Purpose
To correlate aqueous vasoactive protein changes with macular edema after dexamethasone implant in retinal vein occlusion (RVO).
Design
Prospective, interventional case series.
Methods
Twenty-three central RVO (CRVO) and 17 branch RVO (BRVO) subjects with edema despite prior anti–vascular endothelial growth factor (VEGF) treatment had aqueous taps at baseline and 4 and 16 weeks after dexamethasone implant. Best-corrected visual acuity (BCVA) and center subfield thickness were measured every 4 weeks. Aqueous vasoactive protein levels were measured by protein array or enzyme-linked immunosorbent assay.
Results
Thirty-two vasoactive proteins were detected in aqueous in untreated eyes with macular edema due to RVO. Reduction in excess foveal thickness after dexamethasone implant correlated with reduction in persephin and pentraxin 3 (Pearson correlation coefficients = 0.682 and 0.638, P = .014 and P = .003). Other protein changes differed among RVO patients as edema decreased, but ≥50% of patients showed reductions in hepatocyte growth factor, endocrine gland VEGF, insulin-like growth factor binding proteins, or endostatin by ≥30%. Enzyme-linked immunosorbent assay in 18 eyes (12 CRVO, 6 BRVO) showed baseline levels of hepatocyte growth factor and VEGF of 168.2 ± 20.1 pg/mL and 78.7 ± 10.0 pg/mL, and each was reduced in 12 eyes after dexamethasone implant.
Conclusions
Dexamethasone implants reduce several pro-permeability proteins providing a multitargeted approach in RVO. No single protein in addition to VEGF can be implicated as a contributor in all patients. Candidates for contribution to chronic edema in subgroups of patients that deserve further study include persephin, hepatocyte growth factor, and endocrine gland VEGF.
Central retinal vein occlusion (CRVO) is initiated by thrombotic occlusion of the main outflow vessel of the retina resulting in retinal hemorrhages, variable amounts of retinal nonperfusion, and macular edema. Branch vein occlusion (BRVO) is initiated from thrombotic occlusion of a proximal branch of the central retinal vein that drains ≤50% of the retina. Retinal hemorrhages, variable amounts of retinal nonperfusion, and macular edema also occur after BRVO but on average tend to be less severe, because less of the retina is involved by the occlusion compared to CRVO. While ischemic damage to the macula may contribute, the major cause of reduced visual acuity is macular edema. In patients with relatively recent-onset CRVO or BRVO, intraocular injections of a specific antagonist of vascular endothelial growth factor (VEGF) results in dramatic reductions in macular edema and improvements in visual acuity. This indicates that VEGF is a major cause of macular edema in patients with RVO. This was confirmed by large multicenter trials, and intraocular injections of a VEGF antagonist has become first-line therapy for patients with CRVO or BRVO. Frequent injections of a VEGF antagonist are able to completely eliminate edema in some patients, suggesting that VEGF is necessary for edema in those patients; however, it is difficult to maintain a dry retina, because recurrences often occur when the duration between injections is increased. In other patients, it is not possible to achieve complete elimination of the edema despite monthly injections of a VEGF antagonist, suggesting an inability to neutralize all VEGF or contributions from other pro-permeability factors.
It was initially thought that the goal of treatment in RVO would be to control edema and maintain vision until recanalization of the occluded vessel allowed for normalization of the underlying disease process and elimination of the need for injections. However, it appears that the occlusion is merely the initiator of a dynamic disease process that is driven by retinal ischemia and high levels of VEGF, which promote leukostasis, progression of capillary closure, and increased ischemia. This progression of disease makes continued injections necessary to control edema and some patients experience permanent loss of vision from ischemic damage to the macula or damage from chronic/recurrent edema. In the RETAIN study, with a mean follow-up of 49 months after the initiation of anti-VEGF treatment, only 50% of BRVO patients and 44% of CRVO patients no longer required injections to control edema. In many patients, injections of a VEGF antagonist seemed less effective over time, suggesting evolution or change in the disease process such that other pro-permeability factors may play a more important role.
Corticosteroids bind to cytoplasmic receptors that translocate to the nucleus and cause transcriptional repression of a large number of genes whose products participate in inflammation, vascular leakage, and angiogenesis. The dexamethasone implant reduces edema and improves vision in patients with RVO and has a longer duration of effect than intraocular injection of currently available VEGF antagonists. It is an appealing alternative in patients who have residual edema despite anti-VEGF injections or who require frequent injections to control edema. While it is assumed that it reduces many factors that might contribute to edema, there are little data regarding this point. In this study, a vasoactive protein array was used to measure levels of aqueous proteins known to influence vascular cells prior to and after injection of a dexamethasone implant, and changes in protein levels were correlated with changes in edema.
Methods
Study Procedures
The Ozurdex for Retinal Vein Occlusion (ORVO) Study was an investigator-initiated study funded by Allergan, Inc (Irvine, California, USA). The protocol was approved by the Institutional Review Board of the Johns Hopkins Medical Institutions and was conducted in compliance with the Declaration of Helsinki, US Code 21 of Federal Regulations, and the Harmonized “Tripartite Guidelines for Good Clinical Practice (1996). The study was registered on February 8, 2013 at www.clinicaltrials.gov ( NCT01790685 ). All patients provided informed consent. Forty subjects with RVO (17 with BRVO and 23 with CRVO) were enrolled. Disease duration for each patient was calculated from when the patient first developed macular edema until the baseline visit. Because patient reporting is often unreliable, only injections documented in records were used to determine the number of prior anti-VEGF injections. Response to prior anti-VEGF therapy was graded as good, moderate, or poor depending on whether all intraretinal fluid could be eliminated by monthly injections or how frequently anti-VEGF injections had to be given to maintain a dry macula. Qualitative measurement of nonperfusion at baseline were made using ultra-widefield fluorescein angiography (Optos 200Tx, Optos, Dunfermline, Scotland, UK) done at or prior to the baseline visit. At baseline and at all subsequent visits, subjects had measurement of best-corrected visual acuity (BCVA) using the Early Treatment Diabetic Retinopathy Study (ETDRS) protocol, ophthalmologic examination including measurement of intraocular pressure, spectral-domain optical coherence tomography (SD OCT) using the Spectralis machine (Heidelberg Engineering, Inc, Carlsbad, California, USA), and an anterior chamber tap. Aqueous samples were stored at −80 C. Patients were given an intraocular injection of a dexamethasone implant in the study eye at baseline. Povidone-iodine was used to clean the conjunctiva and 2% lidocaine was injected under the conjunctiva. The 22 gauge needle of the injector was inserted through the pars plana and the dexamethasone implant was injected into the vitreous cavity.
Functional and Anatomic Outcomes
The major functional outcome measure was change from baseline BCVA in ETDRS letter score at weeks 4, 8, 12, and 16. One anatomic outcome was the change from baseline center subfield thickness at weeks 4, 8, 12, and 16. Excess foveal thickness (EFT) was calculated for each subject by subtracting the minimum foveal thickness during the course of the disease (edema-free thickness, which in each case was <320 μm) from the foveal thickness at baseline and at weeks 4, 8, 12, and 16. Percent change in EFT at each time point was calculated using the following formula: % change in EFT at a time point = (EFT at baseline–EFT at time point)/EFT at baseline. In addition, qualitative changes in intraretinal fluid were graded by side-to-side comparisons of baseline, week 4, and week 16 SD OCT scans.
Vasoactive Protein Arrays
The levels of vasoactive proteins in aqueous at baseline and at week 4 were measured in 11 eyes with BRVO and 11 eyes with CRVO at IMGENEX Corporation (San Diego, California, USA) using the Human Angiogenesis Antibody Array Kit (catalog number ARY007; R&D Systems, Inc, Minneapolis, Minnesota, USA). Samples obtained at week 16 were also included for 3 eyes with BRVO and 1 with CRVO. Aqueous samples (115 μL) were blotted on the membrane, which was stored for 16 hours at 4 C and further processed according to the manufacturer’s instructions. Each array membrane was exposed to x-ray film for short-term and long-term exposures. The positive signals detected on the developed x-ray film were quantitated using TotalLab Quant software (Gentel Biosciences, Inc, Madison, Wisconsin, USA). Results are reported as percent change from baseline.
Enzyme-Linked Immunosorbent Assays
Protein arrays indicated a reduction in aqueous levels of hepatocyte growth factor in several patients between baseline and week 4 and therefore enzyme-linked immunosorbent assay (ELISA) was used to measure aqueous levels of hepatocyte growth factor and VEGF in another cohort of RVO patients at baseline, week 4, and week 16 (when week 16 samples were available). Levels of hepatocyte growth factor and VEGF were measured in aqueous samples using ELISA kits for each (Abcam, Cambridge, Massachusetts, USA) using the manufacturer’s instructions. Briefly, 100 μL of each aqueous sample diluted 1:1 in dilution buffer or 100 μL of hepatocyte growth factor or VEGF protein standard was added to a well of a 96-well plate and incubated at 4 C overnight. After wells were washed 4 times, 100 μL of biotinylated anti–hepatocyte growth factor or anti-VEGF antibody was added to each well and incubated for 1 hour at room temperature. After 4 washes, 100 μL of horseradish peroxidase–streptavidin solution was added to each well and incubated for 45 minutes at room temperature. After wells were washed 4 times, 100 μL of 3,3′–5,5′ tetramethylbenzidine substrate reagent was added to each well and after 30 minutes the reaction was stopped by adding 50 μL of stop solution. Absorbance at 450 nm was measured on a plate reader. The readings from the standards were used to generate standard curves of absorbance vs hepatocyte growth factor or VEGF. The hepatocyte growth factor and VEGF concentration in each sample was calculated by plotting absorbance on the respective standard curve.
Correlation of Changes in Vasoactive Proteins With Changes in Edema
The diversity of protein levels and center subfield thickness were calculated as percentage change in their measurements at week 4 relative to baseline:
FC Protein = ( Protein B − Protein W 4 ) / Protein B
where FC Protein and FC CST are the changes in protein level and center subfield thickness, and E B , E W4 , CST B , and CST W4 represent the protein level and center subfield thickness at baseline and week 4, respectively. In order to estimate the correlation between percent reduction in protein level and percent reduction in excess foveal thickness, the Pearson correlation coefficient was calculated using R.
Results
Demographics and Baseline Characteristics
The ORVO trial enrolled 40 subjects with macular edema, 17 with BRVO and 23 with CRVO. Most subjects had long-standing macular edema with a median duration of 40 months for patients with BRVO and 45 months for patients with CRVO, but there were also a few patients who had a relatively short duration of disease ( Table 1 ). All subjects had previously been treated with anti-VEGF injections, with a median of 13.9 (BRVO, n = 14) or 18.9 (CRVO, n = 21); only documented injections were counted, not those based on patient history, and therefore these means are minimums and actual means may be larger. Detailed information regarding prior response to anti-VEGF injections was available for 12 patients with BRVO and 20 patients with CRVO. During periods of monthly or in some cases less frequent injections, there was minimal residual intraretinal fluid in 4 subjects with BRVO and 11 subjects with CRVO, while in 8 subjects with BRVO and 9 with CRVO there was substantial residual intraretinal fluid even during periods of monthly injections. The median BCVA at baseline in ETDRS letter score (Snellen equivalent) was 60 (20/63) in eyes with BRVO and 54 (20/80) in eyes with CRVO. The median central subfield thickness at baseline was 453 μm in eyes with BRVO and 539 μm in eyes with CRVO, but there were differences among patients with thickening and intraretinal fluid ranging from mild to severe.
