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的结构。人动脉粥样硬化中活化的巨噬细胞过度表达基质降解酶，包括间质胶原酶(MMP一L/胶原酶-1、MMP一13/胶原酶-3和MMP一8/胶原酶-2)。我们最近发现，由于胶原酶裂解部位I型胶原蛋白的突变导致胶原酶抗性，增加了小鼠动脉粥样硬化症中胶原蛋白的积聚。然而，在动脉粥样硬化形成过程中，特定的胶原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重塑胶原的机制。该项目将在体内或使用来自此类动物的细胞或在体外使用基因操纵的人类巨噬细胞来测试三个假说。特定目标1将通过定向删除主要的小鼠间质胶原酶来检验特定胶原酶在小鼠动脉粥样硬化形成过程中调节斑块结构的假设。我们将确定是否缺乏基质金属蛋白酶-8或-13或两种胶原酶会增加动脉粥样硬化中胶原的积聚，并影响动脉粥样硬化易感载脂蛋白E小鼠斑块结构的其他变量。具体目的2将探讨来自骨髓细胞(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堆积和动脉重构中的作用。我们假设，通过从apoE-/-小鼠(野生型)移植骨髓，选择性地恢复缺乏MMP-13或-8的apoE小鼠的MMP活性，将减少胶原含量，在皮损中产生更薄的纤维帽和/或导致局部扩张。我们还将确定在骨髓来源的细胞中选择性删除基质金属蛋白酶-14是否会影响这些变量(基质金属蛋白酶-14-/-鼠具有早期致死性)。特异靶3将验证这一假设，即基质金属蛋白酶-8、-13或-14在体外介导基质降解和血管细胞和巨噬细胞迁移。我们将测试缺乏这些MMPs的小鼠巨噬细胞是否改变了在培养中降解SMC产生的基质中胶原的能力。我们将对人类巨噬细胞进行类似的实验，在siRNA“击倒”实验中减少特定胶原酶的表达。我们将进一步验证这一假说，即间质胶原酶(MMP13、-8或-14)缺乏的SMC和巨噬细胞在体外通过细胞外基质的迁移能力降低。这些实验将为我们在前两个特定目标的活体实验中可能获得的表型提供机械性的见解。总之，这个项目应该有助于理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.