Regulation of Cellular Phenotype Change in Thoracic Aortic Aneurysms

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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.