Mechanisms of Angiogenic Switch Activation During Wound Repair

描述(由申请人提供)：完全了解刺激血管生成或新血管生长的信号，对于促进损伤组织的血管形成至关重要。虽然许多证据支持生长因子是促进血管生成反应的关键，但关于局部环境的其他信号如何将静止的内皮细胞转化为萌芽结构的了解要少得多。这些研究将提供重要的新见解，并回答尚未解决的基本问题，即血管生成生长因子和脂质以及壁切应力的变化如何在三维中启动内皮细胞萌芽反应。我们假设促血管生成因子刺激依赖于钙蛋白酶的波形蛋白的磷酸化和裂解，从而控制膜相关蛋白的下游激活并启动芽的形成。这些研究将利用可区分的、定义的、可量化的三维系统，在这些系统中，原始人类内皮细胞侵入并形成多细胞、毛细血管样的芽。本文提出的研究有望首次证明促血管生成因子和壁切应力通过钙蛋白和细胞内蛋白激酶结合，破坏波形蛋白网络，并允许膜相关的金属蛋白酶转位到膜上。这些信号会导致随后的侵袭和基质改变。我们将利用缺乏波形蛋白的转基因小鼠来证实这种中间丝参与了成年血管的萌发。这些实验代表了一种平衡的、多学科的方法来定义启动损伤组织中血管生成芽形成的细胞内信号。将已定义的体外和体内伤口愈合模型与活体成像相结合，将增强我们对生化和机械信号如何在3D中调控血管新生萌发和细胞-基质通讯事件的基本理解，为未来研究促进生命系统中新血管生长的信号提供平台。

DESCRIPTION (provided by applicant): A complete understanding of signals that stimulate angiogenesis, or new blood vessel growth, is essential for promoting vascularization of wounded tissues. Although much evidence supports that growth factors are key in promoting angiogenic responses, much less is known about how other cues from the local environment convert quiescent endothelial cells to sprouting structures. These studies will provide significant new insights and answer as-yet unresolved fundamental issues of how angiogenic growth factors and lipids as well as changes in wall shear stress initiate endothelial sprouting responses in three dimensions. We hypothesize that pro-angiogenic factors stimulate phosphorylation and calpain-dependent cleavage of vimentin, which controls downstream activation of membrane-associated proteinases and initiates sprout formation. These studies will utilize discriminating, defined, quantifiable, three-dimensional systems where primary human endothelial cells invade and form multicellular, capillary-like sprouts. The studies proposed here are expected to demonstrate for the first time that pro-angiogenic factors and wall shear stress combine via calpain and intracellular kinases to disrupt vimentin networks and allow translocation of membrane- associated metalloproteinase to the membrane. These signals result in ensuing invasion and matrix alterations. We will utilize transgenic mice lacking vimentin to confirm the involvement of this intermediate filament in sprouting angiogenesis in the adult. These experiments represent a balanced, multidisciplinary approach to defining the intracellular signals that initiate angiogenic sprout formation in wounded tissue. Integration of defined in vitro and in vivo models of wound healing with intravital imaging will enhance our fundamental understanding of how biochemical and mechanical signals regulate angiogenic sprouting and cell-matrix communication events in 3D, providing a platform for future studies on signals that promote new blood vessel growth in living systems.