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

描述（由申请人提供）：2型糖尿病（T2 DM）心脏病与冠状动脉疾病（CAD）的严重程度直接相关，导致冠状动脉血流受损和心肌梗死（MI）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的主要原因或死亡率，占糖尿病患者所有死亡的>65%。此外，糖尿病患者发生MI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的已知贡献者，但冠状动脉阻力微血管（CRM）重塑与大血管（管道动脉）重塑对T2 DM诱导的心脏病的相对贡献仍然很大程度上未知。 我们先前表明，从T2 DM db/db小鼠或MetS的临床前Ossabaw猪模型中分离的CRM在面对增加的大血管刚度（教条）时经历与刚度降低相关的向内肥大性重构（新颖和反教条）。CRM重塑伴随着冠状动脉血流量减少，并部分依赖于血管平滑肌细胞（VSMC）增殖，弹性细胞外基质（ECM）的表达增加，并减少表达的粘着斑/细胞骨架蛋白。相反，在T2 DM db/db小鼠的大血管中，粘着斑/细胞骨架和ECM蛋白表达增加。这些发现指出了细胞硬度和大血管和冠状动脉微血管中的ECM之间的密切相互作用，这决定了T2 DM的整体血管硬度。我们还观察到CRM VSMC表现出与晚期糖基化终产物（AGE）/β-内酰胺酶途径的信号增强和富含半胱氨酸的蛋白2（CRP 2）的表达降低相关的增殖表型，CRP 2是VSMC增殖的负调节因子。目前没有研究直接研究僵硬对T2 DM VSMC表型的影响。根据我的初步数据，不太硬的CRM壁促进体内增殖VSMC表型，而更硬的主动脉（大血管）壁有利于合成VSMC表型。这些数据表明，CRM重塑的基础机制是根本不同的，从那些调节大血管重塑，并可能代表病理机制决定VSMC表型，有助于糖尿病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， 和ECM硬度，其控制VSMC表型，并且可以部分地由AGE/CRP和/或CRP 2决定。 本提案中概述的研究将在主动脉和CRM、原代VSMC培养、分离的冠状动脉阻力微血管以及糖尿病小鼠血管重塑和血流的体内分析中全面利用新型脱细胞技术研究这些机制。在指导阶段，我们将确定ECM和VSMC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中的经典VSMC表型。指导的研究将主要集中在确定这些变化的细胞和生物力学机制在原代VSMC培养。在独立的阶段，我将确定是否刚度，AGE/CRP和CRP 2的相互作用调节VSMCs的表型分离自糖尿病小鼠和临床前猪MetS。最后，我将评估一种新的β受体拮抗剂FPS-ZM 1在减少T2 DM小鼠不良血管重塑方面的治疗潜力。 这一过渡奖将使我能够获得额外的指导必要的未来成功作为一个有竞争力的和成功的独立调查员。该提案包括一个全面的培训计划，该计划将由具有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等专业知识的指导委员会成员监督。一旦完成，我将在假设生成，实验设计和数据解释方面获得额外的专业知识，这些知识与体外细胞信号传导，血管力学，ECM和冠状动脉循环有关，这将增强我在体外和体内生理学方面的专业知识。它将进一步为未来的研究提供坚实的基础，我想研究（1）内皮细胞在T2 DM不良血管重塑中的作用，以及（2）冠状动脉循环和糖尿病心脏之间的动态串扰，因为冠状动脉循环位于一个复杂的组织内，该组织在糖尿病和代谢综合征等代谢疾病中表现出舒张功能障碍的迹象。我致力于维持来自NIH等来源的校外资金，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能力、解决问题的能力、职业道德和动力，这些都将使我变得更好。 为成功做好准备

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.