Analysis aqueous humor lipid profile of neovascular glaucoma secondary to diabetic retinopathy and lipidomic alteration response to anti-VEGF treatment.

The objective of this study was to examine the lipid spectrum of aqueous humor (AH) in patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy and to investigate the lipid alteration response to anti-vascular endothelial growth factor (anti-VEGF) treatment. Lipidomic analysis using ultra-high performance liquid chromatography-tandem mass spectrometry was conducted to compare the lipid profiles of the AH in NVG patients with those of a control group. Lipid changes in the AH of NVG patients before and after intravitreal conbercept injections were also evaluated. The identification of lipids showing differential expression was accomplished through both multivariate and univariate analyses. This study included 13 NVG patients and 20 control subjects. Based on LipidSearch software, 639 lipid species across 33 lipid classes were detected in the participants' AH. The combination of univariate and multivariate statistical analyses yielded 53 differentially expressed lipids (VIP >1 and P < 0.05). In addition, 9 lipids were found to be differentially expressed before and after the intravitreal conbercept injections in the NVG patients. Significant alterations in the metabolic pathways of glycerophospholipid and glycerolipid exhibited notable changes. Our results highlighted the lipid changes in patients' AH in relation to the progression of NVG, and indicated that the modified lipids could potentially be utilized as therapeutic targets for NVG.

Elevated soluble vascular endothelial growth factor receptor levels in aqueous humor from patients with different types of glaucoma.

We investigated the aqueous humor levels of vascular endothelial growth factor (VEGF), soluble VEGF receptor (sVEGFR)1, and sVEGFR2 in glaucoma patients and the correlations among them. Aqueous humor was collected from the anterior chamber at the start of glaucoma or cataract surgery. The levels of VEGF and its receptors, sVEGFR1 and sVEGFR2, were measured using multiplex bead-based immunoassays. Aqueous humor samples were obtained from 79 participants: 21 with primary open angle glaucoma (POAG), 22 with uveitic glaucoma (UG), 19 with neovascular glaucoma (NVG), and 17 with cataracts as controls. sVEGFR1 levels were significantly higher in NVG than in the other cases (NVG, 2839.8 pg/mL, P < 0.001). The sVEGFR2 levels of glaucoma patients were significantly higher than those of the controls (POAG, 699.0 pg/mL; UG, 866.2 pg/mL; NVG, 1198.1 pg/mL; P < 0.001). In the aqueous humor of glaucoma patients, sVEGFR1 and sVEGFR2 levels were positively correlated (POAG, P = 0.0196; UG, P = 0.0047; NVG, P = 0.0050). VEGF levels were negatively correlated with both sVEGFR1 (P = 0.0197) and sVEGFR2 (P = 0.0015) in POAG patients. In UG patients, the correlation between VEGF and sVEGFR1 levels was negative (P = 0.0144). sVEGFR2 levels were increased in various glaucomatous eyes. sVEGFR levels were negatively correlated with VEGF levels in some glaucoma types, implying that sVEGFRs may modulate the effects of aqueous VEGF in glaucoma pathogenesis.

Aqueous Inflammation and Ischemia-Related Biomarkers in Neovascular Glaucoma with Stable Iris Neovascularization.

Purpose: To characterize the aqueous levels of inflammation and ischemia-related biomarkers in a spectrum of retinal ischemic conditions, including neovascular glaucoma (NVG) with stable iris neovascularization after pan retinal photocoagulation (PRP) and anti-VEGF treatment. Methods: Aqueous samples were collected from 139 eyes including NVG (n = 12), stable NVG (n = 26), CRVO (n = 11), NPDR (n = 18), PACG (n = 18), PDR (n = 25), BRVO (n = 7) and cataract (n = 22). The levels of VEGF-A, IL-8 and EPO were measured with ELISA. Results: Aqueous VEGF-A significantly decreased after anti-VEGF and PRP, from 983.79 ± 821.16 pg/ml in the NVG group (n = 11) to 256.50 ± 51.14 pg/ml in the stable NVG group (n = 24) (P = .015). Aqueous VEGF-A in stable NVG group (256.50 ± 51.14 pg/ml, n = 24) was significantly higher (ANOVA, P < .001) than in CRVO (212.10 ± 19.84 pg/ml, n = 7, P = .017), NPDR (221.18 ± 38.21 pg/ml, n = 14, P = .015), BRVO (213.14 ± 48.50 pg/ml, n = 6, P = .028) and cataract group (185.30 ± 34.35 pg/ml, n = 22, P < .001). Aqueous IL-8 in stable NVG group (74.82 ± 10.78 pg/ml, n = 24) was significantly higher (ANOVA, P < .001) than in CRVO (65.19 ± 15.34 pg/ml, n = 11, P = .032) and cataract group (54.11 ± 12.28 pg/ml, n = 22, P < .001). Aqueous EPO in stable NVG group (17.48 ± 3.02 pg/ml, n = 24) was significantly higher (ANOVA, P < .001) than in BRVO (14.98 ± 2.57 pg/ml, n = 7, P = .034) and cataract group (13.50 ± 2.65 pg/ml, n = 22, P < .001). Aqueous concentrations of VEGF-A and IL-8 correlated positively with IOP (r = 0.413, P < .001, r = 0.349, P < .001, respectively, r = correlation coefficient). VEGF-A correlated positively with IL-8 and EPO (P < .001, P = .002, respectively). IL-8 correlated positively with EPO (P < .001). Conclusions: The aqueous levels of VEGF-A, IL-8 and EPO in NVG patients with stable iris neovascularization, who had received PRP and anti-VEGF, were still significantly higher than in control groups with some retinal ischemic conditions.

Expression of platelet-derived growth factor-C in aqueous humor of patients with neovascular glaucoma and its correlation with vascular endothelial growth factor.

AIM: The aim of this study was to investigate the expression of platelet-derived growth factor-C in aqueous humor of patients with neovascular glaucoma and its correlation with vascular endothelial growth factor. METHODS: This study involved 62 eyes of 62 patients with advanced neovascular glaucoma requiring transscleral cyclophotocoagulation. Aqueous humor was collected through paracentesis. Samples from 11 eyes of 11 patients with age-related cataract were collected as control. Concentrations of platelet-derived growth factor-C and vascular endothelial growth factor in aqueous humor were quantified by enzyme-linked immunosorbent assay. Meanwhile, the correlations between the concentrations of platelet-derived growth factor-C and vascular endothelial growth factor were analyzed. The elements including retinal photocoagulation treatment, iris neovascularization grade, and primary fundus disease were also studied to find out their roles in the concentrations of the two factors. RESULTS: The vascular endothelial growth factor and platelet-derived growth factor-C concentrations in aqueous humor from controls were (679.54 ± 49.81) pg/mL and (18.60 ± 1.85) pg/mL, respectively. Both of them were significantly lower than neovascular glaucoma patients (p < 0.001). The vascular endothelial growth factor and platelet-derived growth factor-C concentrations of neovascular glaucoma patients treated with retinal photocoagulation were (1095.99 ± 52.71) pg/mL and (28.55 ± 0.94) pg/mL, respectively, which were both significantly lower than those of untreated neovascular glaucoma patients, (1146.28 ± 69.57) pg/mL and (30.04 ± 1.64) pg/mL (p = 0.008, p = 0.034). There was a weak correlation between the expression level of vascular endothelial growth factor and platelet-derived growth factor-C in aqueous humor with neovascular glaucoma (r = 0.346, p = 0.006). However, iris neovascularization grade and primary fundus disease were not significant elements in the expression level of vascular endothelial growth factor and platelet-derived growth factor-C. CONCLUSION: Higher concentrations of vascular endothelial growth factor and platelet-derived growth factor-C were found in aqueous humor of patients with neovascular glaucoma compared with control, which could be lowered by retinal photocoagulation to some extent. Platelet-derived growth factor-C inhibitors may be another potential target for ocular neovascular diseases.

Angiogenic and inflammatory biomarker levels in aqueous humor and vitreous of neovascular glaucoma and proliferative diabetic retinopathy.

PURPOSE: To explore the relationships between the aqueous and vitreous levels of vascular endothelial growth factor-A (VEGF-A), interleukin-8 (IL-8), placental growth factor (PlGF) and erythropoietin (EPO) in proliferative diabetic retinopathy (PDR) and neovascular glaucoma (NVG). METHODS: Aqueous and vitreous samples were obtained from patients with PDR and NVG during surgery. Aqueous and vitreous concentrations of VEGF-A, IL-8, PlGF and EPO were measured via enzyme-linked immunosorbent assay. RESULTS: No correlation between the aqueous and vitreous levels of VEGF-A, IL-8, PlGF or EPO was found in both the PDR and the NVG eyes. Aqueous VEGF-A was significantly higher in the NVG group (317.55 ± 36.25 pg/ml, n = 15) than that in the PDR group (256.23 ± 46.11 pg/ml, n = 17, P < 0.001). The level of VEGF-A in aqueous (317.55 ± 36.25 pg/ml, n = 15) was significantly higher than that in vitreous (224.74 ± 60.32 pg/ml, n = 15, P < 0.001) in NVG patients. The level of IL-8 in aqueous (76.55 ± 10.88 pg/ml, n = 17) was significantly higher than that in vitreous (63.55 ± 10.74 pg/ml, n = 17, P = 0.001) in PDR patients. The level of EPO in aqueous (18.62 ± 2.87 mIU/ml, n = 15) was significantly higher than that in vitreous (15.97 ± 3.11 mIU/ml, n = 15, P = 0.022) in NVG patients. The ratio of aqueous versus vitreous for VEGF-A was significantly higher in the NVG group (1.475 ± 0.289, n = 15) than that in the PDR group (0.996 ± 0.227, n = 17, P < 0.001). CONCLUSION: Aqueous levels of VEGF-A, IL-8, PlGF and EPO do not correlate with vitreous levels of those proteins. The relationship between protein levels in aqueous humor and vitreous might be dependent on different disease status or protein types investigated.

Increased expression of periostin and tenascin-C in eyes with neovascular glaucoma secondary to PDR.

PURPOSE: To investigate periostin (PN) and tenascin-C (TNC) expression in the aqueous humor and trabeculectomy specimens of patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy (PDR). METHODS: This study enrolled 37 eyes of 37 patients who were grouped into (1) NVG secondary to PDR (NVG; n = 8); (2) PDR without NVG (PDR; n = 9); (3) primary open-angle glaucoma (POAG; n = 11); and (4) cataract surgery patients as a control group (CG; n = 9). Aqueous humor samples were collected from the anterior chamber at the start of surgery or intravitreal injection of anti-VEGF drug. The concentrations of PN, TNC, VEGF, and TGF-ß2 (transforming growth factor-beta 2) were measured by ELISA. Sclerostomy tissues containing trabecular meshwork were obtained from two NVG patients and a POAG patient who underwent trabeculectomy surgery. Immunohistochemical analyses were performed to determine the localization of PN and TNC expression in the sclerostomy tissues. RESULTS: PN and TNC-C levels were below detection threshold in the POAG and CG groups. The NVG group had significantly higher levels of PN and TNC compared with the PDR group (84.7 ng/ml vs 2.2 ng/ml and 18.5 ng/ml vs 4.6 ng/ml, respectively; p < 0.05). There was a significant correlation between the levels of PN and TNC-C in the NVG group (r = 0.86, p < 0.05). We found significant expression of PN in the trabecular meshwork and Schlemm's canal of sclerostomy tissues excised from patients with NVG. CONCLUSIONS: Increased PN and TNC expression suggests their possible involvement in the pathogenesis of NVG secondary to PDR.

Comparison of Erythropoietin, Semaphorins 3A and Pigment Epithelium Derived Factor Levels in Serum and Aqueous Humor of Patients with Neovascular Glaucoma and Cataract.

The aim of this study was to compare the levels of erythropoietin (Epo), semaphorins 3A (Sema 3A) and pigment epithelium derived factors (PEDF) in serum and aqueous humor of those with neovascular glaucoma (NVG) and patients with cataract. It was an experimental study carried out from January 2016 to September 2018. Seventy-three NVG patients were selected as NVG group, and 73 cataract patients were selected as cataract group. The levels of Epo and sema 3A in serum and aqueous humor in NVG group were higher than those in cataract group (both p <0.001), and the level of PEDF in serum and aqueous humor in NVG group were lower than those in cataract group (p <0.001). Compared with cataract patients, NVG patients had higher levels of Epo and sema 3A in serum and aqueous humor and lower levels of PEDF.

What is the impact of intravitreal injection of conbercept on neovascular glaucoma patients: a prospective, interventional case series study.

BACKGROUND: The aim of the present study was to evaluate the efficacy and safety of intravitreal conbercept combined with trabeculectomy and panretinal photocoagulation for neovascular glaucoma (NVG). METHODS: Fifty patients (54 eyes) with NVG were included in this prospective study. Fifty-two eyes initially underwent intravitreal conbercept (0.5 mg/0.05 ml) treatment followed by trabeculectomy and panretinal photocoagulation. Preoperative and postoperative best-corrected visual acuity (BCVA), intraocular pressure (IOP), the number of antiglaucoma medications, and surgical complications were recorded. The levels of VEGF-A, TGF-ß1 and PLGF in aqueous humour samples collected during surgery were measured by enzyme-linked immunosorbent assay (ELISA). Light microscopy and transmission electron microscopy were used to observe the surgically excised trabecular tissue; enucleation was performed in 2 eyes, and light microscopy was used as the histopathological control. RESULTS: The follow-up period after trabeculectomy was 1 year. Of the 52 eyes, 39 completed 1 year of follow-up, and 13 were lost to follow-up. Recurrence of iris neovascularization was observed in 5 eyes, 9 had hyphema, 16 had filter-bled scarring, and no eye had complications attributable to the drug. The mean IOP was reduced from 48.1 ± 14.2 to 23.2 ± 8.7 mmHg, and the mean number of antiglaucoma medications used decreased from 3.0 (3.0, 4.0) to 1.0 (0.0, 1.0) after 1 year (both P < 0.05). The complete success rate was 76.9, 76.9, 71.0, 51.6, and 32.3% at 1 week, 1 month, 3 months, 6 months and 12 months, respectively, when the cut-off IOP was 18 mmHg. After patients underwent intravitreal injection, the concentrations of VEGF-A and TGF-ß1 in the aqueous humour in NVG patients decreased from 168.8 ± 13.4 and 159.6 ± 15.4 pg/ml to 160.2 ± 7.6 and 151.9 ± 2.3 pg/ml, respectively (both P < 0.05). Light microscopy revealed neovascularization regression in the iris in specimens treated with intravitreal conbercept. Electron microscopy revealed trabecular endothelial cell degeneration in the conbercept-treated specimens. CONCLUSIONS: Our initial findings suggest that intravitreal conbercept is an effective treatment for managing NVG that has fewer short-term postoperative complications. TRIAL REGISTRATION: Current Controlled Trials ChiCTR1800019918 , 8 December 2018, retrospectively registered.

Puerarin regulates neovascular glaucoma through pigment epithelium­derived growth factor­induced NF­κB signaling pathway.

Neovascular glaucoma is an ophthalmic disease and a potentially blinding secondary glaucoma caused by the formation of abnormal new blood vessels on the iris, which can prevent the normal drainage of water from the anterior segment of the eye. Evidence from China has suggested that puerarin benefits many diseases including myocardial infarction, stable angina, cerebral ischemia and glaucoma in a clinical setting. In the present study, the aim was to investigate the efficacies of puerarin on neovascular glaucoma in a mouse model. The molecular mechanism of puerarin­mediated treatment for neovascular glaucoma was also investigated both in vitro and in vivo. Inflammatory responses in mice with neovascular glaucoma were analyzed by western blotting. Oxidative stress levels were investigated following treatment with puerarin in a mouse model of neovascular glaucoma. The results indicated that puerarin markedly improved growth of vascular endothelial cells. The present study reported that puerarin treatment markedly decreased interleukin (IL)­1ß, IL­17A and tumor necrosis factor­α expression levels in mice with neovascular glaucoma. It was found that puerarin significantly decreased oxidative stress levels by reducing reactive oxygen species, superoxide dismutase and malondialdehyde levels, as well as neuronal nitric oxide synthase (NOS) and inducible NOS expression levels. Results indicated that expression levels of pigment epithelium­derived growth factor were significantly inhibited following treatment with puerarin. Mechanism analysis demonstrated that treatment with puerarin effectively inhibited nuclear factor (NF)­κB activity and its target protein levels p65, inhibitor of NF­κB kinase subunit ß and inhibitor of NF­κB kinase subunit α in vascular endothelial cells. Increasing endothelial­derived growth factor (EDGF) expression levels could stimulate NF­κB activity and abolish the inhibitory effects of puerarin. An animal study reported that puerarin treatment presented therapeutic effects for mice with neovascular glaucoma. Numbers of new vessels in iris were recovered to normal following puerarin treatment. In conclusion, these results indicated that puerarin treatment can inhibit inflammatory responses and oxidative stress, platelet­derived growth factor (PDGF) expression and NF­κB activity, suggesting puerarin may be a potential agent for the treatment of neovascular glaucoma through PDGF­induced NF­κB signaling pathway.

Characteristic Profiles of Inflammatory Cytokines in the Aqueous Humor of Glaucomatous Eyes.

PURPOSE: To analyze cytokine profiles of the aqueous humor of eyes with primary open-angle glaucoma (POAG), neovascular glaucoma (NVG), and cataract (as controls). METHODS: A multiplex bead assay was used to measure concentrations of 27 cytokines in aqueous humor samples from 54 eyes. RESULTS: Detection rates were as follows: IL-7: NVG higher than POAG; IL-10: POAG lower than cataract or NVG; and GM-CSF: cataract higher than POAG or NVG. Concentrations were as follows: IL-8, IP-10, MCP-1, and MIP-1ß: POAG and NVG higher than cataract; IL-9: POAG lower than NVG; IL-12: POAG lower than cataract or NVG; and VEGF: NVG higher than cataract or POAG and POAG lower than cataract. Further analysis showed that IL-8, IP-10, MCP-1, and MIP-1ß were correlated with intraocular pressure and age. CONCLUSIONS: The detection rates and levels of various cytokines had different patterns in POAG and NVG patients, suggesting distinctive alterations in the microenvironment in different types of glaucoma.

A prospective, randomised, placebo-controlled, double-masked, three-armed, multicentre phase II/III trial for the Study of a Topical Treatment of Ischaemic Central Retinal Vein Occlusion to Prevent Neovascular Glaucoma - the STRONG study: study protocol for a randomised controlled trial.

BACKGROUND: Neovascular glaucoma (NVG) is rare, comprising only 3.9% of all glaucoma cases. The most common cause of NVG is ischaemic central retinal vein occlusion (iCRVO). NVG frequently results in blindness and painful end-stage glaucomatous damage leading to the need for enucleation. Currently, there is no preventive therapy for NVG following iCRVO. Rescue treatments have severe drawbacks. Accordingly, there is a great need for preventing the often visually devastating outcomes of NVG. The STRONG study is designed to test whether the topically active anti-angiogenic agent aganirsen is able to inhibit the formation of neovascularisation leading to the development of secondary NVG in eyes with iCRVO. At the same time, STRONG will provide important information on the natural course of iCRVO and NVG in a large and well-characterised cohort of such patients. METHODS/DESIGN: This protocol describes a phase II/III, prospective, randomised, placebo-controlled, double-masked, three-armed multicentre study for the investigation of aganirsen, a new topical treatment for iCRVO in order to prevent NVG. The study will evaluate the efficacy of two different doses of this newly developed antisense oligonucleotide formulated in an eye emulsion to avoid new vessel formation by blocking insulin receptor substrate-1 (IRS)-1. This leads to subsequent down-regulation of both angiogenic as well as proinflammatory growth factors such as vascular endothelial growth factor (VEGF) and tumour necrosis factor (TNF). Eligible patients (n = 333) will be treated with topical aganirsen or placebo for a period of 24 weeks. They will also be invited to participate in substudies involving analysis of gonioscopic images, detection of biomarkers for NVG and risk factors for iCRVO. DISCUSSION: The STRONG study has the potential to offer a new treatment modality for patients suffering from iCRVO with a high risk of developing NVG. The topical administration can reduce patients' burden and risk related to rescue treatment, such as destructive laser treatment or enucleation, but requires a high level of patient compliance. TRIAL REGISTRATION: EudraCT: 2014-000239-18; ClinicalTrials.gov, ID: NCT02947867 . (Registered on 15 October 2016); see also http://strong-nvg.com .

Placenta Growth Factor in Eyes with Neovascular Glaucoma Is Decreased after Intravitreal Ranibizumab Injection.

PURPOSE: To evaluate changes in the concentrations of placental growth factor (PlGF) and vascular endothelial growth factor-A (VEGF-A) in aqueous humor of patients with neovascular glaucoma (NVG) before and after an intravitreal injection of ranibizumab (IVR) and to determine the underlying correlation between the levels. METHODS: The prospective interventional comparative study involved 20 eyes of 20 patients with surgery-required advanced NVG and 20 control subjects from January 2013 to November 2013. The NVG eyes received the IVR treatment before glaucoma surgery. Aqueous humor was collected at the time of the IVR injection (pre- IVR) and at the time of antiglaucomatous surgery (post-IVR). Aqueous humor was also collected at the time of cataract surgery in normal control. Aqueous humor and plasma VEGF-A and PlGF levels were measured with an enzyme-linked immunosorbent assay methods, respectively. RESULTS: The mean aqueous humor PlGF and VEGF-A concentrations in the pre-IVR eyes were significantly higher than in those of the control subjects (p<0.001), whereas the plasma levels showed no significant difference. There was a statistically significant correlation between the aqueous humor PlGF and the VEGF-A concentration (r = 0.612, p = 0.003). The mean aqueous humor PlGF in the post-IVR eyes dramatically decreased from 1078.36 ± 755.83 to 177.64 ± 151.73 pg/mL (p<0.001). The VEGF-A level showed a similar trend from 3697.64 ± 2104.47 pg/mL to 183.54 ± 130.35 pg/mL (p<0.001). CONCLUSIONS: Aqueous humor concentrations of VEGF-A and PlGF were significantly elevated in the eyes with NVG, and there was a positive correlation between the levels. After an IVR treatment, VEGF-A and PlGF were significantly decreased in NVG eyes.

Risk Factors and Preventive Effects of Laser Panretinal Photocoagulation for Neovascular Glaucoma after Ophthalmic Stereotactic Radiotherapy.

Purpose: To evaluate the risk factors and the preventive effects of laser panretinal photocoagulation (PRP) for neovascular glaucoma (NVG) after ophthalmic stereotactic radiotherapy. Methods: Twenty-four patients with ocular malignant tumor (such as uveal malignant melanoma, lacrimal gland cancer) who received stereotactic radiotherapy (such as gamma knife, cyber knife) were retrospectively analyzed. Patients were divided into group A without preventive PRP (n=9), and group B with preventive PRP (n=15). Survival curves were plotted using the Kaplan-Meier method and compared between the two groups with the log-rank test. Results: In group A, NVG occurred in 3 patients. In contrast, in group B, no patient encountered NVG. The significant risk factor for NVG was dose-volumes that irradiated the optic disk (p=0.045). The incidence of NVG was significantly reduced in group B compared with group A (p=0.019). Conclusions: Dose-volumes that irradiated the optic disk were risk factors for NVG. PRP is effective in the prevention of NVG.

Short Communication: Increase of Sialylated N-Glycansin Eyes with Neovascular Glaucoma Secondary to Proliferative Diabetic Retinopathy.

PURPOSE: To investigate the alteration of N-glycans in the vitreous fluid of patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples were collected from 18 patients with PDR (including 7 with NVG and 11 without NVG), and 17 patients without diabetes. Profiles of N-glycans were analyzed by glycoblotting-based high throughput protocol, which we recently developed. Protein levels of vascular endothelial growth factor (VEGF)-A were measured by ELISA. RESULTS: The concentration of total N-glycans and the concentration of N-glycans with sialic acids were significantly higher in NVG group compared with those in non-NVG group or control group, whereas there was no significant difference in concentrations of high-mannose N-glycans among three groups. There was a moderate correlation between the concentrations of sialylated N-glycans and VEGF-A. CONCLUSIONS: Our data demonstrate the distinct changes of N-glycan profile and the increase of sialylated N-glycans in eyes with NVG secondary to PDR.

Angiogenic and Inflammatory Vitreous Biomarkers Associated With Increasing Levels of Retinal Ischemia.

PURPOSE: To characterize the angiogenic and inflammatory vitreous biomarker profiles in a spectrum of ischemic retinopathies, including neovascular glaucoma. METHODS: This institutional review board-approved study retrospectively analyzed 80 undiluted vitreous samples obtained during pars vitrectomy. The specimens were frozen (-80°C) and sent for concentration analysis of 34 proteins by Bio-Plex Pro assays. Specimens were divided into four groups: patients undergoing epiretinal membrane (ERM) peeling and/or macular hole (MH) surgery with no history of diabetes (non-DM group), patients undergoing ERM peeling, and/or MH surgery with a history of diabetes (DM group), patients with proliferative diabetic retinopathy (PDR group), and patients with neovascular glaucoma (NVG group). Parametric and nonparametric analyses of demographics and cytokine levels were performed using SPSS. RESULTS: There were no significant differences in demographics among cohorts. Numerous proteins were significantly elevated between non-DM and DM (G-CSF, sCD40L, Endoglin, IL-6, placental growth factor [PlGF], VEGF-D), DM and PDR (leptin, IL-8, PlGF, VEGF-A), and PDR and NVG (G-CSF, leptin, TIE-2, sCD40L, EGF, HB-EGF, IL-6, IL-8, PlGF, TNF-α). Only PlGF was significantly elevated between each successive cohort. The most potent drivers of NVG were PlGF, VEGF-A, IL-6, and IL-8. CONCLUSIONS: While the role of angioproliferative growth factors is well documented in ischemic retinopathy, our study delineates the importance of inflammatory and previously underreported angiogenic proteins. It also demonstrates a significant incremental increase in certain factors with increasing levels of ischemia. Both of these findings may guide the development of future therapies for ischemic retinopathies.

Simultaneous increase in multiple proinflammatory cytokines in the aqueous humor in neovascular glaucoma with and without intravitreal bevacizumab injection.

PURPOSE: To investigate aqueous humor proinflammatory cytokine levels of patients with neovascular glaucoma (NVG), and to analyze the effects of background factors in the expression of these molecules. METHODS: This cross-sectional study enrolled 137 participants who were grouped into (1) primary open-angle glaucoma (POAG; n = 36) patients; (2) NVG patients (NVG; n = 33); and (3) cataract surgery patients as a comparative group (CG; n = 68). Aqueous humor samples were collected from the anterior chamber at the start of surgery, deposited in CryoTubes, registered, and stored at -80 °C until processing. Multiplex microparticle-based immunodetection was performed by using xMAP and the Human Cytokine/Chemokine Panel I. Bevacizumab was injected into the vitreous cavity 1 to 2 days before surgery in 22 NVG patients (IVB group), whereas 11 NVG patients received no antivascular endothelial growth factor (VEGF) therapy 3 months preoperatively (N group). The Wilcoxon rank sum test or Fisher's exact test for two variables and the Tukey-Kramer honestly significant difference test for multiple variables were used to compare the cytokine levels. RESULTS: The NVG patients had higher levels of interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor-α (TNF-α), and platelet-derived growth factor (PDGF)-AA compared to both the CG and POAG groups. The levels of IL-6, IL-8, MCP-1, and PDGF-AB/BB were higher in the IVB group than the N group, whereas the VEGF level was significantly lower in the IVB group (P < 0.01). CONCLUSIONS: Intravitreal bevacizumab injection decreased VEGF levels, but not those of the other cytokines.

Angiogenesis in glaucoma filtration surgery and neovascular glaucoma: A review.

Angiogenesis may pose a clinical challenge in glaucoma, for example, during the wound healing phase after glaucoma filtration surgery and in the severe secondary glaucoma called neovascular glaucoma (NVG). Upregulation of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, occurs in eyes that have undergone glaucoma filtration surgery, as well as those with NVG. This has led investigation of the ability of anti-vascular endothelial growth factor therapy to improve outcomes, and we examine the findings with respect to the safety and efficacy of anti-vascular endothelial growth factor agents, mainly bevacizumab and ranibizumab, in eyes that have undergone glaucoma filtration surgery or have NVG. Combining conventional therapies-such as antimetabolites after filtration surgery and panretinal photocoagulation in NVG-and anti-vascular endothelial growth factor drugs may produce a synergetic effect, although further studies are required to evaluate the long-term efficacy of combination treatments.

Alteration of UCP2 and ZO-1 expression in trabecular meshwork of neovascular glaucoma patients.

PURPOSE: The aim of this study was to investigate the expression of uncoupling protein 2 (UCP2) and zonula occludens-1 (ZO-1) in the trabecular meshwork (TM) of neovascular glaucoma (NVG) patients treated with trabeculectomy. MATERIALS AND METHODS: Six eyes with NVG underwent trabeculectomy for therapeutic purposes. The data consisted of patient demographics, presurgical and postsurgical visual acuity, intraocular pressure, gonioscopy, and neovascularization of iris and/or the anterior chamber angle. TM samples were obtained from the NVG eyes that had undergone surgery. Immunofluorescence and confocal laser scanning microscopy were carried out to determine the expression of UCP2 and ZO-1 in the TM cells. RESULTS: The baseline median visual acuity was light perception, and the mean intraocular pressure (standard error) was 52.5 (8.3) mm Hg. All eyes displayed neovascularization of the iris and the anterior chamber angle. The expression of UCP2 was significantly decreased in TM cells of NVG compared with the control (P=0.000), whereas increase in ZO-1 expression was detected in staining cells with NVG in comparison with the control (P=0.000). The necrotic cells in the TM were increased (P=0.000), whereas the viable cells were reduced (P=0.000) in comparison with the control. CONCLUSIONS: The decreased UCP2 expression and increased ZO-1 expression suggest that the oxidative stress-induced mitochondrial dysfunction and tight junction formation may play pivotal roles in the progress of NVG.

Short-term effect of intravitreal ranibizumab on intraocular concentrations of vascular endothelial growth factor-A and pigment epithelium-derived factor in neovascular glaucoma.

BACKGROUND: This study aims to evaluate concentrations of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF)-A in aqueous of patients with neovascular glaucoma prior to, and shortly after, an intravitreal ranibizumab injection. DESIGN: Interventional comparative study. PARTICIPANTS: The study included patients undergoing an intravitreal ranibizumab injection about one week before anti-glaucomatous surgery (study group) or who underwent routine cataract surgery (control group). METHODS: Aqueous and blood samples were collected at the occasions of intravitreal injections, anti-glaucomatous surgery or cataract surgery. They were analysed by enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Concentrations of PEDF and VEGF-A in aqueous. RESULTS: At baseline, concentrations VEGF-A (3698 ± 2105 pg/mL vs. 233 ± 98 pg/mL) and PEDF (18.9 ± 11.9 ug/mL vs. 2.2 ± 0.6 ug/mL) were higher (P < 0.001) in the study group (n = 20 patients) than control group (n = 20 patients). The VEGF-A/PEDF concentration ratio was higher in the study group (396 ± 554 vs. 110 ± 49; P = 0.02). One week after the ranibizumab injection, iris neovascularization had completely regressed in 17 (85%) eyes, and VEGF-A concentration decreased significantly (P < 0.001) to 184 ± 130 pg/mL. The PEDF concentration remained unchanged (19 ± 12 ug/mL). The VEGF-A/PEDF concentration ratio decreased to 13.2 ± 13.6. Plasma concentrations of VEGF-A and PEDF did not differ significantly between both groups (P = 0.65 and P = 0.15, respectively) nor were they significantly correlated with the aqueous concentrations (all P > 0.15). CONCLUSIONS: Aqueous concentrations of VEGF-A and PEDF were significantly elevated in eyes with neovascular glaucoma. Within one week after intravitreal injection of ranibizumab, VEGF-A concentration decreased to subnormal levels, while the PEDF concentration remained unchanged and the VEGF-A/PEDF ratio decreased.

NEOVASCULAR SECONDARY GLAUCOMA, ETIOLOGY AND PATHOGENESIS.

RATIONALE: Neovascular secondary glaucoma is a condition characterized by increased intraocular pressure due to the neovascularization occurring at the iridocorneal angle and iris, the most common complication of end-stage ischemic retina. The early diagnosis and treatment of this disease are important, because the functional prognosis is reserved. OBJECTIVE: Knowing and understanding the etiology and pathogenesis of neovascular secondary glaucoma. METHODS AND RESULTS: Review of the angiogenesis theory to understand the etiology and pathogenesis of neovascular secondary glaucoma. VEGF is the most studied proangiogenic factor involved in the neovascular glaucoma pathogenesis. The 9 isoforms contain consensus signal sequences for extracellular secretion, all of them binding to a specific receptor subtype and stimulating tissue specific angiogenesis. VEGF and VEGF-m RNA levels are significantly increased in the ischemic retina. Diabetes mellitus (with diabetic retinopathy), central retinal vein thrombosis and repeated retinal detachments are diseases that cause neovascular glaucoma through ischemia. DISCUSSION: Correct evaluation of the iris neovascularization followed by a proper treatment is the most important in a case of secondary neovascular glaucoma. Repeated gonioscopy is indicated in cases with high risk of developing neovascular glaucoma. Close monitoring of a patient with high thromboembolic risk: valvular heart disease, open-heart surgery, other angioplasties.