HCAEC) in a time- and dose-dependent manner. P2Y2 antisense oligonucleotides inhibited VCAM-1 expression induced by UTP but not by tumor necrosis factor-α. Furthermore, UTP induced VCAM-1 expression in human 1321N1 astrocytoma cell transfectants expressing the recombinant P2Y2 receptor, whereas vector-transfected control cells did not respond to UTP. The effect of UTP on VCAM-1 expression in HCAEC was prevented by depletion of intracellular calcium stores with thapsigargin or by inhibition of p38 mitogen-activated protein kinase or Rho kinase, but was not affected by inhibitors of the mitogen-activated protein/extracellular signal-regulated kinase pathway (i.e. MEK1/2). Consistent with a role for VCAM-1 in the recruitment of monocytes, UTP or ATP increased the adherence of monocytic U937 cells to HCAEC, an effect that was inhibited by anti-VCAM-1 antibodies. These findings suggest a novel role for the P2Y2 receptor in the p38- and Rho kinase-dependent expression of VCAM-1 that mediates the recruitment of monocytes by vascular endothelium associated with the development of atherosclerosis. P2Y2 receptor up-regulation and activation induces intimal hyperplasia and monocyte/macrophage infiltration in the collared rabbit carotid artery model of vascular injury, suggesting a potential role for P2Y2 receptors in monocyte recruitment by vascular endothelium. In this study, we addressed the hypothesis that activation of P2Y2 receptors by extracellular nucleotides modulates the expression of adhesion molecules on vascular endothelial cells that are important for monocyte recruitment. Results indicated that the equipotent P2Y2 receptor agonists UTP or ATP (1–100 μm) stimulated the expression of vascular cell adhesion molecule-1 (VCAM-1) in human coronary artery endothelial cells (HCAEC) in a time- and dose-dependent manner. P2Y2 antisense oligonucleotides inhibited VCAM-1 expression induced by UTP but not by tumor necrosis factor-α. Furthermore, UTP induced VCAM-1 expression in human 1321N1 astrocytoma cell transfectants expressing the recombinant P2Y2 receptor, whereas vector-transfected control cells did not respond to UTP. The effect of UTP on VCAM-1 expression in HCAEC was prevented by depletion of intracellular calcium stores with thapsigargin or by inhibition of p38 mitogen-activated protein kinase or Rho kinase, but was not affected by inhibitors of the mitogen-activated protein/extracellular signal-regulated kinase pathway (i.e. MEK1/2). Consistent with a role for VCAM-1 in the recruitment of monocytes, UTP or ATP increased the adherence of monocytic U937 cells to HCAEC, an effect that was inhibited by anti-VCAM-1 antibodies. These findings suggest a novel role for the P2Y2 receptor in the p38- and Rho kinase-dependent expression of VCAM-1 that mediates the recruitment of monocytes by vascular endothelium associated with the development of atherosclerosis. Extracellular nucleotides cause a wide range of cellular responses and appear to play a role in the regulation of many vascular functions (1Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Google Scholar, 2Erlinge D. Gen. Pharmacol. 1998; 31: 1-8Google Scholar). Vascular cells release nucleotides when exposed to stimuli such as ischemia, hypoxia, and chemical or mechanical stress (3Grierson J.P. Meldolesi J. J. Biol. Chem. 1995; 270: 4451-4456Google Scholar, 4Pearson J.D. Gordon J.L. Nature. 1979; 281: 384-386Google Scholar). It also is becoming apparent that extracellular nucleotides can promote the development of a variety of pathologies including disorders of the immune system, and neurodegenerative and vascular diseases (1Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Google Scholar). Indeed, ATP or UTP induces proliferation and migration of vascular smooth muscle cells, two processes involved in the development of intimal lesions found in atherosclerosis and post-angioplasty restenosis. The biological effects of extracellular nucleotides are mediated through activation of P1 and P2 purinergic receptors. P1 receptors are responsive to adenosine, whereas P2 receptors are activated by a variety of nucleotides including ATP and UTP (5Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Google Scholar, 6Burnstock G. Turner J.T. Weisman G.A. Fedan J.S. The P2 Nucleotide Receptors. Humana Press, Totowa, NJ1998: 3-42Google Scholar). The P2 receptors are subdivided into two distinct categories, the metabotropic G-protein-coupled (P2Y) receptors and the ionotropic ligandgated channel (P2X) receptors (6Burnstock G. Turner J.T. Weisman G.A. Fedan J.S. The P2 Nucleotide Receptors. Humana Press, Totowa, NJ1998: 3-42Google Scholar, 7Zhang F.L. Luo L. Gustafson E. Palmer K. Qiao X. Fan X. Yang S. Laz T.M. Bayne M. Monsma F. J. Pharmacol. Exp. Ther. 2002; 301: 705-713Google Scholar). Vascular cells have been shown to express metabotropic P2Y and ionotropic P2X receptors (5Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Google Scholar, 8Radisavljevic Z. Avraham H. Avraham S. J. Biol. Chem. 2000; 275: 20770-20774Google Scholar). It has been reported that P2Y2 receptors are up-regulated in cells of rat intimal lesions following balloon angioplasty (9Seye C.I. Gadeau A.P. Daret D. Dupuch F. Alzieu P. Capron L. Desgranges C. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3602-3610Google Scholar). Our recent studies showed that placement of a silicone collar around the rabbit carotid artery promoted up-regulation of P2Y2 receptors in vascular smooth muscle cells and endothelium (10Seye C.I. Kong Q. Erb L. Garrad R. Wang M. Turner J. Sturek M. Gonzalez F.A. Weisman G.A. Circulation. 2002; 106: 2720-2726Google Scholar). Subsequently, local infusion of UTP was shown to stimulate intimal hyperplasia and increase intimal monocyte infiltration (10Seye C.I. Kong Q. Erb L. Garrad R. Wang M. Turner J. Sturek M. Gonzalez F.A. Weisman G.A. Circulation. 2002; 106: 2720-2726Google Scholar), suggesting a role for P2Y2 receptors in the recruitment of blood monocytes leading to inflammation in atherosclerosis. Monocyte recruitment into the vessel wall is a complex process that includes cell rolling, firm attachment, and directed migration. It is now becoming evident that adhesion molecules such as VCAM-1 play an important role in leukocyte adherence to vascular endothelial cells (11Faruqi R.M. DiCorleto P.E. Br. Heart J. 1993; 69: S19-S29Google Scholar, 12Cybulsky M.I. Liyama K. Li H. Zhu S. Chen M. Davis V. Gutierrez-Ramos J.-C. Connelly P.W. Milstone D.S. J. Clin. Invest. 2001; 107: 1255-1262Google Scholar). VCAM-1 expression is induced or up-regulated by proinflammatory cytokines such as TNF-α 1The abbreviations used are: TNF-α, tumor necrosis factor-α; HCAEC, human coronary artery endothelial cells; VCAM-1, vascular cell adhesion molecule-1; EBM-2, endothelial cell basal medium-2; PBS, phosphate-buffered saline; [Ca2+]i, intracellular Ca2+; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; DOTAP, N-[1-(2,3-dioleoyloxy)propy]-N,N,N,-trimethylammonium salt; 2-MeS-ATP, 2-methyl-thio-ATP. 1The abbreviations used are: TNF-α, tumor necrosis factor-α; HCAEC, human coronary artery endothelial cells; VCAM-1, vascular cell adhesion molecule-1; EBM-2, endothelial cell basal medium-2; PBS, phosphate-buffered saline; [Ca2+]i, intracellular Ca2+; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; DOTAP, N-[1-(2,3-dioleoyloxy)propy]-N,N,N,-trimethylammonium salt; 2-MeS-ATP, 2-methyl-thio-ATP. and interleukin 1-β on cellular components of the arterial wall including endothelial cells, smooth muscle cells, and fibroblasts (13Rice G.E. Munro J.M. Belavicqua M.P. J. Exp. Med. 1990; 171: 1369-1374Google Scholar, 14Iademarco M.F. Barks J.L. Dean D.C. J. Clin. Invest. 1995; 95: 264-271Google Scholar, 15Braun M. Pietsch P. Felix S.B. Baumann G. J. Mol. Cell. Cardiol. 1995; 27: 2571-2579Google Scholar). ATP and UTP have been shown to induce cell-cell adhesion in a monocyte/macrophage lineage and neutrophil adherence to an endothelial cell monolayer (16Ventura M.A. Thomopoulus P. Mol. Pharmacol. 1995; 47: 104-107Google Scholar, 17Parker A.L. Likar L.L. Dawicki D.D. Rounds S. Am. J. Physiol. 1996; 270: L695-L703Google Scholar), raising the possibility that released ATP and UTP could induce endothelial cell activation by an autocrine/paracrine mechanism to regulate leukocyte adherence. The purpose of this study was to test the hypothesis that extracellular nucleotides stimulate the expression of adhesion molecules on vascular endothelial cells and promote monocyte adherence. Results showed that the P2Y2 receptor agonists UTP or ATP induced the expression of VCAM-1 in human coronary artery endothelial cells (HCAEC). The ability of P2Y2 antisense oligonucleotides to inhibit UTP-induced VCAM-1 expression strongly suggests that this response is mediated via P2Y2 receptors. The effects of UTP were dependent upon release of calcium from intracellular stores and activation of the p38 kinase pathway. Finally, results indicated that nucleotide-induced VCAM-1 expression increased monocyte binding to endothelial cells, supporting the hypothesis that nucleotides released during vascular injury contribute to leukocyte/endothelial cell interactions and promote inflammatory responses involved in the development of atherosclerosis. Materials—HCAEC and endothelial cell basal medium-2 (EBM-2) were purchased from Clonetics (Walkerville, MD). U937 cells were obtained from American Type Cell Collection (ATCC), and RPMI 1640 medium was purchased from Invitrogen. Anti-VCAM-1 (P3C4), anti-ICAM-1 (P2A4), and anti-E-selectin (P2H3) antibodies used in blocking studies were purchased from Chemicon International (Temecula, CA). Anti-ICAM-1 (clone G-5), anti-β-actin (rabbit polyclonal), anti-VCAM-1 (c-19), and anti-VCAM-1 (HAE-2z) antibodies used for Western analysis and immunofluorescence, respectively, were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Oregon green-labeled goat anti-rabbit IgG antibody and Hoechst 33258 were purchased from Molecular Probes (Eugene, OR). The polyclonal rabbit anti-phospho-p38 MAP kinase (Thr180/Tyr182) antibody was obtained from Cell Signaling (Beverly, MA). The p38 inhibitor SB203580, MEK1/2 inhibitor U0126, and Rho kinase 1/2 inhibitor Y27632 were purchased from Calbiochem (Indianapolis, IN). DOTAP liposomal reagent was purchased from Roche Diagnostics. Phosphorothioate-modified oligonucleotides to human P2Y2 were synthesized and purified by Integrated DNA Technologies. All other reagents including nucleotides and the PKH2 green fluorescent cell linker were obtained from Sigma, unless otherwise specified. Cell Cultures—HCAEC were cultured in EBM-2 supplemented with vascular endothelial growth factor, human fibroblast growth factor, epidermal growth factor, hydrocortisone, ascorbic acid, insulin-like growth factor, GA 1000, and 5% bovine fetal serum at 37 °C in a humidified atmosphere of 5% CO2. HCAEC were used between the third and tenth passages. Monocytic U937 cells were grown in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum. The retroviral vector pLXSN was used for stable expression of the human P2Y2 receptor in human 1321N1 astrocytoma cells, as described previously (18Erb L. Garrad R.C. Wang T. Quinn T. Turner J.T. Weisman G.A. J. Biol. Chem. 1995; 270: 4185-4188Google Scholar). Briefly, the recombinant P2Y2-pLXSN construct or pLXSN (control) was used to transfect PA317 amphotropic packaging cells for production of the viral vectors. Then, 1321N1 cells were infected with the viral vectors and cultured in Dulbecco's modified Eagle's medium supplemented with 5% (v/v) fetal bovine serum, 100 units/ml penicillin, and 1 mg/ml G418 (Invitrogen) for neomycin resistance selection. P2Y2Antisense S-oligonucleotides—Sequences including translation initiation site were as follows: sense, 5′-GGCGATGGCAGCAGACCTGGCCCCTGGA-3′; antisense, 5′-TCCAGGGGCCAGGTCTGCTGCCATCGCC-3′. The sequences were checked for uniqueness using the National Center for Biotechnology Information's Local Alignment Search tool (BLAST). 2Available at www.ncbi.nlm.nih.gov/BLAST. Briefly, HCAEC were incubated with 2 μm P2Y2 sense or antisense S-oligonucleotides for6hat37 °C in EBM-2 medium containing 1.4% (v/v) DOTAP liposomal reagent. Then, fresh EBM-2 medium was added and cells were cultured for an additional 20 h, as previously reported (19Warny M. Aboudola S. Robson S.C. Sevigny J. Communi D. Soltoff S.P. Kelly C.P. J. Biol. Chem. 2001; 276: 26051-26056Google Scholar). Fluorescein isothionate-conjugated P2Y2 antisense oligonucleotides were used to monitor cellular oligonucleotide uptake by HCAEC. Immunofluorescence Staining—HCAEC were made quiescent by incubation in EBM-2 for 24 h at 37 °C before exposure to UTP. After treatment with UTP (100 μm) or TNF-α (10 ng/ml) for 8 h at 37 °C, the cells were fixed in acetone for 15 min and treated with 0.2% (v/v) Triton X-100 in PBS at room temperature. After washing in phosphate-buffered saline (PBS), VCAM-1 expression was detected in cells by incubation for 2 h at room temperature with a rabbit polyclonal anti-VCAM-1 (HAE-2z) antibody (1:200 dilution in PBS containing 5% (v/v) normal goat serum), followed by Oregon green-labeled goat anti-rabbit IgG antibody (1:200 dilution in PBS). Then, the cells were incubated in PBS containing 2 μm Hoechst 33258 for nuclear counterstaining. Binding of U937 Cells to Human Endothelial Cells—HCAEC were grown to confluence in 24-well plates in EBM-2 supplemented with 5% (v/v) fetal bovine serum, and then the cells were incubated for 24 h at 37 °C in EBM-2 followed by stimulation with 100 μm ADP, ATP, UTP, adenosine, or 2MeS-ATP for an additional 8 h at 37 °C. In some experiments, HCAEC were preincubated with BAPTA/AM (10 μm) for 30 min before the addition of U937 cells were with the green fluorescent as described previously S. S. F. P. M. J. Clin. Invest. 1998; Scholar). U937 cells were added to of HCAEC for 1 h at 37 °C. The cells were in PBS, and U937 cell were by using In to the effect of blocking antibodies on binding of U937 cells, HCAEC were treated with UTP for 8 h at 37 °C and then or anti-E-selectin antibody was added to HCAEC for min before addition of U937 cell were in of in Western of protein (100 in of HCAEC were to and to for of VCAM-1 and was using goat VCAM-1 or goat antibodies followed by IgG were with polyclonal anti-β-actin antibody antibody and a rabbit polyclonal MAP kinase antibody were used to and p38 are as were by analysis of and test ATP and UTP VCAM-1 in Vascular Endothelial of HCAEC with UTP or ATP for 8 h a dose-dependent increase in the expression of VCAM-1 and In ADP, or effect at as as 100 μm VCAM-1 expression was induced 2 h of UTP or ATP a h, and through h and increase in VCAM-1 expression when HCAEC were exposed to 100 μm UTP for as as min before the was of HCAEC with the that and inhibited UTP or VCAM-1 expression In to VCAM-1 ATP or UTP did not increase expression in quiescent HCAEC These suggest a effect of UTP and ATP on VCAM-1 expression in human endothelial Immunofluorescence of quiescent HCAEC detected a basal of VCAM-1 expression with a In cells treated with UTP (100 was VCAM-1 the cell VCAM-1 was when cells were treated with TNF-α expression in HCAEC is by UTP or endothelial cells were stimulated with the indicated of UTP or ATP °C, and then protein were by Western analysis with anti-ICAM-1 as described in the to Results shown are of of VCAM-1 expression in endothelial cells by HCAEC were for 24 h at 37 °C and then incubated in the or of 100 μm UTP for 8 h at 37 °C. a control HCAEC were stimulated with TNF-α (10 ng/ml) for 8 h at 37 °C and for VCAM-1 VCAM-1 was obtained by immunofluorescence, as described were with Hoechst and the were of are from UTP-induced VCAM-1 an increase in the intracellular calcium [Ca2+]i, in HCAEC, a response that was inhibited by the intracellular calcium BAPTA/AM VCAM-1 expression induced by UTP also was inhibited in that were preincubated with μm BAPTA/AM for 30 min at 37 °C Furthermore, thapsigargin an that intracellular calcium also inhibited UTP-induced VCAM-1 expression a role for UTP-induced in in the expression of treatment apparent effect on as by UTP-induced VCAM-1 by (100 an inhibitor of the effects of UTP on VCAM-1 expression in HCAEC the P2Y2 receptor has been shown to to L. J. J. Kong Q. R.C. K. C. Gonzalez F.A. Turner J.T. Weisman G.A. J. Cell Biol. 2001; Scholar), we the effect on VCAM-1 expression in HCAEC for inhibition of P2Y2 receptor with P2Y2 antisense of HCAEC with P2Y2 antisense S-oligonucleotides to the P2Y2 translation initiation inhibited UTP-induced VCAM-1 expression by and with cells treated with P2Y2 sense In P2Y2 antisense S-oligonucleotides effect on VCAM-1 expression in HCAEC and the of P2Y2 receptors in UTP-induced VCAM-1 expression in HCAEC, we P2Y2 receptor in 1321N1 cells that P2 receptors Erb L. Weisman G.A. R.C. Turner J.T. S. A. Scholar). Cells with P2Y2 receptor and treated with UTP for 8 h at 37 °C VCAM-1, whereas vector-transfected control cells to respond to UTP results that P2Y2 receptors UTP-induced VCAM-1 receptors VCAM-1 expression in 1321N1 astrocytoma Human 1321N1 cells expressing the P2Y2 receptor or vector were incubated for the indicated at 37 °C with 100 μm UTP and VCAM-1 expression was by Western as described in the to Results shown are of Rho and p38 UTP-induced VCAM-1 UTP-induced increase in mediates mitogen-activated protein kinase in vascular cell including endothelial cells H. Desgranges C. M.A. Dupuch F. G. F. G. P. Gadeau A.P. 2001; Scholar, Pharmacol. 1998; Scholar). In HCAEC, UTP (100 μm) the of and p38 that was inhibited by the MEK1/2 inhibitor μm) or the p38 inhibitor (10 of HCAEC with μm prevented UTP-induced VCAM-1 expression in HCAEC, whereas the MEK1/2 inhibitor μm) effect P2Y receptors have been shown to to activation of the V. H. C. Gadeau A.P. Desgranges C. E. P. P. G. Am. J. Physiol. 2000; Scholar), we the effect of the Rho kinase inhibitor Y27632 on UTP-induced VCAM-1 of HCAEC with μm Y27632 inhibited the expression of VCAM-1 induced by UTP of Rho kinase and but not is involved in the intracellular leading to VCAM-1 expression induced by UTP. VCAM-1 with Monocytic Cell to Endothelial of HCAEC with UTP or ATP (100 μm) for 8 h increased the binding of U937 monocytes to HCAEC by and respectively, with HCAEC incubated in the of nucleotides In treatment of HCAEC with adenosine, or 2MeS-ATP or did not increase U937 cell adherence to HCAEC. of BAPTA/AM (10 μm) into HCAEC to treatment with UTP U937 cell binding to endothelial cells of HCAEC with anti-VCAM-1 but not anti-ICAM-1 or anti-E-selectin U937 cell binding to HCAEC These antibodies did not U937 cell adherence to HCAEC in the of UTP The study that UTP or ATP stimulate VCAM-1 expression via P2Y2 receptors in HCAEC. It also that the P2Y2 increase in VCAM-1 expression the adherence of U937 monocytes to HCAEC. These for the P2Y purinergic receptor to the the expression of an endothelial cell adhesion involved in monocyte recruitment in atherosclerosis. Vascular diseases such as atherosclerosis and post-angioplasty are by vascular that of nucleotides are released into the extracellular in response to vascular stress including and mechanical (3Grierson J.P. Meldolesi J. J. Biol. Chem. 1995; 270: 4451-4456Google Scholar, 4Pearson J.D. Gordon J.L. Nature. 1979; 281: 384-386Google Scholar). In of extracellular ATP and UTP in by or cell that during angioplasty increase the of nucleotides in the vessel the of extracellular nucleotides by a role in the regulation of purinergic recent studies that exposure of artery endothelial cells can the of S. J. Biol. Chem. 2002; Scholar). In also has been reported that associated with endothelial cell can S.C. E. D. K. M. J. Exp. Med. 1997; Scholar), suggesting that blood contribute to local in in the vessel The P2Y2 receptor is up-regulated in activated M. S. P. Chen P. Erb L. Turner J.T. Weisman G.A. S. A. 1997; and in cells in response to of the J.T. M. J.M. Weisman G.A. 1998; Scholar). It is now becoming apparent that P2Y2 receptor up-regulation in of vascular injury including balloon of the rat (9Seye C.I. Gadeau A.P. Daret D. Dupuch F. Alzieu P. Capron L. Desgranges C. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3602-3610Google and placement of a silicone collar around the rabbit carotid artery (10Seye C.I. Kong Q. Erb L. Garrad R. Wang M. Turner J. Sturek M. Gonzalez F.A. Weisman G.A. Circulation. 2002; 106: 2720-2726Google Scholar). In the we showed that activation of the P2Y2 receptor by UTP stimulated intimal hyperplasia and induced monocyte/macrophage infiltration into the vessel suggesting a potential role for this receptor in monocyte recruitment by vascular endothelium. The and of UTP and ATP for of VCAM-1 expression in HCAEC strongly suggests the of P2Y2 receptors. the ability of antisense, but not sense, P2Y2 oligonucleotides to inhibit UTP-induced VCAM-1 expression the role of P2Y2 receptors. Furthermore, UTP stimulated the expression of VCAM-1 in human 1321N1 astrocytoma cells expressing recombinant P2Y2 receptors but not in control 1321N1 cells that P2 receptors Erb L. Weisman G.A. R.C. Turner J.T. S. A. Scholar). the of 2MeS-ATP and to induce VCAM-1 expression in HCAEC suggests that activation of and receptors is not of the P2Y2 receptor in endothelial cells is to a variety of molecules including the and protein and kinase, the and the release of the into the H. Desgranges C. M.A. Dupuch F. G. F. G. P. Gadeau A.P. 2001; Scholar, Pharmacol. 1998; Scholar, V. H. C. Gadeau A.P. Desgranges C. E. P. P. G. Am. J. Physiol. 2000; Scholar). In this study, UTP-induced VCAM-1 expression in HCAEC was prevented by the calcium or by depletion of intracellular calcium stores with thapsigargin a role for P2Y2 in in VCAM-1 UTP-induced VCAM-1 expression also was by inhibitors of p38 and Rho kinase but not by inhibition of suggesting that p38 and are involved in the increased expression of VCAM-1 mediated by P2Y2 receptors. Vascular endothelial growth induces VCAM-1, and expression in endothelial cells through a pathway the activation of S. J. Biol. Chem. 2001; 276: Scholar). of the P2Y2 receptor has been reported to increase and release from endothelial cells Erb L. X. Weisman G.A. Scholar, J. Biol. Chem. Scholar). have shown that modulates the of VCAM-1 protein expression in endothelial cells R. P. M.A. J. Clin. Invest. 1995; Scholar). did not play a role in vascular endothelial growth adhesion expression S. J. Biol. Chem. 2001; 276: Scholar), could the effects of UTP on VCAM-1 expression in endothelial studies are to the involved in nucleotide-induced VCAM-1 expression at the and Consistent with that ATP and UTP stimulate cell-cell adhesion in a monocyte/macrophage lineage and neutrophil adherence to endothelial cell (16Ventura M.A. Thomopoulus P. Mol. Pharmacol. 1995; 47: 104-107Google Scholar), that ATP and UTP stimulate U937 monocyte adherence to HCAEC The P2Y2 receptor the binding in extracellular L. J. J. Kong Q. R.C. K. C. Gonzalez F.A. Turner J.T. Weisman G.A. J. Cell Biol. 2001; Scholar). The has been shown to the for many including and Cell. 69: Scholar). studies have indicated that the in the P2Y2 receptor mediates with and is for L. J. J. Kong Q. R.C. K. C. Gonzalez F.A. Turner J.T. Weisman G.A. J. Cell Biol. 2001; Scholar). It has been reported previously that the on monocytes mediates adherence to endothelial and cells, an in the inflammatory response H. R. J. Biol. 1993; Scholar). protein complex between and P2Y2 receptors also involved in the regulation of monocyte binding to endothelial cells mediated by P2Y2 receptors. In this study is the to that extracellular nucleotides and P2Y2 receptor activation can induce VCAM-1 expression in endothelial cells and promote the adherence of In with results that P2Y2 receptor up-regulation in vascular cells mediates the proliferation of smooth muscle cells and the adherence of monocytes to responses associated with intimal hyperplasia and inflammation in atherosclerosis. These results the development of to P2Y2 receptor up-regulation and activation as a to arterial for