Mechanisms of microvascular remodeling progression

描述（申请人提供）：血管重构是一种长期改变血管直径的适应性机制。在高血压中，向内重构，即阻力血管管腔直径的结构性缩小，与心肌梗死和中风的风险增加有关。然而，尽管它与危及生命的心血管事件有关，但对启动和引导阻力微血管向内重塑进程的机制知之甚少。在这方面，我们认为重塑过程是一个连续的事件，最终在结构改变的血管。我们独特的新颖和具有争议性的假设是，持续的小动脉血管收缩是对长时间的体液和/或机械刺激的反应，启动了重塑机制，其特征是：1)血管壁细胞外基质（ECM）成分的部分降解（周转）；2)血管平滑肌（VSM）细胞骨架重排；3)通过依赖于细胞产生活性氧（ROS）的过程重新定位VSM细胞附着物。利用我们实验室开发的高度创新的多光子成像技术，我们最近证明，在血管收缩延长（高血压的标志）期间，分离的小动脉中的VSM细胞会重新延长并迅速改变位置，而小动脉直径的缩小则保持不变。这种现象发生在短短四个小时内，我们认为这是一种与内向重塑相关的早期机制。我们进一步假设其他机制同时发生，包括：1)ros依赖性的基质金属蛋白酶（MMP）激活以降解ECM；2) ros依赖性调节小G蛋白Rho诱导钙敏化，重塑VSM细胞骨架；3)整合素依赖性VSM细胞附着物的ros依赖性调节。我们将使用最先进的成像和分子方法在三个体内和两个体外模型中测试我们的假设。通过活体显微镜，我们将监测体内血管重构，通过多光子显微镜，我们将确定VSM细胞行为和孤立小动脉的ECM变化。通过原子力显微镜（AFM）和荧光成像，我们将对新分离的VSM细胞施加离散力，并监测病灶粘附（细胞附着物）和细胞骨架重塑。这些方法结合分子和药理学技术将用于我们的特定目标，以确定ROS， MMPs， Rho和整合素在重塑中的作用。这些方法将为检验我们的假设和整合我们的结果提供有力的策略。我们的长期目标是描述导致高血压患者抵抗血管结构改变的机制。这些至关重要的机制研究将使我们能够制定新的策略来预防、停止和/或逆转重塑以及与之相关的危及生命的事件。在高血压患者中，被称为抵抗性小动脉的小血管会经历结构重塑过程，从而减小内径，增加心脏病发作和中风的风险。该项目的目标是了解控制这种重塑的机制。这种理解将使我们能够制定新的策略来预防、停止和/或逆转重塑过程以及与之相关的危及生命的事件。

DESCRIPTION (provided by applicant): Vascular remodeling is an adaptive mechanism for long-term modification of vascular diameter. In hypertension, inward remodeling, that is, the structural reduction of the lumen diameter in resistance vessels, is associated with an increased risk for myocardial infarction and stroke. However, despite its association with life threatening cardiovascular events, little is known about the mechanisms that initiate and guide the progression of inward remodeling in the resistance microvessels. In this regard, we view the remodeling process as a continuum of events that culminate in the structurally altered vessel. Our singularly novel and provocative hypothesis is that sustained arteriolar vasoconstriction in response to prolonged humoral, and/or mechanical stimuli initiates remodeling mechanisms characterized by: 1) partial degradation (turnover) of the extracellular matrix (ECM) components of the vessel wall; 2) rearrangement of the vascular smooth muscle (VSM) cytoskeleton; and 3) repositioning of the VSM cellular attachments via processes that depend on the cellular production of reactive oxygen species (ROS). Using a highly innovative multiphoton imaging technique developed in our laboratories, we recently demonstrated that VSM cells in isolated arterioles re-lengthen and rapidly change position during prolonged vasoconstriction (a hallmark of hypertension) while the reduced arteriolar diameter is maintained. This phenomenon occurs in as little as four hours, and we propose is an early mechanism associated with inward remodeling. We further hypothesize that other mechanisms occur concurrently, including: 1) ROS-dependent activation of matrix metalloproteinases (MMP) to degrade the ECM; 2) ROS-dependent modulation of the small G protein Rho to induce calcium sensitization and remodel the VSM cytoskeleton; and 3) ROS-dependent modulation of integrin-dependent VSM cell attachments. We will test our hypotheses in three in vivo and two in vitro models using state of the art imaging and molecular approaches. With intravital microscopy we will monitor vascular remodeling in vivo, and with multiphoton microscopy, we will determine VSM cell behavior and ECM changes in isolated arterioles. With atomic force microscopy (AFM) and fluorescence imaging we will apply discrete forces to freshly isolated VSM cells and monitor focal adhesion (cellular attachments) and cytoskeletal remodeling. These methodologies combined with molecular and pharmacological techniques will be used in our Specific Aims to determine the role of ROS, MMPs, Rho, and integrins on remodeling. These approaches will provide a powerful strategy for testing our hypotheses and integrating our results. Our long-term goal is to characterize the mechanisms leading to the structural modification of resistance vessels in hypertension. These fundamentally important mechanistic studies will allow us to develop new strategies to prevent, stop, and/or reverse remodeling and the life threatening events associated with it. PUBLIC HEALTH RELEVANCE: Public Health Relevance Statement In people with high blood pressure, the small blood vessels known as resistance arterioles undergo a process of structural remodeling that reduces their internal diameter and increases the risk for heart attacks and stroke. The goal of this project is to understand the mechanisms that control this remodeling. This understanding will allow us to develop novel strategies for preventing, stopping, and/or reversing the remodeling process and the life threatening events that are associated with it.