Mechanisms of Angiogenic Switch Activation During Wound Repair

描述（由申请人提供）：完全理解刺激血管生成或新血管生长的信号对于促进受伤组织的血管化至关重要。尽管许多证据支持生长因子是促进血管生成反应的关键，但对来自局部环境的其他线索如何将静止的内皮细胞转化为发芽结构知之甚少。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面剪切应力的变化如何在三维上启动内皮发芽反应。我们假设，促血管生成因子刺激血管蛋白的磷酸化和钙蛋白酶依赖性裂解，从而控制下游膜相关蛋白酶的激活并启动芽的形成。这些研究将利用有区别的、明确的、可量化的三维系统，在该系统中，原代人内皮细胞侵入并形成多细胞、毛细血管样芽。本文提出的研究有望首次证明，促血管生成因子和壁剪切应力通过钙蛋白酶和细胞内激酶结合，破坏静脉蛋白网络，并允许膜相关金属蛋白酶转移到膜上。这些信号导致随后的侵袭和基质改变。我们将利用缺乏vimentin的转基因小鼠来证实这种中间细丝在成人血管生成中的作用。这些实验代表了一种平衡的、多学科的方法来定义损伤组织中启动血管新生芽形成的细胞内信号。将定义的体外和体内伤口愈合模型与活体成像相结合，将增强我们对生物化学和机械信号如何在3D中调节血管生成发芽和细胞-基质通信事件的基本理解，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. PUBLIC HEALTH RELEVANCE: Chronic wounds elicit significant social and economic burdens; therefore, a complete understanding of these events is warranted. This proposal will significantly advance our understanding of how various physiological forces and biochemical signals initiate new blood vessel growth, which is a required step in wound healing. Such knowledge will ultimately aid to decrease the socio-economic burdens associated with aberrations or deficiencies in the process.