Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy.

BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.

The absence of angiopoietin-2 leads to abnormal vascular maturation and persistent proliferative retinopathy

Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.

Obstructive sleep apnoea is associated with sight threatening retinopathy and predicts the developement of preproliferative and proliferative retinopathy in patients with type 2 diabetes:a longitudinal analysis

troduct I on . An observational longitudinal study. P ur P ose . Assess the relationship between obstructive sleep apnoea (OSA) and DR cross-sectionally and longitudinally. M ethods . Adults with Type 2 diabetes mellitus (T2DM), who were re - cruited from a hospital-based diabetes clinic in the UK. Patients with pre-existing OSA, end-stage renal disease and non-diabetic retinopa - thy were excluded. OSA (apnoea hypopnea index ≥ 5 events/hour) was assessed by a single overnight home-based cardio-respiratory study (Alice PDX, Philips Respironics, USA). DR was assessed us - ing retinal images between 2007 and 2012. Sight threatening diabetic retinopathy (STDR) was defined as presence of pre-proliferative or proliferative DR, maculopathy or photocoagulation. Advanced DR was defined as pre-proliferative or proliferative DR. r esults . 199 patients were included (57.3% (n=114) men, 47.7% (n=95) White Europeans). STDR and OSA prevalence were 38.7% (n=77) and 62.8% respectively. A t b A sel I ne . STR prevalence was higher in patients with OSA (OSA+) compared to those without OSA (OSA-) [48.8% n=61 vs. 21.6% n=16, p<0.001]. After adjustment for confounders, OSA remained independently associated with STR (OR 3.7, 95% CI 1.6-8.9, p=0.006), maculopathy (OR 4.5, 95% CI 1.8-11.4, p=0.002) and advanced DR (OR 3.9, 95% CI 1.02-15.3, p=0.047). Mild and moderate to severe OSA were independently associated with STR and maculopathy and only moderate to severe OSA was associated with advanced DR following adjustment for con - founders. l ong I tud I n A lly . Over the follow-up period of (4.4±1 years), more OSA+ patients progressed from no or background DR to advanced DR (15.3% (n=17) vs. 3% (n=2), p=0.01). OSA was an independent pre - dictor of advanced DR development after adjustment for confounders (OR 6.6, 95% CI 1.2-35.1, p=0.03). OSA did not predict the develop - ment of maculopathy. c onclus I ons . OSA is independently associated with STR and predicts the development of preproliferative and proliferative DR. Intervention - al studies are needed to assess the impact of OSA treatment on DR.

Obstructive sleep apnoea predicts the development of preproliferative and proliferative retinopathy in patients with type 2 diabetes:a longitudinal analysis

Background and aims: Diabetic Retinopathy (DR) is a leading cause of blindness. OSA is associated with increased oxidative and nitrosative stress and endothelial dysfunction in patients with type 2 diabetes (T2DM). Hence, it is plausible that OSA can promote the development and progression of DR. Materials and methods: An observational longitudinal study in adults with T2DM. Patients with pre-existing OSA, end-stage renal disease and non-diabetic retinopathy were excluded. OSA (apnoea hypopnea index ≥ 5 events/hour) was assessed by a single overnight home-based cardio-respiratory monitoring (Alice PDX, etc.). DR was assesses using retinal images between 2007 and 2012. Sight threatening retinopathy (STR) was defined as pre-proliferative or proliferative DR, maculopathy or photocoagulation. Advanced DR was defined as pre-proliferative or proliferative DR. Results: 199 patients were included (57.3% men, 47.7% White Europeans). STR and OSA prevalence were 38.7 % and 62.8% respectively. STR preva-lence was higher in patients with OSA (OSA+) compared to those with-out (OSA-) [48.8% vs. 21.6%, p <0.001]. After adjustment for confounders, OSA remained independently associated with STR (OR 3.7, 95%CI 1.6-8.9, p=0.006, maculopathy (OR 4.5, 1.8-11.4, p=0.002) and advanced DR (OR 3.9, 1.02-15.3, p=0.047). Over 4.4±1 years, more OSA+ patients progressed from no or background DR to advanced DR (15.3% vs. 3%, p=0.01). OSA was an independent predictor of advanced DR development after adjustment (OR 6.6, 95%CI 1.2-35.1, p=0.03). OSA did not predict the development of maculopathy. Patients received continuous positive airway pressure treatment were less likely to develop advanced DR. Conclusion: OSA is independently associated with STR and predicts the development of preproliferative and proliferative DR. Interventional studies are needed to assess the impact of OSA treatment on DR.Supported by: NIHR (UK) and The UK Novo Nordisk Research Foundation.

Painless Indirect Argon Laser in High Risk Proliferative Diabetic Retinopathy.

Background: Proliferative retinopathy is an important cause of vision loss in diabetic patients. Incomplete panretinal photocoagulation (PRP) can lead to recurrent proliferation of new vessels. Patients and Methods: We retrospectively analysed the outcome of patients with high risk proliferative diabetic retinopathy (PDR) previously treated with slit lamp PRP who underwent indirect fill in argon laser treatment with scleral indentation under anesthesia for persistent neovascular proliferation. Results: Seventeen eyes of ten patients were included. The mean age at diabetes onset was 17.3 years SD 16.2 (range 2-44). All patients reported long standing poor glycemic control (mean HbA1c: 8.5 % SD 1.3 range 5.9-10.2). The area of retinal ischemia decreased significantly from 15 ± 7.5 disk areas (DA) before fill-in laser to 3.2 ± 4.2 DA after fill-in laser (p = 0.001). The new vessels also regressed significantly after laser treatment 8.6 ± 6.1 DA before treatment versus 6.5 ± 6.4 DA after laser treatment, (p = 0.044). Quiescent PDR was reached in 10 eyes (58.8 %) at the last visit. Conclusions: Fill-in indirect argon laser under general anesthesia should be considered to achieve further new vessels regression in high risk PDR patients. Scleral indentation and absence of pain may allow for more extensive laser application.

Pericytes and the pathogenesis of diabetic retinopathy

Pericytes provide vascular stability and control endothelial proliferation. Pericyte loss, microaneurysms, and acellular capillaries are characteristic for the diabetic retina. Platelet-derived growth factor (PDGF)-B is involved in pericyte recruitment, and brain capillaries of mice with a genetic ablation of PDGF-B show pericyte loss and microaneurysms. We investigated the role of capillary coverage with pericytes in early diabetic retinopathy and the contribution to proliferative retinopathy using mice with a single functional allele of PDGF-B (PDGF-B(+/-) mice). As assessed by quantitative morphometry of retinal digest preparations, pericyte numbers in nondiabetic PDGF-B(+/-) mice were reduced by 30% compared with wild-type mice, together with a small but significant increase in acellular capillaries. Pericyte numbers were reduced by 40% in diabetic wild-type mice compared with nondiabetic wild-type controls. Pericyte numbers were decreased by 50% in diabetic PDGF-B(+/-) mice compared with nondiabetic wild-type littermates, and the incidence of acellular capillaries was increased 3.5-fold when compared with nondiabetic PDGF-B(+/-) mice. To investigate the effect of pericyte loss in the context of ongoing angiogenesis, we subjected mice to hypoxia-induced proliferative retinopathy. As a result, PDGF-B(+/-) mice developed twice as many new blood vessels as their wild-type littermates. We conclude that retinal capillary coverage with pericytes is crucial for the survival of endothelial cells, particularly under stress conditions such as diabetes. At high vascular endothelial growth factor levels, such as those in the retinopathy of prematurity model, pericyte deficiency leads to reduced inhibition of endothelial proliferation in vivo.

Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy:effects of laser photocoagulation and angiotensin receptor blockade

Background: Proliferative diabetic retinopathy (PDR) may be a response to abnormal angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and the soluble angiopoietin receptor tie-2. The authors hypothesised the following: (a) there are differences in plasma levels of these growth factors in different grades of diabetic retinopathy; and (b) that the effects of intervention with panretinal laser photocoagulation (PRP) for PDR, and angiotensin receptor blockade (using eprosartan) for patients with other grades of diabetic retinopathy will be to reduce levels of the growth factors. Methods: Cross sectional and interventional study (using PRP and eprosartan) in diabetic patients. VEGF, Ang-2, and tie-2 were measured by ELISA. Results: VEGF (p<0.001) and Ang-2 levels (p<0.001) were significantly higher in 93 diabetic patients compared to 20 healthy controls, with the highest levels in grade 2 and grade 3 diabetic retinopathy (p<0.05). Tie-2 was lower in diabetics compared to controls (p = 0.008), with no significant differences between the diabetic subgroups. Overall, VEGF significantly correlated with Ang-2 (p<0.001) and tie-2 (p = 0.004) but the correlation between Ang-2 and tie-2 levels was not significant (p = 0.065). Among diabetic patients only, VEGF levels were significantly correlated with Ang-2 (p<0.001) and tie-2 (p<0.001); the correlation between Ang-2 and tie-2 levels was also significant (p<0.001). There were no statistically significant effects of laser photocoagulation on plasma VEGF, Ang-2, and tie-2 in the 19 patients with PDR, or any effects of eprosartan in the 28 patients with non-proliferative diabetic retinopathy. Conclusion: Increased plasma levels of VEGF and Ang-2, as well as lower soluble tie-2, were found in diabetic patients. The highest VEGF and Ang-2 levels were seen among patients with pre-proliferative and proliferative retinopathy, but there was no relation of tie-2 to the severity of retinopathy. As the majority of previous research into Ang-2 and tie-2 has been in relation to angiogenesis and malignancy, the present study would suggest that Ang-2 and tie-2 may be used as potential indices of angiogenesis in diabetes mellitus (in addition to VEGF) and may help elucidate the role of the angiopoietin/tie-2 system in this condition.

Natural history of retinopathy in children and young people with type 1 diabetes

Purpose: To describe the prevalence and natural history of retinopathy in a cohort of children and young people with type 1 diabetes attending a tertiary hospital diabetes clinic. Methods: We analysed retinopathy screening data from 2008 to 2010 on all eligible children using the 'Twinkle' diabetes database and the regional retinal screening database. Results: A total of 88% (149/169) of eligible children were screened in 2008, median age 14 years, 52% male. The prevalence of retinopathy was 19.5% (30/149). All children had background retinopathy grade R1. There was significant difference in median (range) duration of diabetes, 7.7 years (0.6–13.7) vs 5 years (0.2–12.5) (P<0.001) and median (range) HbA1C, 9.1% (7.2–14) vs 8.6% (5.6–13.1) (P=0.02), between the groups with and without retinopathy. At 2- years follow-up, 12/30 (40%) had unchanged retinopathy grade R1, 10/30 (33.3%) showed resolution of changes (R0), 1/30 progressed to maculopathy, and 7/30 had no follow-up data. Median (range) HbA1C in 2008 and 2010 for the groups with stable vs resolved changes was similar, 9.1% (7.2–14.0) and 9.2% (7–14.0) vs 9.5% (7.8–14.0) and 9.2% (8.7–14.0). Of the 119 without retinopathy in 2008, 27 (22.5%) had developed retinopathy within 2 years, including 1 with pre-proliferative retinopathy and 1 with maculopathy. There was no significant difference in HbA1c between those who progressed to retinopathy (8.7% (7.1–13.1)) (8.7% (7.1–13.1)), and those who did not (8.6% (6.3–12.2)). Conclusions: Prevalence of background retinopathy in our cohort was comparable to the previously published reports, with higher HbA1c and longer duration of diabetes being significant risk factors. On short-term follow-up, Grade 1 retinopathy is likely to resolve in a third of patients and remain unchanged in just over a third.

Key Endothelial Cell Angiogenic Mechanisms Are Stimulated By The Circulating Milieu In Sickle Cell Disease And Attenuated By Hydroxyurea.

As hypoxia-induced inflammatory angiogenesis may contribute to sickle cell disease manifestations, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from steady-state sickle cell anemia patients presented elevated concentrations of pro-angiogenic factors (Angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly augmenting endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice, compared with non-sickle cell disease mice, consistent with an upregulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy displayed a pro-angiogenic profile and had more significant effects on endothelial cell proliferation and capillary formation than plasma of patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factor profile, in association with an inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, sickle cell anemia and retinopathic hemoglobin SC individuals present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy for preventing progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

(106)Ruthenium brachytherapy for retinoblastoma.

PURPOSE: To evaluate the efficacy of (106)Ru plaque brachytherapy for the treatment of retinoblastoma. METHODS AND MATERIALS: We reviewed a retrospective, noncomparative case series of 39 children with retinoblastoma treated with (106)Ru plaques at the Jules-Gonin Eye Hospital between October 1992 and July 2006, with 12 months of follow-up. RESULTS: A total of 63 tumors were treated with (106)Ru brachytherapy in 41 eyes. The median patient age was 27 months. (106)Ru brachytherapy was the first-line treatment for 3 tumors (4.8%), second-line treatment for 13 (20.6%), and salvage treatment for 47 tumors (74.6%) resistant to other treatment modalities. Overall tumor control was achieved in 73% at 1 year. Tumor recurrence at 12 months was observed in 2 (12.5%) of 16 tumors for which (106)Ru brachytherapy was used as the first- or second-line treatment and in 15 (31.9%) of 47 tumors for which (106)Ru brachytherapy was used as salvage treatment. Eye retention was achieved in 76% of cases (31 of 41 eyes). Univariate and multivariate analyses revealed no statistically significant risk factors for tumor recurrence. Radiation complications included retinal detachment in 7 (17.1%), proliferative retinopathy in 1 (2.4%), and subcapsular cataract in 4 (9.7%) of 41 eyes. CONCLUSION: (106)Ru brachytherapy is an effective treatment for retinoblastoma, with few secondary complications. Local vitreous seeding can be successfully treated with (106)Ru brachytherapy.

Vascular endothelial growth factor-B and retinal vascular development in the mouse

Purpose: Vascular endothelial growth factor-A (VEGF-A) is crucial to retinal vascular growth, both normal and pathological. VEGF-B, recently characterized, is reported to be expressed in retinal tissues, but the importance of VEGF-B to retinal vascular development remained unknown. The aim of this study was to analyse retinal vascular growth in the Vegfb (-/-) knockout mouse. Methods: Retinal vascular growth was measured in Vegfb (-/-) knockout mice raised under normal conditions, and Vegfb (-/-) knockout mice with an oxygen-induced proliferative retinopathy. Wild type Vegfb (+/+) mice served as controls. Vessels were perfused with ink and retinal flatmounts secondarily labelled with FITC-lectin (BS-1, Griffonia simplicifolia ). Area and diameter of retinal growth and retinal vascular growth were recorded over days 0-20, and capillary density and mean diameter recorded from day 17 pups. Results: A variety of techniques confirmed that Vegfb (+/+) mice expressed VEGF-B and that VEGF-B expression was absent in Vegfb (-/-) mice. Vegfb (-/-) mice raised in room air showed no significant differences from Vegfb (+/+) controls. No differences were found in oxygen-induced retinopathy between Vegfb (-/-) and Vegfb (+/+) pups in either the extent of the initial oxygen-induced ablation, or in the regrowth of retinal vessels or vitreal (neovascular) sprouts; vitreal sprouts are important markers of the abnormal proliferative response, and are maximally expressed on day 17 in this model of oxygen-induced retinopathy. Conclusions: These results indicate that a lack of VEGF-B does not significantly affect development of the retinal vasculature under normal conditions, nor does it appear to affect the proliferative retinal responses seen in oxygen-induced retinopathy.

Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys

Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFIt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The expression of sFIt-1 was associated with the long-term (8 months) regression of neovascular vessels in 85% of trVEGF029 eyes. In addition, it resulted in the maintenance of retinal morphology, as the majority of the treated trVEGF029 eyes (75%) retained high numbers of photoreceptors, and in retinal function as measured by electroretinography. AAV-mediated expression of sFIt-1 prevented the development of laser photocoagulation-incluced choroidal neovascularization in all treated monkey eyes. There were no clinically or histologically detectable signs of toxicity present in either animal model following AAV.sFlt injection. These results suggest that AAV-mediated secretion gene therapy could be considered for treatment of retinal and choroidal neovascularizations.

Pain response and follow-up of patients undergoing panretinal laser photocoagulation with reduced exposure times

Purpose - We performed a study of laser panretinal photocoagulation in 20 patients with proliferative retinopathy. We compared short exposure, high-energy laser settings with conventional settings, using a 532?nm, frequency doubled, Neodymium–Yag laser and assessed the patients in terms of pain experienced and effectiveness of treatment. Methods - Twenty patients having panretinal photocoagulation for the first time underwent random allocation to treatment of the superior and inferior hemi-retina. Treatment A used ‘conventional’ parameters: exposure time 0.1?s, power sufficient to produce a visible grey-white burns, spot size 300?µm. The other hemi- retina was treated with treatment B using exposure 0.02?s, 300?µm and sufficient power to have similar endpoint. All patients were asked to evaluate severity of pain on a visual analogue scale. (0=no pain, 10=most severe pain). All patients were masked as to the type of treatment and the order of carrying out the treatment on each patient was randomised. Patients underwent fundus photography and were followed up for 6–45 months. Results - Seventeen patients had proliferative diabetic retinopathy, two had ischaemic central retinal vein occlusion and one had ocular ischaemic syndrome. The mean response to treatment A was 5.11, compared to 1.40 treatment B, on the visual analogue scale, which was statistically significant (P=0.001). All patients preferred treatment B. Further treatments, if required, were performed with treatment B parameters and long-term follow-up has shown no evidence of undertreatment. Conclusions - Shortening exposure time of retinal laser is significantly less painful but equally effective as conventional parameters.

How accurate are photographic surrogate markers used to detect macular edema in the English national screening programme?

DESIGN. Retrospective analysis PURPOSE. Macular oedema is not directly visible on two dimensional digital photographs such that surrogate markers need to be used. In the English National Screening Programme these are exudate within one optic disc diameter (DD) of the fovea, group of exudates within two DD of the fovea and haemorrhages or microaneurysms (HMA) within one DD of the fovea with best corrected visual acuity (VA) worse than 6/9. All patients who present with any of these surrogate markers at screening are referred to an ophthalmology clinic for slit lamp examination. The purpose of this audit was to determine how many patients with positive maculopathy diagnosis on photography were truly identified by optical coherence tomography (OCT) with macular oedema. METHODS. Data was collected from patients attending digital diabetic retinopathy screening. Patients who presented with surrogate markers for macular oedema also had an OCT scan. The fast macula scan on the Stratus OCT was used and an ophthalmologist reviewed the scans to determine whether macular oedema was present. RESULTS. Maculopathy by exudates: Of 155 patients 45 (29%) showed thickening on the OCT of these 12 required laser. Those who also had pre-proliferative retinopathy (n=20) were more likely to have macular oedema (75%) than those with background diabetic retinopathy. Maculopathy by HMA and VA worse than 6/9: Of 66 patients 11 (16.7%) showed thickening on the OCT. 5 (7.6%) of these had macular oedema, 5 (7.6%) epi-retinal membrane, and 1 (1.5%) age related macular degeneration. None of these patients required laser. CONCLUSIONS. The likelihood of the presence of macular oedema and requiring laser treatment is greater with macular exudation than HMA within one DD and reduced VA. Overall the surrogate markers used show low specificity for macular oedema, however combining OCT with photography does identify those with macular oedema who require a true referral for an ophthalmological slit lamp examination.