Differential Macro- and Micro-Vascular Remodeling in Type 2 Diabetes and Metabolic Syndrome

描述(申请人提供)：2型糖尿病(T2 DM)的心脏病与冠状动脉疾病(CAD)的严重程度直接相关，冠心病会导致冠状动脉血流受损和心肌梗死(MI)的风险增加。这在美国和世界范围内造成了非常严重的健康和经济负担，因为心血管疾病是T2 DM的主要原因或死亡原因，占糖尿病患者死亡总数的65%。此外，糖尿病患者发生心肌梗死的可能性是非糖尿病患者的2-4倍。虽然动脉粥样硬化和内皮功能障碍是MI的已知因素，但冠状动脉阻力微血管(CRM)重构与大血管(导管动脉)重构在T2 DM诱发心脏病中的相对贡献仍很大程度上尚不清楚。CRM重塑伴随冠脉血流量减少，部分依赖于血管平滑肌细胞(VSMC)增殖、弹性细胞外基质(ECM)表达增加、局灶性黏附/细胞骨架蛋白表达降低。T2 DM db/db小鼠大血管局部黏附/细胞骨架和细胞外基质蛋白表达增加。这些发现指出细胞硬度与大血管和冠状微血管ECM之间的密切相互作用，这决定了T2 DM患者的整体血管硬度。目前还没有直接研究僵直对T2 DM VSMC表型影响的研究。根据我的初步数据，较硬的CRM管壁促进体内增殖的VSMC表型，而较硬的主动脉(大血管)管壁有利于合成的VSMC表型。这些数据表明，CRM重塑的机制与调节大血管重塑的机制是根本不同的，可能代表了决定VSMC表型的病理机制，从而导致糖尿病MI。因此，我的总体假设是，大血管和冠状动脉微血管重塑和僵硬的改变反映了细胞的不同贡献。 以及控制VSMC表型的ECM硬度，可能部分由年龄/愤怒和/或CRP2决定。这项建议中概述的研究将利用一种新的去细胞技术在主动脉和CRM、原代培养的VSMC、分离的冠脉阻力微血管以及糖尿病小鼠血管重构和血流的体内分析中全面研究这些机制。在指导阶段，我们将确定ECM和VSMCs对整体血管硬度的相对贡献，进一步评估硬度是否可以调节大血管和CRM中经典的VSMC表型。指导研究将主要集中在确定在原代VSMC培养中这些变化背后的细胞和生物力学机制。在独立阶段，我将确定僵硬、年龄/RAGE和CRP2的相互作用是否调节了从糖尿病小鼠和临床前患有甲硫氨酸的猪分离的VSMCs的表型。最后，我将评估一种新型RAGE拮抗剂FPS-ZM1在减少T2 DM小鼠不良血管重构方面的治疗潜力。这一过渡奖项将使我能够获得作为一名有竞争力和成功的独立调查员未来取得成功所必需的额外指导。这项提案包括一个全面的培训计划，该计划将由一个指导委员会的成员监督，该委员会拥有涵盖血管信号和生物力学、细胞外基质以及糖尿病和代谢综合征的冠状动脉并发症的专业知识。一旦完成，我将获得与体外细胞信号、血管力学、细胞外基质和冠状循环相关的假说生成、实验设计和数据解释方面的额外专业知识，这将增强我在体外和体内生理学方面已经很完善的专业知识。这将进一步为今后的研究提供坚实的基础，我希望在这些研究中调查(1)内皮细胞在T2 DM不良血管重构中的作用，以及(2)冠脉循环和糖尿病心脏之间的动态串扰，因为冠脉循环位于一个复杂的组织中，在糖尿病和代谢综合征等代谢条件下显示出舒张期功能障碍的迹象。我致力于保持来自NIH等来源的外部资金，以维持独立的实验室和培训环境，同时继续在代谢性疾病的心血管并发症方面取得进展。我过去的表现证明了我出色的技术技能、解决问题的能力、职业道德和干劲，这些都会让我变得更好 做好了成功的准备。

DESCRIPTION (provided by applicant): Heart disease in type 2 diabetes mellitus (T2DM) is directly related to the severity of coronary artery disease (CAD), which results in impaired coronary flow and increased risk of myocardial infarction (MI). This poses a very significant health and economic burden in the U.S. and worldwide since cardiovascular disease is the leading cause or mortality in T2DM, accounting for >65% of all deaths in diabetic patients. Moreover, diabetic patients are 2-4 times more likely to experience MI than non-diabetic patients. While atherosclerosis and endothelial dysfunction are known contributors to MI, the relative contribution of coronary resistance microvessel (CRM) remodeling versus macrovessel (conduit artery) remodeling to T2DM-induced cardiac disease remains largely unknown. We previously showed that CRMs isolated from either T2DM db/db mice or the pre-clinical Ossabaw porcine model of MetS undergo inward hypertrophic remodeling associated with reduced stiffness (novel and anti-dogmatic) in the face of increased macrovascular stiffness (dogmatic). CRM remodeling was accompanied by reduced coronary blood flow and was partially dependent upon vascular smooth muscle cell (VSMC) proliferation, increased expression of elastic extracellular matrix (ECM), and decreased expression of focal adhesion/cytoskeletal proteins. In contrast, focal adhesion/cytoskeletal and ECM protein expression were increased in macrovessels of T2DM db/db mice. These findings point to an intimate interaction between cellular stiffness and the ECM in macro- and coronary micro-vessels that dictates overall vascular stiffness in T2DM. We also observed that CRM VSMCs exhibit a proliferative phenotype that is associated with enhanced signaling by the advanced glycation end products (AGE)/RAGE pathway and decreased expression of cysteine rich-protein 2 (CRP2), which is a negative regulator of VSMC proliferation. There are currently no studies that have directly investigated the effect of stiffness on T2DM VSMC phenotype. Based on my preliminary data, a less stiff CRM wall promotes an in vivo proliferative VSMC phenotype, while the stiffer aortic (macrovascular) wall favors a synthetic VSMC phenotype. These data demonstrate that mechanisms underlying CRM remodeling are fundamentally different from those that regulate macrovascular remodeling and may represent pathologic mechanisms dictating VSMC phenotype, contributing to diabetic MI. Thus, my overall hypothesis is that alterations in macro- and coronary micro-vascular remodeling and stiffness reflect differential contributions of cellular and ECM stiffness that controls VSMC phenotype and may be dictated in part by AGE/RAGE and/or CRP2. The studies outlined in this proposal will investigate these mechanisms comprehensively utilizing a novel decellularization technique in aorta and CRMs, primary VSMC cultures, isolated coronary resistance microvessels, and in vivo analysis of vascular remodeling and blood flow in diabetic mice. During the mentored phase, we will determine the relative contribution of both the ECM and VSMCs to overall vascular stiffness further assess whether stiffness can modulate classical VSMC phenotypes in both macro-vessels and CRMs. The mentored studies will focus heavily on determining the cellular and biomechanical mechanisms underlying these alterations in primary VSMC cultures. During the independent phase, I will determine whether the interplay of stiffness, AGE/RAGE, and CRP2 modulates the phenotype of VSMCs isolated from both diabetic mice and pre-clinical pigs with MetS. Finally, I will evaluate the therapeutic potential of a novel RAGE antagonist, FPS-ZM1, in reducing adverse vascular remodeling in T2DM mice. This transition award will allow me access to additional mentoring necessary for future success as a competitive and successful independent investigator. This proposal includes a comprehensive training plan that will be overseen by members of a mentoring committee with expertise that encompasses vascular signaling and biomechanics, extracellular matrix, and coronary complications of diabetes and metabolic syndrome. Once complete, I will have gained additional expertise in hypothesis generation, experimental design, and data interpretation related to in vitro cellular signaling, vascular mechanics, ECM, and the coronary circulation that will enhance my already sound expertise in ex vivo and in vivo physiology. It will further provide a solid foundation for future studies in whch I would like to investigate (1) the role of endothelial cells in adverse vascular remodeling in T2DM, and (2) the dynamic cross-talk between the coronary circulation and the diabetic heart, since the coronary circulation lies within a complex tissue that exhibits signs of diastolic dysfunction in metabolic conditions such as diabetes and metabolic syndrome. I am committed to maintaining extramural funding from sources such as the NIH to maintain an independent laboratory and training environment, while continuing to make inroads in the cardiovascular complications of metabolic diseases. My past performance is a testament to my excellent technical skills, problem-solving skills, work ethic, and drive that will collectively make me well poised for success.