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Eiichi Hasegawa,
Koh-Hei Sonoda,
Takashi Shichita,
Rimpei Morita,
Takashi Sekiya,
Akihiro Kimura, Yuji Oshima,
Atsunobu Takeda,
Takeru Yoshimura,
Shigeo Yoshida,
Tatsuro Ishibashi,
Akihiko Yoshimura
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ABSTRACT: Choroidal neovascularization (CNV) is a characteristic of age-related macular degeneration. Genome-wide association studies have provided evidence that the immune system is involved in the pathogenesis of age-related macular degeneration; however, the role of inflammatory cytokines in CNV has not been established. In this study, we demonstrated that IL-17 had a strong potential for promoting neovascularization in a vascular endothelial growth factor-independent manner in laser-induced experimental CNV in mice. Infiltrated γδT cells and Thy-1(+) innate lymphoid cells, but not Th17 cells, were the main sources of IL-17 in injured eyes. IL-23 was dispensable for IL-17 induction in the eye. Instead, we found that IL-1β and high-mobility group box 1 strongly promoted IL-17 expression by γδT cells. Suppression of IL-1β and high-mobility group box 1, as well as depletion of γδT cells, reduced IL-17 levels and ameliorated experimental CNV. Our findings suggest the existence of a novel inflammatory cytokine network that promotes neovascularization in the eye.
The Journal of Immunology 01/2013; · 5.79 Impact Factor
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Ryo Asato,
Shigeo Yoshida,
Atsushi Ogura,
Takahito Nakama,
Keijiro Ishikawa,
Shintaro Nakao,
Yukio Sassa,
Hiroshi Enaida, Yuji Oshima,
Kazuho Ikeo,
Takashi Gojobori,
Toshihiro Kono,
Tatsuro Ishibashi
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ABSTRACT: BACKGROUND: Proliferative vitreoretinopathy (PVR) is a destructive complication of retinal detachment and vitreoretinal surgery which can lead to severe vision reduction by tractional retinal detachments. The purpose of this study was to determine the gene expression profile of epiretinal membranes (ERMs) associated with a PVR (PVR-ERM) and to compare it to the expression profile of less-aggressive secondary ERMs. METHODOLOGY/PRINCIPAL FINDINGS: A PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from ERMs obtained during vitrectomy. The sequence from the 5' end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). We obtained 1116 nonredundant clusters representing individual genes expressed in PVR-ERMs, and 799 clusters representing the genes expressed in secondary ERMs. The transcriptome of the PVR-ERMs was subdivided by functional subsets of genes related to metabolism, cell adhesion, cytoskeleton, signaling, and other functions, by FatiGo analysis. The genes highly expressed in PVR-ERMs were compared to those expressed in the secondary ERMs, and these were subdivided by cell adhesion, proliferation, and other functions. Querying 10 cell adhesion-related genes against the STRING database yielded 70 possible physical relationships to other genes/proteins, which included an additional 60 genes that were not detected in the PVR-ERM library. Of these, soluble CD44 and soluble vascular cellular adhesion molecule-1 were significantly increased in the vitreous of patients with PVR. CONCLUSIONS/SIGNIFICANCE: Our results support an earlier hypothesis that a PVR-ERM, even from genomic points of view, is an aberrant form of wound healing response. Genes preferentially expressed in PVR-ERMs may play an important role in the progression of PVR and could be served as therapeutic targets.
PLoS ONE 01/2013; 8(1):e54191. · 4.09 Impact Factor
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Shigeo Yoshida,
Takahito Nakama,
Keijiro Ishikawa,
Mitsuru Arima,
Takashi Tachibana,
Shintaro Nakao,
Yukio Sassa,
Miho Yasuda,
Hiroshi Enaida, Yuji Oshima,
Toshihiro Kono,
Tatsuro Ishibashi
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ABSTRACT: Purpose. We determined whether the concentrations of VEGF, erythropoietin, and endostatin in the vitreous are altered after vitrectomy in patient with proliferative diabetic retinopathy (PDR). Methods. We measured the levels of VEGF, erythropoietin, and endostatin by sandwich ELISA in vitreous samples collected from 38 eyes of 33 patients with PDR before pars plana vitrectomy (without IOL implantation) and the same 38 eyes during IOL implantation 3.1 to 25.7 (mean 6.7) months after the initial vitrectomy. Results. The mean vitreous levels of VEGF (964.5 pg/mL) and erythropoietin (1359.5 pg/mL) in the samples collected before vitrectomy were significantly higher in patients with PDR than in the control patients (0.68 and 70.7 pg/mL, respectively; P < 0.01). The levels of VEGF (292.5 pg/mL) and erythropoietin (557.9 pg/mL) in the samples from eyes with PDR collected at the time of IOL implantation were significantly lower than those collected before vitrectomy (P < 0.01). In contrast, the changes in the level of endostatin were not significant after vitrectomy. The VEGF and erythropoietin levels in the vitreous fluid from patients with PDR were correlated inversely with the interval between the initial vitrectomy and the time of the IOL implantation. Conclusions. The significant decrease in the intravitreal concentration of VEGF and erythropoietin, and an absence of a significant change in the endostatin indicated a shift in the antiangiogenic balance in the vitreous of patients with PDR after successful vitrectomy.
Investigative ophthalmology & visual science 09/2012; 53(11):6997-7003. · 3.43 Impact Factor
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Mitsuru Arima,
Shigeo Yoshida,
Takahito Nakama,
Keijiro Ishikawa,
Shintaro Nakao,
Takeru Yoshimura,
Ryo Asato,
Yukio Sassa,
Takeshi Kita,
Hiroshi Enaida, Yuji Oshima,
Akira Matsuda,
Akira Kudo,
Tatsuro Ishibashi
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ABSTRACT: Purpose. A timely regression of the hyaloid vascular system (HVS) is required for the normal ocular development. Although macrophages have a critical role in this process, the exact mechanism remains undetermined. Periostin is a matricellular protein involved in tissue and vascular remodeling. The purpose of our study was to determine whether periostin is involved in the HVS regression. Methods. We used wild type (WT) and periostin knockout (KO) mice. Indocyanine green angiography and immunohistochemistry with isolectin B4 were used to evaluate the HVS regression. TUNEL-labeling was used to quantify the number of apoptotic hyaloid vascular endothelial cells. F4/80 and Iba-1 staining was performed to determine the number and location of macrophages in the vitreous. The location of periostin also was investigated by immunohistochemistry. To determine the functional role of periostin, the degree of adhesion of human monocytes to fibronectin was measured by an adhesion assay. Results. The HVS regression and peak in the number of TUNEL-positive apoptotic endothelial cells were delayed in periostin KO mice. The number of F4/80 positive cells in the vitreous was higher in periostin KO mice. Only a small number of Iba-1-positive cells near the hyaloid vessels was co-stained with periostin, and peripheral blood monocytes were not stained with periostin. Adhesion assay showed that periostin increased the degree of attachment of monocytes to fibronectin. Conclusions. These results suggest that periostin, which is secreted by the intraocular macrophages, enhances the HVS regression by intensifying the adhesion of macrophages to hyaloid vessels.
Investigative ophthalmology & visual science 08/2012; 53(10):6495-503. · 3.43 Impact Factor
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ABSTRACT: AMD is the most common disease leading to acquired blindness in developed countries. CNV is the foremost cause of AMD and is thought to be induced by regional inflammation as a result of age-related conformational changes of the chorioretinal interface. Here, we show that IL-27, a member of the IL-6/IL-12 cytokine family, has an angiostatic effect and regulates the development of laser-induced experimental CNV in mice. In this model, IL-27 expression increased in the damaged choroid and peaked at the 24 h-time-point. IL-27 neutralization, induced by inoculating an antagonistic antibody into the vitreous cavity, enhanced VEGF production and the extent of CNV. By contrast, the administration of rIL-27 reduced VEGF production and the extent of CNV. Mice deficient in the EBI3, which lack IL-27, also showed more CNV than C57BL/6 mice, and this was reduced by IL-27 supplementation. We additionally investigated the effect of IL-27 on the function of macrophages, which play a critical role in CNV. IL-27 did not affect macrophage migration but inhibited its VEGF production. IL-27 therefore appears to regulate CNV and is a promising candidate target for treating sight-threatening diseases caused by ocular neovascularization.
Journal of leukocyte biology 11/2011; 91(2):267-73. · 4.99 Impact Factor
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Satoshi Arakawa,
Atsushi Takahashi,
Kyota Ashikawa,
Naoya Hosono,
Tomomi Aoi,
Miho Yasuda, Yuji Oshima,
Shigeo Yoshida,
Hiroshi Enaida,
Takashi Tsuchihashi,
Keisuke Mori,
Shigeru Honda,
Akira Negi,
Akira Arakawa,
Kazuaki Kadonosono,
Yutaka Kiyohara,
Naoyuki Kamatani,
Yusuke Nakamura,
Tatsuro Ishibashi,
Michiaki Kubo
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ABSTRACT: Age-related macular degeneration (AMD), the leading cause of irreversible blindness in the world, is a complex disease caused by multiple environmental and genetic risk factors. To identify genetic factors that modify the risk of exudative AMD in the Japanese population, we conducted a genome-wide association study and a replication study using a total of 1,536 individuals with exudative AMD and 18,894 controls. In addition to CFH (rs800292, P = 4.23 × 10(-15)) and ARMS2 (rs3750847, P = 8.67 × 10(-29)) loci, we identified two new susceptibility loci for exudative AMD: TNFRSF10A-LOC389641 on chromosome 8p21 (rs13278062, combined P = 1.03 × 10(-12), odds ratio = 0.73) and REST-C4orf14-POLR2B-IGFBP7 on chromosome 4q12 (rs1713985, combined P = 2.34 × 10(-8), odds ratio = 1.30). Fine mapping revealed that rs13278062, which is known to alter TNFRSF10A transcriptional activity, had the most significant association in 8p21 region. Our results provide new insights into the pathophysiology of exudative AMD.
Nature Genetics 09/2011; 43(10):1001-4. · 35.53 Impact Factor
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Young-Joon Jo,
Koh-Hei Sonoda, Yuji Oshima,
Atsunobu Takeda,
Ri-ichiro Kohno,
Jun Yamada,
Junji Hamuro,
Yang Yang,
Shoji Notomi,
Toshio Hisatomi,
Tatsuro Ishibashi
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ABSTRACT: Subretinal fibrosis causes damage to visual acuity, especially if the lesion is in the macula, as is frequently observed in advanced age-related macular degeneration. Exudate leukocytes form abnormal vessels that initiate regional inflammation accompanied with local glial proliferation and matrix production. The purpose of this study was to establish an animal model of focal subretinal fibrosis.
Macrophage-rich peritoneal exudate cells (PECs) were injected into the subretinal space of C57BL/6 or MCP-1 knockout (KO) mice. Seven days later, the size of the subretinal fibrotic tissue was evaluated by the adherent area of glial fibrillary acidic protein (GFAP)-positive retinal glial cells on choroidal flat mounts. Myofibroblastic changes and collagen synthesis were detected by α-smooth muscle actin (α-SMA) and Masson trichrome staining of the histologic section, respectively. α-SMA expression was also examined on retinal pigment epithelium (RPE) cells during co-culture with activated macrophages.
Subretinal fibrous tissue was observed by funduscopy in PEC-injected mice after 7 days. The tissue consisted of a monotonous, low-cell-density area that expressed α-SMA with collagen synthesis. Both steroid and antioxidant treatment can reduce residual glia. Because PEC-injected MCP-1 KO mice showed less residual glia, not only exogenous macrophages, but also intrinsic macrophages were activated. The macrophages directly induced myofibrotic changes in RPE cells in vitro.
Activated macrophages form subretinal fibrosis when they are placed in the subretinal space and induce myofibrotic changes in RPE cells.
Investigative ophthalmology & visual science 11/2010; 52(9):6089-95. · 3.43 Impact Factor
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ABSTRACT: Choroidal neovascularization (CNV) is directly related to visual loss in persons with age-related macular degeneration (AMD) and other macular disorders. Chlamydia pneumoniae, a prokaryotic pathogen that causes chronic inflammation, is recognized as a risk factor for cardiovascular diseases. In this study, the authors investigated the association between C. pneumoniae infection and AMD using a laser-induced CNV model in mice.
C57BL/6 mice, myeloid differentiation factor (MyD) 88 knockout (KO) mice, Toll-like receptor (TLR) 2 KO mice, and TLR4 KO mice were used. Experimental CNV was induced by rupturing the Bruch's membrane by laser photocoagulation (PC). Seven days after PC, the eyes were enucleated and the areas of CNV were measured in choroidal flat mounts. Cytokine gene expression by quantitative real-time PCR in the primary cultured retinal pigment epithelium (RPE) cells was also examined.
Vitreous injection of the C. pneumoniae antigen increased the size of CNV. Although lipopolysaccharide stimulation can induce multiple cytokines, cultured mouse RPE cells from C57BL/6 mice expressed IL-6 and VEGF, but not TNF-alpha mRNA, in response to C. pneumoniae antigen. RPE cells from either MyD88 KO mice or TLR2 KO mice did not respond to the C. pneumoniae antigen. TLR2 KO mice did not augment the size increase of experimental CNV by C. pneumoniae antigen in vivo.
C. pneumoniae can trigger inflammatory responses in the eye and promote experimental CNV in a TLR2-dependent manner. These data provide experimental evidence to imply persistent C. pneumoniae infection is a risk factor for AMD.
Investigative ophthalmology & visual science 09/2010; 51(9):4694-702. · 3.43 Impact Factor
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ABSTRACT: PURPOSE. The effect of antiproliferative agents with high voltage electric pulses (EP) on the proliferation of retinal pigment epithelial (RPE) cells was investigated. METHODS. Bovine RPE cells were cultured and exposed briefly to an antiproliferative agent: 0.005 to 500 µM of bleomycin (BLM), 0.005 to 500 µM of mitomycin C (MMC) or 0.05 to 5000 µM of 5-fluorouracil (5-FU) with or without high voltage electric pulses (EP; 2,000V/cm, 98 µsec, 8 pulses). Streptomycin (SM) was used as a negative control. Cell proliferation with or without antiproliferative agents was assessed on day 3. DNA fragmentation was assessed by flow cytometric analysis. RESULTS. Treatment with BLM, MMC or 5-FU inhibited cell proliferation in a dose-dependent manner, with and without EP (p < 0.05). EP enhanced the inhibitory effect of BLM as much as 3,000 times (ID50: BLM without EP; 163.7 µM, BLM with EP; 0.0574 µM, MMC without EP; 132.4 µM, MMC with EP; 26.2 µM, 5-FU without EP; 616.4 µM, 5-FU with EP; 873.8 µM). EP treatment showed an inhibitory effect of BLM on cell proliferation more prominently than BLM alone. The cell numbers of 0.5 µM BLM treatment without EP were 87.4 ± 11.7% (mean ± SD), whereas the cell numbers of 0.5 µM BLM with EP were 21.1 ± 2.16% (p < 0.005). BLM treatment with EP increased the apoptosis like DNA fragmentation in the flow cytometric DNA histogram, showing dominant accumulation in the A 0 region, which is the population of DNA fragmentation. (The cell population of the A 0 region: control, 3.4%; EP alone, 18.9%; BLM treatment without EP, 32.0%; with EP, 93.8%.) CONCLUSIONS. EP treatment enhances the inhibitory effect of BLM on RPE cell proliferation. The combination of electric pulse and BLM treatment can localize the drug effect and reduce the necessary dose of the antiproliferative agent in comparison to BLM treatment alone.
07/2009; 16(1):64-70.
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ABSTRACT: Ocular neovascularization (NV) is the primary cause of blindness in a wide range of ocular diseases. The exact mechanism underlying the pathogenesis of ocular NV is not yet well understood, and so there is no satisfactory therapy for ocular NV. Here, we describe a strategy targeting Flk-1, a self-antigen overexpressed on proliferating endothelial cells in ocular NV, by antiangiogenic immunotherapy—DNA vaccine and adoptive T cell therapy. An oral DNA vaccine encoding Flk-1 carried by attenuated Salmonella typhimurium markedly suppressed development of laser-induced choroidal NV. We further demonstrated that adoptive transfer of vaccine-induced CD8+ T cells reduced pathological preretinal NV, with a concomitant facilitation of physiological revascularization after oxygen-induced retinal vessel obliteration. However, physiological retinal vascular development was unaffected in neonatal mice transferred with vaccine-induced CD8+ T cells. These findings suggested that antiangiogenic immunotherapy targeting Flk-1 such as vaccination and adoptive immunotherapy may contribute to future therapies for ocular NV.
Biochemical and Biophysical Research Communications 03/2009; · 2.48 Impact Factor
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Aya Takeuchi,
Masaru Takeuchi,
Kosuke Oikawa,
Koh-hei Sonoda,
Yoshihiko Usui,
Yoko Okunuki,
Atsunobu Takeda, Yuji Oshima,
Keiichi Yoshida,
Masahiko Usui,
Hiroshi Goto,
Masahiko Kuroda
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ABSTRACT: Cigarette smoking is the most consistent risk factor for age-related macular degeneration (AMD), especially the choroidal neovascularization (CNV)-mediated exudative type. Dioxins and dioxin-like compounds have various effects on living organisms and are also contained in cigarette smoke. However, the effects of dioxins on the eye remain elusive. In this study, the authors examined the association between dioxins and neovascularization in the eye.
C57BL/6 mice were injected intraperitoneally with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every other day for 14 days. Messenger RNA expression of cytochrome P450 (CYP)1A1, CYP1B1, vascular endothelial growth factor (VEGF)-A and VEGF-B, and VEGF production were examined in the eyes of TCDD-treated mice and in human retinal pigment epithelial cell lines (ARPE-19) exposed to TCDD. In addition, CNV was induced by photocoagulation in mice injected with TCDD, and the volume of CNV was compared by fluorescence-labeled choroidal flat mount.
TCDD injected intraperitoneally increased CYP1A1 mRNA expression in the iris/ciliary body and retina, indicating that TCDD acts directly on ocular tissues through the aryl hydrocarbon receptor (AhR) to promote the transcription of target genes. TCDD also promoted VEGF-A mRNA expression in the retina and the retinal pigment epithelium. TCDD-induced VEGF production at the molecular level was also observed in vivo by immunohistochemistry and in vitro using ARPE-19. Moreover, the injection of TCDD significantly exacerbated photocoagulation-induced CNV in mice.
The authors demonstrate that dioxins are among the factors inducing abnormal vascularization in the eye through VEGF production mediated by AhR signaling.
Investigative ophthalmology & visual science 02/2009; 50(7):3410-6. · 3.43 Impact Factor
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ABSTRACT: Inflammation affects the formation and the progression of various vitreoretinal diseases. We performed a comprehensive analysis of inflammatory immune mediators in the vitreous fluids from total of 345 patients with diabetic macular edema (DME, n = 92), proliferative diabetic retinopathy (PDR, n = 147), branch retinal vein occlusion (BRVO, n = 30), central retinal vein occlusion (CRVO, n = 13) and rhegmatogenous retinal detachment (RRD, n = 63). As a control, we selected a total of 83 patients with either idiopathic macular hole (MH) or idiopathic epiretinal membrane (ERM) that were free of major pathogenic intraocular changes, such as ischemic retina and proliferative membranes. The concentrations of 20 soluble factors (nine cytokines, six chemokines, and five growth factors) were measured simultaneously by multiplex bead analysis system. Out of 20 soluble factors, three factors: interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were significantly elevated in all groups of vitreoretinal diseases (DME, PDR, BRVO, CRVO, and RRD) compared with control group. According to the correlation analysis in the individual patient's level, these three factors that were simultaneously increased, did not show any independent upregulation in all the examined diseases. Vascular endothelial growth factor (VEGF) was significantly elevated in patients with PDR and CRVO. In PDR patients, the elevation of VEGF was significantly correlated with the three factors: IL-6, IL-8, and MCP-1, while no significant correlation was observed in CRVO patients. In conclusion, multiplex bead system enabled a comprehensive soluble factor analysis in vitreous fluid derived from variety of patients. Major three factors: IL-6, IL-8, and MCP-1 were strongly correlated with each other indicating a common pathway involved in inflammation process in vitreoretinal diseases.
PLoS ONE 01/2009; 4(12):e8158. · 4.09 Impact Factor
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ABSTRACT: Choroidal neovascularization (CNV) is directly related to visual loss in age-related macular degeneration and other macular disorders. We have investigated the role of CD1d-restricted invariant natural killer T (NKT) cells in laser-induced experimental CNV. Quantitative real-time PCR detected increased expression of NKT cell-related genes (Valpha14 and CXCL16) in whole eyes undergoing CNV, indicating local accumulation of NKT cells. We found a significant reduction of CNV and lower concentrations of vascular endothelial growth factor (VEGF) in ocular fluid in two different NKT cell-deficient mice, CD1d knockout (KO) and Jalpha18 KO mice. We also established in vitro co-cultures of retinal pigment epithelial cells and splenic NKT cells, and confirmed NKT cells could produce VEGF in the dish. Moreover, inoculating alpha-galactosylceramide, the ligand for NKT cells, into the vitreous cavity of C57BL/6 mice promoted CNV. We concluded that NKT cells play an important role in CNV as an inducer of VEGF.
Biochemical and Biophysical Research Communications 08/2008; 374(1):38-43. · 2.48 Impact Factor
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ABSTRACT: Retinal neovascularization (NV) and macular edema, resulting from blood-retinal barrier (BRB) breakdown, are major causes of visual loss in ischemic retinopathies. Choroidal NV (CNV) occurs in diseases of the retinal pigmented epithelium/Bruch's membrane complex and is another extremely prevalent cause of visual loss. We used mice in which the hypoxia response element (HRE) is deleted from the vascular endothelial growth factor (vegf) promoter (Vegf(delta/delta) mice) to explore the role of induction of VEGF through the HRE in these disease processes. Compared to wild type (Vegf+/+) mice with oxygen-induced ischemic retinopathy (OIR) in which vegf mRNA levels were increased and prominent retinal NV and BRB breakdown occurred, Vegf(delta/delta) littermates with OIR failed to increase vegf mRNA levels in the retina and had significantly less retinal NV and BRB breakdown, but showed prominent dilation of some superficial retinal vessels. Vegf(+/delta) littermates with ischemic retinopathy developed comparable retinal NV to Vegf+/+ mice, exhibited intermediate levels of BRB breakdown, and did not show vasodilation. In a mouse model of CNV, due to laser-induced rupture of Bruch's membrane, the area of CNV at Bruch's membrane rupture sites was more than tenfold greater in Vegf+/+ mice than in Vegf(delta/delta) littermates. In contrast to these dramatic differences in pathologic ocular NV, Vegf(delta/delta) mice showed subtle differences in retinal vascular development compared to Vegf+/+ mice; it was slightly delayed, but otherwise normal. These data suggest that induction of VEGF through the HRE in its promoter is critical for retinal and CNV, but not for retinal vascular development.
Journal of Cellular Physiology 04/2006; 206(3):749-58. · 3.87 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF) plays a central role in vasoproliferative diseases in the retina, however, other gene products modulate its effects. The angiopoietins are particularly important in this regard. Angiopoietin 2 (Ang2) collaborates with VEGF to stimulate neovascularization (NV) in some situations, but in other situations causes regression of NV. Ang2 also causes a transient increase in vascular density during retinal vascular development. In this study, we sought to determine if Ang1 has similar activities. The effects of Ang1 were tested in double transgenic mice with inducible expression of Ang1. Increased expression of Ang1 in the retina during retinal vascular development did not cause a detectable alteration in vascular density. Also, unlike Ang2, increased expression of Ang1 had no effect on established retinal or choroidal NV. However, when Ang1 expression was initiated simultaneously with that of VEGF, it strongly suppressed VEGF-induced NV and prevented retinal detachment. These data indicate that the timing of Ang1 expression is a critical determinate of its effects on VEGF-induced NV in the retina; it effectively blocks the initiation and progression of NV, but cannot reverse established NV or reduce leakage from NV. These data suggest that increased expression of Ang1 may be a good strategy for prophylaxis of retinal NV, but is unlikely to be effective as monotherapy of established NV.
Journal of Cellular Physiology 08/2005; 204(1):227-35. · 3.87 Impact Factor
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Yuji Oshima,
Sachiko Oshima,
Hiroyuki Nambu,
Shu Kachi,
Kyoichi Takahashi,
Naoyasu Umeda,
JiKui Shen,
Aling Dong,
Rajendra S Apte,
Elia Duh,
Sean F Hackett,
Godwin Okoye,
Kazuki Ishibashi,
James Handa,
Michele Melia,
Stanley Wiegand,
George Yancopoulos,
Donald J Zack,
Peter A Campochiaro
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ABSTRACT: In this study, we used double transgenic mice with inducible expression of angiopoietin-2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal-appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF-induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.
The FASEB Journal 07/2005; 19(8):963-5. · 5.71 Impact Factor
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Yuji Oshima,
Sachiko Oshima,
Hiroyuki Nambu,
Shu Kachi,
Sean F Hackett,
Michele Melia,
Michael Kaleko,
Sheila Connelly,
Noriko Esumi,
Donald J Zack,
Peter A Campochiaro
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ABSTRACT: Increased expression of vascular endothelial cell growth factor (VEGF) in the retina is sufficient to stimulate sprouting of neovascularization from the deep capillary bed of the retina, but not the superficial retinal capillaries or the choriocapillaris. Coexpression of VEGF and angiopoietin 2 (Ang2) results in sprouting of neovascularization from superficial and deep retinal capillaries, but not the choriocapillaris. However, retina-derived VEGF and Ang2 may not reach the choriocapillaris, because of tight junctions between retinal pigmented epithelial (RPE) cells. To eliminate this possible confounding factor, we used the human vitelliform macular dystrophy 2 (VMD2) promoter, an RPE-specific promoter, combined with the tetracycline-inducible promoter system, to generate double transgenic mice with inducible expression of VEGF in RPE cells. Adult mice with increased expression of VEGF in RPE cells had normal retinas and choroids with no choroidal neovascularization (CNV), but when increased expression of VEGF in RPE cells was combined with subretinal injection of a gutless adenoviral vector containing an expression construct for Ang2 (AGVAng2), CNV consistently occurred. In contrast, triple transgenic mice with induced expression of Ang2 and VEGF in RPE cells, did not develop CNV. These data suggest that increased expression of VEGF and/or Ang2 in RPE cells is not sufficient to cause CNV unless it is combined with a subretinal injection of a gutless adenoviral vector, which is likely to perturb RPE cells. These data also suggest that the effects of angiogenic proteins may vary among vascular beds, even those that are closely related, and, therefore, generalizations should be avoided.
Journal of Cellular Physiology 01/2005; 201(3):393-400. · 3.87 Impact Factor
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Yuji Oshima,
Kyoichi Takahashi,
Sachiko Oshima,
Yoshitsugu Saishin,
Yumiko Saishin,
Raquel Lima Silva,
Xaoling Liang,
P Seshidhar Reddy,
Shanthi Ganesh,
Terrence Brann,
Gene Liau,
Michael Kaleko,
Sheila Connelly,
Peter A Campochiaro
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ABSTRACT: Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.
Journal of Cellular Physiology 07/2004; 199(3):399-411. · 3.87 Impact Factor
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ABSTRACT: Increased expression of vascular endothelial growth factor (VEGF) in the retina starting after postnatal day (P)7 results in neovascularization originating from deep retinal capillaries, but not those in the superficial capillary bed. Doxycycline was administered starting P0 to double transgenic mice with inducible expression of VEGF in the retina. These mice showed proliferation and dilation of superficial retinal capillaries, indicating that at this stage of development, the superficial capillaries are sensitive to the effects of VEGF. Angiopoietin-2 (Ang2) is expressed along the surface of the retina for several days after birth, but by P7 and later, Ang2 is only expressed in the region of the deep capillary bed. In mice with ubiquitous doxycycline-inducible expression of Ang2, in the absence of doxycycline, intravitreous injection of a gutless adenoviral vector expressing VEGF (AGV.VEGF) resulted in neovascularization of the cornea and iris, but no retinal neovascularization. After treatment with doxycycline to induce Ang2 expression, intravitreous injection of AGV.VEGF caused retinal neovascularization in addition to corneal and iris neovascularization. The retinal neovascularization originated from both the superficial and deep capillary beds. These data suggest that Ang2 promotes sensitivity to the angiogenic effects of VEGF in retinal vessels.
Journal of Cellular Physiology 07/2004; 199(3):412-7. · 3.87 Impact Factor
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ABSTRACT: The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photo-receptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5'-up-stream region of human VMD2, a gene that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Transgenic mouse studies with VMD2 promoter/lacZ constructs demonstrated that a-253 to +38 bp fragment is sufficient to direct RPE-specific expression in the eye. Transient transfection assays using the D407 human RPE cell line with VMD2 promoter/luciferase reporter constructs identified two positive regulatory regions, -585 to -541 bp for high level expression and -56 to -42 bp for low level expression. Mutation of a canonical E-box located in the -56 to -42 bp region greatly diminished luciferase expression in D407 cells and abolished the bands shifted with bovine RPE nuclear extract in electrophoretic mobility shift assays. Independently a candidate approach was used to select microphthalmia-associated transcription factor (MITF) for testing because it is expressed in the RPE and associated with RPE abnormalities when mutated. MITF-M significantly increased luciferase expression in D407 cells in an E-box-dependent manner. These studies define the VMD2 promoter region sufficient to drive RPE-specific expression in the eye, identify positive regulatory regions in vitro, and suggest that MITF as well as other E-box binding factors may act as positive regulators of VMD2 expression.
Journal of Biological Chemistry 05/2004; 279(18):19064-73. · 4.77 Impact Factor
