Regulation of Cellular Phenotype Change in Thoracic Aortic Aneurysms

胸主动脉瘤(TAA)的发生是由于主动脉细胞外细胞外的异常重构。 基质(ECM)。10这通常是无症状的过程，导致主动脉壁变弱，表现为粗大 进展为破裂的扩张。目前的治疗方法包括血压管理，直到出现 破裂的风险超过了手术或血管内介入的风险；这两者都不能解决潜在的 导致这种毁灭性疾病的途径。11 TAA的发展受到一系列相互关联的 机制，如基质金属蛋白酶(MMPs)12-15，以及生产和 16重要的是，这些机制在一定程度上是通过改变细胞外基质蛋白来调节的。 17，18转化生长因子-β(转化生长因子-β)，可溶性β 多肽生长因子能够调节主动脉ECM的结构和组成，是一种描述良好的 19目前的数据显示转化生长因子-β信号的改变导致I型 转化生长因子-β受体从转化生长因子-β-R1主导信号切换到ALK-1主导信号。转化生长因子-β被隔离 在细胞外基质内，由潜在的转化生长因子-β结合蛋白(LTBP)结合。21，22这些潜在的复合体是 TAA过程中诱导的关键MMPs的蛋白水解靶，如膜型MMP1(MT1-MMPs) 发育。8，23结果表明，在MT1-MMP杂合子中TAA的发育受到抑制 基因缺陷小鼠，针对转化生长因子-β配体(转化生长因子-β-核糖核酸)或MT1-基质金属蛋白酶的中和抗体治疗 活性(MT1-MMPInhAb)足以减弱主动脉扩张；提示MT1-MMPs是一种重要的 TAA形成和发展的中介物。新的数据显示，成熟婴儿的数量有所增加 TAA诱导后8周和16周的巨噬细胞(F4/80)；提示巨噬细胞来源的MT1-MMP 也可能有助于TAA的发展。本提案将探讨与时间相关的和细胞类型 利用已建立的和特征良好的TAA诱导小鼠模型特异性表达MT1-MMPs， 和几个独特的转基因小鼠品系。这项研究的中心假设是MT1-基质金属蛋白酶依赖 转化生长因子-β信号的激活既是时间依赖性的，又是细胞特异性的，它将通过三个特定的 目的：(1)证明成纤维细胞来源的基质金属蛋白酶是转化生长因子β释放和成纤维细胞所必需的 转分化，TAA发育早期。使用有效的他莫昔芬诱导的、成纤维细胞特异性的Cre- 成纤维细胞中依赖的(Col1A2-Cre-ERT2)FLOXED-MT1-MMPMT1-MMP1基因敲除将在 TAA诱导(早期)，或在TAA发育4周后(晚期)；(2)证明巨噬细胞起源于 基质金属蛋白酶是转化生长因子-β的释放和成纤维细胞表型维持所必需的，在成纤维细胞发育的晚期。 利用他莫昔芬诱导的单核/巨噬细胞特异性Cre依赖(LysM-Cre-ERT2)基因敲除 FLOXED-MT1-基质金属蛋白酶、巨噬细胞衍生的MT1-基质金属蛋白酶将被敲除。A)在TAA诱导之前(早期)，或B)之后 -TAA发育的4周(晚期)；以及(3)证明MT1-MMP活性的有效性-中和 抗体作为一种潜在的治疗TAA疾病的方法。小鼠将在注射前注射该抗体 至TAA诱导(早期)，或在TAA发育4周后(晚期)。在每个目标中，对主动脉几何形状的影响 和结构，转化生长因子-β信号的激活(pSmad-1/5/8)，成纤维细胞的表型/功能及其定位 记录主动脉壁细胞类型特异性标记物和MT1-MMPs的表达。这组研究将结合在一起 建立MT1-MMP在TAA形成过程中的时间依赖性和细胞类型特异性表达 并确定其在TAA发展中的机制作用。

Thoracic aortic aneurysms (TAAs) develop as a consequence to abnormal remodeling of the aortic extracellular matrix (ECM).10 This usually asymptomatic process results in a weakened aortic wall manifested as gross dilatation that progresses to rupture. Current treatment includes blood pressure management until the risk of rupture outweighs the risk of surgical or endovascular intervention; neither of which address the underlying pathways which drive this devastating disease.11 TAA development is influenced by a series of interrelated mechanisms such as the matrix metalloproteinases (MMPs) 12-15, and dysregulation of the production and deposition of ECM proteins.16 Importantly, these mechanisms are mediated in part through changes in the resident cellular constituents within the aortic wall.17, 18 Transforming growth factor-beta (TGF-β), a soluble peptide growth factor capable of regulating the structure and composition of the aortic ECM, is a well described mediator of fibroblast phenotype.19 Current data shows that the alterations in TGF-β signaling result in a type-I TGF-β receptor switch, from a TGF-β-R1 dominant signal, to an ALK-1 dominant signal. TGF-β is sequestered within the extracellular matrix, bound by latent TGF-β binding proteins (LTBPs).21, 22 These latent complexes are proteolytic targets for key MMPs, such as membrane type-I MMP (MT1-MMP), which is induced during TAA development.8, 23 Results demonstrated that TAA development was attenuated in MT1-MMP heterozygous deficient mice, and neutralizing antibody treatment targeting either TGF-β ligands (TGF-β-NAb) or MT1-MMP activity (MT1-MMP-InhAb) was sufficient to attenuate aortic dilatation; suggesting MT1-MMP as an important mediator of TAA formation and progression. New data demonstrate an increase in the number of mature macrophages (F4/80+) at 8- and 16- weeks post-TAA induction; suggesting that macrophage-derived MT1-MMP may also contribute to TAA development. The present proposal will explore the time-dependent and cell-type specific expression of MT1-MMP using an established and well-characterized mouse model of TAA induction, and several unique transgenic mouse strains. The central hypothesis of this study is that MT1-MMP-dependent activation of TGF-β signaling is both time-dependent and cell-specific, and it will be tested through three specific aims: (1) Demonstrate that fibroblast-derived MT1-MMP is required for TGF-β release and fibroblast transdifferentiation, early in TAA development. Using a validated Tamoxifen-inducible, fibroblast-specific Cre- dependent (Col1A2-Cre-ERT2) knockout of floxed-MT1-MMP, MT1-MMP will be deleted in fibroblasts prior to TAA induction (Early), or after 4-weeks of TAA development (Late); (2) Demonstrate that macrophage-derived MT1-MMP is required for TGF-β release and the maintenance of fibroblast phenotype, late in TAA development. Using a Tamoxifen-inducible, monocyte/macrophage-specific Cre-dependent (LysM-Cre-ERT2) knockout of floxed-MT1-MMP, macrophage-derived MT1-MMP will be knockout. A) prior to TAA induction (Early), or B) after 4-weeks of TAA development (Late); and (3) Demonstrate the efficacy of the MT1-MMP activity-neutralizing antibody as a potential therapeutic for the treatment of TAA disease. Mice will be treated with the antibody prior to TAA induction (Early), or after 4-weeks of TAA development (Late). In each aim, the effects on aortic geometry and structure, the activation of TGF-β signaling (pSmad-1/5/8), fibroblast phenotype/function, and the localization of cell-type specific markers, and MT1-MMP, in the aortic wall will be recorded. Together this set of studies will establish the time-dependent and cell-type-specific expression of MT1-MMP during TAA formation and progression and define its mechanistic role in TAA development.