Role of the P2Y2 receptor/tissue factor axis in endotoxemia and atherothrombosis
Type of DegreePhD Dissertation
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Inflammation and coagulation play a critical role in the pathogenesis of thrombosis. Biologically active substances initiate the inflammatory response in vascular endothelial cells (ECs) and immune cells, including monocytes/macrophages, which involves the inflammatory response due to the overwhelming production of many inflammatory mediators, such as GM-CSF, TNF-α, and extracellular nucleotides. Recently we reported that in human coronary artery endothelial cells (HCAEC), activation of the P2Y2 receptor (P2Y2R) induces up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. Our previous study also demonstrated positive cell signaling pathways initiated by the P2Y2R inducing TF expression, namely P2Y2R-Rho-JNK-c-Jun/ATF-2 and P2Y2R-Src-p38-MK2. Furthermore, we discovered an original repressor, Fra-1 for the TF gene, which is controlled by P2Y2R signaling via the ERK1/2 pathway in human coronary artery endothelial cells. However, it is unclear if we could generate new ligands that would guide the P2Y2R to transduce only the TF-suppressing ERK1/2 pathway without turning on the other pathways so that TF expression can be well-controlled under the vascular inflammatory condition. In addition, others have shown that monocyte TF is more important than endothelial TF in terms of provoking an inflammatory pro-thrombotic state. Based on these findings, we hypothesized that manipulating P2Y2R is a new strategy in controlling inflammatory/pro-thrombotic TF expression through transcriptional and post-transcriptional mechanisms. 1) Here, using human monocytic cells, we show that P2Y2R mediates TF expression. First, we found the P2Y2 was selectively and functionally expressed in human primary monocytes and monocytic THP-1 cells. Consistent with this, ATP or UTP at micromolar concentration dramatically increased TF protein expression, which was abolished by AR-C118925, a selective P2Y2R antagonist, suggesting a key role for P2Y2R. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA, and preceded with an increased TF pre-mRNA, indicating enhanced TF gene transcription. Moreover, activation of the monocyte P2Y2R by UTP or ATP significantly activated the MAPK pathways including ERK1/2, JNK, p38, and AKT pathway, along with their downstream transcription factors including Fra-1, c-Jun, c-Fos, and ATF-2, whereas blocking the ERK1/2, JNK, p38 or AKT pathways respectively, all significantly suppressed P2Y2R-mediated TF expression. Furthermore, we found that endotoxin LPS induced a dramatic ATP release in THP-1 cells and P2Y2R knockout significantly rescued mice challenged by a lethal dose of LPS. These findings demonstrate for the first time that the P2Y2R mediates TF expression through new mechanisms involving ERK1/2, JNK and p38, and that both transcriptional and posttranscriptional mechanisms contribute to a pro-thrombotic status through the P2Y2-TF axis in monocytes (Chapter 2). 2) We also demonstrate that deletion of P2Y2R leads to decreased atherothrombosis via mechanisms involving TF expression and macrophage function without affecting platelet aggregation in ApoE-null mice maintained on Western diet for 16 weeks. We show that P2Y2R knockout delayed FeCl3 (ferric chloride)-induced blood clot formation in P2Y2R−/−ApoE-/- mice compared to P2Y2R+/+ ApoE−/− mice. In addition, P2Y2R knockout significantly reduced serum TF and plasma TAT levels without affecting blood platelet activation compared to P2Y2R+/+ ApoE−/− mice. Besides, P2Y2R−/−ApoE−/− showed higher HDL levels and other anti-thrombosis components, fewer cell adhesion molecules and inflammatory chemokines, and maintained the platelet count in a physiological level. In addition, P2Y2R−/−ApoE−/− had significantly less TF expression and macrophages staining in aortic atherosclerotic plaque than P2Y2R+/+ ApoE−/− (Chapter 3). 3) We further identified an ATP analog, APPNHP acted as a biased agonist for human P2Y2R, in which it selectively activates the ERK1/2-Fra1 pathway, leading to suppression of agonists-induced TF expression in HCAEC. UTP/ATP activated all p-ERK, p-JNK, p-p38 MAPK kinase and caused Ca2+ mobilization in HCAEC in a dose-dependent manner. In comparison to UTP/ATP, APPNHP selectively induced p-ERK1/2 signaling, with negligible activation of p-JNK and no stimulation of p-p38 and Ca2+ mobilization, suggesting APPNHP induced a biased ERK1/2 signaling via P2Y2R in HCAEC. This notion was further supported by the fact that a P2Y2R-selective antagonist ARC-118925 inhibited APPNHP-induced MAPK signaling. Moreover, APPNHP alone did not affect TF mRNA and protein expression, but it unexpectedly inhibited UTP/ATP-induced TF protein expression in HCAEC. In addition, APPNHP selectively activated the TF gene repressor Fra-1, with much less effective on AP-1 subunits c-Jun and ATF-2 than UTP treatment. Finally, UTP/ATP protected TF mature mRNA decay, but APPNHP did not change the TF mRNA stability in HCAEC (Chapter 4). In summary, this dissertation revealed the new roles of P2Y2R in human monocytes TF expression. and a mouse model of thrombosis. In addition, we conclude that P2Y2R biased signaling in human vascular endothelial cells is possible, indicating the potential to establish a new therapeutic target and to produce new therapeutic drugs in combating inflammatory and thrombotic diseases such as endotoxemia and atherothrombosis.