Cellular response in subretinal neovascularization induced by bFGF-impregnated microspheres (original) (raw)
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Subchoroidal Release of VEGF and bFGF Produces Choroidal Neovascularization in Rabbit
Current Eye Research, 2016
Purpose: Intravitreal vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) produced florid retinal neovascularization and hemorrhage in the rabbit. This study seeks to determine whether sustained subchoroidal release of both VEGF and bFGF can induce robust choroidal neovascularization (CNV) in the rabbit. Methods: Subchoroidal implantation through the sclera of polymeric pellets containing both 15 μg VEGF and 15 μg bFGF was performed on adult pigmented male Dutch belted rabbits (N = 6) and NZW albinos (N = 8). As negative controls, blank pellets with no growth factors were implanted in both Dutch belted rabbits (N = 6) and NZW albino rabbits (N = 4). Development of CNV was documented weekly over a 4week period with indirect ophthalmoscopy, color fundus photography, and fluorescein angiography. Eyes were enucleated and prepared for histologic and immunohistochemical analyses at the end of the study. Amounts of VEGF and bFGF that were released in vitro from the pellets were measured by ELISA. Results: In all eyes with subchoroidal implants containing both VEGF and bFGF, strong fluorescein leakage was observed at 2, 3, and 4 weeks (P < 0.005); no leakage was seen initially in week 1. Negative control groups with blank implants showed no fluorescein leakage throughout the 4-week study period. Histologic analysis confirmed the presence of experimental CNV. New subretinal blood vessel growth occurred in all eyes with VEGF/bFGF implants. Negative control eyes with blank implants showed no vascular changes. In vitro sustained release of both VEGF and bFGF was confirmed by ELISA. Conclusion: Sustained subchoroidal release of both VEGF and bFGF produced experimental CNV rapidly in the rabbit. Understanding how these growth factors induce CNV may suggest novel therapeutic strategies in the large rabbit eye.
Fate of Biotinylated Basic Fibroblast Growth Factor in the Retina Following Intravitreal Injection
Experimental Eye Research, 1996
Exogenous basic fibroblast growth factor (bFGF) stimulates proliferation of non-neuronal retinal cells in vivo. To help understand how this proliferative effect is mediated, we followed the fate of biotinylated bFGF after injection into the vitreous of normal rabbit eyes. The retinal distributions, binding, and processing of biotinylated bFGF (bFGF-biotin) was examined from 2 hr to 7 days after intravitreal injection using laser scanning confocal microscopy, electron microscopy and Western blot analysis. At 2 hr, bFGF-biotin was detected throughout the extracellular space and on retinal basement membranes. At 6 hr, discrete punctate material first appeared within the cytoplasm of Mu$ ller cells, astrocytes, endothelial cells, retinal pigment epithelial (RPE) cells, and ganglion cells. Labeling was also present in the invaginations of the photoreceptor synaptic terminals at this time. This general pattern persisted up to 4 days after injection but was greatly attenuated by post-injection day 7. Labeling in the inner retina decreased progressively over the seven days ; whereas labeling in the outer retina, primarily within the RPE, increased at 4 days post-injection and then gradually decreased to nearly undetectable levels by 7 days. Western analysis of retinal protein homogenates following injection showed that an 18 kDa component representing intact bFGF, can be identified up to 1 week following injection. This component, as well as a 15 and 9 kDa biotinylated fragment, showed a progressive reduction during the one week post-injection period. Cross-linking experiments demonstrated that bFGF-biotin binds to three putative receptors with approximate molecular weights of 54, 62, and 110 kDa. These data are consistent with binding of exogenous bFGF to : (a) low affinity bFGF receptors associated with retinal basement membranes ; (b) invaginations at the base of photoreceptor synapses ; and (c) putative high affinity bFGF receptors on the plasma membranes of glial cells, endothelial cells, RPE cells and ganglion cells. bFGFbiotin apparently binds to, and is then internalized by, the same non-neuronal cell types that are stimulated to proliferate following retinal injuries such as detachment.
PLoS ONE, 2013
Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Mü ller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Mü ller cells of the rat retina and in acutely isolated Mü ller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Mü ller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Mü ller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Mü ller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK21/22) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Mü ller cells are major sources of bFGF in the ischemic retina. Mü ller cells under physiological conditions or transient hypoxia seem to provide an anti-angiogenic environment, but long-lasting hypoxia causes the release of bFGF, which might significantly co-stimulate neovascularization in the retina.
Experimental corneal neovascularisation using sucralfate and basic fibroblast growth factor
Australian and New Zealand Journal of Ophthalmology, 1996
Purpose: To develop a non-inflammatory model of both acute and chronic angiogenesis in the rabbit cornea using a known directly angiogenic cytokine.Methods: Pellets made of the slow-release polymer Hydron (polyhydroxyethylmethacrylate) and containing sucralfate and/or basic fibroblast growth factor (basic-FGF) were implanted into rabbit corneas. The neovascular response to the implantation of pellets containing basic-FGF alone, sucralfate alone or a titration of basic-FGF in the presence of a constant amount of sucralfate was measured. The role of inflammation in the neovascular response was also investigated.Results: The addition of sucralfate to the pellets led to the sustained release of basic-FGF resulting in a predictable and aggressive neovascular response with a low dose of basic-FGF that by itself was unable to elicit neovascularisation. At a dose of 500ng per pellet, approximately one-third of the surface area of the cornea was vascularised within eight days of implantation. Minimal or no vascularisation occurred with the same dose of basic-FGF without sucralfate. While this dose of basic-FGF induced corneal oedema, only minimal inflammation was observed and the response was unaffected by ionising radiation. A less aggressive though still robust neovascular response with no or only minimal oedema was observed when the dose was lowered to 50ng of basic-FGF per pellet. Some induced vessels persisted for more than three months.Conclusion: This is an inexpensive in vivo model of angiogenesis with the advantages of the neovascularisation being aggressive, predictable, persistent, unassociated with an obvious inflammatory response and induced by the sustained release of an agent known to have a direct stimulatory action on endothelial cells.
Intravitreal VEGF and bFGF produce florid retinal neovascularization and hemorrhage in the rabbit
Current Eye Research, 2001
Purpose. Vascular endothelial growth factor (VEGF) causes widespread retinal vascular dilation, produces breakdown of the blood-retinal barrier, and is implicated in ocular neovascularization (NV). Basic fibroblast growth factor (bFGF) also has been implicated in the production of ocular NV. This study was performed to investigate the ability of simultaneous sustained intravitreal release of both VEGF and bFGF to induce robust retinal NV in the rabbit.
American journal of ophthalmology, 1989
Injection of activated macrophages into the posterior vitreous of the rabbit induced vigorous fibrovascular proliferation over the optic disk and medullary rays, as demonstrated by 3H-thymidine autoradiography. One week after injection, endothelial cells and pericytes of the capillaries near the inner surface of the optic disk and rays were labeled; fibroblast-like cells, which were also labeled, migrated and formed vitreous strands. By the second week after injection, the fibrovascular tissue proliferated most actively, and traction medullary ray detachment and peripapillary retinal fold formation were observed. The cellular proliferation was accompanied by inflammatory cell infiltration. Glial cells within the optic disk, as well as retinal pigment epithelial cells beneath the detached retina, were labeled by 3H-thymidine. These results demonstrate that the fibrovascular proliferation originates from the vessel complex of the optic disk and medullary rays in this experimental mode...
Proceedings of the National Academy of Sciences, 1999
We developed an experimental approach with genetically engineered and encapsulated mouse NIH 3T3 fibroblasts to delay the progressive degeneration of photoreceptor cells in dark-eyed Royal College of Surgeons rats. These xenogeneic fibroblasts can survive in 1.5-mm-long microcapsules made of the biocompatible polymer AN69 for at least 90 days under in vitro and in vivo conditions because of their stable transfection with the gene for the 18-kDa form of the human basic fibroblast growth factor (hFGF-2). Furthermore, when transferred surgically into the vitreous cavity of 21-day-old Royal College of Surgeons rats, the microencapsulated hFGF-2-secreting fibroblasts provoked a local delay of photoreceptor cell degeneration, as seen at 45 days and 90 days after transplantation. This effect was limited to 2.08 mm 2 (45 days) and 0.95 mm 2 (90 days) of the retinal surface. In both untreated eyes and control globes with encapsulated hFGF-2-deficient fibroblasts, the rescued area (of at most 0.08 mm 2 ) was significantly smaller at both time points. Although, in a few ocular globes, surgical trauma induced a reorganization of the retinal cytoarchitecture, neither microcapsule rejection nor hFGF-2-mediated tumor formation were detected in any treated eyes. These findings indicate that encapsulated fibroblasts secreting hFGF-2 or perhaps other agents can be applied as potential therapeutic tools to treat retinal dystrophies.
A new model of experimental subretinal neovascularization in the rabbit
Experimental Eye Research, 2006
Existing animal models of choroidal neovascularization (CNV) present several problems: they are hard to reproduce, they are inefficient, and the CNV created is not sustainable. The purpose of this study is to develop a highly efficient, reliable, sustainable rabbit model of CNV to facilitate the study of anti-angiogenic and anti-proliferative therapies for ocular diseases. Twenty-two pigmented rabbits were used in this study. Eleven rabbits received subretinal injections of either 10 ml of Matrigel with 500 ng of vascular endothelial growth factor (VEGF) or 20 ml of Matrigel with 750 ng of VEGF; eight rabbits received subretinal injections of either 10 or 20 ml of Matrigel only; three rabbits used as controls received subretinal injections of 20 ml phosphate-buffered saline (PBS) alone. Fundus photography, fluorescein angiography, optical coherence tomography, and histologic examinations were performed 1, 2, 4, and 9 weeks after injection. All experimental eyes showed angiographic leakage within this localized area 1 week after injection. The amount of leakage usually increased at weeks 2 and 4 and, in most cases, persisted at week 9. Control eyes demonstrated no leakage at any time point. Optical coherence tomography of treated eyes showed subretinal fluid and the presence of a lesion, possibly vascular or fibrotic, at the site of the leakage. Histologic analysis confirmed the presence of new subretinal blood vessels in the areas of Matrigel deposit. In conclusion, this novel method provides a highly reproducible, reliable, and sustainable rabbit model of experimental choroidal neovascularization. Such a model may prove useful for screening new anti-angiogenic therapies in a larger animal eye.