Wang H, Haeger SM, Kloxin AM et al.Redirecting valvular myofibroblasts into dormant fibroblasts through light-mediated reduction in substrate modulus. PLoS One 7:e39969

Brigham and Women's Hospital, Harvard Medical School, United States of America
PLoS ONE (Impact Factor: 3.23). 07/2012; 7(7):e39969. DOI: 10.1371/journal.pone.0039969
Source: PubMed


Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young's modulus of the substrate was reduced from ~32 kPa, mimicking pre-calcified diseased tissue, to ~7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in α-smooth muscle actin (α-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including α-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-β1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.

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Available from: Leslie A Leinwand
    • "Thus there is an urgent need to construct an in vitro cardiac fibrosis model for successful discovery of therapy. Berry et al [38] reported that a significant difference of myocardium compliance existed between fibrosis area (∼10 kPa) and surrounding intact area post-infarction (∼40 kPa), and recent evidences [39] [40] [41] suggested the transition between fibroblasts and myofibroblasts could be orchestrated biophysically by varying the stiffness of cell matrices. Therefore, we anticipate that the construction of an in vitro cardiac fibrosis model could be realized by regulating mechanotransduction of cardiac fibroblasts via patterning the stiffness of extracellular matrix. "
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    ABSTRACT: Cardiac fibrosis greatly impairs normal heart function post infarction and there is no effective anti-fibrotic drug developed at present. The current therapies for cardiac infarction mainly take effect by eliminating occlusion in coronary artery by thrombolysis drugs, vascular stent grafting or heart bypass operation, which are capable to provide sufficient blood flow for intact myocardium yet showed subtle efficacy in ameliorating fibrosis condition. The advances of in vitro cell/tissue models open new avenues for drug assessment due to the low cost, good controllability and availability as well as the convenience for operation as compared to the animal models. To our knowledge, no proper biomimetic in vitro cardiac fibrosis model has been reported yet. Here we engineered an in vitro cardiac fibrosis model using heart-derived fibroblasts, and the fibrogenesis was recapitulated by patterning the substrate rigidity which mimicked the mechanical heterogeneity of myocardium post-infarction. Various biomarkers for cardiac fibrosis were assayed to validate the biomimicry of the engineered platform. Subsequent addition of Rho-associated protein kinase (ROCK) pathway inhibitor reduced the ratio of myofibroblasts, indicating the feasibility of applying this platform in screening anti-fibrosis drugs.
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    • "We show that myofibroblastic and fibroblastic phenotypes obtained from ADSCs are not terminal and can be modulated reversibly and the contrasting interplay observed between bFGF and TGF-β in this modulation may have relevance to in vivo tissue repair. Studies have shown that in other cell types α-SMA expression can be reversed by changing cell density [39], [40], by manipulating tensional forces [41], [42], [43], by changing stiffness [44], or by growth factors [7], [8]. Here, bFGF completely reversed the TGF-β-differentiated myofibroblastic phenotype in ADSCs. "
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    ABSTRACT: Unregulated activity of myofibroblasts, highly contractile cells that deposit abundant extracellular matrix (ECM), leads to fibrosis. To study the modulation of myofibroblast activity, we used human adipose-derived mesenchymal stem cells (ADSCs), which have much potential in regenerative medicine. We found that ADSCs treated with TGF-β developed a myofibroblastic phenotype with increases in α-smooth muscle actin (α-SMA), a myofibroblast marker, and ECM proteins type I collagen and fibronectin. In contrast, treatment with bFGF had the opposite effect. bFGF-differentiated ADSCs showed marked down-regulation of α-SMA expression, collagen I, and fibronectin, and loss of focal adhesions and stress fibers. Functionally, bFGF-differentiated ADSCs were significantly more migratory, which correlated with up-regulation of tenascin-C, an anti-adhesive ECM protein, and vimentin, a pro-migratory cytoskeletal protein. On the other hand, TGF-β-differentiated ADSCs were significantly more contractile than bFGF-differentiated cells. Interestingly, cells completely reversed their morphologies, marker expression, signaling pathways, and contractility versus migratory profiles when switched from culture with one growth factor to the other, demonstrating that the myofibroblast differentiation process is not terminal. Cell differentiation was associated with activation of Smad2 downstream of TGF-β and of ERK/MAP kinase downstream of bFGF. Reversibility of the TGF-β-induced myofibroblastic phenotype depends, in part, on bFGF-induced ERK/MAP kinase signaling. These findings show that ADSC differentiation into myofibroblasts and re-differentiation into fibroblast-like cells can be manipulated with growth factors, which may have implications in the development of novel therapeutic strategies to reduce the risk of fibrosis.
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    • "However, the progeny of the ABCG2+ cells still had higher calcification capacities than the ABCG2- cells, suggesting that progeny of the progenitor-like cells may still maintain different properties than the rest of the population. As progenitor/stem cells require appropriate chemical cues and physical cues for preserving their innate and primitive phenotypes [46,47], in the future, sorted ABCG2+ cells might be introduced to extracellular matrix-coated surfaces of defined elasticities, closer to valve tissue in stiffness, to enhance cell survival and maintain a more native-like phenotype [48–50]. Our study here presents an early effort in dissecting the complexity of cells residing in porcine aortic valves and provides evidence for heterogeneity not only in cellular composition, but also in molecular functions. "
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    ABSTRACT: Valvular interstitial cells (VICs) are the main population of cells found in cardiac valves. These resident fibroblastic cells play important roles in maintaining proper valve function, and their dysregulation has been linked to disease progression in humans. Despite the critical functions of VICs, their cellular composition is still not well defined for humans and other mammals. Given the limited availability of healthy human valves and the similarity in valve structure and function between humans and pigs, we characterized porcine VICs (pVICs) based on expression of cell surface proteins and sorted a specific subpopulation of pVICs to study its functions. We found that small percentages of pVICs express the progenitor cell markers ABCG2 (~5%), NG2 (~5%) or SSEA-4 (~7%), whereas another subpopulation (~5%) expresses OB-CDH, a type of cadherin expressed by myofibroblasts or osteo-progenitors. pVICs isolated from either aortic or pulmonary valves express most of these protein markers at similar levels. Interestingly, OB-CDH, NG2 and SSEA-4 all label distinct valvular subpopulations relative to each other; however, NG2 and ABCG2 are co-expressed in the same cells. ABCG2(+) cells were further characterized and found to deposit more calcified matrix than ABCG2(-) cells upon osteogenic induction, suggesting that they may be involved in the development of osteogenic VICs during valve pathology. Cell profiling based on flow cytometry and functional studies with sorted primary cells provide not only new and quantitative information about the cellular composition of porcine cardiac valves, but also contribute to our understanding of how a subpopulation of valvular cells (ABCG2(+) cells) may participate in tissue repair and disease progression.
    Full-text · Article · Jul 2013 · PLoS ONE
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