TGFβ-Induced Contraction Is Not Promoted by Fibronectin-Fibronectin Receptor Interaction, or αSMA Expression

Dawes, Lucy J.; Eldred, Julie A.; Anderson, Ian K.; Sleeman, Matthew A.; Reddan, John R.; Duncan, G.; Wormstone, I. Michael

doi:10.1167/iovs.07-0586

Cited by 32 publications

(49 citation statements)

References 43 publications

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“…1B). This decrease in patch area was not attributable to losses in cell population by apoptosis or cell detachment from the culture dish (data not shown), but by matrix configuration and distribution changes in association with cells as previously determined by Dawes et al 28 using periodic acid Schiff (PAS) or aniline blue collagen staining. The addition of the broad-spectrum MMP inhibitor GM6001 (25 lM) resulted in no changes in patch area when compared with untreated control (Figs.…”

Section: Broad-spectrum Mmp Inhibition By Gm6001 Prevents Tgfb2-inducsupporting

confidence: 78%

“…The assay previously described by Dawes et al 28 was employed to assess matrix contraction. FHL-124 cells were seeded at four distinct sites on a 35-mm tissue culture dish (Corning Incorporated Life Sciences, Corning, NY) at a density of 5000 cells/25 lL Eagle's minimum essential medium (EMEM) supplemented with 5% fetal calf serum (FCS, Gibco, Paisley, Scotland, UK) and 0.05 mg/mL gentamicin and maintained in a 358C, 5% CO 2 incubator until confluent patches spanning 5 mm developed.…”

Section: Patch Contraction Assaymentioning

confidence: 99%

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MMP2 Activity is Critical for TGFβ2-Induced Matrix Contraction—Implications for Fibrosis

Eldred

Hodgkinson

Dawes

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. The fibrotic lens disorder posterior capsule opacification (PCO) develops in millions of patients following cataract surgery. PCO characteristics are extensive extracellular matrix (ECM) production and contraction of the posterior lens capsule, resulting in light-scattering ECM modification (wrinkling). The pro-fibrotic cytokine transforming growth factor beta (TGFb) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFb-mediated PCO formation. METHODS.The human lens epithelial cell-line FHL-124 and human capsular bag models were employed. Gene expression of MMP family members was determined by oligonucleotide microarray and quantitative real-time RT-PCR. MMP2 and MT1-MMP protein levels were analyzed by ELISA and Western blotting, respectively. Matrix contraction was determined using an FHL-124 patch contraction assay; at end-point, cells were stained with Coomassie brilliant blue and area was determined using image analysis software. Cell coverage and wrinkle formation on the posterior capsule were also assessed using human capsular bag models.RESULTS. Active TGFb2 (10 ng/mL) increased gene and protein levels of MMP2 and MT1-MMP and induced matrix contraction in FHL-124 cells. Specific siRNA inhibition of MT1-MMP did not suppress TGFb2-induced matrix contraction. Active TGFb2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 lM), MMP2 siRNA, and MMP2 neutralizing antibody (4 lg/mL). TGFb2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 lg/mL).CONCLUSIONS. MMP2 plays a critical role in TGFb2-mediated matrix contraction, which appears to be independent of MT1-MMP. MMP2 inhibition provides a novel strategy for the treatment of PCO and potentially other fibrotic disorders. (Invest Ophthalmol Vis Sci. 2012;53:4085-4098)

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Section: Broad-spectrum Mmp Inhibition By Gm6001 Prevents Tgfb2-inducsupporting

confidence: 78%

Section: Patch Contraction Assaymentioning

confidence: 99%

MMP2 Activity is Critical for TGFβ2-Induced Matrix Contraction—Implications for Fibrosis

Eldred

Hodgkinson

Dawes

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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“…To permit thorough investigations of the signalling pathways driving fibrotic events, human lens cell lines have been employed. Using this biological tool, specific assays have been developed to assess matrix production, transdifferentiation and matrix contraction (figures 6 and 7) [5,12,30]. Inhibition studies could then be employed to tease out the pathways mediating TGFb-induced fibrosis.…”

Section: Tgfb: the Orchestrator Of Fibrosismentioning

confidence: 99%

“…Moreover, it has been proposed that a5b1 integrin and fibronectin form a putative contractile apparatus with aSMA [132,133]. A recent study has investigated the putative role of fibronectin/fibronectin receptor interaction in relation to TGFb-induced matrix contraction by human lens epithelial cells [5]; application of an RGDS peptide to block the RGD binding site of a5 integrin revealed that the fibronectin/fibronectin receptor interaction was not required to promote matrix contraction by TGFb [5]. In addition, Marcantonio et al [134] demonstrated that a5b1 integrin expression was altered in FHL 124 cells following TGFb exposure, such that a diffuse pattern across the cell was observed, with membrane bound a5b1 integrin having no association with actin filaments.…”

Section: Extracellular Matrix/cell Communication-integrinsmentioning

confidence: 99%

“…The expression of aVb5 integrin is upregulated in scleroderma fibroblasts [142] and interstitial renal fibroblasts [136]. With respect to the lens, TGFb treatment of lens epithelial cells promotes aVb5 expression [30] and is a potent inducer of transdifferentiation of lens epithelial cells to myofibroblasts [5,17]. These investigations have led to speculation over the bidirectional role of aVb5 integrin in the formation of lens fibrosis [143], which could occur through (i) mediating transdifferentiation induced by increasing levels of active TGFb that result from trauma to the lens or (ii) mediating signals from myofibroblasts back to the ECM that activate matrix-associated TGFb.…”

Section: Extracellular Matrix/cell Communication-integrinsmentioning

confidence: 99%

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The lens as a model for fibrotic disease

Eldred

Dawes

Wormstone

2011

Phil. Trans. R. Soc. B

103

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Fibrosis affects multiple organs and is associated with hyperproliferation, cell transdifferentiation, matrix modification and contraction. It is therefore essential to discover the key drivers of fibrotic events, which in turn will facilitate the development of appropriate therapeutic strategies. The lens is an elegant experimental model to study the processes that give rise to fibrosis. The molecular and cellular organization of the lens is well defined and consequently modifications associated with fibrosis can be clearly assessed. Moreover, the avascular and non-innervated properties of the lens allow effective in vitro studies to be employed that complement in vivo systems and relate to clinical data. Using the lens as a model for fibrosis has direct relevance to millions affected by lens disorders, but also serves as a valuable experimental tool to understand fibrosis per se.

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Endothelial cells enhance adipose mesenchymal stromal cell‐mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis

Monsuur

Broek

Koolwijk³

et al. 2018

Journal Cellular Physiology

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Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross‐talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full‐thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue‐derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF‐β pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC‐mediated matrix contraction via regulation of fibrosis‐related proteins.

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TGFβ-Induced Contraction Is Not Promoted by Fibronectin-Fibronectin Receptor Interaction, or αSMA Expression

Abstract: A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGFbeta-induced matrix contraction.

Cited by 32 publications

References 43 publications

MMP2 Activity is Critical for TGFβ2-Induced Matrix Contraction—Implications for Fibrosis

MMP2 Activity is Critical for TGFβ2-Induced Matrix Contraction—Implications for Fibrosis

The lens as a model for fibrotic disease

Endothelial cells enhance adipose mesenchymal stromal cell‐mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis

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