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Smad3 deficiency alters key structural elements of the extracellular matrix and mechanotransduction of wound closure.

ABSTRACT: The loss of TGFbeta or its downstream mediator, Smad3, key players in tissue repair, accelerates closure of incisional wounds in mice. In contrast, we now report that excisional ear wounds in mice lacking Smad3 enlarge compared with wild-type controls resulting from changes in extracellular matrix molecules, which alter the mechanotransduction properties of these wounds. Specifically, levels of elastin and glycosoaminoglycans are increased, collagen fibers are more compactly organized, and matrix modulators like integrins, TGFbeta1, and matrix metalloproteinases (MMPs) are altered both basally and after wounding in Smad3 knockout mice. Mechanical testing of dorsal skin correlates these changes in matrix composition with functional parameters, specifically an increased elastic modulus, suggesting an imbalance of tissue forces. We propose that the altered mechanical elastic properties translate into a persistent retractile force that is opposed by decreased wound contractile forces contributing to the enlarging ear wound in Smad3 knockout mice. These studies highlight a previously undescribed role for Smad3 in the mechanotransduction of matrix unsupported ear wound closure.

SUBMITTER: Arany PR

PROVIDER: S-EPMC1474013 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Smad3 deficiency alters key structural elements of the extracellular matrix and mechanotransduction of wound closure.

Arany Praveen R PR Flanders Kathleen C KC Kobayashi Tetsu T Kuo Catherine K CK Stuelten Christina C Desai Kartiki V KV Tuan Rocky R Rennard Stephen I SI Roberts Anita B AB

Proceedings of the National Academy of Sciences of the United States of America 20060605 24

The loss of TGFbeta or its downstream mediator, Smad3, key players in tissue repair, accelerates closure of incisional wounds in mice. In contrast, we now report that excisional ear wounds in mice lacking Smad3 enlarge compared with wild-type controls resulting from changes in extracellular matrix molecules, which alter the mechanotransduction properties of these wounds. Specifically, levels of elastin and glycosoaminoglycans are increased, collagen fibers are more compactly organized, and matri ...[more]

PMID: 16754864

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Project description:Extracellular matrix (ECM) is comprised of different types of proteins, which change in composition and ratios during morphogenesis and disease progression. ECM proteins provide cell adhesion and impart mechanical cues to the cells. Increasing substrate stiffness has been shown to induce Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet these mechanistic studies used fibronectin only as the biochemical cue. How varying the types of ECM modulates mechanotransduction of stem cells remains largely unknown. Using polyacrylamide hydrogels with tunable stiffness as substrates, here we conjugated four major ECM proteins commonly used for cell adhesion: fibronectin, collagen I, collagen IV and laminin, and assessed the effects of varying ECM type and density on YAP translocation in human mesenchymal stem cells (hMSCs). For all four ECM types, increasing ECM ligand density alone can induce YAP nuclear translocation without changing substrate stiffness. The ligand threshold for such biochemical ligand-induced YAP translocation differs across ECM types. While stiffness-dependent YAP translocation can be induced by all four ECM types, each ECM requires a different optimized ligand density for this to occur. Using antibody blocking, we further identified integrin subunits specifically involved in mechanotransduction of different ECM types. Finally, we demonstrated that altering ECM type further modulates hMSC osteogenesis without changing substrate stiffness. These findings highlight the important role of ECM type in modulating mechanotransduction and differentiation of stem cells, and call for future mechanistic studies to further elucidate the role of changes in ECM compositions in mediating mechanotransduction during morphogenesis and disease progression. STATEMENT OF SIGNIFICANCE: Our study addresses a critical gap of knowledge in mechanobiology. Increasing substrate stiffness has been shown to induce nuclear YAP translocation, yet only on fibronectin-coated substrates. However, extracellular matrix (ECM) is comprised of different protein types. How varying the type of ECM modulates stem cell mechanotransduction remains largely unknown. We here reveal that the choice of ECM type can directly modulate stem cell mechanotransduction, filling this critical gap. This work has broad impacts in mechanobiology and biomaterials, as it provides the first evidence that varying ECM type can impact YAP translocation independent of substrate stiffness, opening doors for a more rational biomaterials design tuning ECM properties to control cell fate for promoting normal development and for preventing disease progression.

Project description:Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. Impact statement Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

Project description:BackgroundProper inflammation resolution is critical for cutaneous wound healing and disordered inflammation resolution results in chronic nonhealing wounds. However, the cellular and molecular mechanisms for resolution of inflammation during skin wound healing are not well understood. MicroRNA-34a is regarded as one tumor suppressor with complexed immune regulatory effects, yet its role during skin wound repair is still unclear.MethodsCircular full thickness excisional wounds were made on the dorsal skin of C57 mice and miR-34a expression pattern was examined by real time RT-PCR and in situ hybridization. The wound healing rates and histologic morphometric analysis were quantified and compared between wounds treated with antagomir-34a and autologous control antagomir-NC wounds, as well as wounds between miR-34a knockout (KO) and wild type (WT) mice. Immunohistochemistry (IHC) for both MPO and F4/80 were performed to examine the infiltrative neutrophils and macrophages in wounds from miR-34a KO and WT mice. Cytokines including IL-1β, IL-6, TNF-α and IL-10, were detected and analyzed by real time RT-PCR during wound healing. IHC for IL-6 and p-STAT3 were quantified, and WB for p-STAT3 and IL-6R were examined in wounds of miR-34a KO and WT mice.ResultsWe found miR-34a was significantly downregulated in the inflammatory phase and back to normal levels in the proliferative phase. Both topical knockdown wounds miR-34a levels by antagomir gel and systematic knockout miR-34a using KO mice resulted in impaired wound healing with delayed re-epithelialization and augmented inflammation. IHC results indicated that there were more residual infiltrative inflammatory cells in the proliferative phase. Moreover, over-activated IL-6/STAT3 signal pathway was identified in the wounds of miR-34a KO mice.ConclusionsOur findings reveal that miR-34a deficiency augments skin wound inflammation response and leads to impaired wound healing, which suggest that targeted inhibition of miR-34a for tissue repair/regeneration should be with serious consideration.

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Smad3 deficiency alters key structural elements of the extracellular matrix and mechanotransduction of wound closure.

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Smad3 deficiency alters key structural elements of the extracellular matrix and mechanotransduction of wound closure.

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