Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture

Abstract

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.

Doxycycline Hydrogels as a Potential Therapy for Ocular Vesicant Injury

“…Using these facilities makes it difficult to do extensive drug screening because animal studies using the vesicant are costly. Because air-liquid interface corneal organ cultures have been used for a variety of experimental purposes, 13,16,17,[27][28][29][30][31] we adapted an organ culture model system and tested whether it could serve as a preliminary screening method for potential SM therapies. SM exposures cause variable injury according to the exposure time, concentration of the vesicant, and the exposed individual's susceptibility.…”

Corneal Sensitivity Is Reduced and Relates to the Severity of Neuropathy in Patients With Diabetes

“…In addition to serving a protective function, corneal nerves regulate corneal epithelial integrity, proliferation, and wound healing (3). In diabetic patients, corneal sensitivity is reduced (4), due to a loss of corneal nerve fibers (5), which leads to corneal keratopathy (6,7) and a susceptibility to injury, with recurrent erosions and ulcers (8). Thus, these changes are analogous to the diabetic foot, but because the cornea is not exposed to high pressures, ulceration infrequently occurs.…”

Maintenance of the Keratocyte Phenotype during Cell Proliferation Stimulated by Insulin

“…Patients with type I and II diabetes mellitus have abnormal changes that occur at the anterior segment of the eye (33). Patients with diabetes show a thickening of the corneal stroma (33,34) due to accumulation of water and elevated stromal mRNA and protein levels of MMP3 and MMP10 (stromelysin-1 and 2, respectively) (34 -36). Fetal bovine serum also contains FGF-2 (37,38), and FGF-2 has been shown to promote keratocyte proliferation as well as keratan sulfate proteoglycan secretion into the medium when cells were cultured in 0.1% platelet-poor horse serum (39).…”