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Vascular disease-causing mutation, smooth muscle ?-actin R258C, dominantly suppresses functions of ?-actin in human patient fibroblasts.

ABSTRACT: The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) ?-actin encoded by ACTA2 We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM ?-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM ?-actin. Telomerase-immortalized lines of control and R258C human dermal fibroblasts were established and SM ?-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2 Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM ?-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM ?-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM ?-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM ?-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.

SUBMITTER: Liu Z

PROVIDER: S-EPMC5514740 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts.

Liu Zhenan Z Chang Audrey N AN Grinnell Frederick F Trybus Kathleen M KM Milewicz Dianna M DM Stull James T JT Kamm Kristine E KE

Proceedings of the National Academy of Sciences of the United States of America 20170626 28

The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by <i>ACTA2</i> We focus here on <i>ACTA2-</i>R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model ...[more]

PMID: 28652363

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Project description:IntroductionVascular smooth muscle cell (VSMC) senescence in the vasculature results in vascular aging as well as age-related diseases, while metformin improves the inflamm-aging profile by enhancing autophagy. However, metformin's impact on VSMC senescence is largely undefined.ObjectivesTo test the hypothesis that metformin exerts an anti-senescence role by restoring autophagic activity in VSMCs and vascular tissues.MethodsAnimal models established by angiotensin II (Ang II) induction and physiological aging and senescent primary VSMCs from the aortas of elderly patients were treated with metformin. Cellular and vascular senescence were assessed by measuring the amounts of senescence-associated β-galactosidase and senescence markers, including p21 and p53. Autophagy levels were assessed by autophagy-related protein expression, transmission electron microscope, and autolysosome staining. In order to explore the underlying mechanism of the anti-senescence effects of metformin, 4D label-free quantitative proteomics and bioinformatic analyses were conducted, with subsequent experiments validating these findings.ResultsAng II-dependent senescence was suppressed by metformin in VSMCs and vascular tissues. Metformin also significantly improved arterial stiffness and alleviated structural changes in aged arteries, reduced senescence-associated secretory phenotype (SASP), and improved proliferation and migration of senescent VSMCs. Mechanistically, the proteomic analysis indicated that autophagy might contribute to metformin's anti-senescence effects. Reduced autophagic flux was observed in Ang II-induced cellular and vascular senescence; this reduction was reversed by metformin. Specifically, metformin enhanced the autophagic flux at the autophagosome-lysosome fusion level, whereas blockade of autophagosome-lysosome fusion inhibited the anti-senescence effects of metformin.ConclusionsMetformin prevents VSMC and vascular senescence by promoting autolysosome formation.

Dataset Information

Vascular disease-causing mutation, smooth muscle ?-actin R258C, dominantly suppresses functions of ?-actin in human patient fibroblasts.

Publications

Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts.

Similar Datasets

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