Epithelial basement membrane proteins perlecan and nidogen-2 are up-regulated in stromal cells after epithelial injury in human corneas

Abstract

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-min epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins-important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 were expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

Keywords: Apoptosis; Cornea; Epithelial basement membrane; Haze; Keratocyte; Myofibroblast; Nidogen-2; Perlecan; Stroma; Wound healing.

Figure 1

Representative immunohistochemistry for perlecan in human corneas. Column A. Perlecan protein was noted in the epithelium and epithelial basement membrane (arrows) and at low levels in keratocytes (arrowheads) in unwounded control corneas. Column B. Negative control in unwounded cornea with pre-absorption with blocking peptide shows specific blocking of antigen-antibody interaction. Column C. In all corneas at 30 minutes after epithelial scrape, strong perlecan immunoreactivity was noted in keratocytes (arrowheads), as shown in this representative section from one cornea, including in keratocytes in the early stages of apoptosis that label with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (not shown). Staining near the stromal surface (arrows) could represent BM that was not completely removed by scrape or expression in keratocytes undergoing apoptosis—which cannot be distinguished at this level of magnification. Column D. Pre-absorption with blocking peptide shows specific blocking of antigen-antibody interaction in the negative control cornea after epithelial scrape. Blue is DAPI staining of cell nuclei. e is epithelium. Magnification 400x.

Figure 2

Representative immunohistochemistry for nidogen-2 in human corneas. Column A. Nidogen-2 protein was noted in the epithelial basement membrane (arrows) and epithelium (e), as well as at low levels in keratocytes (arrowheads) in unwounded control corneas. Column B. Negative control in unwounded cornea with isotypic control antibody showed no non-specific immunostaining for nidogen-2. Column C. In a cornea at 30 minutes after epithelial scrape, nidogen-2 protein was up-regulated in stromal keratocytes (arrowheads), including anterior stromal keratocytes in the early stages of apoptosis detected with the TUNEL assay (not shown). Staining near the stromal surface (arrows) could represent BM that was not completely removed by scrape or expression in keratocytes undergoing apoptosis. Column D. Isotypic control shows specific antigen-antibody interaction in the control cornea after epithelial scrape. Blue is DAPI staining of cell nuclei. e is epithelium. Magnification 400x.