Exercise, Diabetes, & Coronary Smooth Muscle Ca2+

描述（由申请人提供）：长期目标是确定冠状动脉平滑肌（CSM）腺苷A1和P2Y2受体信号传导机制，通过这种机制，肾素-血管紧张素-醛酮系统（RAAS）的拮抗和运动训练可以减少冠状动脉疾病（CAD）和代谢综合征（MetS）的再狭窄。RAAS调节CSM A1和P2Y2受体信号，调节激酶和下游炎症尚不清楚。虽然药物洗脱支架可以减少再狭窄，但由于再狭窄并发症和支架周围CAD的进展，即支架周围CAD，需要进一步研究。我们之前拨款的主要发现为这个项目提供了出色的理论基础。Ossabaw猪临床上有明显的动脉粥样硬化（bbb50%狭窄），我们首次对自然病变进行支架置入，而不仅仅是球囊损伤的健康动脉。我们克隆了猪的4种腺苷受体（A1, A2A, A2B, A3），发现A1受体（A1R）在CSM中选择性表达，并且在动脉粥样硬化和冠脉支架中增加。A1R的直接激活诱导CSM增殖，提示新的调控机制。我们克隆了3个猪P2核苷酸受体亚型，发现P2Y2亚型仅在病变CSM中表达，并在冠脉支架中选择性上调。总体假设：在MetS中，RAAS（主要是醛固酮）增加冠状动脉A1R和P2Y2R信号，这是CSM生长的关键信号。综合实验设计比较lean和MetS Ossabaw RAAS拮抗作用。动脉粥样硬化病变支架置入后进行恢复，有或没有运动。结果与较简单的体外器官培养进行了比较。具体目的是验证MetS与lean Ossabaw的6个特定假设：1)动脉粥样硬化、支架周围CAD和支架内狭窄在MetS中增加，并通过RAAS拮抗剂和运动减弱。血管内超声评估体内CAD，光学成像和组织病理学评估体外CAD。2) MetS增加了A1R和P2Y2R的分子表达。蛋白质和mRNA将在5个不同的冠状动脉段中测定：健康、动脉粥样硬化、支架周围、支架内新生内膜和支架内介质。3) MetS通过激活ERK、JNK和AKT增加A1R和P2Y2R的功能性表达。磷酸化ERK、JNK和AKT的免疫印迹和免疫细胞化学；店家经营的Ca流入；炎症和氧化应激量化了功能激活。4)血管紧张素II和醛固酮直接、独立、协同地增加A1R和P2Y2R的分子和功能表达，这种作用因血脂异常而增强。体内处理包括几个猪组，以与体外器官培养进行比较。5)运动将逆转met中A1R和P2Y2R分子和功能表达的增加。该Aim评估更成熟的CAD和支架植入术。6)阻断A1R和P2Y2R可减轻过度扩张损伤和原生动脉粥样硬化的支架内狭窄。瘦猪和MetS猪将接受药物和基因洗脱支架的体内冠脉支架植入，以选择性阻断A1R和P2Y2R。

DESCRIPTION (provided by applicant): The long-term goal is to determine the coronary smooth muscle (CSM) adenosine A1 and P2Y2 receptor signaling mechanisms by which antagonism of the renin-angiotensin-aldosterone system (RAAS) and exercise training decrease coronary artery disease (CAD) and restenosis in metabolic syndrome (MetS). RAAS regulation of CSM A1 and P2Y2 receptor signaling that modulates kinases and downstream inflammation is unknown. Although drug-eluting stents decrease restenosis, further study is needed because of restenosis complications and progression of CAD adjacent to the stent, i.e. peri-stent CAD. Major findings in our previous grant provide outstanding rationale for this project. Ossabaw swine have clinically significant atherosclerosis (>50% stenosis) and we have for the first time stented natural lesions, not just balloon-injured healthy arteries. We cloned all 4 adenosine receptor isoforms (A1, A2A, A2B, A3) from pig and found A1 receptors (A1R) selectively expressed in CSM and increased in atherosclerotic and stented coronary. Direct activation of A1R induces CSM proliferation, indicating novel regulation. We cloned 3 porcine P2 nucleotide receptor isoforms and found the P2Y2 subtype is only expressed in diseased CSM and is selectively up-regulated in stented coronary. Overall hypothesis: in MetS RAAS (mainly aldosterone) increases coronary A1R and P2Y2R signaling, pivotal signals for CSM growth. The integrative experimental design compares lean vs. MetS Ossabaw RAAS antagonism. Atherosclerotic lesions are stented followed by recovery with or without exercise. Results are compared to simpler in vitro organ culture. Specific Aims are to test 6 specific hypotheses in MetS vs. lean Ossabaw: 1) Atherosclerosis, peri-stent CAD, and in-stent stenosis are increased in MetS and attenuated by RAAS antagonism and exercise. Intravascular ultrasound assesses CAD in vivo and optical imaging and histopathology assess CAD in vitro. 2) MetS increases molecular expression of the A1R and P2Y2R. Protein and mRNA will be determined in 5 different coronary segments: healthy, atherosclerotic, peri-stent, in-stent neointima, and in-stent media. 3) MetS increases functional expression of the A1R and P2Y2R by ERK, JNK, and AKT activation. Immunoblots and immunocytochemistry for phospho ERK, JNK, and AKT; store-operated Ca influx; and inflammation and oxidant stress quantify functional activation. 4) Angiotensin II and aldosterone directly, independently, and synergistically increase molecular and functional expression of A1R and P2Y2R, effects that are potentiated by dyslipidemia. In vivo treatments include several pig groups to be compared with in vitro organ culture. 5) Exercise will reverse the increased molecular and functional expression of A1R and P2Y2R in MetS. This Aim assesses more established CAD and stenting. 6) Blockade of A1R and P2Y2R attenuates in-stent stenosis in over-expansion injury and native atherosclerosis. Lean and MetS pigs will undergo in vivo coronary stenting with drug- and gene-eluting stents to selectively block A1R and P2Y2R.