Pericyte proteinase inhibitors and EC tube stabilization

描述(申请人提供)：在这份续订建议书中，我们继续我们的工作，展示周细胞和内皮细胞(EC)衍生的蛋白酶抑制物在3D细胞外基质环境中控制内皮管稳定过程的关键功能作用。我们的总体假设是，蛋白酶抑制剂TIMP-3和TIMP-2具有稳定性质，因为它们能够抑制前形态刺激和前回归刺激，而前形态刺激和前回归刺激是稳定输卵管所必需的。在上一次资助期间，我们在展示周细胞稳定内皮细胞衬里的小管的能力和TIMP在这一过程中的作用方面取得了重要进展，开发了新的EC-周细胞共培养模型来确定这些事件的分子需求，并确定了TIMP-3和TIMP-2的关键EC靶点，如MT1-MMP1、MMP1、MMP10和ADAM-15，这些靶点介导了它们的影响。此外，我们在以下方面取得了重大进展：分析内皮细胞和周细胞的基因表达和功能以确定这一过程的分子需求；分析细胞外基质重塑变化的影响，例如确定内皮细胞与周细胞相互作用催化血管基底膜基质组装的需求；确定周细胞通过PDGF-BB和HB-EGF介导的信号被招募到内皮细胞衬里的小管的机制；以及确定关键生长因子(如转化生长因子-β)如何影响这一过程。我们提出了三个特定的目标：特定的目标1：确定周细胞能够催化内皮细胞基底膜基质组装以促进血管稳定的分子机制。具体目标#2：明确控制周细胞如何被特异性地招募到内皮细胞衬里的管上，以及它们如何通过迁移事件动态扫描内皮细胞基底面以调节管的稳定的机制。具体目标#3：明确周细胞如何通过减弱无处不在的细胞因子转化生长因子-β介导的回归刺激而稳定发育中的血管管。 公共卫生相关性：这项工作的重点是周细胞，一种围绕着小血管的细胞类型，支持这些细胞衬里的管状结构的完整性。这些支持细胞产生一系列促进这种支持机制的蛋白质。对血管形成和稳定的基本机制的基本了解，对于在各种人类疾病，如心血管疾病、糖尿病或癌症的背景下，努力刺激或抑制这一过程至关重要。

DESCRIPTION (provided by applicant): In this renewal proposal, we continue our work demonstrating the critical functional role of pericyte and endothelial cell (EC)-derived proteinase inhibitors to control the process of endothelial tube stabilization in 3D extracellular matrix environments. Our overall hypothesis is that the proteinase inhibitors, TIMP-3 and TIMP-2, possess stabilizing properties due to their ability to suppress both promorphogenic and proregression stimuli which are necessary for tube stabilization. During this last funding period, we have made essential progress in demonstrating the ability of pericytes to stabilize EC-lined tubes and the role of TIMPs during this process, have developed novel EC-pericyte tube coculture models to identify molecular requirements for these events, and have identified critical EC targets of TIMP-3 and TIMP-2 such as MT1-MMP, MMP-1, MMP-10 and ADAM- 15 which mediate their influence. In addition, we have made significant advances in our ability to; analyze gene expression and function in ECs versus pericytes to identify the molecular requirements for this process; analyze the influence of extracellular matrix remodeling changes such as identifying a requirement for EC- pericyte interactions to catalyze vascular basement membrane matrix assembly; identify mechanisms whereby pericytes are recruited to EC-lined tubes through PDGF-BB and HB-EGF mediated signaling; and determine how critical growth factors such as TGF-beta affect this process. We propose three specific aims which are; Specific Aim #1: To determine the molecular mechanisms by which pericytes are able to catalyze endothelial basement membrane matrix assembly to facilitate vascular stabilization. Specific Aim #2: To define the mechanisms controlling how pericytes are specifically recruited to endothelial cell-lined tubes and how they dynamically scan the endothelial basal surface through migratory events to regulate tube stabilization. Specific Aim #3: To define how pericyte recruitment to developing vascular tubes leads to stabilization through attenuation of regression stimuli mediated by the ubiquitous cytokine, TGF-beta. PUBLIC HEALTH RELEVANCE: This work focuses on the ability of pericytes, a cell type surrounding small blood vessels, to support the integrity of these cell-lined tube structures. These support cells produce a series of proteins that facilitate this support mechanism. A basic understanding of the mechanisms underlying how blood vessels form and become stabilized is critical in efforts to stimulate or inhibit the process in the context of various human diseases such as cardiovascular disease, diabetes or cancer.