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-β (TGFβ). 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 TGFβ signaling, on corneal myofibroblast transformation by analyzing the expression of α-smooth muscle actin (αSMA) 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 TGFβ1 ± HGF. The expression of αSMA 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 αSMA showed a clear dose response to TGFβ1. The expression was significantly suppressed when cells were incubated with 20 ng/ml HGF in the presence of 2 ng/ml of TGFβ1. The protein expression of αSMA induced by 5 ng/ml TGFβ1 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 αSMA expression in the presence or absence of TGFβ1. Cellular stiffness was decreased by HGF in the presence of TGFβ1 as measured by AFM. In this study, we have demonstrated that HGF can suppress the myofibroblast phenotype promoted by TGFβ1 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.

A Systematic Review on Cornea Epithelial-Stromal Homeostasis

“…HGF has been proven to counteract aforementioned TGF-β effects to achieve anti-fibrotic function in different organs such as lung [32], liver [33] and heart [34]. The addition of TGF-β1 to HCFs for 24 h was shown to increase mRNA expression of α-SMA [35], proving that TGF-β1 induces transformation of HCFs to myofibroblasts. In addition, HGF at 5–50 ng/mL could suppress TGF-β1 induced α-SMA expression, with 20 ng/mL HGF having the maximal inhibitory effect on keratocyte differentiation into myofibroblasts.…”

“…To solve this problem, in the long-term, novel topical drugs, gene or cell therapies can be developed by targeting different proteins, genes or cells that can reduce fibrosis or repair epithelial damage. A particular study highlighted that HGF was shown to have the potential ability to reverse the differentiation of human myofibroblasts back into keratocytes [35], providing hope for patients suffering from scarring to regain visual clarity through pharmaceutical means. The effects of other aforementioned proteins on the reversed differentiation of myofibroblasts could also be investigated, and further studies on post-treatment of keratocytes after injury are warranted.…”

The Efficacy of Topical HGF on Corneal Fibrosis and Epithelial Healing after Scar-Producing PRK Injury in Rabbits

“…Thus major differences in the injuries to the corneas and HGF application could explain the differences in results. Miyagi et al 23 also demonstrated that HGF can suppress the myofibroblast phenotype promoted by TGF-β1 in human corneal stromal cells in vitro, and suggested HGF had the potential for use as a therapeutic agent to decrease corneal fibrosis after corneal injury.…”

“… 20 , 21 Also, Mittal et al 13 found myofibroblasts present in the superficial corneal stroma after the lamellar keratectomy involving the limbus after only a few days and ended their study at 7 days, whereas prior studies of central corneal injuries in C57Bl/6 mice found that, although small numbers of α-SMA-positive cells may be seen in the cornea the first few days after injury, many more of these myofibroblasts developed at 2 to 4 weeks after injury. 22 Mittal et al 13 also only applied topical HGF two times per day. Thus major differences in the injuries to the corneas and HGF application could explain the differences in results.…”

Exosomal miR-19a from adipose-derived stem cells suppresses differentiation of corneal keratocytes into myofibroblasts

“…Previous investigations show that corneal keratocytes incubated with 10% FBS differentiate into myofibroblasts [32]. Moreover, ADSCs can be induced to differentiate into functional keratocytes under specific growth conditions [1,33].…”