Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells.

Enhancer of zeste homolog 2 (EZH2) enhances tumorigenesis and is commonly overexpressed in several types of cancer. To investigate the anticancer effects of EZH2 inhibitors, microRNA (miRNA) expression profiles were examined in gastric and liver cancer cells treated with suberoylanilide hydroxamic acid (SAHA) and 3-deazaneplanocin A (DZNep). We confirmed that SAHA and DZNep suppressed EZH2 expression in AGS and HepG2 cells and inhibited their proliferation. The results of microarray analyses demonstrated that miR-1246 was commonly upregulated in cancer cells by treatment with SAHA and DZNep. MiR-302a and miR-4448 were markedly upregulated by treatment with SAHA and DZNep, respectively. DYRK1A, CDK2, BMI-1 and Girdin, which are targets of miR-1246, miR-302a and miR-4448, were suppressed by treatment with SAHA and DZNep, leading to apoptosis, cell cycle arrest and reduced migration of AGS and HepG2 cells. ChIP assay revealed that SAHA and DZNep inhibited the binding of EZH2 to the promoter regions of miR-1246, miR-302a and miR-4448. These findings suggest that EZH2 inhibitors such as SAHA and DZNep exert multiple anticancer effects through activation of tumor-suppressor miRNAs.

Longitudinal analysis of the peripapillary retinal nerve fiber layer thinning in patients with retinitis pigmentosa.

PURPOSE: To investigate longitudinal changes in peripapillary retinal nerve fiber layer (RNFL) thickness in patients with retinitis pigmentosa (RP). METHODS: We re-examined 103 RP patients whose RNFL thickness was previously examined and reported. RNFL thickness was measured using Stratus optical coherence tomography and was compared with the previous measurements. The results were also compared with that of previously reported normal subjects. Association between the decrease rate and visual acuity, and visual field was also investigated. RESULTS: The mean follow-up period was 56.9 months. After excluding the patients in whom RNFL images were of poor quality, 88 patients were eventually analyzed. The average RNFL thickness decreased from 105.8 to 98.2 µm during the period, with the average rate of decrease being 1.6 µm/year. The decrease in RNFL was more evident in superior and inferior sectors. Cross-sectional linear regression analysis also revealed an age-dependent decrease in RNFL, with the slower rate of decrease being 0.94 µm/year. The decrease in RNFL thickness was significantly faster than that reported in normal subjects. The decrease rate was not associated with visual functions. CONCLUSION: Age-dependent RNFL thinning occurs at a faster rate in RP patients as compared with that in normal subjects. The result supports the notion that pathologic changes involve inner retina as well as outer retina in eyes with RP. Considering the discrepancy in the rate of RNFL thinning estimated from trend analysis and longitudinal measurement, care should be taken when interpreting the result of cross-sectional analysis.

Prognostic factors for visual outcomes 2-years after intravitreal bevacizumab for myopic choroidal neovascularization.

PURPOSE: To determine the pre-treatment ocular factors significantly associated with the visual outcome 24 months after intravitreal bevacizumab (IVB) for myopic choroidal neovascularization (mCNV). METHODS: A total of 23 eyes of 23 patients with mCNV were treated with IVB followed by as needed therapy. The efficacy of IVB was evaluated by the best-corrected visual acuity (BCVA) at 24 months after the initial treatment. Forward stepwise multiple linear regression analyses were performed to evaluate the influence of pre-treatment factors on the BCVA and the improvement of the BCVA at 24 months. RESULTS: The mean pre-IVB BCVA was 0.74 ± 0.30 logarithm of the minimum angle of resolution (logMAR) units, and it improved to 0.43 ± 0.31 logMAR units after 1 month (P < 0.001, paired t-test). The improvement was maintained at 24 months (0.46 ± 0.40, P < 0.005). The mean number of IVB performed during the 24 months was 1.35 ± 0.71. Forward stepwise regression analysis showed that the pre-IVB CNV size (standardized ß = 0.52, P < 0.01) and BCVA (standardized ß = -0.44, P < 0.05) significantly affected the visual acuity change after 24 months. The CNV size was the only factor that significantly affected the BCVA after 24 months (standardized ß=0.56, P < 0.01). CONCLUSIONS: IVB with as needed therapy for mCNV led to a rapid and sustained visual improvement. Smaller CNV size was a significant prognostic factor that predicts better visual acuity. Patients with lower pre-treatment BCVA had better visual recovery than those with better pre-treatment BCVA, however, this may be due to a ceiling/floor effect.

Intravitreal bevacizumab for type 1 idiopathic macular telangiectasia.

PURPOSE: To evaluate the effect of intravitreal bevacizumab injection for treating type 1 idiopathic macular telangiectasia (IMT). METHODS: Retrospective case series of five eyes of five male patients with type 1 IMT that were treated with 2-3 injections of intravitreal bevacizumab. Best-corrected visual acuity, foveal thickness obtained by optical coherence tomography, and fluorescein angiography (FA) were monitored over a period of up to 12 months. RESULTS: The average follow-up period was 17.0 months (range, 12-21 months). The mean logarithm of the minimal angle of resolution visual acuity was 0.295 at baseline and 0.254 (P=0.194) and 0.311 (P=0.461) at 3 and 12 months, respectively, after the initial injection. At 12 months, visual acuity had improved in one eye, remained stable in three eyes, and decreased in one eye. The mean foveal thickness was 479 microm at baseline; at 1 month after the therapy, marked reduction of macular oedema was seen only in one eye. The mean foveal thickness was 418 microm (P=0.287) and 473 microm (P=0.482) at 3 and 12 months after the initial injection, respectively. At 12 months, the foveal thickness had decreased by >100 microm in one eye, but had increased by >100 microm in two eyes. FA did not show a reduction in late leakage. CONCLUSIONS: Treatment with intravitreal bevacizumab does not appear to improve visual acuity or retinal oedema in type 1 IMT.

ARMS2/HTRA1 and CFH polymorphisms are not associated with choroidal neovascularization in highly myopic eyes of the elderly Japanese population.

PURPOSE: The purpose of this study was to investigate whether the genetic risk factors of age-related macular degeneration (AMD) are associated with the development of choroidal neovascularization (CNV) in highly myopic eyes of elderly Japanese. METHODS: Highly myopic elderly Japanese patients with and without CNV were genotyped for three AMD-associated single nucleotide polymorphisms (SNPs), namely rs10490924 (A69S) of ARMS2, rs11200638 of HTRA1, and rs1061170 (Y402H) of complement factor H (CFH), with the TaqMan SNP assay. One hundred and eighty-three unrelated highly myopic (axial lengths>26.00 mm or refractive errors>-6.0 diopters) Japanese patients with CNV who were >or=50 years of age (mean age+/-standard deviation of 62.7+/-6.3 years) and 170 highly myopic patients without CNV who were >or=50 years old (62.3+/-7.1 years) were studied. The differences in the genotypic distributions for the three SNPs between the two groups were tested with the Trend chi2 test, and logistic regression analyses were performed for age and gender adjustment. RESULTS: No significant difference was detected in the distribution of the three SNPs, rs10490924 (P>0.1), rs11200638 (P>0.1), and rs1061170 (P>0.5), between the two groups even after adjustments for age and gender differences. CONCLUSION: The genetic risk factors of AMD related to these SNPs do not contribute significantly to the development of CNV in a highly myopic elderly Japanese population.

Circulating hematopoietic stem cells in patients with choroidal neovascularization secondary to pathologic myopia.

BACKGROUND: Emerging evidences suggest that circulating hematopoietic stem cells (HSCs) affect the pathogenesis of choroidal neovascularization (CNV), however, the roles of HSCs in CNV remain unclear in human population. The current study was designed to investigate the role of HSCs in the pathogenesis of CNV secondary to pathologic myopia (PM). METHODS: We clinically documented 78 patients with CNV in PM, and 35 of 78 patients and 28 age-matched controls were experimentally analysed. Functional analyses of HSCs were performed using an ex vivo culture system. RESULTS: We disclosed colony-forming units of endothelial cell (CFU-EC) were markedly lower in patients with bilateral CNV compared to those with unilateral CNV (13.8+/-3.7 vs 45.9+/-7.8, P<0.001). Systemic characteristics between both groups showed no significant difference. To identify local ocular factors that may affect the occurrence of CNV, clinical parameters were compared with the following groups in all enrolled subjects: eyes with CNV vs without CNV in unilateral affected patients, and primary affected eyes vs secondary affected eyes in patients with bilateral CNV. However, no statistically significant factors were identified in any of the groups. CONCLUSIONS: Circulating HSCs may play a role in the bilateral involvement of CNV in PM patients as one of the systemic factors. Further prospective and longitudinal studies are required.

Photoreceptor integrity and visual acuity in cystoid macular oedema associated with retinitis pigmentosa.

AIMS: To assess the correlation between macular morphology and visual acuity in retinitis pigmentosa (RP) patients with cystoid macular oedema (CME). DESIGN: Retrospective cross-sectional study. PATIENTS AND METHODS: Forty-one eyes of 25 RP patients with CME. Patients underwent cross-sectional scans with optical coherence tomography (Stratus OCT). Age, total retinal thickness, photoreceptor thickness, and the transverse and vertical lengths of the cystoid space were measured. Correlation between visual acuity and each of the measurements were examined. Additionally, the status of the inner segment/outer segment junction (IS/OS) was classified as being absent, discontinuous, or distinct. Measurements were then compared among the three groups. RESULTS: Total retinal thickness or photoreceptor thickness was not correlated with visual acuity. There was a correlation between the transverse length of the cystoid space and visual acuity, although the correlation coefficient was weak (r=0.30). The logMAR visual acuity in the IS/OS absent group (0.67+/-0.43) was worse than that seen in the IS/OS discontinuous (0.22+/-0.19) or IS/OS distinct groups (0.07+/-0.16) (P<0.001). CONCLUSIONS: When monitoring CME associated with RP, the status of IS/OS is the essential parameter that needs to be examined.

Retinal nerve fiber layer thickness in patients with retinitis pigmentosa.

PURPOSE: To study retinal nerve fiber layer (RNFL) thickness in patients with retinitis pigmentosa (RP). DESIGN: Cross-sectional observational study. METHODS: One hundred and thirty-seven eyes of 137 patients with RP were examined. The effect of age, gender, laterality, inheritance trait, spherical equivalent refractive error, visual acuity, and the extent of visual field defect on RNFL thickness measured with optical coherence tomography were analyzed by a multiple linear regression model. RESULTS: The average RNFL thickness was 104.1+/-21.7 microm. The multiple R(2) for the model was 0.349. Among the variables studied, ageing and being male were significant risk factors for thinner RNFL thickness. RNFL thickness was not correlated with inheritance trait, laterality, refractive error, visual acuity, or the extent of visual field defect. CONCLUSION: RNFL thickness in RP patients was not correlated with visual function but ageing as in the normal subjects. Currently proposed therapies, including photoreceptor rescue/transplantation and visual prosthesis, are based on the premise that the inner retinal structures are relatively retained despite the profound loss of photoreceptors. The present result supports this notion.

Optical coherence tomographic pattern and focal electroretinogram in patients with retinitis pigmentosa.

PURPOSE: The foveal function of patients with retinitis pigmentosa (RP) has been estimated by visual acuity (VA) or visual field (VF) tests. In the present study, the potential of optical coherence tomography (OCT) and focal electroretinogram (fERG) for monitoring macular function in RP patients was investigated. DESIGN: Cross-sectional observational study. METHODS: A total of 56 eyes of 56 patients with RP underwent ophthalmic examination including VA, VF, fERG, and OCT. Patients were morphologically divided into three groups by the appearance of photoreceptor inner/outer segment junction (IS/OS) that were depicted with OCT; type 1: no IS/OS visible, type 2: IS/OS was visible but the length was < or =2 mm, and type 3: IS/OS >2 mm was confirmed. Functional results for VA and fERG were compared and analysed based on the three groups. RESULTS: The average VA of type 1 patients was significantly lower than that of types 2 or 3 patients (P<0.001). There were no significant VA differences detected between types 2 and 3 patients. While most of the type 1 patients (21/22) showed non-recordable fERG, 3 out of 18 type 2 patients and none of type 3 patients showed non-recordable fERG. Significant differences of the fERG amplitudes were observed among the three groups (a-wave, b-wave, and OP, P<0.001 in all three components). However, the implicit time showed no difference between type 2 and 3. CONCLUSIONS: Analysing the IS/OS with OCT and the amplitudes of fERG may be helpful for monitoring RP patients in addition to VA and VF.

Photodynamic therapy combined with low-dose intravitreal triamcinolone acetonide for age-related macular degeneration refractory to photodynamic therapy alone.

AIM: To examine the effects of photodynamic therapy (PDT) with verteporfin combined with low-dose intravitreal triamcinolone acetonide (IVTA) for exudative age-related macular degeneration (AMD) that is resistant to PDT alone. DESIGN: Retrospective case series. METHODS: A retrospective review was performed, using the medical records of 22 eyes of 21 patients who consecutively received combined PDT and 2 mg of IVTA for exudative AMD with a suspected chorioretinal anastomosis or for AMD that was resistant to prior PDT alone. Only those patients who could be followed up for more than 12 months after this combined therapy were enrolled in the study. Best corrected visual acuity and intraocular pressure measurements were taken during each examination. Colour photography, fluorescein/indocyanine green angiography and optical coherence tomography were carried out at baseline and every 3 months thereafter. Need for retreatment was based on dye leakage and the presence of serous retinal detachement (SRD) seen by optical coherence tomography. RESULTS: Visual acuity improved or was maintained in the majority of patients, with the mean change between baseline and the last visit being an improvement of 0.94 lines (p = 0.45). Seventeen (77%) of the 22 eyes showed improved or maintained visual acuity after 12 months of follow-up. Eight (36%) of the 22 eyes continued to show an SRD at the 12-month follow-up; this corresponded to unchanged or even decreased leakage of dye. The mean number of retreatments was 1.36, but the incidence of side effects accompanying treatment was not as high as that reported previously for combined therapy that utilised higher-dose IVTA. CONCLUSIONS: PDT combined with low-dose IVTA for exudative AMD seems to be as effective and safe as combined therapy with the higher-dose IVTA that was reported previously.

Angiotensin II induces expression of the Tie2 receptor ligand, angiopoietin-2, in bovine retinal endothelial cells.

Recent studies have shown that angiopoietins (Angs) and their receptor, Tie2, play a role in vascular integrity and neovascularization. The renin-angiotensin system has been hypothesized to contribute to the development of diabetic retinopathy. In this study, we investigated the effect of angiotensin II (AII) on Ang1 and Ang2 expression in cultured bovine retinal endothelial cells (BRECs). AII stimulated Ang2 but not Ang1 mRNA expression in a dose- and time-dependent manner. This response was inhibited completely by angiotensin type 1 receptor (AT1) antagonist. AII increased the transcription of Ang2 mRNA, but did not change the half-life. Protein kinase C (PKC) inhibitor completely inhibited AII-induced Ang2 expression, and the mitogen-activated protein kinase (MAPK) inhibitor also inhibited it by 69.4+/-15.6%. In addition, we confirmed the upregulation of Ang2 in an AII-induced in vivo rat corneal neovascularization model. These data suggest that AII stimulates Ang2 expression through AT1 receptor-mediated PKC and MAPK pathways in BREC, and AII may play a novel role in retinal neovascularization.

Angiotensin II-stimulated vascular endothelial growth factor expression in bovine retinal pericytes.

PURPOSE: Angiotensin II (AII) has been shown to play a role in many vascular diseases. In the study described, the effect of AII on vascular endothelial growth factor (VEGF) expression and related intracellular signaling mechanism was investigated in bovine retinal microcapillary pericytes. METHODS: Cultured bovine retinal microvascular endothelial cells and pericytes were prepared. VEGF expression was determined by Northern blot analysis and immunoprecipitation assay. Cell proliferation was assessed by DNA content growth assay. Reporter gene studies were performed to identify the AII responsible transcription-activating region of VEGF gene. RESULTS: Angiotensin II induced a significant increase in VEGF mRNA in a time- and dose-dependent manner. Angiotensin II type I receptor antagonist inhibited this effect. Angiotensin II activates the transcription of VEGF gene without changing the mRNA half-life, and the AII responsible region was found in the 5'-flanking region of the VEGF gene. Angiotensin II also increased the expression of c-fos and c-jun mRNA, and antisense oligonucleotides against c-Fos blocked the AII-induced VEGF mRNA expression. The conditioned media of AII-stimulated pericyte cultures had a growth-promoting effect on endothelial cells, and this effect was inhibited almost completely by VEGF neutralizing antibody. CONCLUSIONS: These findings suggest that AII might induce angiogenic activity through a paracrine function of VEGF in retinal microvascular cells.

The potential angiogenic role of macrophages in the formation of choroidal neovascular membranes.

PURPOSE: To investigate the distribution of inflammatory mediators such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and angiogenic cytokines such as vascular endothelial growth factor (VEGF) and to identify their cellular source in surgically excised choroidal neovascular membranes (CNVMs) of various origins. METHODS: Immunoperoxidase staining was performed on paraffin-embedded sections of 11 surgically excised CNVMs to identify cellular distribution and localization of cytokines. Immunofluorescent double staining was performed to detect the cellular source of cytokines. RESULTS: Cytokeratin-positive cells were detected in the RPE layer, in stromal cells, and around neovascular vessels. Macrophages identified by their cellular marker CD68 showed almost the same distribution as cytokeratin-positive cells, although they were most prominent in the stroma. A substantial number of neovascular vessels were also immunoreactive to IL-1beta and TNF-alpha. Immunofluorescent double staining revealed that the RPE layers immunopositive for cytokeratin were also immunopositive for all cytokines, whereas stromal cells immunostained for CD68 were positive for IL-1beta and TNF-alpha, but not for VEGF. CONCLUSIONS: These results indicate that IL-1beta and TNF-alpha secreted by macrophages may promote, at least in part, angiogenesis in CNVMs by stimulating VEGF production in RPE cells.

Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes.

PURPOSE: To elucidate the potential role of angiopoietins and the Tie2 system in choroidal neovascularization. METHODS: Surgically excised choroidal neovascular membranes (CNVMs) were obtained at vitrectomy from five eyes with age-related macular degeneration, three eyes with idiopathic neovascular maculopathy, and two eyes had degenerative myopia and two eyes had angioid streaks. Light microscopic immunohistochemistry was performed to detect cytokines such as vascular endothelial growth factor (VEGF), Ang1, and Ang2 and cellular components such as retinal pigment epithelial (RPE) cells, macrophages, and endothelial cells. Immunofluorescent double staining using confocal microscopy was performed to identify the cell types that secrete specific cytokines. RESULTS: Ang1 and Ang2 were positive in all surgically excised CNVMs, regardless of the primary disease. Double staining revealed that many of the cytokeratin, CD68 and factor VIII positive cells also had Ang1 and Ang2 immunoreactivities. In contrast to Ang1, Ang2 immunoreactivity tends to be higher in the highly vascularized regions of many CNVMs, and the localization was very similar to that of VEGF staining. Almost all vascular structures had prominent immunoreactivity for Tie2, which was confirmed by double staining for Tie2 and factor VIII. Tie2 immunoreactivity was also observed in the RPE monolayer and in pigmented, polygonal, and fibroblast-like cells in the stroma. CONCLUSIONS: Present findings that Ang2 and VEGF are co-upregulated and that Tie2 is expressed in a variety of cell types in CNVMs further support a crucial role of the interaction between VEGF and Ang2 in pathologic angiogenesis of CNVM formation.

17 Beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells.

PURPOSE: Estrogen is known to promote angiogenesis in gonads. The presence of estrogen receptors in the vascular endothelium of organs other than gonads has been reported. The goal of this study was to determine whether estrogen promotes the proliferation of retinal microvascular endothelial cells and to explore the mechanism of it. METHODS: DNA was quantitated using primary cultures of bovine retinal endothelial cells that were incubated with different doses of 17 beta-estradiol (E2), VEGF, or both. The changes in expression level of VEGF and VEGF receptor-2 (VEGFR2) were measured using northern blot analysis after treatment with E2. The presence of estrogen receptors in the endothelial cells was studied by immunohistochemistry and western blot analysis. RESULTS: 17 Beta-estradiol (E2) increased the DNA level in bovine retinal capillary endothelial cells (BRECs) by 177% at 1 nM (P < 0.05) and 150% at 10 nM (P < 0.05) by comparison with unstimulated BREC. One hundred nanomole tamoxifen completely blocked the E2-induced DNA synthesis in BRECs. Ten nanomole E2 augmented vascular endothelial growth factor (VEGF)-induced DNA synthesis in BRECs significantly (160%, P < 0.01). Ten nanomole E2 also increased VEGF mRNA expression, which peaked after 24 hours (6.7 times, P < 0.05), and VEGF receptor-2 (VEGFR2) mRNA expression, which peaked after 9 hours (2.4 times, P < 0.05). The mRNA expression level of VEGFR2 peaked with 10 nM E2 (P < 0.05) and that of VEGF reached maximum with 1 nM E2 (15 times, P < 0.001). VEGFR2 and VEGF proteins increased in parallel with their mRNA levels. Immunocytochemistry showed estrogen receptor expression in BRECs, and western blot analysis indicated the presence of a 67-kDa protein that was compatible with the estrogen receptor. CONCLUSIONS: These findings suggest that E2 may stimulate BREC growth by the receptor-mediated pathway and that E2 may augment the VEGF-dependent angiogenesis partly through the upregulation of VEGFR2.

Tranilast inhibits protein kinase C-dependent signalling pathway linked to angiogenic activities and gene expression of retinal microcapillary endothelial cells.

1. Tranilast, first developed as an anti-allergic drug, has been reported to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis and vasopermeability. To further clarify the inhibitory mechanism, we investigated the effects of tranilast on VEGF binding and subsequent intracellular signalling pathway linked to angiogenic activities and gene expression of bovine retinal microcapillary endothelial cells. 2. Tranilast significantly (P<0.01) inhibited VEGF, basic fibroblast growth factor (bFGF), and hypoxia conditioned media-induced BREC proliferation in a dose dependent manner with IC50's of 22, 82 and 10 microM, respectively. 3. VEGF-induced migration was also inhibited by tranilast in a dose dependent manner, with IC50 of 18 microM, and complete inhibition was observed at 300 microM (P<0.01). Tranilast suppressed VEGF-induced tube formation in a dose dependent manner with maximum (46%) inhibition observed at 300 microM (P<0.05). 4. Tranilast inhibited phorbol myristate acetate (PMA)-dependent stimulation of [3H]-thymidine incorporation and VEGF- and PMA-induced gene expression of integrin alpha v and c-fos in BREC. 5. Tranilast suppressed VEGF- and PMA-stimulated PKC activity in BREC. 6. Tranilast did not affect VEGF binding or VEGF-induced phosphorylation of tyrosine residues of VEGF receptor- and phospholipase Cgamma and their associated proteins. 7. These data suggest that tranilast might prove an effective inhibitor to prevent retinal neovascularization in ischaemic retinal diseases, and that its inhibitory effect might be through suppression of PKC-dependent signal transduction in BREC.

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.

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.

New surgical approach for removing massive foveal hard exudates in diabetic macular edema.

OBJECTIVE: To examine the efficacy of surgical removal of foveal hard exudates in diabetic macular edema and to determine the expression of vascular endothelial growth factor (VEGF) in the excised specimens. DESIGN: Cohort study. PARTICIPANTS: Seven eyes of six patients with massive subfoveal hard exudate due to diabetic macular edema were examined. The average age of the patient was 56 years (range, 46-60 years). INTERVENTION: Pars plana vitrectomy for removal of massive foveal exudates was performed. MAIN OUTCOME MEASURES: Preoperative and postoperative visual acuity and complications were recorded; immunohistochemical staining for VEGF and other cell markers for macrophage and pigment epithelial cells in excised specimens was performed. RESULTS: Postoperative best-corrected visual acuity improved by two or more lines of Snellen equivalent in five eyes (71%) (P = 0.0061). VEGF, identified by anticytokeratin and CD68 antibodies, was expressed in pigment epithelial cells and macrophages invading the hard exudates. CONCLUSION: Surgical removal of foveal hard exudates might be effective in low-vision patients with diabetic maculopathy. VEGF might play a role in the formation and persistence of foveal hard exudates in diabetic macular edema.