Exercise, Diabetes, & Coronary Smooth Muscle Ca2+

描述（由申请方提供）：长期目标是确定冠状动脉平滑肌（CSM）腺苷A1和P2 Y2受体信号传导机制，通过该机制，拮抗肾素-血管紧张素-醛固酮系统（RAAS）和运动训练可减少代谢综合征（MetS）中的冠状动脉疾病（CAD）和再狭窄。RAAS对调节激酶和下游炎症的CSM A1和P2 Y2受体信号传导的调节是未知的。尽管药物洗脱支架可减少再狭窄，但由于再狭窄并发症和支架附近CAD（即支架周围CAD）的进展，需要进一步研究。我们在上一次资助中的主要发现为这个项目提供了出色的理由。Ossabaw猪具有临床显著的动脉粥样硬化（>50%狭窄），我们首次对自然病变进行了支架植入，而不仅仅是球囊损伤的健康动脉。我们克隆了猪腺苷受体的4种亚型（A1，A2 A，A2 B，A3），发现A1受体（A1 R）在CSM中选择性表达，在动脉粥样硬化和支架植入的冠状动脉中表达增加。A1 R的直接激活诱导CSM增殖，表明新的调节。我们克隆了3个猪P2核苷酸受体亚型，发现P2 Y2亚型仅在病变的CSM中表达，并且在支架植入的冠状动脉中选择性上调。总体假设：在MetS RAAS（主要是醛固酮）增加冠状动脉A1 R和P2 Y2 R信号，CSM生长的关键信号。综合实验设计比较瘦型与MetS Ossabaw RAAS拮抗作用。动脉粥样硬化病变是支架，然后恢复或不运动。结果与更简单的体外器官培养进行了比较。具体目的是检验MetS与lean Ossabaw的6个具体假设：1）MetS中的动脉粥样硬化、支架周围CAD和支架内狭窄增加，RAAS拮抗作用和运动减弱。血管内超声在体内评估CAD，光学成像和组织病理学在体外评估CAD。2)MetS增加A1 R和P2 Y2 R的分子表达。将在5个不同的冠状动脉节段中测定蛋白质和mRNA：健康、动脉粥样硬化、支架周围、支架内新生内膜和支架内中膜。3)MetS通过ERK、JNK和AKT激活增加A1 R和P2 Y2 R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;钙池操作的钙内流;以及炎症和氧化应激量化功能激活。4)血管紧张素II和醛固酮直接、独立和协同地增加A1 R和P2 Y2 R的分子和功能表达，这种作用由血脂异常增强。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MetS中A1 R和P2 Y2 R的分子和功能表达增加。该目标评估了更成熟的CAD和支架植入术。6)阻断A1 R和P2 Y2 R可减轻过度扩张损伤和自体动脉粥样硬化中的支架内狭窄。瘦肉猪和MetS猪将接受药物和基因洗脱支架的体内冠状动脉支架植入术，以选择性阻断A1 R和P2 Y2 R。 公共卫生相关性：我们的研究涉及Ossabaw小型猪的高度临床相关的冠状动脉疾病，其极好地模拟了人类的复杂代谢综合征（“前驱糖尿病”）和2型糖尿病。因此，我们的研究结果将促进新的治疗干预措施对人类的转化，包括生物工程设备，药物洗脱支架（支架打开闭塞的动脉）和全身药物治疗。目前批准用于人类的药物很可能在治疗冠心病方面找到新的用途。我们处于独特的地位，有利于预防和逆转冠状动脉疾病，这将改善美国的健康。

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. PUBLIC HEALTH RELEVANCE: Our studies involve highly clinically relevant coronary artery disease in the Ossabaw miniature pig, which superbly mimics complex metabolic syndrome ("pre-diabetes") and type 2 diabetes in humans. Thus, our findings will catalyze the translation of novel therapeutic interventions to humans, including bioengineered devices, drug-eluting stents (scaffold to open occluded artery), and systemic drug therapy. Drugs currently approved for humans are very likely to find new uses in treatment of coronary disease. We are uniquely positioned to benefit the prevention and regression of coronary disease, which will improve health in the U.S.