E P Kay1, X Gu, Y Ninomiya, R E Smith. 1. Department of Ophthalmology, University of Southern California School of Medicine, Los Angeles.
Abstract
PURPOSE: We have previously reported that corneal endothelial modulation takes place when rabbit corneal endothelial (CE) cells are exposed to corneal endothelium modulation factor (CEMF) released by polymorphonuclear leukocytes (PMN) (Kay, E. P., L. Rivela, and Y. G. He, 1990. Invest Ophthalmol Vis Sci. 31:313-322). The modulation was involved in phenotypic switches from polygonal cell shape to fibroblastic morphology and from basement membrane collagen (type IV-rich) synthesis to fibrillar collagen (type I-rich) synthesis. In the current study, we tested the effect of several growth-modulating factors on corneal endothelial modulation. METHODS: The effect of basic fibroblast growth factor (bFGF) on cell proliferation was measured by [3H]thymidine incorporation into DNA and cell numbers. Collagen expression was determined by SDS-polyacrylamide gel electrophoresis and by Northern blot analysis. Transcription rate was determined by nuclear run-off assay. Basic fibroblast growth factor synthesis was analyzed by immunoblot assay and quantitated by ELISA assay. Immunofluorescent staining was used for in vivo localization of bFGF and its receptors. RESULTS: Basic fibroblast growth factor (bFGF) supplemented with heparin is able to modulate the same phenotypes as observed in CEMF-induced modulation. Basic fibroblast growth factor has a marked stimulatory effect on cell proliferation, as shown by increased cell numbers and [3H]thymidine incorporation into DNA. It also has a strong effect on modulation of cell morphology and collagen phenotypes; the polygonal endothelial cells are induced to assume an elongated shape, and fibrillar collagen synthesis (types I and V) is turned on by bFGF, whereas type IV synthesis is markedly reduced. Such modulating effects of bFGF are augmented by CEMF. Furthermore, CEMF significantly increases production of bFGF in CE cells; the CEMF-treated CE cells synthesized bFGF seven times more than did the control cells. The induced bFGF has a major peptide band of 18.4 kD. Immunohistochemical analysis demonstrates that rabbit corneal endothelium in vivo stains for bFGF, while Descemet's membrane requires prior digestion with proteinase K. In situ localization of bFGF receptors demonstrates that high affinity receptors for bFGF are present in corneal endothelium. However, neither transforming growth factor (TGF-beta), epidermal growth factor (EGF), nor retinoic acid (RA) alters qualitative collagen phenotypes; rabbit CE cells continue to synthesize type IV collagen as a predominant species under the influence of these factors. Unlike rabbit CE cells, bovine CE cells in culture produce predominantly fibrillar collagens (I, III, and V). Transforming growth factor enhances type III collagen synthesis and induces type I collagen, but none of these factors affects type IV collagen synthesis by bovine cells. Neither steady-state levels of collagen RNA nor relative transcription rates of the collagen genes are changed significantly by TGF-beta, EGF, or RA in either rabbit or bovine CE cells. CONCLUSIONS: These findings suggest that bFGF is able to simultaneously modulate three phenotypic aspects of rabbit CE cells (cell proliferation, cell shape, and collagen expression). Furthermore, CEMF induces de novo synthesis of biologically active bFGF, indicating that bFGF, through the action of CEMF, is the key molecule during corneal endothelial modulation, which ultimately leads to corneal fibrosis (retrocorneal fibrous membrane).
PURPOSE: We have previously reported that corneal endothelial modulation takes place when rabbit corneal endothelial (CE) cells are exposed to corneal endothelium modulation factor (CEMF) released by polymorphonuclear leukocytes (PMN) (Kay, E. P., L. Rivela, and Y. G. He, 1990. Invest Ophthalmol Vis Sci. 31:313-322). The modulation was involved in phenotypic switches from polygonal cell shape to fibroblastic morphology and from basement membrane collagen (type IV-rich) synthesis to fibrillar collagen (type I-rich) synthesis. In the current study, we tested the effect of several growth-modulating factors on corneal endothelial modulation. METHODS: The effect of basic fibroblast growth factor (bFGF) on cell proliferation was measured by [3H]thymidine incorporation into DNA and cell numbers. Collagen expression was determined by SDS-polyacrylamide gel electrophoresis and by Northern blot analysis. Transcription rate was determined by nuclear run-off assay. Basic fibroblast growth factor synthesis was analyzed by immunoblot assay and quantitated by ELISA assay. Immunofluorescent staining was used for in vivo localization of bFGF and its receptors. RESULTS: Basic fibroblast growth factor (bFGF) supplemented with heparin is able to modulate the same phenotypes as observed in CEMF-induced modulation. Basic fibroblast growth factor has a marked stimulatory effect on cell proliferation, as shown by increased cell numbers and [3H]thymidine incorporation into DNA. It also has a strong effect on modulation of cell morphology and collagen phenotypes; the polygonal endothelial cells are induced to assume an elongated shape, and fibrillar collagen synthesis (types I and V) is turned on by bFGF, whereas type IV synthesis is markedly reduced. Such modulating effects of bFGF are augmented by CEMF. Furthermore, CEMF significantly increases production of bFGF in CE cells; the CEMF-treated CE cells synthesized bFGF seven times more than did the control cells. The induced bFGF has a major peptide band of 18.4 kD. Immunohistochemical analysis demonstrates that rabbit corneal endothelium in vivo stains for bFGF, while Descemet's membrane requires prior digestion with proteinase K. In situ localization of bFGF receptors demonstrates that high affinity receptors for bFGF are present in corneal endothelium. However, neither transforming growth factor (TGF-beta), epidermal growth factor (EGF), nor retinoic acid (RA) alters qualitative collagen phenotypes; rabbit CE cells continue to synthesize type IV collagen as a predominant species under the influence of these factors. Unlike rabbit CE cells, bovine CE cells in culture produce predominantly fibrillar collagens (I, III, and V). Transforming growth factor enhances type III collagen synthesis and induces type I collagen, but none of these factors affects type IV collagen synthesis by bovine cells. Neither steady-state levels of collagen RNA nor relative transcription rates of the collagen genes are changed significantly by TGF-beta, EGF, or RA in either rabbit or bovine CE cells. CONCLUSIONS: These findings suggest that bFGF is able to simultaneously modulate three phenotypic aspects of rabbit CE cells (cell proliferation, cell shape, and collagen expression). Furthermore, CEMF induces de novo synthesis of biologically active bFGF, indicating that bFGF, through the action of CEMF, is the key molecule during corneal endothelial modulation, which ultimately leads to corneal fibrosis (retrocorneal fibrous membrane).
Authors: Maryam Ali; VijayKrishna Raghunathan; Jennifer Y Li; Christopher J Murphy; Sara M Thomasy Journal: Exp Eye Res Date: 2016-09-14 Impact factor: 3.467