Kay E P, Gu X, Ninomiya Y, Smith R E
Department of Ophthalmology, University of Southern California School of Medicine, Los Angeles.
Invest Ophthalmol Vis Sci. 1993 Mar;34(3):663-72.
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.
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.
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.
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).
我们之前报道过，当兔角膜内皮（CE）细胞暴露于多形核白细胞（PMN）释放的角膜内皮调节因子（CEMF）时，会发生角膜内皮调节（Kay, E. P., L. Rivela, and Y. G. He, 1990. Invest Ophthalmol Vis Sci. 31:313 - 322）。这种调节涉及细胞表型的转变，从多边形细胞形态转变为成纤维细胞形态，以及从基底膜胶原蛋白（富含IV型）合成转变为纤维状胶原蛋白（富含I型）合成。在本研究中，我们测试了几种生长调节因子对角膜内皮调节的影响。
通过[³H]胸腺嘧啶核苷掺入DNA和细胞计数来测量碱性成纤维细胞生长因子（bFGF）对细胞增殖的影响。通过SDS - 聚丙烯酰胺凝胶电泳和Northern印迹分析来确定胶原蛋白表达。通过核转录分析来确定转录速率。通过免疫印迹分析来分析碱性成纤维细胞生长因子的合成，并通过ELISA测定法定量。免疫荧光染色用于bFGF及其受体的体内定位。
补充肝素的碱性成纤维细胞生长因子（bFGF）能够调节与CEMF诱导调节中观察到的相同表型。碱性成纤维细胞生长因子对细胞增殖有显著的刺激作用，表现为细胞数量增加和[³H]胸腺嘧啶核苷掺入DNA增加。它对细胞形态和胶原蛋白表型的调节也有很强的作用；多边形内皮细胞被诱导呈现细长形状，bFGF开启纤维状胶原蛋白（I型和V型）的合成，而IV型合成则显著减少。CEMF增强了bFGF的这种调节作用。此外，CEMF显著增加CE细胞中bFGF的产生；经CEMF处理的CE细胞合成的bFGF比对照细胞多七倍。诱导产生的bFGF有一条主要的18.4 kD肽带。免疫组织化学分析表明，兔角膜内皮在体内可被bFGF染色，而Descemet膜需要先用蛋白酶K消化。bFGF受体的原位定位表明角膜内皮中存在bFGF的高亲和力受体。然而，转化生长因子（TGF - β）、表皮生长因子（EGF）或视黄酸（RA）均未改变胶原蛋白的定性表型；在这些因子的影响下，兔CE细胞继续以IV型胶原蛋白作为主要合成产物。与兔CE细胞不同，培养的牛CE细胞主要产生纤维状胶原蛋白（I型、III型和V型）。转化生长因子增强III型胶原蛋白的合成并诱导I型胶原蛋白的产生，但这些因子均不影响牛细胞IV型胶原蛋白的合成。在兔或牛CE细胞中，TGF - β、EGF或RA均未显著改变胶原蛋白RNA的稳态水平或胶原蛋白基因的相对转录速率。
这些发现表明bFGF能够同时调节兔CE细胞的三个表型方面（细胞增殖、细胞形状和胶原蛋白表达）。此外，CEMF诱导生物活性bFGF的从头合成，表明bFGF通过CEMF的作用，是角膜内皮调节过程中的关键分子，最终导致角膜纤维化（角膜后纤维膜）。
