P2Y12-dependent regulation of emergency hematopoiesis after myocardial infarction

Abstract

Abstract Introduction Inflammation is essential for wound healing after myocardial infarction (MI). Leukocytes, especially neutrophils and monocytes, orchestrate removal of necrosis and regulation of tissue remodeling. Beside local recruitment from the blood, leukocyte supply via increased hematopoiesis is of major relevance for post-ischemic myocardial inflammation. Little is known about the pathways that carry the signals for increased demand for leukocytes from the site of injury to upstream hematopoietic stem cells (LSK) in the bone marrow (BM). In this study, we investigate the role of the P2Y12-ADP-receptor mediated regulation for emergency hematopoiesis after MI. Methods For standardized MI, a model of permanent coronary ligation was used in C57/Bl6 and P2Y12−/− mice. Changes in plasmatic ADP levels after MI were screened using ELISA, whereas the expression of the P2Y12-ADP-receptor in cell populations isolated from the BM was investigated by qPCR. CFU assays added further functional insight on hematopoietic proliferation in vitro. The effect of P2Y12 on the hematopoietic system after MI was investigated by inhibiting the P2Y12-receptor via prasugrel and compared to inhibition of the thromboxane pathway via acetylsalicylic acid (ASA). Proliferation of LSK in BM and leukocyte composition in blood, BM and infarct tissue after MI were assessed via FACS. Leukocyte composition in the infarcted myocardium was validated by immunohistochemistry. Finally, left ventricular function (LV-EF) and remodeling were investigated by echocardiography. Results 24 h after MI, we found a peak of plasmatic ADP levels. LSK as upstream hematopoietic progenitors in the BM express a P2Y12-receptor, which was validated on transcriptional and protein level. Whereas ADP stimulation of LSK led to significantly larger colony growth in vitro on the one hand, percentage of cycling LSK were significantly reduced 48 h after MI in P2Y12−/− mice compared to WT mice, assessed by Ki67/DAPI cell cycle analysis. Prasugrel treatment showed similar effects, translating into reduced numbers of downstream hematopoietic progenitors GMP and MDP 72 h after MI. Treatment with ASA however had no significant effect neither on cycling LSK nor progenitor populations. Consequently, decreased medullary hematopoiesis under P2Y12-inhibition led to reduced infiltration of inflammatory cells in the infarct tissue 7 days after MI, finally resulting in significantly improved outcome in terms of LV-EF 3 weeks after MI. Conclusion In this study, we demonstrate that P2Y12-mediated signaling is involved in emergency hematopoiesis after MI and advocates post-MI inflammation. In turn, inhibition of P2Y12-mediated signaling contributes to improved wound healing and prevention of adverse cardiac remodeling after MI, which adds a yet unknown mechanism to the success story of modern P2Y12-receptor blockers. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft