Apoptosis induced by a corneal-endothelium-derived cytokine.

PURPOSE: The purpose of this study was to isolate and characterize cDNA clones encoding target proteins for autoantibodies in patients at high risk for transplant rejection. METHODS: A pool of 10 sera from patients at high risk for rejection who had undergone corneal transplantation was used for immunoscreening of an endothelial cDNA library, and the cDNA fragments were subcloned into prokaryotic expression vectors to generate recombinant fusion proteins. Cytotoxicity of recombinant protein was determined by a modified 51Cr-release assay. Apoptosis induced by recombinant protein was determined by fluorescent dye-chromatin fragmentation assay and by TdT-dUTP terminal nick-end labeling (TUNEL) assay. An enzyme-linked immunosorbent assay was used to detect the presence of antibodies to recombinant protein in the sera of high-risk patients undergoing corneal transplantation and of control subjects. RESULTS: Screening of 500,000 plaques identified six positive clones, one of which demonstrated extensive homology with a novel tumor-derived cytokine termed endothelial monocyte-activating polypeptide (EMAP). EMAP was synthesized as a 39-kDa precursor that was proteolytically cleaved to generate an active 22-kDa cytokine. The mature peptide of EMAP alone was capable of inducing the death of cultured endothelial cells, whereas the propeptide was inactive. The protein synthesis inhibitor cycloheximide potentiated EMAP-induced apoptosis in endothelial cells. Cell death by apoptosis was evidenced by DNA fragmentation, extensive surface bleb formation, and chromatin condensation. A statistically significant difference was found in the level of antibodies specific to EMAP between patients at high risk for corneal transplant rejection and control subjects (P<0.001). The antibody levels were elevated in patients with severe graft reaction when compared with patients with no graft reaction (P<0.001). CONCLUSIONS: These studies demonstrated that EMAP is a novel protein in corneal endothelial cells that is capable of inducing programmed cell death. Overexpression of this cytokine could initiate endothelial cell damage leading to stromal edema and corneal decompensation.

Large-scale sequencing of the rabbit corneal endothelial cDNA library.

PURPOSE: This study sought to identify novel or active genes in corneal endothelial cells with description of the gene-expression profile. METHODS: We performed the single-pass sequencing of 1,000 clones from a rabbit corneal endothelial cDNA library. Inserts of the library were amplified by polymerase chain reaction (PCR), sequenced, and compared with several databases. We used four database similarity search programs: FASTA, BLASTN, TBLASTX, and BLASTX. RESULTS: Of the sequences generated, 618 (61.8%) showed sequence homology with known genes, whereas 192 (19.2%) matched previous reported expression sequence tags (ESTs) and 174 (17.4%) showed no sequence similarity. Among the homologous clones to known genes are collagen type VIII, secreted protein acidic and rich in cysteine (SPARC), lysyl oxidase, phosphatidylcholine-2-acylhydrolase, and thrombospondin. Several matched ESTs, and no matched clones that showed high frequency were also detected. CONCLUSION: Large-scale sequencing can be useful in obtaining a profile of the active genes. Several ESTs showed relatively frequent expression, suggesting that these genes may have important functions in the corneal endothelium.

The corneal stroma: an inhomogeneous structure.

PURPOSE: This study was conducted to determine the elemental composition of the human cornea. Special attention was paid to corneal stroma inhomogeneity. METHODS: Seventy human corneas were examined by means of energy-dispersive X-ray analysis. Epithelium, subepithelium, middle stroma, sub-Descemet layer, Descemet's membrane and endothelium were subjected to repeated measurements. RESULTS: In the cellular layers the phosphorus concentrations were high [0.35 mol/kg dry weight (dw) in the epithelium and 0.403 mol/kg dw in the endothelium]. Similar concentrations were found for sulphur (0.38 mol/kg dw in the epithelium). Stromal layers showed high contents of sulphur: 0.26 mol/kg dw. The phosphorus concentration was found to be higher in the subepithelium than in the middle stroma. Sulphur concentrations were highest in Descemet's membrane, followed by the subepithelium and the middle stroma. DISCUSSION: Nucleic acids and energy-containing phosphates explain the high levels of phosphorus in the cellular layers. The high sulphur concentrations may be related to the phosphoadenosinphosphosulfate and protein turnover in the epithelium. We interpret the inhomogeneous distribution of phosphorus in the stroma as a function of the density of keratocytes. An evaluation of all known sulphur-containing biochemical components of the stroma (0.217 mol sulphur/kg dw) corresponds to our measurements. In contrast to former results we find the corneal stroma to be an inhomogeneous structure.

Advances in corneal preservation.

The functional status of the endothelium and sustained corneal deturgescence after corneal preservation are of great clinical importance and have been primary goals in the development of corneal storage media. In our investigational studies we have specifically addressed the improvement of the quality of donor tissue after 4 degrees C storage, the extension of corneal preservation time, the enhancement of corneal wound healing, and the reduction of the normal progressive loss of endothelial cells postkeratoplasty. Specifically we have developed in vitro HCE cell and epithelial cell culture models that can accurately reflect the response of human corneal tissue in vivo. These models have been utilized to study the effects of growth factors and medium components in relation to their biocompatibility and efficacy in the development of improved corneal preservation solutions. Our laboratory investigated in vitro conditions that allowed human corneal endothelium to shift from a nonproliferative state, in which they remain viable and metabolically active, to a proliferative, mitotically active state. Isolation techniques developed in our laboratory have enabled the establishment of primary and subsequent subcultures of human corneal endothelium that retain the attributes of native endothelium. These in vitro conditions maintain HCE cells in a proliferative state, actively undergoing mitosis. A quantitative bioassay has been developed to determine the effects of various test medium in the stimulation or inhibition of DNA synthesis. In attempting to learn more about the events that occur during in vitro endothelial cell isolation, cell reattachment, extracellular matrix interaction and migrating during subculture, SEM was done on isolated HCE cells incubated in CSM. These studies suggest that the components of the extracellular matrix modulate the growth response of HCE cells, and play a role in regulating proliferation and migration. These observations are important in view of the fact that anterior chamber environment limits cell regeneration of the endothelium, and supports wound healing via cell migration. In vivo, it is the complex interaction of the HCE cell and the extracellular matrix that signal the cell to respond to cell loss in this manner. As our knowledge of human corneal endothelium has increased so has our anticipation of developing the "optimum" medium. Thus additional components have been added to this basic medium to address specific complications encountered with 4 degrees C corneal preservation. Antioxidants, additional energy sources, and other nutritive substrates have been used to supplement and further define a chondroitin sulfate-based medium. These changes have been a part of our new awareness that, even at 4 degrees C, the cornea is metabolically active.(ABSTRACT TRUNCATED AT 400 WORDS)