Chikako Yamamoto

Vascular endothelial growth factor expression in choroidal neovascularization in rats

Background: The pathogenesis of choroidal neovascularization is largely unknown. We investigated vascular endothelial growth factor (VEGF) expression in laser-induced choroidal neovascularization (CNV) in rats.Methods: Intense krypton laser photocoagulation was applied to the posterior poles of the eyes of pigmented rats to induce CNV, which was confirmed by fluorescein angiography and histopathology. The eyeballs were enucleated 1, 3, 7, 14 and 28 days after laser photocoagulation. Cryostat sections were prepared for immunofluorescence staining using anti-VEGF and macrophage marker (ED1) antibodies. The posterior segments of eyeballs pooled from photocoagulated and control rats were submitted for immunoprecipitation and immunoblotting by the anti-VEGF antibody, and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VEGF mRNA.Results: Very weak immunoreactivity for anti-VEGF antibody was found in the ganglion cell layer, inner nuclear layer, and retinal pigment epithelium (RPE) in the normal retina. In the development of CNV, strong positive staining for anti-VEGF antibody was found in photocoagulated areas in the subretinal space and choroid. Double immunofluorescence staining showed that many cells in lasered lesions were positive both for anti-VEGF and macrophage marker ED1 antibody staining in the early stage of this model. Immunoblots showed a positive band for the VEGF molecule in treated but not control animals. RT-PCR results demonstrated upregulation of VEGF transcripts in the CNV model compared with normal animals.Conclusions: Our findings showed the upregulation of VEGF expression in experimentally induced CNV, where it may be involved in promoting choroidal angiogenesis. Macrophages may be one of the main sources of VEGF in the early stage of the disease.

Expression of basic fibroblast growth factor mRNA in developing choroidal neovascularization

PURPOSE Basic fibroblast growth factor (bFGF) is an angiogenic peptide that may be important in the pathogenesis of choroidal neovascularization. We attempted to determine the transcription of the bFGF gene during the development of experimentally induced choroidal neovascularization. METHODS Rat bFGF cDNA was inserted in the pBluescript to prepare antisense and sense riboprobes. Multiple krypton laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing subretinal neovascularization in these animals. At intervals of up to 4 weeks after photocoagulation, the eyes were removed and cut into thin sections. The sections were subjected to histopathological analysis, cell proliferation study, or in situ hybridization with digoxigenin (DIG)-labeled single-strand riboprobes synthesized from rat bFGF cDNA. RESULTS In normal adult rat retinas, bFGF mRNA expression was mainly observed in the ganglion cell layer and the inner nuclear layer. After laser photocoagulation, proliferation of RPE cells, fibroblast-like cells and cells in the choroid in the lesions were observed. Expression of bFGF mRNA was observed in the lesions 3 days to 2 weeks after laser treatment. Signals of bFGF mRNA were detected in the proliferating RPE-like cells, choroidal vascular endothelial cells and fibroblast-like cells, all of which are essential for neovascularization. However, bFGF mRNA expression was no longer detectable in these cells 4 weeks after photocoagulation. CONCLUSIONS Our findings indicate that bFGF is normally transcribed in ganglion cells and the inner nuclear cell layer. During the neovascularization that followed laser photocoagulation, bFGF mRNA expression was detected within the laser lesions. It is thus probable that bFGF acts as a mediator in the neovascularization process.

Immunolocalization of basic fibroblast growth factor during wound repair in rat retina after laser photocoagulation.

Abstract•Background: Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key role in wound repair. We studied the immunohistochemical localization of bFGF during wound repair in the rat retina after laser photocoagulation.• Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were enucleated on days 1, 3, 7, 14 and 28 after the photocoagulation, and the immunohistochemical localization of bFGF was assessed. Two different monoclonal antibodies and one polyclonal antibody against bFGF as first antibodies were used.• Results: Marked immunoreactivity for bFGF was found in the ganglion cell layer, and weak immunoreactivity for bFGF was found in the retinal pigment epithelial (RPE) cells of the normal adult rat retina. On day 3 after laser photocoagulation, the nuclei and cytoplasm of proliferating RPE cells at the center of the photocoagulated lesion showed intense bFGF immunoreactivity. The nuclei of RPE cells around the lesion showed intense bFGF immunoreactivity. Macrophages that migrated into the lesion showed positive staining for bFGF. These immunoreactivity decreased with time. Controls (0.05 M Tris-HCl buffer, normal serum, or these same antibodies preabsorbed with bFGF) did not show positive staining.• Conclusion: The finding of an elevated expression of bFGF immunoreactivity in the photocoagulated lesion suggests that bFGF may play a role in wound repair in the rat retina after laser photocoagulation.

Immunolocalization of transforming growth factor β during wound repair in rat retina after laser photocoagulation

Abstract• Background: Scatter photocoagulation induces regression of retinal neovascularization, but the mechanism of its therapeutic effect is incompletely understood. To elucidate the mechanism of therapeutic effect of photocoagulation is the main focus of our research. We have already demonstrated basic fibroblast growth factor (bFGF) immunolocalization during retinal wound repair following laser photocoagulation. Transforming growth factor beta (TGF β) reportedly inhibits endothelial cell growth and bFGF-induced cell proliferation in vitro. In the present study, we evaluated the immunohistochemical localization of TGF-β1 and -β2 during wound repair in the rat retina following laser photocoagulation. • Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were then enucleated on day 1, 3, 7, 14, 28 or 56 following the photocoagulation and enrolled into the analysis of immunohistochemical localization of TGF-β1 and -β2. • Results: Immunoreactivity for TGF-β1 and -β2 was present in the ganglion cell layer and photoreceptor outer segments of the normal adult rat retina. The cytoplasm of RPE cells at the photocoagulated lesion showed intense TGF-β1 and -β2 immunoreactivity on day 3 after laser photocoagulation. Macrophages that migrated into the lesion lacked positive staining for TGF-β1 and -β2. TGF-β immunoreactivity in RPE cells continued to be upregulated for more than 1 month compared with that in normal RPE cells. Controls did not exhibit any positive staining. • Conclusion: An elevated expression of TGF-β immunoreactivity for a longer period of time than bFGF was observed in RPE cells at the photocoagulated lesion in vivo. In the late phase of retinal wound repair, TGF-β may inhibit cell proliferation induced by mitogens, introduce an end stage of cellular events, and induce extracellular matrix induction.

Expression of basic fibroblast growth factor and its receptor mRNA in retinal tissue following ischemic injury in the rat

Abstract · Background: Our purpose was to determine the time-dependent changes of expression of basic fibroblast growth factor (bFGF) and its receptor in pressure-induced retinal ischemia in rats. · Methods: Retinal ischemia was induced in Wistar rats by increasing the intraocular pressure to 110 mmHg for 45 min by cannulation into the eyes. At the end of the ischemic period, reperfusion of the retinal vasculature was confirmed. Localization of bFGF and FGF receptor-1 (FGF-R) mRNAs were evaluated by in situ hybridization at various times after reperfusion. The reverse-transcription polymerase chain reaction (RT-PCR) method was used to detect the expression of bFGF and FGF-R mRNA in the sensory retina. · Results: In normal sensory retina, bFGF and FGF-R mRNAs were observed in the ganglion cell layer and inner nuclear layer. bFGF gene expression in the sensory retina increased within 24 h, particularly at 6–12 h. FGF-R gene expression increased earlier than that of bFGF. By RT-PCR, expression of bFGF gene reached a peak at 6–24 h, and FGF-R reached a peak at 3–12 h. These RT-PCR results are comparable to those of in situ hybridization. · Conclusions: These results demonstrate that transient retinal ischemia leads to the induction of bFGF mRNA synthesis, and suggest that bFGF has a protective role, e.g., a defense mechanism for the sensory retina.

Expression of Transforming Growth Factor-β Receptors in Normal Rat Retina and Experimental Choroidal Neovascularization

PURPOSE Transforming growth factor-beta (TGF-beta) plays an important role in the development of choroidal neovascularization. TGF-beta transduces signals through the mediation of type I and type II receptors. We investigated the expression of TGF-beta receptors in a normal rat retina and a model of experimentally induced choroidal neovascularization. METHODS Choroidal neovascularization was induced by laser photocoagulation in rat eyes. The expression of TGF-beta receptors was determined using immunohistochemical and in situ hybridization methods. RESULTS In normal adult rat retinas, immunoreactivity and mRNA expression of TGF-beta receptor type I (TbetaRI) and TGF-beta receptor type II (TbetaRII) were found in the ganglion cells. During the process of neovascularization, immunoreactivity and mRNA expression of TbetaRI and TbetaRII were widely distributed in laser lesions soon after photocoagulation; thereafter, these receptors were specifically detected in the endothelial cells of choroidal neovascularization. CONCLUSIONS The expression of TGF-beta receptors in normal rat retinas suggests that TGF-beta plays an important role in the homeostasis of normal retina. The upregulation of TGF-beta receptors in choroidal neovascularization strongly suggests that TGF-beta is most likely transduced through specific receptors and plays an important role in the development of choroidal neovascularization.