Mechanisms of microvascular remodeling progression

描述(由申请人提供)：血管重塑是一种适应血管直径长期改变的机制。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，但人们对启动和引导阻力微血管向内重构进展的机制知之甚少。在这一点上，我们认为重塑过程是一个连续的事件，最终导致结构改变的血管。我们独特的新颖和挑衅性的假说是，持续的小动脉血管收缩响应于持续的体液和/或机械刺激，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(翻转)；2)血管平滑肌(VSM)细胞骨架的重排；以及3)VSM细胞附着物的重新定位依赖于细胞内活性氧物种(ROS)的产生。使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，在持续的血管收缩(高血压的一个标志)期间，孤立小动脉中的VSM细胞重新延长并迅速改变位置，同时保持缩小的小动脉直径。这种现象在短短四个小时内就会发生，我们认为这是一种与内向重构有关的早期机制。我们进一步假设，其他机制同时发生，包括：1)依赖ROS激活基质金属蛋白酶(MMPs)以降解ECM；2)依赖ROS调节小G蛋白Rho以诱导钙敏化和重塑VSM细胞骨架；以及3)依赖ROS调节整合素依赖的VSM细胞附着。我们将使用最先进的成像和分子方法在三个体内模型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塑，利用多光子显微镜，我们将确定VSM细胞的行为和分离小动脉的ECM变化。通过原子力显微镜(AFM)和荧光成像，我们将对新鲜分离的VSM细胞施加离散的力，并监测焦点黏附(细胞黏附)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。这些方法将为测试我们的假设和整合我们的结果提供一个强大的策略。我们的长期目标是确定导致高血压患者阻力血管结构改变的机制。这些根本性的重要机制研究将使我们能够开发新的策略来预防、阻止和/或逆转重塑和与之相关的危及生命的事件。公共健康相关性：公共健康相关性声明在高血压患者中，被称为阻力小动脉的小血管经历了一个结构重塑的过程，缩小了其内径，增加了心脏病发作和中风的风险。这个项目的目标是了解控制这种重塑的机制。这一理解将使我们能够开发新的策略来预防、阻止和/或逆转重塑过程和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.