A selective inhibitor of the Rho kinase pathway, Y-27632, and its influence on wound healing in the corneal stroma

作者：Yamamoto, Mayumi; Quantock, Andrew J.; Young, Robert D.; Okumura, Naoki; Ueno, Morio; Sakamoto, Yuji; Kinoshita, Shigeru; Koizumi, Noriko^*

来源：Molecular Vision, 2012, 18(178-80): 1727-1739.

• GROWTH-FACTOR-BETA
• SMOOTH MUSCLE ACTIN
• TGF-BETA
• TRANSFORMING GROWTH-FACTOR-BETA-1
• MYOFIBROBLAST DIFFERENTIATION
• MATRIX CONTRACTION
• REFRACTIVE SURGERY
• GRANULATION TISSUE
• RADIAL KERATOTOMY
• ROCK INHIBITOR

Purpose: Our study examined the effect of a selective Rho kinase inhibitor, Y-27632, on corneal wound healing and potential stromal scarring after superficial keratectomy.
Methods: Rabbit keratocytes were induced into myofibroblasts by transforming growth factor beta 1 (TGF beta 1) either with or without Y-27632. Then alpha-smooth muscle actin (alpha-SMA) was examined by immunohistochemistry and western blotting, and the contractility of the seeded collagen gels was measured. Y-27632 eye drops (or vehicle only) were administered to eyes after a superficial keratectomy, and the tissue was examined by immunohistochemistry for alpha-SMA, collagen types I, II, and III, and keratan sulfate. Electron microscopy was conducted with and without histochemical contrasting of sulfated proteoglycans.
Results: Spindle-like cells in culture constituted 99.5 +/- 1.1% with TGF beta 1 stimulation, but 3.5 +/- 1.0% after TGF beta 1 and Y-27632 treatment (p<0.01, n=6). alpha-SMA was seen in 4% of TGF beta 1-treated cells, but in only 0.3% of cells with Y-27632 added (p<0.01, n=6), which was confirmed by western blotting. Y-27632 also inhibited the TGF beta 1-induced contraction of seeded collagen gels. After superficial keratectomies, collagen type I and keratan sulfate were unchanged by Y-27632 application. Collagen type II was not detected in Y-27632 or vehicle-only corneas. With Y-27632 treatment, alpha-SMA expression increased and the collagen type III signal became in the weaker subepithelial area. Interestingly, bundles of aligned and uniformly spaced collagen fibrils were more prevalent in keratocytes in Y-27632-treated corneas, which is reminiscent of fibripositor-like structures that have been proposed as a mechanism of matrix deposition in embryonic connective tissues.
Conclusions: Y-27632 inhibits keratocyte-to-myofibroblast transition, and its topical application after a superficial lamellar keratectomy elicits an altered wound healing response, with evidence of an embryonic-type deposition of collagen fibrils.

• 出版日期2012-6-27