Robert L. Avery

Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders

BACKGROUND Retinal ischemia induces intraocular neovascularization, which often leads to glaucoma, vitreous hemorrhage, and retinal detachment, presumably by stimulating the release of angiogenic molecules. Vascular endothelial growth factor (VEGF) is an endothelial-cell-specific angiogenic factor whose production is increased by hypoxia. METHODS We measured the concentration of VEGF in 210 specimens of ocular fluid obtained from 164 patients undergoing intraocular surgery, using both radioimmuno-assays and radioreceptor assays. Vitreous proliferative potential was measured with in vitro assays of the growth of retinal endothelial cells and with VEGF-neutralizing antibody. RESULTS VEGF was detected in 69 of 136 ocular-fluid samples from patients with diabetic retinopathy, 29 of 38 samples from patients with neovascularization of the iris, and 3 of 4 samples from patients with ischemic occlusion of the central retinal vein, as compared with 2 of 31 samples from patients with no neovascular disorders (P < 0.001, P < 0.001, and P = 0.006, respectively). The mean (+/- SD) VEGF concentration in 70 samples of ocular fluid from patients with active proliferative diabetic retinopathy (3.6 +/- 6.3 ng per milliliter) was higher than that in 25 samples from patients with nonproliferative diabetic retinopathy (0.1 +/- 0.1 ng per milliliter, P = 0.008), 41 samples from patients with quiescent proliferative diabetic retinopathy (0.2 +/- 0.6 ng per milliliter, P < 0.001), or 31 samples from nondiabetic patients (0.1 +/- 0.2 ng per milliliter, P = 0.003). Concentrations of VEGF in vitreous fluid (8.8 +/- 9.9 ng per milliliter) were higher than those in aqueous fluid (5.6 +/- 8.6 ng per milliliter, P = 0.033) in all 10 pairs of samples obtained simultaneously from the same patient; VEGF concentrations in vitreous fluid declined after successful laser photocoagulation. VEGF stimulated the growth of retinal endothelial cells in vitro, as did vitreous fluid containing measurable VEGF. Stimulation was inhibited by VEGF-neutralizing antibodies. CONCLUSIONS Our data suggest that VEGF plays a major part in mediating active intraocular neovascularization in patients with ischemic retinal diseases, such as diabetic retinopathy and retinal-vein occlusion.

Electrophysiologic and retinal penetration studies following intravitreal injection of bevacizumab (Avastin).

Purpose: Intravitreal bevacizumab (Avastin; Genentech Inc., San Francisco, CA) is a new treatment for age-related macular degeneration. The aim of this study was to evaluate retinal penetration and toxicity of bevacizumab. Methods: Ten albino rabbits were injected intravitreally with 0.1 mL (2.5 mg) of Avastin into one eye and 0.1 mL saline into the fellow eye. The electroretinogram (ERG) was recorded after 3 hours, 3 days, and 1, 2, and 4 weeks. The visual evoked potential (VEP) was recorded after 4 weeks. Confocal immunohistochemistry was used to assess retinal penetration. Results: The ERG responses of the control and experimental eyes were similar in amplitude and pattern throughout the follow-up period. The flash VEP responses of the experimental eyes were of normal pattern and amplitude and did not differ from those recorded by stimulation of the control eye alone. Full thickness retinal penetration was present at 24 hours and was essentially absent at 4 weeks. Conclusions: Bevacizumab was found to be nontoxic to the retina of rabbits based on electrophysiologic studies. Full thickness retinal penetration may explain observed clinical effects of intravitreal bevacizumab. Although it is difficult to directly extrapolate to humans, our study supports the safe use of intravitreal bevacizumab injection.

Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation

Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy–based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I–induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I–induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.

Promoter polymorphism of the erythropoietin gene in severe diabetic eye and kidney complications

Significant morbidity and mortality among patients with diabetes mellitus result largely from a greatly increased incidence of microvascular complications. Proliferative diabetic retinopathy (PDR) and end stage renal disease (ESRD) are two of the most common and severe microvascular complications of diabetes. A high concordance exists in the development of PDR and ESRD in diabetic patients, as well as strong familial aggregation of these complications, suggesting a common underlying genetic mechanism. However, the precise gene(s) and genetic variant(s) involved remain largely unknown. Erythropoietin (EPO) is a potent angiogenic factor observed in the diabetic human and mouse eye. By a combination of case–control association and functional studies, we demonstrate that the T allele of SNP rs1617640 in the promoter of the EPO gene is significantly associated with PDR and ESRD in three European-American cohorts [Utah: P = 1.91 × 10−3; Genetics of Kidneys in Diabetes (GoKinD) Study: P = 2.66 × 10−8; and Boston: P = 2.1 × 10−2]. The EPO concentration in human vitreous body was 7.5-fold higher in normal subjects with the TT risk genotype than in those with the GG genotype. Computational analysis suggests that the risk allele (T) of rs1617640 creates a matrix match with the EVI1/MEL1 or AP1 binding site, accounting for an observed 25-fold enhancement of luciferase reporter expression as compared with the G allele. These results suggest that rs1617640 in the EPO promoter is significantly associated with PDR and ESRD. This study identifies a disease risk-associated gene and potential pathway mediating severe diabetic microvascular complications.

Systemic pharmacokinetics following intravitreal injections of ranibizumab, bevacizumab or aflibercept in patients with neovascular AMD

Background Data comparing systemic exposure and systemic vascular endothelial growth factor (VEGF) suppression of ranibizumab, bevacizumab and aflibercept following intravitreal injection are lacking. Methods Fifty-six patients with wet age-related macular degeneration received intravitreal ranibizumab (0.5 mg), bevacizumab (1.25 mg), or aflibercept (2.0 mg). Serum pharmacokinetics and plasma free VEGF were evaluated after the first and third injections. Results Following the first dose, systemic exposure to aflibercept was 5-, 37-, and 9-fold higher than ranibizumab, whereas, bevacizumab was 9-, 310-, and 35-fold higher than ranibizumab, based on geometric mean ratio of peak and trough concentrations and area under the curve, respectively. The third dose showed accumulation of bevacizumab and aflibercept but not ranibizumab. Aflibercept substantially suppressed plasma free VEGF, with mean levels below lower limit of quantitation (10 pg/mL) as early as 3 h postdose until ≥7 days postdose. Mean free (unbound) VEGF levels with ranibizumab were largely unchanged, with mean trough level of 14.4 pg/mL compared with baseline of 17 pg/mL. Conclusions There are notable differences in systemic pharmacokinetics and pharmacodynamics among anti-VEGF treatments after intravitreal administration. All three agents rapidly moved into the bloodstream, but ranibizumab very quickly cleared, whereas bevacizumab and aflibercept demonstrated greater systemic exposure and produced a marked reduction in plasma free VEGF. Trial registration number NCT02118831.

Ranibizumab for the Treatment of Macular Edema Associated with Perfused Central Retinal Vein Occlusions

PURPOSE Assessment of biological effect, visual acuity changes, and safety of intravitreal (IVT) ranibizumab in patients with macular edema associated with perfused central retinal vein occlusion (CRVO). DESIGN Ongoing, prospective, open-label, single-center, uncontrolled study. PARTICIPANTS Ten adult patients with macular edema associated with perfused CRVO. METHODS Patients were randomly assigned to receive 3 monthly IVT injections of either 0.3 or 0.5 mg ranibizumab (n = 5 at each dose). Additional injections were administered quarterly as needed over the ensuing 21 months at the physicians discretion for recurrent or persistent macular edema. MAIN OUTCOME MEASURES The predetermined primary endpoint was the percentage of patients gaining >or=15 letters of best-corrected Early Treatment of Diabetic Retinopathy Study visual acuity (BCVA). The secondary endpoints include the mean change in BCVA and central retinal thickness (CRT) measured by optical coherence tomography, the rate of progression to ischemic CRVO, extent of intraocular hemorrhage, retinal vein diameter, optic nerve head swelling, and the incidence and severity of ocular and nonocular adverse events. RESULTS After 3, 6, and 9 months of follow-up, 40%, 10%, and 30% of patients, respectively, gained >or=15 letters in BCVA; mean BCVA improved by 12+/-20 letters, 3+/-21 letters, and 1+/-24 letters, respectively, compared with baseline; CRT showed a mean decrease of 272+/-244 microm, 88+/-178 microm, and 119+/-153 microm, compared with baseline. No significant differences were observed between the 0.3- and 0.5-mg doses. Most patients experienced decreases in the extent of retinal hemorrhage, retinal vein diameter, and optic nerve head swelling at months 3 and 6 compared with baseline. No patients progressed to ischemic CRVO or experienced a severe adverse event that was attributed to ranibizumab. CONCLUSIONS Ranibizumab is generally well-tolerated and may improve BCVA and decrease CRT. The improvements in BCVA and CRT observed during the initial monthly injection period (0 to 3 months) were possibly lost to the recurrence of macular edema in between ranibizumab injection during the quarterly treatments (3 to 9 months). The extent of retinal hemorrhage, retinal vein diameter, and nerve swelling continued to normalize for most of the patients from baseline to 6 months. Follow-up is ongoing, and alternative dosing regimens are being evaluated.

Intravitreal injection technique and monitoring: updated guidelines of an expert panel.

Purpose: To review evidence and provide updated guidelines on intravitreal (IVT) injection technique and monitoring. Methods: A review of the published literature on IVT injection from 2004 to 2014 formed the basis for round table deliberations by an expert panel of ophthalmologists. Results: The dramatic increase in the number of IVT injections has been accompanied by a comparable increase in evidence surrounding IVT practice patterns and techniques. The expert panel identified a number of areas that have evolved since publication of the original IVT injection guidelines in 2004, the most notable of which were a lack of evidence to support the routine use of pre-, peri-, and postinjection antibiotics to reduce the risk of endophthalmitis, and the role of aerosolized droplets containing oral contaminants from the patient and/or providers as a potential source of infection. The panel emphasized the continued importance of applying povidone–iodine to and avoiding eyelid contact with the intended injection site and needle. Conclusion: Updated guidelines on IVT injection technique and monitoring are proposed based on a review of published literature and expert panel deliberations.

Systemic Safety of Prolonged Monthly Anti-Vascular Endothelial Growth Factor Therapy for Diabetic Macular Edema: A Systematic Review and Meta-analysis.

IMPORTANCE Anti-vascular endothelial growth factor (VEGF) therapy is commonly used to treat numerous retinal conditions and appears safe, yet controversy remains regarding systemic safety. OBJECTIVE To evaluate the systemic safety of intravitreous anti-VEGF injections in high-risk patients with diabetic macular edema (DME) and to investigate separately the subgroup of these patients with the highest level of exposure to anti-VEGF monthly treatment for 2 years. DATA SOURCES A search of MEDLINE, Cochrane Central Register of Controlled Trials, clincaltrials.gov, and ophthalmology congress abstracts January 1, 1947, to May 19, 2015. STUDY SELECTION Randomized clinical trials were selected that evaluated monthly anti-VEGF injections for DME for 2 years and reported the outcome measures of cerebrovascular accidents, myocardial infarctions, arteriothrombotic events, and mortality. DATA EXTRACTION AND SYNTHESIS Two reviewers collected data independently from each study for the meta-analysis. Data were pooled using a fixed-effects model and analyzed from November 6, 2014, to June 28, 2015. Peto odds ratios with 95% CIs were calculated. MAIN OUTCOMES AND MEASURES Primary end points included cerebrovascular accidents and all-cause mortality in the highest-dose arms. Secondary outcomes included myocardial infarctions, arteriothrombotic events, and vascular-related death. RESULTS Of 1126 articles reviewed, 598 were removed as duplicate studies and 524, for lack of monthly treatment data for 2 years, leaving 4 studies for the meta-analysis that met the search criteria: 2 trials using monthly aflibercept and 2 using monthly ranibizumab, representing 1328 patients. The primary evaluation (1078 patients) combined the monthly aflibercept and the 0.5-mg ranibizumab arms and yielded an increased risk for death compared with sham and laser treatments (odds ratio [OR], 2.98; 95% CI, 1.44-6.14; P = .003). Analysis including monthly aflibercept and 0.5-mg ranibizumab yielded an increased risk for cerebrovascular accidents (OR, 2.33; 95% CI, 1.04-5.22; P = .04) and vascular death (OR, 2.51; 95% CI, 1.08-5.82; P = .03). No definitive increased risk for myocardial infarctions and arteriothrombotic events was seen with all dose combinations. CONCLUSIONS AND RELEVANCE In this meta-analysis of anti-VEGF agents for patients with DME, assessment of the highest-level exposure group (those high-risk patients with DME who received 2 years of monthly treatment) revealed a possible increased risk for death and potentially for cerebrovascular accidents. Consideration of total exposure to anti-VEGF agents when treating those at high risk for vascular disease may be important.

PARS PLANA IMPLANT AND VITRECTOMY FOR TREATMENT OF NEOVASCULAR GLAUCOMA

Purpose: To evaluate the effectiveness of pars plana implants in the treatment of neovascular glaucoma. Patients and Methods: Twenty‐two consecutive pars plana implant procedures performed for neovascular glaucoma were retrospectively reviewed. Results: Preoperative intraocular pressures ranged from 22 to 80 mmHg (mean 46 mmHg) on a mean of three glaucoma medications. With a mean follow‐up interval of 16 months, final postoperative intraocular pressures ranged 9‐21 mmHg (mean 16) on a mean 0.7 medications. One patient required a second implant to achieve final intraocular pressure control. Visual acuity was stabilized or improved in 19 of 22 cases (86%). Complications included retinal detachment in two cases (9%). Macular pucker, vitreous hemorrhage, hyphema, choroidal hemorrhage, diplopia, and loss of light perception occurred in one case (5%) each. None developed phthisis bulbi. Conclusion: Pars plana drainage tube implants are effective in the treatment of neovascular glaucoma.

Ranibizumab: treatment in patients with neovascular age-related macular degeneration

Vascular endothelial growth factor (VEGF)-A is a major regulator of angiogenesis and vascular permeability implicated in the development of diseases involving pathological angiogenesis and increased vascular permeability, such as neovascular age-related macular degeneration (AMD). LUCENTIS™ (ranibizumab), a humanised antigen-binding fragment (Fab) that neutralises all VEGF-A isoforms and their biologically active degradation products, was recently approved by the FDA. Ranibizumab is the first FDA-approved treatment for neovascular AMD that maintains or improves vision in ≥ 90% patients and provides a ≥ 15-letter improvement in visual acuity for a quarter to a third of patients with all choroidal neovascularisation subtypes. Ranibizumab was associated with a ≤ 1.7% rate of key serious ocular adverse events, such as endophthalmitis and uveitis, in two pivotal Phase III trials.