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BACKGROUND: Using a rabbit cornea model, our recent study demonstrated that Chen Medium (CM), an isotonic media enriched with nonlactate-generating high-energy substrates, is very effective for organ preservation. In the present study, the efficacy of CM is further evaluated with human corneas METHODS: The effectiveness of CM and Optisol for preserving the endothelial integrity of human corneas in vitro was evaluated by scanning electron microscopy. Clinical efficacy was evaluated in a total of 83 patients: 10 patients with keratoconus grafted randomly with either CM- or Optisol-stored cornea of the same donor, and 73 patients with various conditions grafted with CM-stored corneas. After surgery, visual acuity and quality of the graft were monitored for up to 4.6 years. RESULTS: The scanning electron microscopic study revealed that after 11-day storage at 4 degrees C, the CM-stored cornea had only marginal disruptive changes, 9.4+/-1.1%, in endothelial cells, as opposed to 42.4+/-4.6% of the Optisol-stored cornea. All 78 CM-stored corneas, including 67 with 12.2- to 17.7-hr death-to-storage time, 3-7.6 days of storage time, and initial marginal quality before storage, were successfully transplanted. These grafts were thin and clear, with an excellent epithelial integrity and without significant changes in endothelial cell density. Five Optisol-stored corneas were also successfully grafted; one of them, however, was edematous for about 4 weeks, and all the grafts were slightly thicker with substantial endothelial cell loss. CONCLUSION: Using a cornea model, present and recent studies show that CM is very effective for preserving tissue viability and endothelial integrity. Previous study revealed that CM-stored tissues maintained high levels of ATP and metabolic function, with suppression of lactate formation and accumulation. Thus, these findings support the concept that preservation of tissue viability is closely associated with the ability of the tissues to retain metabolic activity, to generate ATP efficiently, and to prevent acidosis effectively during storage.

Studies on the intact avascular cornea reveal two types of lactate effluxes: exogenous glucose-elicited and spontaneous. The former type exhibits characteristics resembling the proton-lactate symport system previously found in tumor cells and erythrocytes, including an enhanced lactate efflux at a higher extracellular pH and in the presence of H+ and K+ ionophores, and an inhibition by mersalyl with subsequent lactate accumulation in the tissue and cessation of glycolytic activity. The latter type occurs immediately following the incubation of freshly isolated cornea in a medium containing no exogenous glucose, with a rate about 10 times that of exogenous glucose-elicited lactate efflux. It is insensitive to 10 m m iodoacetate and lacks the characteristics of the proton-lactate symport system. Findings reveal that about 50% of corneal glucose utilization occurs in the epithelium, with the stroma and endothelium sharing the other 50% approximately equally. Of the glucose utilized, the lactate formation to pyruvate oxidation rate ratios are approximately 1:1 in the epithelium, 2:1 in the stroma, and 1:2 in the endothelium. About 79% of total tissue lactate is formed in the epithelium and stroma, and in vivo, this is probably pumped into the stromal extracellular space (about 90% of total tissue volume) via the proton-lactate symport system, with spontaneous release into the aqueous humor via a simple diffusion process. The H+ and K+ ionophores facilitate lactate efflux at the expense of the cellular pyruvate pool, without significant effect on the glucose uptake and glycolytic activity. These findings suggest that the ionophore-mediated lactate efflux favors the reduction of low pyruvate concentration in the tissue, rather than parallel increases in glycolytic activity.

The scope of this study includes investigations on uptake of dextran and chondroitin sulfate in human donor corneas, and effects of chondroitin sulfate on adhesion and growth of rabbit and cat corneal endothelial cells. Fibronectin and dextran sulfate served as controls. Nuclear magnetic resonance spectroscopy revealed no detectable osmotic agents in the corneas stored in either Dexsol or Optisol at 4 degrees C for 6 days. The study showed that chondroitin sulfate enhanced cell adhesion marginally at 2.5%, but >40% at 0.5%, comparable to that of 10 mu g/ml fibronectin, whereas 2% dextran sulfate abolished approximately 70% of cell adhesion capability. In cultures with fibronectin present, the duration for cells to reach confluence was extended from 1.75 to 3 days. Chondroitin sulfate (2%) elicited no apparent cytotoxic effect, with cells becoming polygonal and reaching confluence in approximately 9 days. Cell growth was retarded by 2% dextran sulfate, with signs of senescence on day 3 and clear evidence of cell degeneration on day 9. The cells did not survive without serum in cultures, especially at 36 degrees C. These findings suggest that corneal deterioration probably is linked primarily to serum-free storage conditions, not uptake of osmotic agents in the cornea. The highly negatively charged sulfate group, however, may have rendered chondroitin sulfate less effective as an additive.

A study of subcellular acid phosphatase distribution in mammalian tissues shows that isozymes with specific functions are compartmentalized in the cells. The enzyme may be generalized into two types: type A and type B. They are shown by several means to be distinct entities. Type A is confined to the cytoplasm and is inhibited by Cu2+, HCHO, and the coupling agents (for enzyme staining) fast blue RR salt and fast Garnet GBC salt (newly discovered inhibitors), but is insensitive to fluoride and L-(+)-tartrate. Type B is localized in the organelles, presumably lysosomes, in both soluble form and membrane-bound form, with inhibitor sensitivity exactly opposite to that of type A enzyme. Types A and B consist of different sets of isozymes, with sensitivities to inhibitors resembling those observed with the crude extracts of subcellular fractions. Acid phosphatase that exhibits a phosphoryl transfer property was identified as type A enzyme. Type A enzyme has a slightly higher optimal pH and is inhibited by alloxan, whereas for type B, the addition of alloxan broadens the optimal pH to a higher range and elevates the activity of pH 7.4 from negligible to about 30-40% of that obtained under optimal conditions. The alloxan-mediated elevation of type B enzyme activity to this level at the physiological pH may be of considerable significance. Type B enzyme has a high affinity for metabolic intermediates and nucleotides, while type A has an extremely low affinity for these substrates. Cytoplasmic acid phosphatase (type A) is a significant enzyme population and its activity is not related to the lysosome density in the cells. Type A enzyme in the cytoplasm is thus shown to be an entity distinctly different from type B enzyme in the lysosomes. These findings suggest that the physiological functions of type A acid phosphatase, such as metabolic regulatory processes, merit further studies because of the phosphoryl transfer activity and cytoplasmic localization of the enzyme.

The purification and characterization of uridine and thymidine, two previously isolated vascular endothelium effectors, are described. These two effectors were purified from all dialyzable fractions of fetal bovine serum (FBS), vitreous, and retina. The present study demonstrates the stimulatory characteristic of uridine and thymidine on the proliferation of aortic endothelial cells. The stimulation appears to link to their utilization by the cells in a dialyzed FBS-dependent manner. In the presence of optimal concentrations of uridine (10 microM), thymidine (0.8 microM), and dialyzed FBS (3 mg ml-1), a markedly enhanced vascular endothelial cell proliferation is attainable; uridine and thymidine alone had no significant stimulatory effect. The presence of both nucleosides, however, increased the concentration of dialyzed FBS required for half-maximum proliferation activity of fetal bovine aortic endothelial cells and also increased maximum activity. Studies with fetal bovine aortic endothelial cells showed that the activity of uridine kinase in the cells was activated by dialyzed FBS, but not by uridine and thymidine. Uridine and thymidine exerted no marked stimulatory effect on Balb/c 3T3, Swiss 3T3, and SV-T2 as noted with endothelial cells. The significance of the present findings to the proliferation of vascular endothelial cells in the retina is discussed.

The effect of fetal bovine retinal extract (FBR) and dialyzed fetal bovine serum (FBS) on in vitro proliferation of vascular endothelial cells was studied. Quantitatively, FBR, both dialyzable and nondialyzable, and dialyzed FBS exhibited a synergism in bringing about an enhanced proliferation of vascular endothelial cells. Assays on selected samples using both aortic and retinal vascular endothelial cells yielded similar results. In the absence of dialyzed FBS, FBR had only a negligible stimulatory effect. When compared at a protein concentration of 3 mg ml−1, dialyzed FBS elicited vascular endothelial proliferation at a rate only 25% that of FBS (not dialyzed). However, under experimental conditions using 3 mg ml−1 of dialyzed FBS and 0·5 mg ml−1 of dialyzed retinal extract, a six-fold increase in the activity of vascular endothelial proliferation was demonstrated. The observed synergistic effect was dependent on the concentrations of both dialyzed FBS and FBR. The observed synergism rules out the possibility that the stimulatory effect of FBR was due to blood contamination in sample preparation. Qualitatively, major components of dialyzed FBS and dialyzed FBR that exerted a synergistic effect on the stimulation of vascular endothelial proliferation were isolated by permeation chromatography using Sephadex G-100, with Mr estimated at 150 000 and 80 000, respectively. One minor component in FBS and one in FBR were also present.

The presence of a vascular endothelial cell growth factor (VEGF) in the retina was reported in a previous study. The present experiments show that VEGF exhibits a pronounced synergism with the serum-derived factor and the vascular endothelium (VE) effectors in stimulating the proliferation of vascular VE cells. VEGF shows a chromatographic multiplicity with the 25,000-D component as the smallest subunit. Mg2+ is the specific divalent cation that retains the VEGF molecule in the aggregated form and enhances the activity, both total and specific. In addition, VEGF is highly specific for endothelial cells and is distinctly different from FGF, EGF, and insulin in terms of molecular weight (MW) and cell specificity. Under our assay conditions, VEGF has no stimulatory effect on other cell lines examined, including lens epithelial cells, corneal epithelial cells, corneal keratocytes, Walker 256 carcinoma, and fibroblasts. These findings indicate that VEGF possesses characteristic properties not reported for other growth factors, and that VEGF is distinctly different from the growth factors isolated from the retina in other laboratories. The present study suggests that VEGF in the retina represents a new type of growth factor. The need to employ a highly defined assay condition could have eluded the detection of this factor in other laboratories.