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Modulation of human corneal stromal cell differentiation by hepatocyte growth factor and substratum compliance.

ABSTRACT: Corneal wound healing is a complex process that consists of cellular integration of multiple soluble biochemical cues and cellular responses to biophysical attributes associated with the matrix of the wound space. Upon corneal stromal wounding, the transformation of corneal fibroblasts to myofibroblasts is promoted by transforming growth factor-? (TGF?). This process is critical for wound healing; however, excessive persistence of myofibroblasts in the wound space has been associated with corneal fibrosis resulting in severe vision loss. The objective of this study was to determine the effect of hepatocyte growth factor (HGF), which can modulate TGF? signaling, on corneal myofibroblast transformation by analyzing the expression of ?-smooth muscle actin (?SMA) as a marker of myofibroblast phenotype particularly as it relates to biomechanical cues. Human corneal fibroblasts were cultured on tissue culture plastic (>1?GPa) or hydrogel substrates mimicking human normal or wounded corneal stiffness (25 and 75?kPa) in media containing TGF?1?±?HGF. The expression of ?SMA was analyzed by quantitative PCR, Western blot and immunocytochemistry. Cellular stiffness, which is correlated with cellular phenotype, was measured by atomic force microscopy (AFM). In primary human corneal fibroblasts, the mRNA expression of ?SMA showed a clear dose response to TGF?1. The expression was significantly suppressed when cells were incubated with 20?ng/ml HGF in the presence of 2?ng/ml of TGF?1. The protein expression of ?SMA induced by 5?ng/ml TGF?1 was also decreased by 20?ng/ml of HGF. Cells cultured on hydrogels mimicking human normal (25?kPa) and fibrotic (75?kPa) cornea also showed an inhibitory effect of HGF on ?SMA expression in the presence or absence of TGF?1. Cellular stiffness was decreased by HGF in the presence of TGF?1 as measured by AFM. In this study, we have demonstrated that HGF can suppress the myofibroblast phenotype promoted by TGF?1 in human corneal stromal cells. These data suggest that HGF holds the potential as a therapeutic agent to improve wound healing outcomes by minimizing corneal fibrosis.

SUBMITTER: Miyagi H

PROVIDER: S-EPMC6339683 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Modulation of human corneal stromal cell differentiation by hepatocyte growth factor and substratum compliance.

Miyagi Hidetaka H Jalilian Iman I Murphy Christopher J CJ Thomasy Sara M SM

Experimental eye research 20180905

Corneal wound healing is a complex process that consists of cellular integration of multiple soluble biochemical cues and cellular responses to biophysical attributes associated with the matrix of the wound space. Upon corneal stromal wounding, the transformation of corneal fibroblasts to myofibroblasts is promoted by transforming growth factor-β (TGFβ). This process is critical for wound healing; however, excessive persistence of myofibroblasts in the wound space has been associated with cornea ...[more]

PMID: 30193807

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Project description:Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGF?)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGF?-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation.Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGF?1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGF?-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (?-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis.Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGF?1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in ?-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGF?1 showed the expected low expression of ?-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGF?-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGF?-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein.Conclusions: Id3 regulates TGF?-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.

Project description:: The incidence of idiopathic pulmonary fibrosis is on the rise and existing treatments have failed to halt or reverse disease progression. Mesenchymal stromal cells (MSCs) have potent cytoprotective effects, can promote tissue repair, and have demonstrated efficacy in a range of fibrotic lung diseases; however, the exact mechanisms of action remain to be elucidated. Chemical antagonists and short hairpin RNA knockdown were used to identify the mechanisms of action used by MSCs in promoting wound healing, proliferation, and inhibiting apoptosis. Using the bleomycin induced fibrosis model, the protective effects of early or late MSC administration were examined. The role for hepatocyte growth factor (HGF) in MSC protection against bleomycin lung injury was examined using HGF knockdown MSC. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling assay was performed on ex vivo lung sections to examine the effects of MSC on apoptosis. MSC conditioned media (CM) enhanced wound closure and inhibited apoptosis of pulmonary cells in vitro. HGF was required for MSC CM enhancement of epithelial cell proliferation and inhibition of apoptosis. In contrast, MSC required COX-2 for CM to inhibit fibroblast proliferation. In a murine model, early administration of MSC protected against bleomycin induced lung fibrosis and correlated with reduced levels of the proinflammatory cytokine interleukin-1?, reduced levels of apoptosis, and significantly increased levels of HGF. These protective effects were in part mediated by MSC derived HGF as HGF knockdown MSC were unable to protect against fibrosis in vivo. These findings delineate the mechanisms of MSC protection in a preclinical model of fibrotic lung disease.SignificanceThe mechanisms used by mesenchymal stromal cells (MSCs) in mediating protective effects in chronic models of lung disease are not understood and remain to be elucidated. These findings from in vitro studies highlight an important role for the MSC-derived soluble factors hepatocyte growth factor (HGF) and prostaglandin E2 in promoting wound healing and inhibiting apoptosis. Furthermore, this study translates these findings demonstrating an important role for HGF in the protective effects mediated by MSC in vivo in the bleomycin model. These findings support a targeted approach to enhancing MSC therapy for fibrotic disease and highlight the importance of timing of MSC therapy.

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Modulation of human corneal stromal cell differentiation by hepatocyte growth factor and substratum compliance.

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Modulation of human corneal stromal cell differentiation by hepatocyte growth factor and substratum compliance.

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