Role of Cyp2j-epoxygenases, sEH and PPARs in adenosine-induced vascular response

描述（由申请人提供）：内皮功能障碍与相应的血管张力变化和收缩力增加相关，这种情况可能导致易感个体高血压，这些易感个体可能具有cyp2j-环氧合酶、可溶性环氧化物水解酶（sEH）、A2A和A1腺苷受体（A2A AR和A1 AR）基因的等位基因变异。这些等位基因变异可能与我们的转基因小鼠有相似之处，可以调节血管张力和血压。我们的初步数据表明腺苷诱导的松弛和通过A2A AR-cyp2j-PKA-PPARγ通路打开KATP通道之间可能存在联系。此外，腺苷诱导的KATP通道的收缩和关闭可能通过A1 AR-sEH-cyp4a- PPARα途径存在联系。药物工具和转基因小鼠的结合将使我们能够确定可能的目标，作为治疗可能具有导致高血压的等位基因变异的人群的长期目标。因此，迫切需要探索腺苷诱导血管反应中涉及cyp2j2-环氧合酶、sEH/cyp4a、A1 AR/A2A AR、PPARα/γ、PKA/PKCα/ λ和KATP通道的可能机制。我们的中心假设是腺苷通过cyp2j-环氧合酶通过A2A ar -cyp2j- pka - ppar信号通路导致KATP通道打开，从而诱导血管松弛和血压降低。另一方面，腺苷通过A1 AR-sEH-cyp4a-PPARα通路诱导血管收缩和血压升高，导致KATP通道关闭。为了验证这一假设，我们拟通过A2A AR-/-、A1 AR-/-、cyp2j5-/-、sEH-/-、Tie2-cyp2j2Tr（内皮-cyp2j2过表达）、Tie2-sEHTr（内皮-sEH过表达）、野生型小鼠、H-2Kb- tsA58小鼠肾内皮细胞系、小鼠主动脉内皮细胞（MAEC）和小鼠主动脉平滑肌细胞（MASMC）深入探讨机制。此外，我们还将探索在饮用水（或灌胃）中使用sEH抑制剂（AUDA/t-AUCB）治疗可能存在高血压的A2A AR-/-、cyp2j5-/-和Tie2-sEHTr小鼠的可能性。我们将测量血压，我们将使用主动脉/肾动脉（器官液/ dmt -钢丝肌图）进行治疗（腺苷受体激动剂和拮抗剂）、环氧化酶、sEH、腺苷酸环化酶、PKCα/柱、MAPK和PKA抑制剂、PPARα/γ、EETs和KATP通道（激活剂和抑制剂）。EETs和DHETs将被分析（UPLC-MS/MS）。Western blot和RT-PCR将用于蛋白质和mRNA的表达。我们提出了3个具体目标来确定：(1)cyp2j-环氧合酶或sEH是否影响血压、腺苷诱导的血管反应和eet /DHETs；(2) A2A AR的存在或缺失是否影响腺苷诱导的血管反应，通过cyp2j-环氧合酶，sEH (3) cyp2j-环氧合酶的存在/过表达或sEH是否通过A2A AR-cyp2j- pka - ppar γ/A1 AR-sEH-cyp4a- ppar α途径在腺苷诱导的血管反应中调节KATP通道。这些结果可能具有积极的影响，因为确定的成分有望为抑制与内皮功能障碍相关的临床问题提供新的靶点，从而导致高血压。

DESCRIPTION (provided by applicant): Endothelial dysfunction is associated with corresponding changes in vascular tone and increases in contractility, a condition that may cause hypertension in susceptible individuals which may have allelic variants in cyp2j-epoxygenases, soluble epoxide hydrolase (sEH), A2A, and A1 adenosine receptors (A2A AR & A1 AR) genes. These allelic variants may have similarities to our transgenic mice which may regulate vascular tone and blood pressure (BP). Our preliminary data have suggested a possible link between adenosine-induced relaxation and opening of KATP channels through A2A AR-cyp2j-PKA-PPARγ pathway. Also, it is possible that a link may exist between adenosine-induced contraction and closing of KATP channels through A1 AR-sEH-cyp4a- PPARα pathway. A combination of pharmacological tools and transgenic mice would allow us to identify the possible targets as a long term goal to treat population which may have allelic variants leading to hypertension. Therefore, there is a critical need to explore the possible mechanism involving cyp2j2-epoxygenases, sEH/cyp4a, A1 AR/A2A AR, PPARα/γ, PKA/PKCα/ϵ and KATP channels in adenosine-induced vascular responses. Our central hypothesis is that adenosine induces vascular relaxation and decreases in BP through cyp2j-epoxygenases via A2A AR-cyp2j-PKA-PPARγ signaling leading to opening of KATP channels. On the other hand, adenosine induces vascular contraction and increases in BP through sEH via A1 AR-sEH-cyp4a-PPARα pathway leading to closing of KATP channels. To test this hypothesis, we propose to explore in depth mechanism(s) using A2A AR-/-, A1 AR-/-, cyp2j5-/-, sEH-/-, Tie2-cyp2j2Tr (endothelial-cyp2j2 overexpressed), Tie2-sEHTr (endothelial-sEH overexpressed), wild-type mice, immortal renal endothelial cell line from H-2Kb- tsA58 mouse, mouse aortic endothelial cells (MAEC) and mouse aortic smooth muscle cells (MASMC). Further, we will also explore the possible treatment with sEH inhibitors (AUDA/t-AUCB) in drinking water (or gavage) for A2A AR-/-, cyp2j5-/- and Tie2-sEHTr mice which may have high BP. We will measure BP, and we will use aortas/renal arteries (organ bath/DMT-wire myograph) with treatments (adenosine-receptors agonists & antagonists), cyp-epoxygenases, sEH, adenylyl cyclase, PKCα/ϵ, MAPK and PKA inhibitors, PPARα/γ, EETs and KATP channel (activators & inhibitors). EETs & DHETs will be analyzed (UPLC-MS/MS). Western blot & RT-PCR will be used for proteins & mRNA expression. We propose 3 specific aims to determine: (1) whether the cyp2j-epxygenases or sEH affects BP, adenosine-induced vascular response and EETs/DHETs; (2) whether the presence or absence of A2A AR affects adenosine-induced vascular response through PPARs via cyp2j-epoxygenases, sEH (3) whether the presence /overexpression of cyp2j-epoxygenases or sEH regulate KATP channels through A2A AR-cyp2j-PKA-PPARγ/A1 AR-sEH-cyp4a-PPARα pathway in adenosine-induced vascular response. Such results can have a positive impact, as the identified components are expected to provide new targets to curb clinical problems linked with dysfunctional endothelium leading to hypertension.