Calcium Sparklers-Induced Vascular Dysfunction During Diabetes

 描述(申请人提供)：高血压、中风和冠状动脉疾病的发病率在非胰岛素依赖型2型糖尿病患者中急剧增加。通过L钙通道(LTCC)的钙离子异常内流导致血管平滑肌收缩、肌张力和血流/血压增强，参与了糖尿病高血糖(HG)引起血管并发症的一系列事件。然而，这种病理改变涉及的机制尚不清楚。我们最近发现了令人兴奋的新数据，表明在高血压和糖尿病期间，支架蛋白AKAP150(AKAP79是人类同源蛋白)锚定蛋白激酶A(PKA)是刺激LTCC活性和血管收缩所必需的。这与cAMP/PKA活性增加导致VSM松弛和血管扩张的传统观念形成鲜明对比。因此，这一竞争性更新应用的总体目标是解决我们新观察到的知识中的两个基本空白：1)HG期间VSM中肌膜cAMP/PKA信号的空间限制是否会促进血管收缩？2)在HG/糖尿病过程中，哪些上游机制触发cAMP/PKA信号转导？初步数据表明，在HG/糖尿病过程中，空间离散和异质性的cAMP/PKA信号促进LTCC增强和VSM收缩。我们还提供了令人信服的证据表明，一种新的AKAP150锚定的P2Y11受体(P2Y11)与GS偶联，是PKA介导的CaV1.2磷酸化、通道活性功能上调、钙内流增强、钙调神经磷酸酶(CaN)/NFATc3依赖的信号转导激活以及HG/糖尿病血管收缩的重要组成部分。令人信服的结果再现了来自非糖尿病和糖尿病患者的天然人类VSM/动脉的这些病理变化，突显了我们数据的翻译意义。除了GS偶联的P2Y11信号在血管生理学中的意想不到的作用外，在HG/糖尿病期间，区域化的腺苷环化酶(AC)/PKA信号导致VSM收缩的病理性诱导是我们模型的一个高度创新的概念。两个目的将被用来检验这一中心假设，即涉及P2Y11、AKAP150、AC和PKA的大分子复合体支撑着CaV1.2的磷酸化和LTCC介导的钙内流，以响应HG/糖尿病。目的1将验证空间受限的AC/PKA信号刺激LTCC在HG/糖尿病期间触发PKA介导的VSM收缩和血管收缩的假说。目的2探讨AKAP150锚定的P2Y11复合体在HG/糖尿病过程中介导AC/PKA活性、LTCC上调和血管收缩的假说。用于检验这些假说的方法将包括我们团队开发的光学技术、超分辨率显微镜、光遗传学、遗传编码的生物传感器、最先进的电生理学、分子生物学、遥测和血流测量。这项变革性/转化性研究中提出的实验将提供宝贵的机制信息，为糖尿病血管功能障碍的早期干预治疗策略奠定基础。

 DESCRIPTION (provided by applicant): The incidence of hypertension, stroke and coronary artery disease increases dramatically in patients with non- insulin dependent type 2 diabetes. Aberrant Ca2+ influx via L-type CaV1.2 channels (LTCCs) leading to enhanced vascular smooth muscle (VSM) contraction, myogenic tone and blood flow/pressure has been implicated in the chain of events contributing to hyperglycemia (HG)-induced vascular complications during diabetes. Yet, the mechanisms involved in this pathological alteration are unclear. We recently discovered exciting new data indicating that anchoring of protein kinase A (PKA) by the scaffolding protein AKAP150 (AKAP79 is the human ortholog) is required for stimulation of LTCC activity and vasoconstriction during HG and diabetes. This contrasts the conventional notion that increases in cAMP/PKA activity leads to VSM relaxation and vasodilation. Thus, the overall goal of this competitive renewal application is to address two fundamental gaps in knowledge raised by our novel observations: 1) could spatial confinement of sarcolemmal cAMP/PKA signals in VSM during HG promote vasoconstriction? and 2) what upstream mechanisms trigger cAMP/PKA signaling during HG/diabetes? Preliminary data indicate that spatially discrete and heterogeneous cAMP/PKA signaling foster LTCC potentiation and VSM contraction during HG/diabetes. We also provide compelling evidence implicating a novel AKAP150-anchored P2Y11 receptor (P2Y11), which is coupled to GS, as an essential component of PKA-mediated CaV1.2 phosphorylation, functional upregulation of channel activity, enhanced Ca2+ influx, activation of calcineurin (CaN)/NFATc3-dependent signaling and vasoconstriction in HG/diabetes. Compelling results reproducing these pathological changes in native human VSM/arteries from non-diabetic and diabetic patients underscore the translational significance of our data. Beyond an unexpected role for GS-coupled P2Y11 signaling in vascular physiology, the pathological induction of compartmentalized adenylyl cyclase (AC)/PKA signaling leading to VSM contraction during HG/diabetes is a highly innovative concept of our model. Two aims will be investigated to test the central hypothesis that a macromolecular complex involving the P2Y11, AKAP150, AC and PKA underlies CaV1.2 phosphorylation and LTCC-mediated Ca2+ influx in response to HG/diabetes. Aim 1 will examine the hypothesis that spatially confined AC/PKA signaling stimulates LTCCs to trigger PKA-mediated VSM contraction and vasoconstriction during HG/diabetes. Aim 2 will investigate the hypothesis that an AKAP150-anchored P2Y11 complex mediates compartmentalized AC/PKA activity, LTCC upregulation and vasoconstriction during HG/diabetes. Methods used to test these hypotheses will include optical techniques developed by our group, super-resolution microscopy, optogenetics, genetically encoded biosensors, state-of-the-art electrophysiology, molecular biology, telemetry and blood flow measurements. Experiments proposed in this transformative/translational study will provide invaluable mechanistic information that could lay the foundation for novel early intervention therapeutic strategies during diabetic vascular dysfunction.