Determinants of Arterial Remodeling in Atherogenesis

描述（由申请人提供）：间质胶原严重影响动脉粥样硬化动脉的结构。人动脉粥样硬化中活化的巨噬细胞过表达基质降解酶，包括间质胶原酶（MMP-1/胶原酶-1，MMP- 13/胶原酶-3和MMP-8/胶原酶-2）。我们最近发现，由于I型胶原蛋白在胶原酶裂解位点的突变导致的胶原酶抗性增加了小鼠动脉粥样硬化中胶原蛋白的积累。然而，在动脉粥样硬化形成过程中，特异性胶原溶解酶在动脉重塑中的重要性的直接体内证据仍然很少。因此，我们建议在体内研究胶原酶的胶原重塑机制。该项目将在体内基因改变的小鼠或来自这些动物的细胞或体外基因操作的人巨噬细胞中测试三种假设。特异性目的1将检验以下假设：在靶向缺失主要鼠间质胶原酶的小鼠中，特异性胶原酶调节动脉粥样硬化形成期间的斑块结构。我们将确定是否MMP-8或-13或两种胶原酶的缺乏会增加动脉粥样硬化斑块中的胶原积累，并影响动脉粥样硬化易感apoE小鼠斑块结构的其他变量。具体目标2将探索来自骨髓细胞（主要是巨噬细胞）的间质胶原酶（MMP-8、-13和-14）在动脉粥样硬化中胶原积聚和动脉重塑中的作用。我们假设，通过转移来自apoE-/-小鼠（相应MMP的野生型）的骨髓，选择性恢复缺乏MMP-13或-8的apoE小鼠的MMP活性，将降低胶原蛋白含量，在病变中产生更薄的纤维帽和/或引起局部扩张。我们还将确定在骨髓源性细胞中选择性缺失MMP-14是否会影响这些变量（MMP-14-/-小鼠具有早期致死性）。具体目标3将检验MMP-8、-13或-14介导基质降解以及血管细胞和巨噬细胞体外迁移的假设。我们将测试这些基质金属蛋白酶缺陷的小鼠巨噬细胞是否改变了降解基质中胶原蛋白的能力，基质由培养中的SMC产生。我们将在siRNA“敲低”实验中用特异性胶原酶表达减少的人巨噬细胞进行类似的实验。我们将进一步检验间质胶原酶（MMP-13、-8或-14）缺陷的SMC和巨噬细胞在体外通过细胞外基质迁移的能力降低的假设。这些实验将为我们在前两个特定目标的体内实验中可能获得的表型提供机械见解。总之，这个项目应该有助于了解动脉粥样硬化形成过程中细胞外基质重塑的机制，这是这种疾病临床表现的关键决定因素。

DESCRIPTION (provided by applicant): Intersitial collagen critically influences the structure of atherosclerotic arteries. Activated macrophages in human atheromata overexpress matrix-degrading enzymes including interstitial collagenases (MMP-l/collagenase-1, MMP- 13/collagenase-3, and MMP-8/collagenase-2). We recently showed that collagenase resistance due to mutation of collagen type I at the cleavage site for collagenases yields increased collagen accumulation in mouse atheromata. However, direct in vivo evidence of the importance of specific collagenolytic enzymes in arterial remodeling during atherogenesis remains scant. We therefore propose to study in vivo mechanisms of collagen remodeling by collagenases. This project will test three hypotheses in genetically-altered mice in vivo or with cells derived from such animals or with genetically-manipulated human macrophages in vitro. Specific Aim 1 will test the hypothesis that specific collagenases regulate plaque structure during atherogenesis in mice with targeted deletions of the major murine interstitial collagenases. We will determine whether deficiency of MMP-8 or -13 or both collagenases increases collagen accumulation in atheromata and influences other variables of plaque structure in atherosclerosis-susceptible apoE mice. Specific Aim 2 will explore the roles of interstitial collagenases (MMP-8, -13 and -14) from cells derived from bone-marrow (primarily macrophages) in collagen accumulation and arterial remodeling in atheromata. We hypothesize that selective restoration of MMP activity in apoE mice lacking MMP-13 or -8 by transfer of bone marrow from apoE-/- mice, wild-type for the respective MMP, will decrease collagen content, yield thinner fibrous caps in lesions and/or cause local ectasia. We will also determine whether selective deletion of MMP-14 in bone marrow-derived cells affects these variables (the MMP-14-/- mouse has early lethality). Specific Aim 3 will test the hypothesis that MMP-8, -13 or -14 mediates matrix degradation and vascular cell and macrophage migration in vitro. We will test whether mouse macrophages deficient in these MMPs have altered ability to degrade collagen in matrices generated by SMC in culture. We will perform similar experiments with human macrophages with reduced expression of specific collagenases in siRNA "knock-down" experiments. We will further test the hypothesis that SMC and macrophages deficient in the interstitial collagenases (MMP-13, -8, or -14) have reduced ability to migrate through extracellular matrix in vitro. These experiments will provide mechanistic insights into the phenotypes that we will likely obtain in the in vivo experiments of the first two specific aims. Together, this project should help in understanding the mechanisms of extracellular matrix remodeling during atherogenesis, a key determinant of the clinical expression of this disease.