S1PR1和VEGF受体在急性心肌梗死后血管新生中的交互作用

Acute myocardial infarction (MI) is a major public health problem in the 21st century. Pro-angiogenic therapy is essential to rescue ischemic myocardium after MI, and to prevent the transition to heart failure. Sphingosine 1-phosphate (S1P), a bioactive sphingolipid metabolite, tightly regulates cardiovascular functions via its receptors. Our previous data show that the ventricles express S1P receptor 1 (S1PR1), which plays an essential role in cardiovascular system. Recent investigations suggest that a transactive and complementary relationship between S1PR1 and vascular endothelial growth factor receptors (VEGFRs) signaling, which is known as a key pro-angiogenic factor. More importantly,the crosstalk between S1PR1 and VEGFRs signaling sequentially controls angiogenesis under physiological conditions. Under pathophysiologic conditions, e.g. post-MI, VEGFRs signaling is essential for angiogenesis in ischemic myocardium; however, the effect of S1PR1 on post-MI angiogenesis is unknown. Our preliminary results reveal that S1PR1 antagonist, W146, inhibits post-MI angiogenesis. Hence, we hypothesize that S1PR1 signaling is involved in regulating post-MI angiogenesis, and that the crosstalk between S1PR1 and VEGFRs signaling may contribute to angiogenesis in ischemic myocardium. To clarify this, we plan to induce acute MI in vascular endothelial specific S1PR1-deficient mice. We will apply histological analyses， microSPECT/CT, and cardiovascular ultrasound to examine the effect of S1PR1 on cardiac angiogenesis, cell survival and cardiac function after MI. Furthermore, we will modulate both S1PR1 and VEGFRs signaling to investigate the crosstalk between S1PR1 and VEGFRs in post-MI angiogenesis. Next, we will perform high-throughput screening of the signaling pathways of phosphorylation involved in the crosstalk between S1PR1 and VEGFRs signaling. Last, we will optimize pro-angiogenic therapy by sequentially combining VEGF gene therapy and S1PR1 modulator. Taken together, this project will reveal an unknown function of S1PR1 in post-MI angiogenesis, and uncover the mechanism how S1PR1 and VEGFRs synergistically control angiogenesis following MI. Most importantly, this project will raise a new and promising pro-angiogenic treatment of patients with acute MI.

1-磷酸鞘氨醇 (S1P)是一种脂质活性分子，S1P受体1型（S1PR1）和VEGF受体（VEGFRs）有交互作用，两者有时序性地控制着血管新生和成熟。本课题组新近发现：阻断S1PR1会抑制心肌梗死后的血管新生，说明S1PR1可能对心梗后血管新生有重要影响，其机制可能与VEGFRs密切相关。本研究拟采用血管内皮细胞S1PR1基因缺失小鼠，制作急性心梗模型，运用免疫组化、microSPECT和心超评估S1PR1对心梗后血管新生、心肌存活和心功能的影响；阻断或激活S1PR1信号, 同时在缺血心肌处过表达VEGF或抑制VEGFRs，研究两者在心梗后血管新生中的交互作用；利用蛋白芯片解析S1PR1和VEGFRs交互作用所涉及的信号通路；探索VEGF基因治疗有时序性地辅以S1PR1调控剂来促进心梗后血管新生的方法。本项目将为阐明心梗后血管新生的机制和探索有效的促进血管新生的新方法提供重要的实验依据。

在本项目的资助下，我们通过细胞特异性基因敲除小鼠、急性心梗动物模型和一系列体外机制实验鉴定出血管内皮细胞的1-磷酸鞘氨醇（S1P）/S1P受体1型（S1PR1）信号在维系心梗后“促炎型”和“修复型”单核/巨噬细胞的动态平衡中起重要调控作用。我们首先证明了血管内皮细胞S1P/S1PR1信号在调控血管新生和血管微环境中期关键作用，并且揭示了小分子microRNA302通过调控S1P/S1PR1信号来影响血管新生和血管内皮的屏障功能，该研究已发表于《Circulation Research》。其次, 我们成功了建立他莫昔芬（Tomaxifen）药物诱导型血管内皮细胞S1PR1特异性敲除小鼠和小鼠急性心梗模型，结果显示血管内皮细胞S1PR1特异性基因敲除加重心梗后心室重构的恶化和心功能的下降。通过成功建立多通道流式细胞仪分析法和心梗后心肌中白细胞分离富集法，我们实现了对于心梗后心脏中浸润的白细胞多种亚群的分析和绝对计数，结果显示血管内皮细胞S1PR1特异性基因敲除不影响T细胞和中性粒细胞在心梗后心肌中的浸润，但是在心梗3天轻度减少“促炎型”单核细胞数量，在心梗后7天严重减少 “修复型”巨噬细胞。高通量基因表达芯片进一步证实血管内皮细胞S1PR1基因缺失严重影响心梗后心肌中的“修复型”巨噬细胞相关促进心肌修复基因的表达。进一步机制研究表明，血管内皮细胞S1P/S1PR1可以通过上调MCP-1的表达来促进心梗后单核/巨噬细胞在心肌中的募集，更为重要的是可以通过ERK/CSF1通路以细胞紧密接触的方式促进血液来源的“修复型”巨噬细胞在心肌的原位增殖，从而促进心梗后的心室重构和心功能。基于上述研究，我们研究了应用S1PR1激动剂（SEW2871）在治疗心梗中的效果，结果显示SEW2871可以显著提高 “修复型”巨噬细胞在心肌中的富集，改善心梗后心室重构和促进心功能增强。综上所述，本课题证明了血管内皮细胞S1P/S1PR1信号及其调控小分子microRNA302在调控血管新生和血管微环境中的重要作用，同时率先鉴定出血管内皮细胞调控心梗后“促炎型”和“修复型”单核/巨噬细胞在心肌中的动态平衡的关键分子机制，基于我们对于心梗后心室重构分子病理机制的新发现，我们在动物模型上探索出有效改善心梗后心室重构和促进心肌修复的一种药物新靶点，为临床治疗心血管病提供依据，有一定的临床应用潜在价值。