Novel role of integrins in paracrine regulation of vasculature

抽象的。 这项提议旨在了解血管和常驻天然免疫细胞之间相互干扰的本质。 (小胶质细胞)使功能失调(不足或过度)的血管系统正常化，并形成新的组织 再生和血运重建技术。它阐述了小胶质细胞的基本功能，它扮演着一种 在许多病理中的协调作用从发育异常和行为问题到 神经退行性疾病对HIV神经病理学的影响。利用小胶质细胞特异性的Kindlin3和整合素基因敲除， 我们发现，尽管正常存在于大脑和视网膜，缺乏Kindlin的小胶质细胞的特征是 放松对血管生成因子的生产管制。结果，小胶质细胞特异性KOS中的视网膜血管形成 异常致密和不规则的血管图案，表现出血管过度生长进入正常的 光感受器的无血管区域。我们证明这是由K3KO小胶质细胞过度产生转化生长因子β1所介导的。 因为在K3KO小鼠中敲除小胶质细胞转化生长因子β1可以挽救这种血管表型。机械上，虽然 正常小胶质细胞在具有较高配体密度或较高硬度的基质中“关闭”其转化生长因子β-1的产生。 这种机械开关在缺乏Kindlin3的小胶质细胞中被取消。我们证明了机器人的机械感觉功能 小胶质细胞依赖于膜-皮质连接(MCA)复合体，该复合体因缺乏 Kindlin3.我们证明Kindlin3不仅与整合素、质膜结合，而且还与 细胞骨架(通过直接与亮蛋白/帕西林结合)和K3-帕西林结合的破坏破坏了MCA复合体。 这些结果导致我们提出了一个新的范式转换假说：Kindlin3坐标MCA 复合体，它反过来协调小胶质细胞的机械感觉功能，即它对 ECM的力学性能受血管生成因子表达的影响。机械论的问题：“如何做到？ 小胶质细胞是否能够感知细胞外基质的特性，从而调节血管系统？将会在这里得到回答 求婚。目的1.验证旁分泌调节血管生成受β-1控制的假说。 β2整合素在小胶质细胞上，需要整合素和Kindlin3之间具有全功能的复合体，进而， 调节小胶质细胞转化生长因子β的表达。目的2.检验整合素-Kindlin3-亮蛋白/巴西林的假说 复合体作为膜张力的传感器，因此，ECM的组成和机械 性质，并定义这个复合体的结构-功能决定因素。我们将利用我们最近的 开发了基于3D水凝胶的体系，具有可控的机械特性(配体密度、刚性和 放松时间)。我们的新体外技术将导致新材料和新方法的发展 用于组织再生和小胶质细胞控制的血管重建。对小胶质细胞的认识将导致新的 治疗多发性硬化症、阿尔茨海默病和其他神经和视网膜病变。

Abstract. This proposal aims to understand the nature of the crosstalk between vascular and resident innate immune cells (microglia) in order to normalize dysfunctional (insufficient or excessive) vasculature and to develop novel tissue regeneration and revascularization technologies. It addresses the basic functions of microglia, which plays a coordinating role in numerous pathologies from developmental abnormalities and behavioral problems to neurodegenerative disorders to neuropathology of HIV. Using microglia-specific Kindlin3 and integrin knockouts, we show that despite the normal presence in brain and retinas, Kindlin deficient microglia is characterized by deregulated production of angiogenic factors. As a result, retinal vasculature in microglia-specific KOs has abnormally dense and irregular vascular pattern and exhibits excessive growth of blood vessels into the normally avascular area of photoreceptors. We show that this is mediated by overproduction of TGFβ1 by K3KO microglia, since knockout of microglial TGFβ1 in K3KO mice rescues this vascular phenotype. Mechanistically, while normal microglia “switches off” its production of TGFβ1 in matrix with higher ligand density or higher stiffness, this mechano-switch is abrogated in Kindlin3 deficient microglia. We show that the mechanosensory function of microglia depends on the membrane-to-cortex attachment (MCA) complex, which is disrupted by the lack of Kindlin3. We demonstrate that Kindlin3 binds not only to integrin, to plasma membrane but also connects to cytoskeleton (by direct binding to leupaxin/paxillin) and disruption of K3-paxillin binding disrupts MCA complex. These results led us to formulate a novel paradigm-changing hypothesis that: Kindlin3 coordinates MCA complex, which, in turn, orchestrates the mechanosensory function of microglia, i.e. its ability to respond to the mechanical properties of ECM by the changes in angiogenic factors expression. The mechanistic question: “How exactly microglia is able to sense the ECM properties in order to regulate vasculature?” will be answered in this proposal. Aim 1. To test the hypothesis that paracrine regulation of angiogenesis is controlled by β1 rather than β2 integrin on microglia and requires a fully functional complex between integrin and Kindlin3, which, in turn, regulates microglial expression of TGFβ. Aim 2. To test a hypothesis that integrin-kindlin3-leupaxin/paxillin complex functions as a sensor of membrane tension and as a result, ECM composition and mechanical properties and to define the structure-functional determinants of this complex. We will utilize our recently developed 3D hydrogel-based system with controlled mechanical characteristics (ligand density, stiffness and a relaxation time). Our new in vitro technologies will lead to the development of novel materials and approaches for tissue regeneration and microglia-controlled revascularization. The knowledge of microglia will lead to new treatments for multiple sclerosis, Alzheimer's disease and other neuro- and retinal pathologies.