Beatrice Y. J. T. Yue

Heterogeneity in keratoconus: possible biochemical basis.

Abstract Total protein and collagen content in normal and keratoconus corneas were determined. The protein content (expressed as a function of dry weight) in all keratoconus corneal samples was lower than that found in normal corneas. However, among the 11 keratoconus corneas examined, only 7 (group A) had the same hydroxyproline content (expressed as a function of dry weight) as normal corneas; 4 others (group B) showed significantly less. In tissue culture, four strains derived from keratoconus stroma (group I) produced total protein at the same rate as cells from normal controls. Four other strains (group II), however, had a decreased rate of protein synthesis. The amount of collagenous protein synthesized per microgram DNA by group I strains was similar to that found in normal cultures, whereas it was significantly reduced in group II cultures. We suggest that group I strains represent group A corneas. Group II strains, with a reduced level of both protein and collagen synthesis, may represent group B corneas. The defect in this group appears to be decreased total synthetic activity of corneal cells. The variation in our results suggests that keratoconus is a heterogeneous disease. The heterogeneity may explain the contradictory data that exist in the literature.

Biosynthesis of glycosaminoglycans by trabecular meshwork cells in vitro

We compared the incorporation of radioactive precursors into glycosaminoglycans by cynomolgus monkey trabecular meshwork cells in tissue culture and in organ culture. Hyaluronic acid and a variety of sulfated glycosaminoglycans were synthesized in both systems. In organ cultures, the ratio of chondroitin 6- to chondroitin 4-sulfate was higher than that found in tissue cultures. Also, a greater proportion of total glycosaminoglycans found in the organ culture medium was represented by hyaluronic acid. The higher production of chondroitin 6-sulfate and hyaluronic acid, as noted in some embryonic systems, suggested that cells in organ cultures may resemble an acute wound healing or an early developmental state more closely than did cells in tissue cultures.

Effects of chondroitin sulfate on metabolism of trabecular meshwork

Glycosaminoglycans are believed to play a role in the physiological functions of trabecular meshwork. The effects of chondroitin sulfate on the metabolism of trabecular meshwork were studied by replacing the aqueous humor in the right eye (experimental eye) of 12 albino rabbits with 0.3 ml of chondroitin-4-sulfate solution (10 mg ml-1 in glucose-supplemented phosphate buffered saline). The aqueous humor in the left eye (control eye) was replaced with the phosphate buffered saline-glucose solution in the same manner. The intracameral procedures were performed twice a week for four weeks (initial injection period), and then discontinued for another four weeks (resting period). No significant or prolonged increase in the intraocular pressure was observed during either period in any eyes. Subsequent intracameral injections performed after the resting period, however, caused an intraocular pressure elevation in 10 of the 12 experimental eyes. During this second injection period, the injections were carried out whenever the intraocular pressure of experimental eyes dropped to the control value. The pressure generally remained 5-10 mmHg above that of the control eyes for periods ranging from two days to four weeks. In the eyes with elevated intraocular pressure, light microscopy showed that the trabecular beams were compact and sclerotic and the intertrabecular spaces were narrower. Chamber angle tissues obtained from the eyes with elevated intraocular pressure incorporated more radioactive precursors into glycosaminoglycans than those from the control eyes. The synthesis of glycosaminoglycans and the metabolism of trabecular meshwork seemed to be modified by the long-term chondroitin sulfate treatment.

RNA metabolism in cultures of corneal stromal cells from patients with keratoconus.

Abstract Total cellular RNA was extracted from cultured keratoconus and normal human corneal stromal cells. The translational activity of these RNAs was examined in a cell-free translation system derived from reticulocyte lysate. Results indicated that keratoconus cells can be separated into two groups, as has been shown previously. Group I keratoconus cells contained the same amount of total RNA as normal cells. RNA activity and the rate of mRNA synthesis in this group of keratoconus cells were also normal. By these criteria it seems that the protein synthesizing system is functioning properly, and group I keratoconus cells should have a normal rate of protein synthesis. These results correlate well with previous findings. Group II keratoconus cells, in contrast, contained more RNA than normal cells. The translational efficiency of RNA was so markedly reduced that the elevation in RNA content did not compensate for the decrease in translational efficiency. It is likely that the reduced protein and collagen synthesis in this group of cells is related to the reduction in the RNA activity. An inhibitory component was present in the keratoconus RNA which affected synthesis of all proteins and suppressed translation of normal RNA.

Effects of chronic intracameral injections of chondroitin sulfate on cat eyes

The effect of chondroitin sulfate on the metabolism of trabecular meshwork was studied by repeatedly injecting a chondroitin sulfate solution into the anterior chamber of cat (experimental) eyes for 28–29 weeks. The aqueous humor of the opposite (control) eyes were similarly replaced with a phosphate-buffered saline-glucose solution. The intraocular pressure of the experimental eyes was increased 4–7 mm Hg above that of the control eyes for a prolonged period of time. In addition, collagen fibers, elastic fibers, and basement membrane-like material were moderately increased in the trabecular meshwork of experimental eyes. Three forms of thickened basement membranes of endothelial cells were observed. The compact multilaminated basement membrane was formed concentrically around some of the trabecular beams. The discontinuous, thin basement membrane was seen among bundles of coarse collagen in association with long spacing collagen fibers. Also, fine filaments were noted streaming from the plasma membrane and basement membrane of endothelial cells of angular aqueous veins. Chondroitinase ABC was able to remove ground substance in the elastic fiber of the trabecular meshwork. Biochemically, the chamber angle tissues from these eyes showed a higher synthetic activity of glycosaminoglycans and proteins. Chondroitin sulfate appears to have a direct effect on trabecular meshwork and can influence the physiologic function of these cells.

Effects of chondroitin sulfate on trabecular meshwork in rabbit eyes: an electron microscopic study.

The intraocular pressure of four New Zealand albino rabbit eyes was elevated when we replaced the aqueous humor of these eyes repeatedly with a chondroitin sulfate solution. Seen by electron microscopy, the trabecular meshwork of these eyes showed moderately increased collagen fibers, elastic fibers, and fine fibrils, and thickening of basement membrane. The amount of extracellular material present was markedly increased when compared with control eyes. Three types of basement membranes were noted. Compact multilaminated basement membrane and placoid accumulations of filamentous material with a granular background were found adjacent to the endothelial cells of the trabecular meshwork, and fine fibrils were observed around the angular aqueous veins. The ultrastructural alterations we observed were similar to those seen in some human glaucoma cases. The metabolism of trabecular meshwork cells may be affected by the long-term chondroitin sulfate treatment. As a result, extracellular matrix material appears to be accumulated in the trabecular meshwork, which may then contribute to an increased outflow resistance and a mild intraocular pressure elevation.

CD44 and Primary Open Angle Glaucoma

In our view, primary open-angle glaucoma (POAG) is a common neurodegenerative disease caused by a variety of molecular defects and/or cellular insults that result in cell stress and death of the trabecular meshwork (TM) and retinal ganglion cells (RGC). One potential biological marker of POAG is CD44, which is one of the adhesion/homing molecules. Direct evidence for CD44’s very central role in POAG includes: (1) aqueous humor of patients with POAG contains an increased amount of the soluble extracellular 32-kDa fragment of CD44 (sCD44) in comparison with the aqueous humor of age-matched normal individuals; (2) increased levels of sCD44 in the aqueous correlates with the extent of visual field loss in POAG patients; (3) sCD44, particularly hypo-phosphorylated sCD44, is a potent and specific toxic protein to TM and RGC in vitro; and (4) overexpression of both full-length CD44 and truncated sCD44 in transgenic mouse eyes is sufficient to cause ocular hypertension. The increase in intraocular pressure (IOP) lasted more than 90 days accompanied by optic nerve damage. The overexpression of CD44 may thus be the first documented animal model that closely mimics the human disease POAG. Other models have been cytodestructive and nonphysiologic.