Retinoids enhance the number of EGF receptors in corneal endothelial cells.

Corneal endothelium appears to be an important target tissue for retinoids and epidermal growth factor (EGF). We report here that retinoic acid, or its synthetic analogue CBS-211 A (10(-8)-10(-7) M) dose-dependently enhances the mitogenic effect of EGF on cultured bovine corneal endothelial cells (BCEC). Furthermore, retinoid treatment, especially with CBS-211 A, increases the EGF-binding capacity of BCEC without any modification of EGF receptor affinity. The addition of cycloheximide (0.5 microgram ml-1) to cultures simultaneously with retinoids suppressed the retinoid effect on EGF-binding, whereas this protein synthesis inhibitor was ineffective when added 24 hr later. These results suggest that retinoids could induce the expression of EGF receptors on BCEC as an early event. This study, which demonstrates a cooperation between retinoids and EGF in corneal endothelium, could explain the beneficial effect of retinoic acid previously reported on this tissue repair in vivo. Our data could offer clues for new pharmaceutical strategies in ophthalmology therapy.

A morphological study of the inner surface of the anterior chamber angle in pre and postnatal human eyes.

The morphological changes in the inner surface of the human anterior chamber angle during pre and postnatal development were studied by light microscopy, scanning and transmission electron microscopy. Seventeen human foetal eyes (12-22 weeks) and ten infant and juvenile eyes (2.5 months--8 years) were investigated with the aim of establishing whether the trabecular meshwork is covered by an uninterrupted membrane at any stage in development. In 12-14 week old foetal eyes cuboidal corneal endothelial cells cover the inner surface of the anterior half to one third of the trabecular anlage. In this region there is a transition to more flattened uveal trabecular endothelial cells. Only rarely did corneal endothelial cells extend completely to the angle apex. The transition zone between corneal and uveal trabecular endothelial cells becomes located more anteriorly as development progresses. Intercellular gaps, which occur between uveal trabecular endothelial cells as early as 12-14 weeks in development, enlarge and become more frequent during development thus providing a route of communication between the anterior chamber and the developing intertrabecular spaces or channels. In the first months of life only a few cord-like uveal trabeculae orientated predominantly in a meridional direction overlie the more lamellate corneoscleral trabeculae. By the second year typical uveal trabeculae are more prominent and form a web-like arrangement. This is accompanied by a gradual decrease in the frequency of the cytoplasmic extensions of uveal trabecular endothelial cells which circumscribe the intratrabecular gaps. The timing of this remodelling and maturation appears to be remarkably variable between individuals. The implications of these findings on the theories of the pathogenesis of congenital glaucoma are discussed.

Corneal stromal wound healing in rabbits after 193-nm excimer laser surface ablation.

An argon fluoride excimer laser (193 nm) with a moving slit delivery system was used to perform anterior myopic keratomileusis in both eyes of 24 New Zealand white rabbits. Rabbits were killed immediately after ablation and at intervals up to 100 days. By slit-lamp microscopy, four rabbits at day 100 exhibited four clear corneas and four corneas had central, spotty, subepithelial haze. Light and electron microscopy documented corneal healing. In the early stages a transient acellular zone in the anterior stroma appeared over a period of three weeks, followed by an increased number of fibrocytes. In the corneas with opacification, focal areas of 20-microns-thick subepithelial scarring were present. An unexpected finding was transient damage to posterior stromal keratocytes and endothelial cells. The endothelium produced a layer of granular material that migrated anteriorly across Descemet's membrane. Immunochemistry at day 6 showed a marked staining for collagen IV, proteoglycans, fibronectin, and laminin.

Changes in nuclear DNA content and cell size of injured human corneal endothelium.

To understand how human corneal endothelium compensates for cell loss, nuclear DNA-cytofluorometry and cell morphometry were carried out on injured corneal endothelium. The examined corneas included two cases of keratoconus complicated with acute hydrops and one without acute hydrops, two cases of herpetic keratitis, one case of post-intracapsular cataract extraction (post-ICCE) and one case of luetic keratitis. The endothelial cell layer was separated from Descemet's membrane and double-stained with Rhodamine-labeled wheat germ agglutinin-lectin (WGA) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). The area of each cell was measured with a color image analyser and compared with its cytofluorometric nuclear DNA content. The endothelium in apparently intact regions of the diseased corneas showed the same DNA-ploidy pattern and cell area as the physiological corneas. However, endothelial cells in injured regions had greater area, even in diploidy, than in presumably normal ones and showed a larger number of hyperploid cells ranging from 4C to 36C. Hyperploid cells consisted of many multinucleates and few polyploidies and had extremely large and bizarre cytoplasm. All injured corneas were accompanied by cells with numerous micronuclei. A few asymmetrical 4C-binucleates (with DNA values such as 1.3 plus 2.6C) appeared in the case of the post-ICCE. It is concluded that damage to human corneal endothelial cells in vivo results in cell enlargement with or without DNA synthesis. Those changes appear more severe in diseased corneas than in the situation of physiological aging which we have reported previously. In severe cases, micronuclei, polyploid cells and multinucleated giant cells are frequent, thereby suggesting a possible long-persistent metabolic impairment of the endothelium after severe damage to the cornea.

Heterogeneity of collagens in rabbit cornea: type III collagen.

Whole neonate rabbit corneas and adult corneas containing 2-week-old scars were incubated in the presence of [14C] glycine. Radiolabeled collagen extracted from the corneas and scar tissue were analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography to determine the types and relative quantity of collagen polypeptides present and synthesized by these tissues. In addition to other collagen types, type III was found in both neonate cornea and scar tissue from adult cornea, albeit in relatively small quantities. Type III collagen in normal cornea was associated with the residue after pepsin digestion and formic acid extraction of the tissue, and the same type of collagen was extracted from scar tissue after similar treatment. Type III collagen-specific monoclonal antibody bound to developing normal corneas and healing adult tissue sections, as determined by immunofluorescence. Antibody binding was localized to the endothelium and growing Descemet's membrane in fetal and neonate corneas, and restricted to the most posterior region of the corneal scar tissue. Although monoclonal antibody to keratan sulfate, used as a marker for stromal fibroblasts, bound to most of the scar tissue, the antibody failed to bind to the posterior scar tissue positive for type III collagen. We conclude that endothelial cells from fetal and neonate rabbit cornea and endothelium-derived fibroblasts from healing wounds of adult cornea synthesize and deposit type III collagen. Moreover, this collagen appears to be incorporated into the growing Descemet's membrane of normal corneas and narrow posterior portion of the scar tissue.(ABSTRACT TRUNCATED AT 250 WORDS)