Abstract
When primary corneal endothelial cells were grown in polymorphonuclear leukocyte (PMN)-conditioned medium, a minor population of cells acquired fibroblastic morphology. Such modulated endothelial cells supported by PMN-conditioned medium grew much faster than the major nonresponding polygonal endothelial cell. Upon serial passages, the modulated endothelial cells became the dominant cell type and eventually formed a homogeneous fibroblastic culture. At the same time, phenotypic changes of collagen were observed. The primary endothelial cells grown in PMN-conditioned medium, consisting of responding elongated cells and nonresponding polygonal endothelial cells, produced predominantly type IV collagen with type III collagen as a minor component. As cells were subcultured and fibroblastic cells became dominant, type IV collagen synthesis was dramatically decreased and type I collagen synthesis was increased in parallel. When they reached the fully modulated stage, the cultures synthesized types I and III collagen, with type I accounting for 75-85% of the total. Type I collagen synthesized by the fibroblastic endothelial cells shared common characteristics with known type I collagen, such as migration behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CNBr peptide profiles, and immunologic identity. Thus, PMNs apparently contribute to the modulation of corneal endothelial cells, causing them to acquire characteristics of fibroblasts, cell multilayering, and deposition of interstitial extracellular matrix composed predominantly of interstitial type I collagen.
Highlights
When primary corneal endothelial cells weregrown in polymorphonuclear leukocyte (PMN)-conditioned medium, a minor population of cells acquired fibroblastic morphology
In the previous study [10]we reported thatPMNs modulate corneal endothelialcells into fibroblast-likecells that synthesize predominantly typeI procollagen rather than thephysiologic type IV collagen seen in Descemet’s membrane-corneal endothelium complex
The abbreviations usedare: retrocornealfibrous membrane (RCFM), retrocorneal fibrous mem- resuspended in DMEM supplemented with 10% fetaclalf serum and brane; PMN, polymorphonuclear leukocyte; fibroblastic corneal endothelial cells (FCEC), fibroblastic cor- 50 pg/ml gentamicin (DMEM-10)
Summary
Cell Cultures-Isolation and establishment of primary rabbit corbasemenmt embrane (Descemet’s membranef)requently nealendothelial cells inculture was as previouslydescribed [9]. Cause loss of function of the corneal endothelium, corneal Briefly, we used sequentialtreatments of Descemet’s membrane/. Diverse terms are used to describe corneal fibrosis, the most (DMEM) (GIBCO) supplemented wi1th0% fetal calf serum (GIBCO). The abbreviations usedare: RCFM, retrocorneal fibrous mem- resuspended in DMEM supplemented with 10% fetaclalf serum and brane; PMN, polymorphonuclear leukocyte; FCEC, fibroblastic cor- 50 pg/ml gentamicin (DMEM-10). The supernatant, which resis; NCEC, normal corneal endothelial cells. Twentyml of cell suspension was neal endothelial cells; DMEM, Dulbecco’s minimal essentialmedium; gentlylayered on 1 2 ml of Ficoll-Paque solution (Pharmacia) and SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electropho- centrifuged for 20 min at 2000 X g a t 4 “C.
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