腺苷受体2A介导的内皮间质化对动脉粥样硬化的影响

Atherosclerosis is the major cause for most of cardiovascular diseases. The endothelium, a cellular barrier between circulating blood and vascular vessel wall, plays an important role in the development of atherosclerosis. Adenosine, an endogenous small molecule, regulates the cardiovascular functions via its receptors A1R, A2AR, A2BR, and A3R. Our preliminary data showed that deletion of endothelial A2AR decrease the size of atherosclerotic plaque in apoE-/- mice. The objective of this proposal is to study the cellular and molecular mechanisms underlying the inhibitory effect of endothelial A2AR deletion on atherosclerosis. Endothelial mesenchymal transition (EndoMT) is a new cellular mechanism for the development of atherosclerosis and we found that A2AR deletion can suppresses this transition. Using multiple approaches including in vivo, in vitro, many cellular and molecular techniques, our project try to answer below important questions: (1)does the regulatory effect of A2AR on EndoMT occur in atherosclerotic mice; (2) is the Smad pathway or HIF1a-Snail involved in A2AR-regulated EndoMT? (3) is the A2AR-regulated EndoMT the major cause for increased inflammatory response and permeability for endothelial cells? (4) in addition to genetic approach, is pharmacological approach such as A2AR antagonist able to regulate EndoMT and suppress atherosclerosis? Completion of this project not only elucidates the molecular mechanism for the suppressive effect of A2AR ablation on atherosclerosis, further evaluates the importance of EndoMT in the formation of atherosclerosis, but also provides a solid scientific basis for the use of A2AR antagonist as a promising therapeutic approach in the treatment of atherosclerosis in patients.

动脉粥样硬化是大部分心脑血管疾病的主要诱因。内皮细胞作为屏障血液及血管壁的单细胞层在动脉硬化发生发展中发挥重要作用。腺苷是一重要的内源性心血管系统的调控小分子，主要通过腺苷受体发挥作用。我们前期的研究显示敲除内皮细胞腺苷受体A2AR显著抑制动脉硬化的发展。近几年的研究表明内皮细胞间质化是动脉硬化发病的重要新机制。我们的初步研究结果显示内皮细胞A2AR阻断抑制内皮细胞间质化。运用在体A2AR敲除小鼠，体外A2AR过表达细胞及特异性A2AR拮抗剂KW6002，本项目要重点探索以下几个问题：（1）A2AR对内皮间质化调控是否发生在小鼠动脉硬化在体模型；（2）A2AR介导的内皮间质化是否为内皮炎症反应、通透性增加及动脉硬化加剧的主因；（3）A2AR调控内皮间质化的分子信号机制。本研究通过对这些问题的回答能进一步阐明内皮间质化对动脉硬化的重要性及为A2AR作为动脉硬化的干预靶标提供理论依据。

心脑血管疾病是当前全球头号致死病因，且致死率仍在逐年攀升。血管损伤和炎症导致的动脉粥样硬化是心脑血管疾病的主要诱因。内皮损伤和功能失调是动脉粥样硬化发生的始动环节。内皮损伤刺激因素可导致主动脉和冠状动脉内皮细胞向平滑肌样间充质细胞转化，失去内皮细胞的屏障作用，并伴随内皮细胞炎症反应和功能受损，即发生内皮间质转化（EndoMT）。腺苷受体属于 G 蛋白偶联受体超家族，而大约40%的现代药物都以 G 蛋白偶联受体作为靶点。我们的研究发现，腺苷A2A 受体（ADORA2A）大量表达于血管内皮细胞，并在血流剪切力、转化生长因子(TGFβ)等EndMT刺激因素下表达显著上调。ADORA2A基因沉默或药理学拮抗剂可减少EndMT的发生，抑制TGFβ下游信号通路，下调EndMT标记物的基因转录和蛋白表达；相反，腺病毒转染过表达ADORA2A或使用激动剂可促进EndMT的发生。本文进一步研究了ADORA2A对EndMT的调控在血管炎症及动脉粥样硬化等病理条件下的作用，发现ADORA2A基因沉默或药理学拮抗剂可显著抑制内皮细胞炎症反应，并降低炎症条件下内皮细胞对单核细胞THP1的黏附，改善炎症及EndMT刺激因子导致的单层内皮细胞通透性升高，改善内皮细胞屏障功能的缺失。内皮细胞特异性敲低ADORA2A或腹腔注射ADORA2A药理学拮抗剂KW6002可显著抑制小鼠动脉粥样硬化斑块的形成，并改善斑块各项病理特征和稳定性。在进一步的分子机制研究中我们发现，ADORA2A是通过 cAMP/PKA/ CREB 信号通路，转录因子 CREB1 促进 TGFβR1(ALK5) 的表达及其下游P-Smad2/3信号通路的活性，进而调控 EndMT 标记物的表达和内皮细胞形态及功能，参与慢性血管炎症疾病动脉粥样硬化的发生。综上所述，本研究从体外细胞实验和动物模型两方面揭示了ADORA2A在内皮细胞 EndMT 及其所致的血管炎症疾病中的作用，明确了ADORA2A通过下游 cAMP/PKA/CREB/ALK5/p-Smad2/3 信号通路调控 EndMT 及标记分子的表达及相关信号通路活性，这一结果为ADORA2A作为动脉粥样硬化新的干预靶点提供了理论依据和应用转化基础。