Activation of MRTF-A–dependent gene expression with a small molecule promotes myofibroblast differentiation and wound healing

Velasquez, Lissette S.; Sutherland, Lillian B.; Liu, Zhi; Grinnell, Frederick; Kamm, Kristine E.; Schneider, Jay W.; Olson, Eric N.; Small, Eric M.

doi:10.1073/pnas.1316764110

Cited by 124 publications

(105 citation statements)

References 44 publications

(59 reference statements)

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“…We have implemented a human cell model to interrogate functional effects of mutant SM α-actin by inducing ACTA2 expression from endogenous genes with myocardin-related transcription factor A (MRTF-A), a potent coactivator of smooth muscle contractile genes. This protocol effectively converts fibroblasts to myofibroblasts allowing a specific focus on SM α-actin-dependent properties (21). Our results are generally consistent with biochemical findings, namely expression of R258C SM α-actin inhibits, in an autosomal dominant manner, well-documented functional effects of WT SM α-actin on myofibroblast migration and contraction.…”

supporting

confidence: 89%

“…Stress signals that promote F-actin polymerization lead to nuclear accumulation of MRTF-A where the MRTF-A/ SRF complex binds to consensus elements within promoters of smooth muscle-specific target genes, primary of which is ACTA2 (34). In the present study we have bypassed extracellular signaling to induce ACTA2 by expression of MRTF-A, which was previously shown to be effective in promoting the myofibroblast phenotype (21,25). We found changes in protein expression that were consistent with those made by others (24,25).…”

Section: Discussionmentioning

confidence: 94%

“…A powerful regulator of this modulation is TGF-β1, which induces SM α-actin protein and mRNA expression in both growing and quiescent fibroblasts (26). TGF-β1 induction of the myofibroblast phenotype is dependent upon the serum response factor (SRF) transcriptional coactivator MRTF-A, as MRTF-A null or deficient fibroblasts fail to induce SM α-actin in response to TGF-β1 (21,24,25). MRTF-A is sequestered in the cytoplasm by interactions with G-actin (34).…”

Section: Discussionmentioning

confidence: 99%

“…Immortalized human dermal fibroblasts grown to ∼60% confluence were infected with Ad-MRTF-A or Ad-GFP at 80 multiplicity of infection (MOI) in DMEM containing 10% FBS for 24 h (21,24). Following an additional 24 h in DMEM containing 10% FBS, cells were collected and replated onto six-well plates.…”

Section: Methodsmentioning

confidence: 99%

“…To study effects of the ACTA2-R258C mutation on cytoskeletal functions in dermal fibroblasts, we induced SM α-actin expression by transfecting cells with replication-deficient adenovirus encoding MRTF-A (Ad-MRTF-A). MRTF-A is necessary and sufficient for myofibroblast differentiation in response to the established cytokine inducer, transforming growth factor β1 (TGF-β1) (21,24,25). Ad-MRTF-A was selected over TGF-β1 treatment owing to its shorter time (2 d versus 4-7 d) to achieve similar or greater (more than threefold versus more than twofold) increases in SM α-actin protein expression (21,(24)(25)(26).…”

Section: Immortalization Of Dermal Fibroblasts From Normal Individualmentioning

confidence: 99%

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

Liu

Chang

Grinnell

et al. 2017

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

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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 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 α-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 α-actin. Telomeraseimmortalized lines of control and R258C human dermal fibroblasts were established and SM α-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 α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-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 α-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.thoracic aortic aneurysms | ACTA2 mutation | vascular disease | cell migration | human fibroblasts

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supporting

confidence: 89%

Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Immortalization Of Dermal Fibroblasts From Normal Individualmentioning

confidence: 99%

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

Liu

Chang

Grinnell

et al. 2017

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

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Engineering Platelet‐Rich Plasma Based Dual‐Network Hydrogel as a Bioactive Wound Dressing with Potential Clinical Translational Value

Zhang

Yao

Zhao

et al. 2020

Adv Funct Materials

134

104

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Delayed wound healing has a profound impact on patients, healthcare, and society. Platelet‐rich plasma (PRP) gel, as a preparation for regenerative medicine, has proven to be of clinical value in various wound treatments. Nevertheless, its weak mechanical properties and consequent burst release effect have restricted its application and efficacy. Here, an engineered PRP dual‐network hydrogel (named DN gel) based on sodium alginate is constructed through a simple “one‐step” activation process. Its improved gelling property and sustained release of growth factors may be beneficial for clinical use. Evaluations in rats indicate that DN gel promote wound healing in terms of rapid re‐epithelialization, up‐regulated growth factor levels and early transitions in the wound healing and angiogenesis stages. As a proof of concept, DN gel also exhibits superior healing efficiency in a porcine wound model. These results demonstrate the great potential of transforming this hydrogel into the next generation of PRP‐based bioactive wound dressing.

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Stiffness Mediated‐Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels

Tan,

Wang,

Chin

et al. 2024

Adv Healthcare Materials

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In obstructive airway diseases such as asthma and chronic obstructive pulmonary disease, the extracellular matrix (ECM) protein amount and composition of the airway smooth muscle (ASM) is often remodelled, likely altering tissue stiffness. The underlying mechanism of how human ASM cell (hASMC) mechanosenses the aberrant microenvironment is not well understood. Physiological stiffnesses of the ASM layer were measured by uniaxial compression tester using porcine ASM layers under 0, 5 and 10% longitudinal stretch above in situ length. Linear stiffness gradient hydrogels (2–30 kPa range) were fabricated and functionalized with ECM proteins, collagen I (ColI), fibronectin (Fn) and laminin (Ln), to recapitulate the above‐measured range of stiffnesses with an Atomic Force Microscope. Overall, hASMC mechanosensation exhibited a clear correlation with the underlying hydrogel stiffness with some specificity to ECM protein type. Cell size, nuclear size and contractile marker alpha‐smooth muscle actin (αSMA) expression showed a strong correlation to substrate stiffness. Mechanosensation, assessed by Lamin‐A intensity and YAP nuclear translocalization, exhibited stiffness‐mediated behaviour only on ColI and Fn‐coated hydrogels. Inhibition studies using blebbistatin or Y27632 attenuated most mechanotransduction‐derived cell morphological responses, αSMA and Lamin‐A expression and YAP nuclear translocalization (blebbistatin only). This study highlights the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.This article is protected by copyright. All rights reserved

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Activation of MRTF-A–dependent gene expression with a small molecule promotes myofibroblast differentiation and wound healing

Cited by 124 publications

References 44 publications

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

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

Engineering Platelet‐Rich Plasma Based Dual‐Network Hydrogel as a Bioactive Wound Dressing with Potential Clinical Translational Value

Stiffness Mediated‐Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels

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