Hideyasu Oh

Hypoxia and Vascular Endothelial Growth Factor Selectively Up-regulate Angiopoietin-2 in Bovine Microvascular Endothelial Cells

Recent studies have shown that the angiopoietin-Tie2 system is a predominant regulator of vascular integrity. In this study, we investigated the effect of two known angiogenic stimuli, hypoxia and vascular endothelial growth factor (VEGF), on these molecules. VEGF induced both a time- and concentration-dependent increase in angiopoietin-2 (Ang2) mRNA expression in bovine microvascular endothelial cells. This up-regulation was derived primarily from an increased transcription rate as evidenced by nuclear run-on assay and mRNA decay study. The increased Ang2 expression upon VEGF treatment was almost totally abolished by inhibition of tyrosine kinase or mitogen-activated protein kinase and partially by suppression of protein kinase C. Hypoxia also directly increased Ang2 mRNA expression. In contrast, Ang1 and Tie2 responded to neither of these stimuli. The enhanced Ang2 expression following VEGF stimulation and hypoxia was accompanied by de novo protein synthesis as detected by immunoprecipitation. In a mouse model of ischemia-induced retinal neovascularization, Ang2 mRNA was up-regulated in the ischemic inner retinal layer, and remarkable expression was observed in neovascular vessels. These data suggest that both hypoxia- and VEGF-induced neovascularization might be facilitated by selective induction of Ang2, which deteriorates the integrity of preexisting vasculature.

Selective induction of neuropilin-1 by vascular endothelial growth factor (VEGF): A mechanism contributing to VEGF-induced angiogenesis

Neuropilin (NRP) 1, previously identified as a neuronal receptor that mediates repulsive growth cone guidance, has been shown recently to function also in endothelial cells as an isoform-specific receptor for vascular endothelial growth factor (VEGF)165 and as a coreceptor in vitro of VEGF receptor 2. However, its potential role in pathologic angiogenesis remains unknown. In the present study, we first show that VEGF selectively up-regulates NRP1 but not NRP2 via the VEGF receptor 2-dependent pathway. By NRP1 binding analysis, we showed that its induction by VEGF accompanies functional receptor expression. Endothelial proliferation stimulated by VEGF165 was inhibited significantly by antibody perturbation of NRP1. In a murine model of VEGF-dependent angioproliferative retinopathy, intense NRP1 mRNA expression was observed in the newly formed vessels. Furthermore, selective NRP1 inhibition in this model suppressed neovascular formation substantially. These results suggest that VEGF cannot only activate endothelial cells directly but also can contribute to robust angiogenesis in vivo by a mechanism that involves up-regulation of its cognate receptor expression.

Phosphatidylinositol 3-Kinase/Akt Regulates Angiotensin II–Induced Inhibition of Apoptosis in Microvascular Endothelial Cells by Governing Survivin Expression and Suppression of Caspase-3 Activity

Abstract— Angiotensin II (Ang II) plays essential roles in vascular homeostasis, neointimal formation, and postinfarct remodeling. Although Ang II has been shown to regulate apoptosis in cardiomyocytes and vascular smooth muscle cells, its role in vascular endothelial cells (ECs) remains elusive. To address this issue, we first performed TUNEL and caspase-3 activity assays with porcine microvascular ECs challenged by serum deprivation. Ang II significantly reduced the ratio of apoptotic cells and caspase-3 activity. The Ang II type 1 receptor (AT1) was responsible for these effects. Among the signaling molecules downstream of AT1, we revealed that PI3-kinase/Akt pathway plays a predominant role in the antiapoptotic effect of Ang II. Interestingly, the expression of survivin, a central molecule of cell survival, increased after Ang II stimulation. Overexpression of a dominant-negative form of Akt abolished both Ang II–induced antiapoptosis and survivin protein expression. In a murine model of hyperoxygen-induced retinal vascular regression, AT1a knockout mice showed a significant increase in retinal avascular areas. Our data indicate that Ang II plays a critical antiapoptotic role in vascular ECs by a mechanism involving PI3-kinase/Akt activation, subsequent upregulation of survivin, and suppression of caspase-3 activity.

Serous Retinal Detachment Associated With Retinal Vein Occlusion

PURPOSE To study the pathomorphology of serous retinal detachment (RD) associated with retinal vein occlusion by optical coherence tomography (OCT). DESIGN Retrospective chart review. METHODS Ninety-one eyes of 91 patients with macular edema associated with retinal vein occlusion had undergone a comprehensive ophthalmologic examination, including measurement by spectral-domain OCT. RESULTS Eyes with macular edema associated with retinal vein occlusion typically showed foveal cystoid spaces and marked retinal swelling, especially in the outer retina. In addition, 76 eyes (83.5%) showed serous RD involving the fovea, which ranged in thickness from 64 microm to 871 microm (219.2 +/- 161.6 microm). Fifty-two eyes showed a small pointed RD, with a small base. The point of the RD was always located beneath the fovea, where the outer surface of the swollen neurosensory retina seemed to be contracted inward, resulting in development of the pointed RD. Two eyes with no RD at the initial visit developed such a pointed RD during follow-up. In contrast, 24 eyes showed a more dome-shaped RD, with a large base, and in 18 eyes, a pointed RD seen at the initial visit changed into a dome-shaped RD during follow-up. In some cases, small outer retinal discontinuity was seen on the external surface of the swollen neurosensory retina. CONCLUSIONS In eyes with retinal vein occlusion, a small pointed RD initially developed just beneath the fovea, but subsequently changed into a dome-shaped RD. Based on the findings by OCT, we hypothesize that the foveal architecture, especially that of the Müller cell cone, is involved in the formation of serous RD.

Comparison of the Effect of Ranibizumab and Verteporfin for Polypoidal Choroidal Vasculopathy: 12-Month LAPTOP Study Results

PURPOSE To compare the effect of photodynamic therapy (PDT) and intravitreal ranibizumab in patients with polypoidal choroidal vasculopathy (PCV). DESIGN Randomized clinical trial. METHODS SETTING Multicenter. STUDY POPULATION Total of 93 patients with treatment-naïve PCV. INTERVENTION Patients were randomized to 2 arms. Patients in the PDT arm underwent a single session of PDT with verteporfin, and patients in the ranibizumab arm received 3 monthly ranibizumab injections at baseline. Additional treatment was performed as needed in each arm. MAIN OUTCOME MEASURES Primary outcome measurement was the proportion of patients gaining or losing more than 0.2 logarithm of minimal angle of resolution (logMAR) units from baseline. Mean change of logMAR and central retinal thickness (CRT) were also evaluated. RESULTS In the PDT arm (n = 47), 17.0% achieved visual acuity gain, 55.3% had no change, and 27.7% experienced visual acuity loss. The results were 30.4%, 60.9%, and 8.7%, respectively, in the ranibizumab arm (n = 46), significantly better than the PDT arm (P = .039). In the PDT arm, mean CRT improved (366.8 ± 113.6 μm to 289.1 ± 202.3 μm, P < .001), but logMAR was unchanged (0.57 ± 0.31 to 0.62 ± 0.40). The ranibizumab arm demonstrated improvement in both CRT (418.9 ± 168.6 μm to 311.2 ± 146.9 μm, P < .001) and logMAR (0.48 ± 0.27 to 0.39 ± 0.26, P = .003). Mean change of logMAR was also greater in the ranibizumab arm (P = .011). CONCLUSION Intravitreal injection of ranibizumab is more effective than PDT for treatment-naïve PCV.

Expression of Thrombospondin-1 in Ischemia- Induced Retinal Neovascularization

Thrombospondin-1 is an extracellular matrix protein that inhibits endothelial cell proliferation, migration, and angiogenesis. This study was performed to investigate the role of thrombospondin-1 in ischemic retinal neovascularization. In a murine model of retinal neovascularization, thrombospondin-1 mRNA was increased from postnatal day 13 (P13), with a threefold peak response observed on P15, corresponding to the time of development of retinal neovascularization. Prominent expression of thrombospondin-1 was observed in neovascular cells, specifically, cells adjacent to the area of nonperfusion. It has been suggested that vascular endothelial growth factor (VEGF) plays a major role in ischemia-induced retinal neovascularization of this model, so we studied the effects of VEGF on thrombospondin-1 expression. In bovine retinal microcapillary endothelial cells, VEGF induced a biphasic response of thrombospondin-1 expression; VEGF decreased thrombospondin-1 mRNA 0.41-fold after 4 hours, whereas it increased, with a threefold peak response, after 24 hours. VEGF-induced endothelial cell proliferation was completely inhibited by exogenous thrombospondin-1 and increased by 37.5% with anti-thrombospondin-1 antibody. The present findings suggest that, in the ischemic retina, retinal neovascular cells increase thrombospondin-1 expression, and VEGF may stimulate endogenous thrombospondin-1 induction, which inhibits endothelial cell growth. VEGF-mediated thrombospondin-1 induction in ischemia-induced angiogenesis may be a negative feedback mechanism.

Efficacy of surgical removal of the internal limiting membrane in diabetic cystoid macular edema.

Purpose: To evaluate the efficacy of surgical removal of the internal limiting membrane (ILM) in diabetic cystoid macular edema (CME). Methods: Prospective, noncomparative, interventional case series including 21 eyes of 18 consecutive patients with diabetic CME. Vitrectomy with separation of the posterior hyaloid and induction of posterior vitreous detachment had been performed previously on nine eyes. Pars plana vitrectomy for removal of the ILM was performed. Results: CME resolved in eyes that underwent initial vitrectomy and in those with long-standing (>1 year) CME after previous vitrectomy. Postoperative best-corrected visual acuity improved by ≥2 lines of a Snellen equivalent in 14 eyes (67%) (P < 0.01). The mean foveal thickness (distance between the inner retinal surface and the retinal pigment epithelium) decreased from 553 &mgr;m to 221 &mgr;m at 4 weeks (P < 0.001). No recurrences or deterioration of CME was observed during the entire follow-up period (mean, 17.8 months; range, 8–34 months). Conclusion: Surgical removal of the ILM might be an effective procedure for reducing CME in patients with diabetes. A prospective, randomized, controlled study is necessary to further evaluate the efficacy of the procedure.

Transcription Factor Ets-1 Mediates Ischemia- and Vascular Endothelial Growth Factor-Dependent Retinal Neovascularization

Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases.

Retinal structural changes associated with retinal arterial macroaneurysm examined with optical coherence tomography.

Purpose: To study the retinal structural changes associated with a retinal arterial macroaneurysm and their association with visual prognosis. Methods: We studied retrospectively the medical records of 44 eyes of 44 patients with a retinal arterial macroaneurysm; all eyes showed hemorrhagic and/or exudative changes of the fovea with a visual disturbance. The retinal structural changes were examined by optical coherence tomography (OCT). Results: At the initial visit, 30 eyes (68%) showed severe hemorrhagic complications. In eyes with subretinal hemorrhage beneath the fovea, retinal structure was relatively preserved at the initial visit. At the final visit, however, foveal structure was often degenerated, especially in its outer aspect, with limited visual recovery. The 14 eyes (32%) with minimal hemorrhagic complications showed an extensive exudative change caused by the aneurysm. In 13 of these eyes, OCT examination revealed extensive retinal edema, predominantly in the outer retina. Retinal edema beneath the fovea caused a focal serous retinal detachment in 12 eyes. Eight eyes with extensive exudative change showed an accumulation of hard exudates in the macular area, and visual recovery of these eyes was often limited. Conclusion: Subretinal hemorrhage or extensive exudative change from a retinal arterial macroaneurysm can cause destruction of the foveal outer photoreceptor layer, resulting in a poor visual outcome.

Expression of connective tissue growth factor and its potential role in choroidal neovascularization.

Background: To determine the expression of connective tissue growth factor (CTGF) in choroidal neovascularization. Methods: Surgically excised choroidal neovascular membranes (CNVMs) were obtained at vitrectomy from eight eyes with age-related macular degeneration, five eyes with high myopia, and two eyes with angioid streaks. Light microscopic immunohistochemical analysis was performed to detect CTGF, transforming growth factor β 1 (TGF-β1), vascular endothelial growth factor (VEGF), pancytokeratin, and smooth muscle actin (SMA). Results: CNVMs were classified by fibrotic status as cellular CNVM, moderate fibrous CNVM, and extensive fibrous CNVM. CTGF expression was found in vascular cells, stromal cells, and retinal pigment epithelium (RPE) cells. For the stromal cells, fibroblastlike cells were most strongly positive for CTGF. CTGF immunoreactivity in the stroma was stronger in the fibrous CNVMs than in the cellular CNVMs. Immunohistochemical analysis of serial sections revealed colocalization of CTGF with TGF-β1 and VEGF; colocalization of CTGF with pancytokeratin and SMA was also found. Conclusion: Our findings suggest that transdifferentiated RPE cells and vascular cells possess remarkable CTGF expression in CNVMs. This expression of CTGF may stimulate fibroblasts to produce extracellular matrix and may promote angiogenesis in vascular cells. Colocalized TGF-β1 and VEGF may also contribute the upregulation of CTGF.