Blood vessel endothelial VEGFR-2 delays lymphangiogenesis: an endogenous trapping mechanism links lymph- and angiogenesis.

Angio- and lymphangiogenesis are inherently related processes. However, how blood and lymphatic vessels regulate each other is unknown. This work introduces a novel mechanism explaining the temporal and spatial relation of blood and lymphatic vessels. Vascular endothelial growth factor-A (VEGF-A) surprisingly reduced VEGF-C in the supernatant of blood vessel endothelial cells, suggesting growth factor (GF) clearance by the growing endothelium. The orientation of lymphatic sprouting toward angiogenic vessels and away from exogenous GFs was VEGF-C dependent. In vivo molecular imaging revealed higher VEGF receptor (R)-2 in angiogenic tips compared with normal vessels. Consistently, lymphatic growth was impeded in the angiogenic front. VEGF-C/R-2 complex in the cytoplasm of VEGF-A-treated endothelium indicated that receptor-mediated internalization causes GF clearance from the extracellular matrix. GF clearance by receptor-mediated internalization is a new paradigm explaining various characteristics of lymphatics.

Role of TGF-beta in proliferative vitreoretinal diseases and ROCK as a therapeutic target.

Cicatricial contraction of preretinal fibrous membrane is a cause of severe vision loss in proliferative vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR). TGF-beta is overexpressed in the vitreous of patients with proliferative vitreoretinal diseases and is also detectable in the contractile membranes. Therefore, TGF-beta is presumed to contribute to the cicatricial contraction of the membranes, however, the underlying mechanisms and TGF-beta's importance among various other factors remain to be elucidated. Vitreous samples from PDR or PVR patients caused significantly larger contraction of hyalocyte-containing collagen gels, compared with nonproliferative controls. The contractile effect was strongly correlated with the vitreal concentration of activated TGF-beta2 (r = 0.82, P < 0.0001). PDR or PVR vitreous promoted expression of alpha-smooth muscle actin (alpha-SMA) and phosphorylation of myosin light chain (MLC), a downstream mediator of Rho-kinase (ROCK), both of which were dramatically but incompletely suppressed by TGF-beta blockade. In contrast, fasudil, a potent and selective ROCK inhibitor, almost completely blocked the vitreous-induced MLC phosphorylation and collagen gel contraction. Fasudil disrupted alpha-SMA organization, but it did not affect its vitreal expression. In vivo, fasudil significantly inhibited the progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells by electroretinographic and histological analyses. These results elucidate the critical role of TGF-beta in mediating cicatricial contraction in proliferative vitreoretinal diseases. ROCK, a key downstream mediator of TGF-beta and other factors might become a unique therapeutic target in the treatment of proliferative vitreoretinal diseases.

Antiangiogenic mechanisms of simvastatin in retinal endothelial cells.

BACKGROUND: While statins have an anti-angiogenic property, their underlying mechanisms are not fully understood. We investigated intracellular mechanisms of simvastatin-mediated reduction in VEGF-induced signalings. METHODS: The effects of simvastatin on cell proliferation and viability were evaluated by [(3)H]-thymidine incorporation in retinal endothelial cells (RECs) and cell counting. The impact of simvastatin on VEGF-induced phosphorylation of p44/42 mitogen-activated protein (MAP) kinase, myosin light chain (MLC), and VEGF-receptor (VEGFR) 2 were examined by Western blotting. Involvement of the mevalonate pathway in VEGF-induced signaling was also examined. RESULTS: Simvastatin (1 and 10 microM) suppressed VEGF-induced RECs proliferation in a concentration-dependent manner, without affecting cell viability. Simvastatin significantly inhibited VEGF-induced phosphorylation of VEGFR2 and its downstream mediators, p44/42 MAP kinase and MLC. Mevalonate completely reversed VEGF-induced VEGFR2 phosphorylation, but only partially reversed the phosphorylation of p44/42 MAP kinase and MLC. CONCLUSION: These data indicate that simvastatin exerts its anti-angiogenic effects through the reduction of VEGFR2 phosphorylation in RECs at least in part. However, there seems to be both mevalonate-dependent and independent pathway in simvastatin's anti-angiogenic property.

Retinitis pigmentosa associated with asteroid hyalosis.

BACKGROUND: Asteroid hyalosis (AH) is a condition in which cream-colored or white spherical particles are suspended in the vitreous body. Asteroid hyalosis is considered not to cause decreased vision or any other visual symptoms except in rare cases. There have been a few reports of AH in patients with retinitis pigmentosa (RP). METHODS: To assess the prevalence of AH in patients with RP, 320 patients with typical forms of RP were studied. One patient was offered a standard three-port vitrectomy, and the spherical particles obtained from her vitrectomy sample were analyzed using an energy-dispersive x-ray spectrometer. RESULTS: Ten patients (two men and eight women) developed AH. Among them, four had bilateral AH and two had rapidly increasing vitreous opacity that led to decreased vision. One patient was a 48-year-old woman with progressive AH in the left eye. After treatment with a vitrectomy, her vision improved from 0.4 to 0.8. The spherical particles were composed of mainly calcium and phosphorus. CONCLUSION: The prevalence of AH in RP was higher than in previous reports, and we encountered two rare cases of progressive AH with decreased vision. We conclude that AH might lead to decreased vision in patients with RP.

Nine-year incidence and risk factors for age-related macular degeneration in a defined Japanese population the Hisayama study.

PURPOSE: To estimate the 9-year incidence and risk factors for age-related macular degeneration (AMD) in a general Japanese population. DESIGN: Population-based, cohort study. PARTICIPANTS: In 1998, a total of 1775 Hisayama residents aged >or=40 years underwent a baseline eye examination. Of those, 1401 subjects (78.9%) took part in the follow-up eye examination in 2007 and were enrolled in the present study. METHODS: At both time points, the characteristics of AMD were determined by grading color fundus photographs using the Wisconsin Age-Related Maculopathy Grading System. MAIN OUTCOME MEASURES: Incident early and late AMD. RESULTS: The age-standardized, 9-year cumulative incidence of early AMD was 10.0%, and that of late AMD was 1.4%. Men were found to have a significantly higher incidence of late AMD than women (age-adjusted odds ratio [OR], 2.97; 95% confidence interval [CI], 1.25-7.09). The incidence of both early and late AMD increased significantly with age. Multiple logistic regression analysis showed that older age (per 1 year; OR, 1.10; 95% CI, 1.05-1.16), smoking habits (OR, 3.98; 95% CI, 1.07-14.7), and higher circulating white blood cell (WBC) count (per 1000 cells/mm(3)) (OR, 1.38; 95% CI, 1.07-1.79) were significantly associated with the development of late AMD. CONCLUSIONS: Our findings suggest that the 9-year incidences of late AMD are lower among the Japanese than among white people in Western countries, and it is higher than among black people. Smoking habits and higher circulating WBC count are significant risk factors for the development of late AMD in the Japanese.

An early "reopening" case of idiopathic macular hole; supportive usefulness of fundus autofluorescence.

BACKGROUND: Anatomical closure of macular holes (MH) is now largely confirmed by optical coherence tomography (OCT). Fundus autofluorescence (FAF) is also helpful in diagnosis and anatomical estimation of MH. We report a case of early reopening of anatomically closed MH, 2 days after release from face-down positioning where FAF abnormalities proceeded OCT findings. METHODS: A case report. A 67-year-old woman underwent vitrectomy with brilliant blue G-assisted ILM peeling for the treatment of full-thickness stage 4 MH (diameter 578 microm). FAF and OCT were used to evaluate the patient. RESULTS: On post operative day 3, OCT showed anatomical closure of MH, but FAF persistently demonstrated hyperfluorescence in the fovea. On post operative day 5, 2 days after termination of positioning, OCT showed reopening of the MH. Intra-vitreous injection of 50 % sulfur hexafluoride (SF(6)) gas was performed followed by face-down positioning again. Fourteen days after surgery, we confirmed the findings of both the anatomical closure in OCT and hypofluorescence on FAF. Two months later, MH remained closed. CONCLUSIONS: FAF might be a useful measure as a supportive method to guide release from posture restriction.

Anatomical findings of vitreoretinal interface in eyes with asteroid hyalosis.

PURPOSE: To investigate the anatomical features of vitreoretinal interface in eyes with asteroid hyalosis (AH) with optical coherence tomography (OCT) and intravitreal triamcinolone acetonide (TA) during vitreous surgery. METHODS: This study was an interventional clinical case series. Records relating to ten eyes from ten patients who underwent a TA-assisted vitrectomy for the treatment of diverse vitreoretinal diseases complicated with AH. The posterior vitreoretinal interface was examined by preoperative OCT and by intraoperative visualization of posterior vitreous cortex utilizing TA. RESULTS: In eight of ten AH eyes, preoperative OCT revealed abnormal vitreoretinal adhesions. In four of these eight eyes, posterior vitreoschisis could be seen on OCT. In the other four of these eight eyes, a clear no posterior vitreous detachment (PVD) pattern could be seen on OCT. Although posterior vitreous cortex could not be clearly identified with preoperative OCT in two of ten AH eyes, a complete PVD was refuted by intraoperative visualization of the posterior vitreous cortex with TA identical to the other eight eyes. CONCLUSION: These results indicate that complete PVD appears to be unlikely to occur in eyes with AH. In addition, spontaneous PVD in eyes with AH might lead to vitreoschisis or residual whole layer or posterior vitreous cortex, possibly due to anomalous vitreoretinal adhesion.

Equivalent tamponade by room air as compared with SF(6) after macular hole surgery.

BACKGROUND: To evaluate the effect of tamponade by room air after vitrectomy for the treatment of idiopathic macular hole (MH). METHODS: There were 156 eyes of 151 patients studied. The patients' ages ranged from 35 to 88 years old (mean: 65.1 years). After conventional pars plana vitrectomy with internal limiting membrane peeling, fluid air exchange was performed using 20% SF(6) (Gas group: 91 eyes) or room air (Air group: 65 eyes). Surgical outcomes were retrospectively analyzed. RESULTS: Mean preoperative hole diameter was 352 microm in the Gas group and 370 microm in the Air group (P = 0.558). The closure rate of all cases was 91.0% after first surgery and 98.7% at last follow-up. The primary closure rate was 90.1% in the Gas group after 7.44 +/- 1.66 (mean +/- SD) days prone positioning period, and 92.3% in the Air group after 3.83 +/- 0.97 days of prone positioning. There was significant difference in prone positioning period (P < 0.0001), but not in the first closure rate (P = 0.132). CONCLUSION: This study suggests that room air may have an equivalent tamponade effect, in spite of the shorter prone positioning period, than SF(6) after MH surgery.

[Preventive strategy for the treatment of diabetic vitreoretinopathy].

Despite considerable recent advances in vitreoretinal surgery, generally performed in more advanced stages of diabetic vitreoretinopathy (DVR), a satisfying visual acuity cannot always be achieved. Even in the early DVR stages that might be detected by routine eye exams, management of general factors, such as blood glucose concentration and blood pressure, currently constitutes the only proven preventive measures. New approaches for amelioration and treatment of DVR are needed. The Hisayama study, an ongoing prospective cohort study of cardiovascular disease and its risk factors in a community in Hisayama Town adjoining Fukuoka City, revealed that the cut-off point for diagnostic fasting glucose level is lower (116 mg/dl) than that of the current diagnostic criteria (126 mg/ dl), indicating that more rigid diagnostic criteria might reduce the incidence of DVR in the Japanese population. In early stages of DVR, leukocyte adhesion in the retinal microvasuculature substantially contributes to DVR. We investigated the involvement of the Rho/ ROCK pathway in diabetic microvasculopathy and the therapeutic potential of fasudil, a selective ROCK inhibitor, and demonstrated that the Rho/ROCK pathway plays a critical role in leukocyte adhesion in diabetic retinal microvasculature and endothelial damage. Fasudil protects the vascular endothelium at least in part by inhibiting neutrophil adhesion and reducing neutrophil-induced endothelial injury via endothelial nitric oxide. In later stages of DVR, namely proliferative diabetic retinopathy, tractional retinal detachment associated with a cicatrical contraction of proliferative membranes can cause severe vision loss. We demonstrated the possible involvement of hyalocytes in proliferative membrane formation and its contraction mainly mediated through the function of TGF-beta 2 resulting in myofibroblastic transdifferentiation and phosphorylation of the myosin light chain, a downstream mediator of ROCK. ROCK inhibition by fasudil or statins successfully inhibited cicatrical contraction of the proliferative membranes both in vitro and in vivo. Further studies for direct evidence demonstrating whether altered diagnostic criteria of diabetes may lead to a lower incidence of DVR and determination of the therapeutic potential of ROCK inhibition in the clinic could provide new avenues of intervention for inhibiting DVR.

Transforming growth factor-beta2 and connective tissue growth factor in proliferative vitreoretinal diseases: possible involvement of hyalocytes and therapeutic potential of Rho kinase inhibitor.

The critical association of connective tissue growth factor (CTGF), which is thought to be one of the downstream mediators of transforming growth factor-beta (TGF-beta), with vitreoretinal diseases remains to be clarified. In the current study, we first demonstrated the correlation between the concentrations of TGF-beta2 as well as CTGF in the vitreous and CTGF gene regulation in cultured hyalocytes. Concentrations of TGF-beta2 and CTGF in the vitreous from patients with proliferative vitreoretinal diseases were significantly higher than in those with nonproliferative diseases, and there was a positive correlation between their concentrations (r = 0.320, P < 0.01). Cultured hyalocytes expressed CTGF mRNA, which was enhanced in the presence of TGF-beta2, associated with nuclear accumulation of Smad4. TGF-beta2-dependent Smad4 translocation and CTGF gene expression were mediated through Rho kinase and at least partially via p38 mitogen-activated protein kinase. Finally, fasudil, a Rho kinase inhibitor already in clinical use, inhibited both Smad4 translocation and CTGF gene expression. In conclusion, combined effects of TGF-beta2 and CTGF appear to be involved in the pathogenesis of proliferative vitreoretinal diseases. Hyalocytes may be a possible source of CTGF and thus might play a role in vitreoretinal interface diseases. Furthermore, Rho kinase inhibitors might have therapeutic potential to control fibrotic disorders in the eye.

Interleukin-18 regulates pathological intraocular neovascularization.

Recently, the proinflammatory cytokine IL-18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen-induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL-18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL-18 reduction in oxygen-treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL-18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL-18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL-18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL-18-binding protein was administered during the OIR recovery phase to neutralize endogenous IL-18, OIR was still apparent on Day 24. We therefore concluded that IL-18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans.

Antiangiogenic properties of fasudil, a potent Rho-Kinase inhibitor.

PURPOSE: Vascular endothelial growth factor (VEGF) plays a pivotal role in pathological angiogenesis. In this study, we addressed the therapeutic potential of fasudil, a potent Rho-kinase inhibitor, for VEGF-elicited angiogenesis and also for the intracellular signalings induced by VEGF. METHODS: In vitro, the inhibitory effects of fasudil on the VEGF-dependent VEGF receptor 2 (VEFGR2 or KDR), extracellular signal-related kinase (ERK) 1/2, Akt and myosin light chain (MLC) phosphorylation, as well as on the migration and proliferation of bovine retinal microvascular endothelial cells (BRECs) were analyzed with Western blotting, [3H]-thymidine uptake, and modified Boyden chamber assay. VEGF-elicited in vivo angiogenesis was analyzed with a mouse corneal micropocket assay coembedded with or without fasudil. RESULTS: VEGF caused enhanced MLC phosphorylation of BRECs, which was almost completely attenuated by 10microM fasudil. VEGF-dependent phosphorylation of ERK1/2 and Akt were also partially but significantly attenuated by treatment with fasudil without affecting VEGFR2 (KDR) phosphorylation. Moreover, both VEGF-induced [3H]-thymidine uptake and the migration of BRECs were significantly inhibited in the presence of fasudil. Finally, VEGF-elicited angiogenesis in the corneal micropocket assay was potently attenuated by coembedding with fasudil (P < 0.01). CONCLUSIONS: These findings indicate that fasudil might have a therapeutic potential for ocular angiogenic diseases. The antiangiogenic effect of fasudil appears to be mediated through the blockade not only of Rho-kinase signaling but also of ERK and Akt signaling.

Intracellular events in retinal glial cells exposed to ICG and BBG.

PURPOSE: To investigate the intracellular events in retinal glial cells exposed to indocyanine green (ICG) and brilliant blue G (BBG). METHODS: The human Müller cell line MIO-M1 was exposed to a low dose (0.25 mg/mL) and a clinical dose (2.5 mg/mL) of ICG and a clinical dose (0.25 mg/mL) of BBG for 15 minutes, respectively. To quantify the proliferation and viability of the cells, [(3)H]-thymidine incorporation was measured and cell numbers were counted 24 hours after treatment. Cell morphology was evaluated using phase-contrast microscopy and transmission electron microscopy. The effects of ICG and BBG on phosphorylation of p38 MAPK and cleavage of caspase-9 and caspase-3 were examined by Western blot. RESULTS: ICG and BBG significantly reduced [(3)H]-thymidine incorporation in MIO-M1 cells compared with the vehicle-treated controls (P < 0.01). Cell number significantly decreased after exposure to ICG at 2.5 or 0.25 mg/mL (P < 0.01) but did not decrease after exposure to BBG at 0.25 mg/mL. Transmission electron microscopy revealed apoptotic changes only in the ICG-treated cells. Prominent p38 MAPK phosphorylation was observed in the presence of ICG, even at the low concentration and within a short time exposure; however, no apparent enhancement was observed in the presence of 0.25 mg/mL BBG. Furthermore, ICG, but not BBG, induced the cleavage of caspase-9 and caspase-3, which was inhibited by an inhibitor of p38 MAPK. CONCLUSIONS: ICG is toxic to retinal glial cells because it induces apoptosis, involving induction of the caspase cascade through p38 MAPK phosphorylation. In contrast, BBG does not cause apoptosis and thus could be a safer adjuvant during vitreoretinal surgery.

Reduced incidence of intraoperative complications in a multicenter controlled clinical trial of triamcinolone in vitrectomy.

PURPOSE: To evaluate the benefits and potential complications of using triamcinolone acetonide (TA) in pars plana vitrectomy (PPV). DESIGN: Multicenter, prospective, controlled clinical trial. PARTICIPANTS: In total, 774 patients from 8 Japanese hospitals were enrolled, with 391 patients undergoing TA-assisted PPV and 383 control patients undergoing conventional PPV. INTERVENTION: Intraoperative use of TA to aid visualization of the vitreous. MAIN OUTCOME MEASURES: The incidence of intraoperative complications, including retinal breaks, was evaluated. Early postoperative complications, intraocular pressure (IOP), and adverse events occurring within 3 months of the operation were also monitored. RESULTS: The incidence of both retinal breaks and intraoperative retinal detachment was significantly lower in TA-assisted PPV than in conventional PPV. Retinal breaks were seen in 34 eyes (8.7%) undergoing TA-assisted PPV compared with 54 eyes (14.1%) undergoing conventional PPV (odds ratio [OR], 0.603; 95% confidence interval [CI], 0.381-0.955; P = 0.031). Retinal detachment was seen in only 3 eyes (0.8%) in which TA was used compared with 14 eyes (3.7%) in which TA was not used (OR, 0.204; 95% CI, 0.057-0.727; P = 0.014). In total, 388 eyes in the TA-assisted PPV group (99.2%) and 374 eyes in the conventional PPV group (97.6%) were followed up for 3 months after the operation. Although the mean postoperative IOPs were comparable in both groups, antiglaucoma eye drops were used more frequently by patients in the TA-assisted group than by those in the conventional PPV group (OR, 1.673; 95% CI, 1.126-2.484; P = 0.011). No serious adverse events, such as endophthalmitis or retinal degeneration, were observed in either group. CONCLUSIONS: Intraoperative use of TA reduced the incidence of retinal breaks and retinal detachments in eyes undergoing PPV. There were no serious adverse events related to the intraoperative use of TA. Although antiglaucoma eye drops were required more frequently after TA-assisted PPV than after conventional PPV, IOP was well-controlled in both groups.

Staining ability and biocompatibility of brilliant blue G: preclinical study of brilliant blue G as an adjunct for capsular staining.

OBJECTIVE: To evaluate the effectiveness and biocompatibility of brilliant blue G (BBG) for capsular visualization for continuous curvilinear capsulorrhexis. METHODS: The capsular staining ability of BBG was evaluated at graded concentrations of 10.0, 1.0, 0.5, 0.25, 0.1, and 0.01 mg/mL in enucleated pig's eyes. The biocompatibility of BBG was assessed in rat's eyes for 2 months. The eyes were analyzed using light, fluorescence, transmission electron, and scanning electron microscopy. TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) was used to detect apoptotic cells, and endothelial cell counts were analyzed using scanning electron microscopy. The results were compared using indocyanine green and trypan blue. RESULTS: The BBG improved capsular visualization, and a complete capsulorrhexis could be performed. In the rat model, no apparent toxic effect was observed using biomicroscopy during 2 months. Histologically, BBG showed satisfactory biocompatibility. Apoptotic cell death of the endothelial cells was detected in only the trypan blue group. In contrast to BBG, indocyanine green and trypan blue showed degeneration of corneal endothelial cells using transmission and scanning electron microscopy. CONCLUSION: The BBG contributed to better capsular visualization and caused no apparent complications to the corneal endothelium.Clinical Relevance The BBG is effective and safe capsular staining for continuous curvilinear capsulorrhexis.

Cellular migration associated with macular hole: a new method for comprehensive bird's-eye analysis of the internal limiting membrane.

OBJECTIVE: To elucidate the pathogenesis of macular hole formation, focusing in particular on the possible role of cellular migration on the cortical vitreous and internal limiting membrane (ILM) around the macular hole. METHODS: To gain a comprehensive overview of the ILM excised in macular hole surgery (n = 36), the ILMs were carefully unfolded and spread out onto glass slides as continuous flat sheets that each contained a macular hole. The specimens were observed by light microscopy and transmission electron microscopy (n = 9), and the cellular distribution was analyzed by scanning electron microscopy in a quantitative manner (n = 27). Immunohistochemistry for glial fibrillary acidic protein and cytokeratin 18 was carried out for cellular characterization. Cellular proliferation was assessed by immunohistochemistry for proliferating cell nuclear antigen and Ki-67. RESULTS: Cellular migration was not apparent around the macular hole in the early stage of development of the macular hole (stage 2, 0 microm). As the macular hole passed through the later stages of development, cellular migration developed around the macular hole (stage 3, 84 microm) and the area of cellular migration gradually enlarged (stage 4, 420 microm). The immunophenotypic analysis showed that these cells were mainly glial fibrillary acidic protein-positive glial cells and cytokeratin 18-positive retinal pigment epithelial cells. The proliferating cell nuclear antigen and Ki-67 immunohistochemistry showed that some of these cells were proliferating on the ILM. CONCLUSIONS: Cellular migration on the ILM is not necessary for the initial formation of a macular break. Cellular migration developed after the macular break occurred, and the migration and proliferation increased gradually from the macular hole. CLINICAL RELEVANCE: This study provides a new method for understanding the ultrastructural analysis of the pathogenesis of the macular hole.

Bifunctional properties of peroxisome proliferator-activated receptor gamma1 in KDR gene regulation mediated via interaction with both Sp1 and Sp3.

Vascular endothelial growth factor receptor 2 (KDR) plays a critical role in mediating a variety of vasculogenic and angiogenic processes, including diabetic retinopathy. We previously demonstrated that the promoter activity of the KDR gene in retinal capillary endothelial cells (RCECs) was regulated in part by the relative concentration of positive/negative transcription factors Sp1/Sp3. We also reported that the peroxisome proliferator-activated receptor (PPAR)gamma ligand could inhibit intraocular angiogenesis. In the present study, the role of PPARgamma1 in KDR gene regulation in RCECs was examined. PPARgamma1 protein physically interacted with both Sp1 and Sp3. Transactivation and electrophoretic mobility shift assays clearly demonstrated novel findings that PPARgamma1 increased KDR promoter activity by enhancing the interaction between Sp1, but not Sp3, and KDR promoter region without its ligand in RCECs. The ligand-binding site but not the DNA binding site of PPARgamma1 enhanced the interaction between Sp1 and KDR promoter region. Conversely, PPARgamma1 ligand 15-deoxy Delta (12,14)-prostaglandin J2 dose-dependently suppressed the binding of KDR promoter region with both Sp1 and Sp3, resulting an inhibition of KDR gene expression. In conclusion, PPARgamma1 has bifunctional properties in the regulation of KDR gene expression mediated via interaction with both Sp1 and Sp3.

A new method for comprehensive bird's-eye analysis of the surgically excised internal limiting membrane.

PURPOSE: To investigate the role of cells and extracellular matrix on the internal limiting membrane (ILM), we demonstrated a new method for a comprehensive bird's-eye analysis of surgically excised ILM. DESIGN: Laboratory investigation. METHODS: The ILM of an idiopathic epiretinal membrane was fixed and spread out onto a glass slide, using fine needles under a biomicroscope. The expanded ILM was analyzed by light microscopy, immunohistochemistry, and scanning electron microscopy. RESULTS: The excised ILM could be unfolded and spread out as a flat sheet onto the glass slide. Immunohistochemistry revealed that most migrating cells were glial cells. Scanning electronmicroscopy revealed that the vitreous surface of the ILM was smooth, in contrast to the rough retinal surface. The ILM showed abundant cellular migration as a continuous stratified cellular sheet. CONCLUSIONS: This new bird's-eye-view observation of excised ILM enables us to carry out comprehensive analysis of cellular distribution from a temporal and spatial perspective.