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。人动脉粥样硬化活化的巨噬细胞过度表达基质降解酶，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，由于胶原酶裂解位点的I型胶原突变导致胶原酶抵抗，导致小鼠动脉粥样硬化中胶原积累增加。然而，在动脉粥样硬化过程中，特定的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，我们建议研究胶原酶在体内重塑胶原的机制。该项目将在体内的转基因小鼠或来自这些动物的细胞或体外的基因操纵的人类巨噬细胞中测试三个假设。特异性目的1将验证特异性胶原酶在小鼠动脉粥样硬化过程中调节斑块结构的假设，这些小鼠的主要小鼠间质胶原酶被靶向缺失。我们将确定在易感动脉粥样硬化的apoE小鼠中，缺乏MMP-8或-13或两种胶原酶是否会增加动脉粥样硬化中的胶原积累，并影响斑块结构的其他变量。特异性目标2将探讨骨髓细胞（主要是巨噬细胞）中的间质胶原酶（MMP-8， -13和-14）在动脉粥样硬化中胶原积累和动脉重塑中的作用。我们假设，在缺乏MMP-13或-8的apoE小鼠中，通过转移apoE-/-小鼠（各自的MMP为野生型）的骨髓来选择性地恢复MMP活性，将降低胶原含量，在病变中产生更薄的纤维帽和/或引起局部扩张。我们还将确定骨髓源性细胞中选择性删除MMP-14是否会影响这些变量（MMP-14-/-小鼠具有早期致命性）。特异性Aim 3将在体外验证MMP-8、-13或-14介导基质降解和血管细胞和巨噬细胞迁移的假设。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由SMC生成的基质中降解胶原的能力。我们将在siRNA“敲除”实验中对特异性胶原酶表达降低的人巨噬细胞进行类似的实验。我们将进一步验证SMC和巨噬细胞缺乏间质胶原酶（MMP-13、-8或-14）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制见解。总之，该项目应该有助于理解动脉粥样硬化过程中细胞外基质重塑的机制，这是该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.