ADP Stimulates Human Endothelial Cell Migration via P2Y ₁ Nucleotide Receptor–Mediated Mitogen-Activated Protein Kinase Pathways

Abstract

Abstract-Extensive research on the role of ADP in platelet activation led to the design of new anti-thrombotic drugs, such as clopidogrel (Plavix; sanofi-aventis); however, very little is known about the ADP-preferring nucleotide receptors (P2Y 1 , P2Y 12 , and P2Y 13 ) in endothelium. Here, we show that ADP stimulates migration of cultured human umbilical vein endothelial cells (HUVECs) in both Boyden chamber and in vitro wound repair assays. This promigratory effect was mimicked by 2-MeSADP, but not by AMP, and was inhibited by MRS2179 (P2Y 1 receptor antagonist) but not by AR-C69931MX (P2Y 12/13 receptor antagonist). RT-PCR revealed abundant P2Y 1 , barely detectable P2Y 12 , and absent P2Y 13 receptor message in these cells. In addition, both ADP and 2-MeSADP, but not AMP, activated the mitogen-activated protein kinase pathways as evidenced by increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), and p38 kinase. ADP also stimulated phosphorylation of p90RSK, a downstream substrate of phosphorylated ERK1/2, and induced phosphorylation of such transcription factors downstream of the JNK and p38 pathways as c-Jun and activating transcription factor-2. These signaling events were inhibited by MRS2179 but not by AR-C69931MX. Furthermore, blockade of the ERK or JNK pathways by U0126 and SP600125, respectively, abolished ADP-and 2-MeSADP-stimulated HUVEC migration. However, inhibition of the p38 pathway by SB203580 partially suppressed ADP-and 2-MeSADP-induced HUVEC migration. We conclude that ADP promotes human endothelial cell migration by activating P2Y 1 receptor-mediated MAPK pathways, possibly contributing to reendothelialization and angiogenesis after vascular injury. V ascular endothelial cell migration and proliferation are vital in many physiological and pathological processes, including organ development, angiogenesis, and healing of the injured endothelium. 1,2 Numerous factors produced within the vascular wall or from the circulating blood can stimulate migration and/or proliferation of endothelial cells through their cognate receptors, which are either receptor tyrosine kinases, such as vascular endothelial growth factor (VEGF), or G protein-coupled receptors, such as thrombin. 2,3 In addition to these well-established factors, extracellular nucleotides are relatively newly defined vasoactive substances in the vasculature. 4 -6 ATP and UTP stimulate vascular smooth muscle constriction and proliferation 5,6 ; however, in intact arteries, nucleotides usually cause endotheliumdependent relaxation, 5 suggesting that endothelium has 1 or more functional nucleotide receptors. Indeed, among the 8 cloned P2Y nucleotide receptors (P2Y 1,2,4,6,11,12,13,14 ), 5 a previous study in human vascular endothelial cells showed significant level of P2Y 1 , P2Y 2 , and P2Y 11 receptor mRNAs. The expression levels of the newly cloned P2Y 12 , 13 , 14 receptors were not investigated. 7 It also has been shown that stimulation of the P2Y 2 receptor by ATP or UTP in cultur...

Major Contribution of the P2Y 1 Receptor in Purinergic Regulation of TNFα-Induced Vascular Inflammation

“…Thus, stimulation of P2Y 1 receptors by ADP has been associated with p38 MAPK activation in human platelets 34 and in ECs where this pathway was implicated in the ADP-induced migration of human umbilical vein ECs. 35 These data and our findings suggest that pharmacological inhibition of P2Y 1 might represent an interesting strategy to inhibit p38 MAPK-mediated vascular inflammation. Interestingly, treatment with the p38 MAPK inhibitor SB203580 has been shown to reduce atherosclerotic disease progression in ApoE Ϫ/Ϫ mice 36 and vascular inflammation in patients undergoing percutaneous coronary intervention.…”

Purinergic Signaling and Blood Vessels in Health and Disease

“…ADP promotes human endothelial cell migration by activating P2Y 1 receptor-mediated MAPK pathways, perhaps contributing to re-endothelialization and angiogenesis after vascular injury (Shen and DiCorleto, 2008). Evidence has been presented that stimulation of human endothelial cells via P2Y 1 receptors activates VEGF-2 to stimulate angiogenesis (Rumjahn et al, 2009).…”

New insights regarding the regulation of chemotaxis by nucleotides, adenosine, and their receptors

“…Many different types of endothelial cells exhibit a chemotactic response to extracellular nucleotides; the P2Y 1 receptor has been implicated as a mediator of this process [71,72]. Activation of P2Y 1 receptors by ADP promotes human umbilical vein endothelial cell (HUVEC) migration via mitogen-activated protein kinase pathways (including ERK1/2, JNK, and p38; [71]).…”

“…Many different types of endothelial cells exhibit a chemotactic response to extracellular nucleotides; the P2Y 1 receptor has been implicated as a mediator of this process [71,72]. Activation of P2Y 1 receptors by ADP promotes human umbilical vein endothelial cell (HUVEC) migration via mitogen-activated protein kinase pathways (including ERK1/2, JNK, and p38; [71]). Similarly, in vasa vasorum endothelial cells (VVECs), but not pulmonary artery or aortic endothelial cells, ATP promotes cell migration and potentiates the DNA synthesis-stimulating activity of VEGF and bFGF [73].…”