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.

Project Narrative The vasculature is a key determinant for organism and organ development, its function in health and disease as well as for organ/tissue regeneration. At present there are no safe and efficient therapeutic strategies facilitating vascular regeneration as well as rewiring and restructuring of pathological vasculature. The goal of this proposal is to address the mechanisms by which resident microglia (innate immune cells in Central Nervous System shapes vascular development, function and pattern. We will show how the paracrine function of microglia (which populates brain and retinas prior to vasculature) defines vascular pattern. This knowledge will allow a deep understanding of the tissue-specific mechanisms controlling vascularization and will allow development of strategies aimed to normalize pathological vasculature by influencing microglial responses. Our studies on microglia will be instrumental for the treatment of integrin and kindlin 3 deficient patients. Microglia and its activation recently emerged as a main contributor to age-related macular degeneration, multiple sclerosis, Alzheimer's disease, depression and behavioral abnormalities such as autism, schizophrenia and pathology of AIDS. Our studies on functions of microglia will lead to new therapies for these pathologies. Our studies will also advance artificial tissue design and regeneration tec hnologies .