Characterization of release of basic fibroblast growth factor from bovine retinal endothelial cells in monolayer cultures

Abstract

Release of basic fibroblast growth factor (bFGF) was investigated in bovine retinal endothelial cells (BREC) maintained in monolayer culture. Confluent cells released bFGF into serum-free culture medium or medium containing 5 % serum at rates of up to 105.2 and 61.3 pM/day respectively. bFGF release coincided with a decrease in monolayer cell number and increases in lactate dehydrogenase (LDH) concentration and cells and cell-debris particles in the medium, which suggested that cell damage and lysis were responsible for growth-factor release. Maximum bFGF release at 24 h (230+10 pM) occurred when the cells were treated with lipopolysaccharide (10 /eg/ml), which also produced the greatest changes in parameters of cell damage. Sub-confluent cells showed little overt damage at 24 h, but released bFGF (78 + 20 pM) along with LDH, indicating that some cell lysis had occurred. Insulin-like growth factor 1 (IGF-1) was also released into serum-free culture medium at a rate of 0.34 nM/day, but not into medium containing serum or when the cells were treated with lipopolysaccharide. This implies that the mechanism of IGF-1 release is different from that of bFGF and is not related to cell damage. Culture medium conditioned by BREC stimulated the proliferation of these cells, as measured by an increase in their incorporation of [methyl-3H]

Negative Feedback for Endothelial Apoptosis: A Potential Physiological Role for Fibroblast Growth Factor

“…Further supporting the idea that FGF-2 is pre-loaded in endothelium and released during apoptosis were ELISA data demonstrating the presence of the growth factor in both non-apoptotic and apoptotic HUVEC, as well as increasing levels of FGF-2 in culture supernatants during apoptosis. These observations were consistent with an earlier report demonstrating the presence of FGF-2 in the culture supernatants of bovine endothelium after 6 days of serum deprivation [51]. Exogenously added FGF-2 appeared comparatively inactive compared with that manufactured endogenously by the cells, in that much higher protein levels of exogenous FGF-2 were required to mediate an effect comparable to endogenous FGF-2 detected by ELISA.…”

“…Interestingly, however, neutralizing antibodies against FGF-2 failed to inhibit the earlier reported activity, so that the identity of the reported protective factor is uncertain [52]. Insulin-like growth factor-1 is released along with FGF-2 when bovine retinal endothelial monolayers are wounded by scraping [51], while platelet-derived growth factor has also been detected in media of injured bovine aortic endothelium [39], and it is possible that these cytokines play a similar role to FGF-2 in protecting against endothelial apoptosis. An earlier study demonstrated increased synthesis and release of Fas ligand by non-apoptotic endothelium in hypoxic conditions, with the effect of reducing hypoxia-induced apoptosis [27].…”

Conditioned media of carcinoma cells cultured in hypoxic microenvironment stimulate angiogenesis in vitro; relationship to basic fibroblast growth factor

“…bFGF is well known to have no signal peptide for its secretion [1], and may be passively released by cell damage or death [3,17,33,38,42]. In this study, as the LDH levels in the CM were not increased and no cell damage or death of cancer cells was apparent under hypoxic cultivation.…”

Basic fibroblast growth factor induces expression of VEGF receptor KDR through a protein kinase C and p44/p42 mitogen-activated protein kinase-dependent pathway.

“…10). In ischemic retinopathies where bFGF and VEGF are thought to play significant roles, damaged and dying retinal cells release bFGF (74). In addition, bFGF stored in the extracellular matrix can be released as a bioactive bFGF-glycosaminoglycan complex (75)(76)(77)(78).…”