Slingshot Isoform-Specific Regulation of Cofilin-Mediated Vascular Smooth Muscle Cell Migration and Neointima Formation

Torres, Rebecca A.; Drake, Douglas A.; Solodushko, Viktoriya; Jadhav, Rashmi; Smith, Erika; Ročić, Petra; Weber, David S.

doi:10.1161/atvbaha.111.232769

Cited by 31 publications

(26 citation statements)

References 29 publications

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“…In striated muscle cells, cofilin is thought to be involved in precise length control of the thin filament (28,29). In vascular smooth muscle cells, increased cofilin activation upon dephosphorylation with slingshot phosphatase 1 has been implicated in vascular smooth muscle cell migration and neointima formation following vascular injury (30). Here, we show that R258C actin filaments are considerably more susceptible to cleavage by cofilin and show increased levels of shortening, compared with WT filaments.…”

Section: R258c Mutation Decreases Contractile Function By Perturbing mentioning

confidence: 58%

Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

Fagnant

Bookwalter

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.actin | myosin II | smooth muscle | thoracic aortic aneurysms | vascular disease T horacic aortic aneurysms and dissections (TAAD) are the 18th most common cause of death in individuals in the United States (1). The high degree of mortality is partly due to the fact that aneurysms tend to be asymptomatic until a lifethreatening acute aortic dissection occurs. Familial TAAD is an autosomal dominant disorder with variable penetrance, which is characterized by enlargement or dissection of the thoracic aorta (reviewed in 2). The most prevalent genetic cause of familial TAAD, responsible for ∼15% of all cases, are mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2. More than 40 mutations in ACTA2 have been identified to date (3-5). Intriguingly, ACTA2 mutations also differentially predispose individuals to occlusive vascular diseases, such as premature coronary artery disease and strokes (6). ACTA2 mutations thus can lead to either dilation of large elastic arteries like the aorta or occlusion of smaller muscular arteries.SM α-actin is the most abundant protein in vascular smooth muscle cells, constituting ∼40% of the total protein and ∼70% of the total actin, with the rest composed of β-and γ-cytoplasmic actin. Actin is critical for contraction and force production by smooth muscle cells, as well as for their proliferation and migration. Dissected aortas show s...

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Section: R258c Mutation Decreases Contractile Function By Perturbing mentioning

confidence: 58%

Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

Fagnant

Bookwalter

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The migration of VSMCs was measured using Transwell chambers with 8.0-µm pore polycarbonate membranes as previously described (12). Briefly, the cells were harvested 30 min after electroporation.…”

Section: Methodsmentioning

confidence: 99%

Ras ssDNA aptamer inhibits vascular smooth muscle cell proliferation and migration through MAPK and PI3K pathways

Wang

et al. 2015

Int J Mol Med

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Abstract. Proliferation and migration of vascular smooth muscle cells (VSMCs) mediated by Ras proteins are crucial in restenosis following percutaneous coronary intervention (PCI) and coronary artery bypass graft (CABG). In this study, a novel, single-stranded DNA (ssDNA) aptamer designated as Ras-a1 with high affinity and specificity to human Ras protein was isolated using systematic evolution of ligands by exponential enrichment. Ras-a1 was delivered into VSMCs by electroporation using one square waveform of 200 V for 20 msec. Proliferation of VSMCs was determined using a cell counting kit-8 assay, which revealed the maximal inhibitory rate (40%) was obtained at 24 h after Ras-a1 transfection. The migration of VSMCs, determined using a Transwell assay, was significantly inhibited in Ras-a1 cells in a time-dependent manner. To investigate the potential mechanisms of transfected Ras-a1 on the migration and proliferation of VSMCs, the phosphorylation of MEK1/2, ERK1/2, and Akt was determined using western blot analysis, which showed that a marked downregulation was observed in the phosphorylation of MEK1/2, ERK1/2, and Akt following the delivery of Ras-a1. This result demonstrated that Ras-a1 inhibits the proliferation and migration of VSMCs by inhibiting the phosphorylation of Ras and interrupting signal transduction in the Ras-MEK1/2-ERK1/2 and phosphoinositide-3 kinase/Akt pathways. The novel Ras protein-targeted ssDNA aptamer selected may be applicable for the prevention of restenosis after PCI and CABG.

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“…Scratch Wound Assay. The scratch wound experiment was performed on normal rat kidney (NRK-52E) renal epithelial cells using a protocol modified from Torres et al (2011). The NRK-52E cells were cultured to 80 to 90% confluence.…”

Section: Methodsmentioning

confidence: 99%

G-ProteinβγSubunit Dimers Modulate Kidney Repair after Ischemia-Reperfusion Injury in Rats

White¹,

North²,

Haines³

et al. 2014

Mol Pharmacol

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Heterotrimeric G-proteins play a crucial role in the control of renal epithelial cell function during homeostasis and in response to injury. In this report, G-protein bg subunit (Gbg) dimer activity was evaluated during the process of tubular repair after renal ischemia-reperfusion injury (IRI) in male Sprague Dawley rats. Rats were treated with a small molecule inhibitor of Gbg activity, gallein (30 or 100 mg/kg), 1 hour after reperfusion and every 24 hours for 3 additional days. After IRI, renal dysfunction was prolonged after the high-dose gallein treatment in comparison with vehicle treatment during the 7-day recovery period. Renal tubular repair in the outer medulla 7 days after IRI was significantly (P , 0.001) attenuated after treatment with high-dose gallein (100 mg/kg) in comparison with low-dose gallein (30 mg/kg), or the vehicle and fluorescein control groups. Gallein treatment significantly reduced (P , 0.05) the number of proliferating cell nuclear antigen-positive tubular epithelial cells at 24 hours after the ischemia-reperfusion phase in vivo. In vitro application of gallein on normal rat kidney (NRK-52E) proximal tubule cells significantly reduced (P , 0.05) S-phase cell cycle entry compared with vehicletreated cells as determined by 59-bromo-29-deoxyuridine incorporation. Taken together, these data suggest that Gbg signaling contributes to the maintenance and repair of renal tubular epithelium and may be a novel therapeutic target for the development of drugs to treat acute kidney injury.

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Slingshot Isoform-Specific Regulation of Cofilin-Mediated Vascular Smooth Muscle Cell Migration and Neointima Formation

Cited by 31 publications

References 29 publications

Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

Ras ssDNA aptamer inhibits vascular smooth muscle cell proliferation and migration through MAPK and PI3K pathways

G-ProteinβγSubunit Dimers Modulate Kidney Repair after Ischemia-Reperfusion Injury in Rats

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