CLINICAL CHARACTERIZATION OF AQUEOUS AND VITREOUS RETINOL-BINDING PROTEIN 3 CONCENTRATIONS IN RELATION TO DIABETIC RETINOPATHY SEVERITY, RETINAL STRUCTURES, AND SYSTEMIC COMPLICATIONS.

PURPOSE: To evaluate Retinol-Binding Protein 3 (RBP3) from photoreceptors in aqueous and its association with vitreous concentrations, diabetic retinopathy (DR) severity, retinal layer thickness, and clinical characteristics in people with diabetes. METHODS: RBP3 concentration was measured by custom-developed enzyme-linked immunosorbent assay in aqueous and correlated with vitreous concentrations in patients from the 50-Year Medalist study and Beetham Eye Institute at Joslin Diabetes Center. RESULTS: Aqueous RBP3 concentration (N = 131) was elevated in eyes with no to mild DR (mean ± SD 0.7 nM ± 0.2) and decreased in eyes with moderate to severe DR (0.65 nM ± 0.3) and proliferative DR (0.5 nM ± 0.2, P < 0.001) compared to eyes without diabetes. Aqueous and vitreous RBP3 concentrations correlated with each other (r = 0.34, P = 0.001) and between fellow eyes (P < 0.0001). History of retinal surgery did not affect aqueous RBP3 concentrations, but cataract surgery affected both vitreous and aqueous levels. Elevated aqueous RBP3 concentration associated with increased thickness of the outer nuclear layer (P = 0.004) and correlated with hemoglobin A1c, whereas vitreous RBP3 concentrations correlated with diabetic systemic complications. CONCLUSION: These findings suggest that aqueous RBP3 concentration may be an important endogenous clinical retinal protective factor, a biomarker for DR severity, and a promising VEGF-independent clinical intervention target in DR.

Elevated Retinol Binding Protein 3 Concentrations Are Associated With Decreased Vitreous Inflammatory Cytokines, VEGF, and Progression of Diabetic Retinopathy.

OBJECTIVE: To correlate inflammatory cytokines and vascular endothelial growth factor (VEGF) in vitreous and plasma with vitreous retinol binding protein 3 (RBP3), diabetic retinopathy (DR) severity, and DR worsening in a population with type 1 and type 2 diabetes. RESEARCH DESIGN AND METHODS: RBP3, VEGF, and inflammatory cytokines were measured in plasma and vitreous samples (n = 205) from subjects of the Joslin Medalist Study and Beetham Eye Institute. RESULTS: Higher vitreous RBP3 concentrations were associated with less severe DR (P < 0.0001) and a reduced risk of developing proliferative DR (PDR) (P < 0.0001). Higher RBP3 correlated with increased photoreceptor segment thickness and lower vitreous interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and TNF-ß (P < 0.05). PDR was associated with lower vitreous interferon-γ and IL-10 and higher VEGF, IL-6, and IL-15 (P < 0.05), but was not associated with their plasma concentrations. CONCLUSIONS: Higher vitreous RBP3 concentrations are associated with less severe DR and slower rates of progression to PDR, supporting its potential as a biomarker and therapeutic agent for preventing DR worsening, possibly by lowering retinal VEGF and inflammatory cytokines.

Retinol binding protein 3 is increased in the retina of patients with diabetes resistant to diabetic retinopathy.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

Retinal proteome associated with bradykinin-induced edema.

The plasma kallikrein-stimulated generation of bradykinin (BK) has been implicated in diabetic macular edema (DME). This study characterizes the effects of BK on the ultrastructure and proteome of the rat retina. The effects of intravitreal injection of BK on retinal thickness and vascular ultrastructure in Sprague Dawley rats were analyzed and compared with the effects of VEGF using spectral-domain optical coherence tomography. At 24 h post intravitreal injection of BK or saline vehicle retina were harvested and solubilized proteins were analyzed by mass spectrometry-based proteomics. Proteins were identified using X!Tandem and spectral counts were used as a semiquantitative measurement of protein abundance. Proteins identified from retinal extracts were annotated by Gene Ontology (GO) slim terms and compared with a human DME vitreous proteome. Intravitreal injection of BK and VEGF induced transient increases in retinal thickness of 46 µm (24.6%, p = 0.015) and 39 µm (20.3%, p = 0.004), respectively at 24 h, which were resolved to baseline thicknesses at 96 h post injection. BK and VEGF also increased retinal vessel diameters and tortuosity at 24 h post intravitreal injection. Proteomic analyses identified 1757 non-redundant proteins in the rat retina, including 1739 and 1725 proteins from BK- and saline control-injected eyes, respectively. Eighteen proteins, including two proteins associated with intercellular junctions, filamin A and actinin alpha 4, were decreased by at least 50% (p < 0.05) in retina from BK-injected eyes compare with retina from eyes injected with saline. In addition, 32 proteins were increased by > 2-fold (p < 0.05) in retina from BK-injected eyes. Eight proteins, including complement C3, were identified to be increased in both BK-stimulated rat retina and in human DME vitreous. Western blot analysis showed that Complement 3 levels in vitreous from BK-injected eyes in rats and clinical DME samples were increased by 6.6-fold (p = 0.039) and 4.3-fold (p = 0.02), compared with their respective controls. In summary, this study identifies protein changes in rat retina that are associated with BK-induced retinal thickening, including 8 proteins that were previously reported to be increased in the human DME vitreous proteome.

PREDICTIVE VALUE OF OPTICAL COHERENCE TOMOGRAPHIC FEATURES IN THE BEVACIZUMAB AND RANIBIZUMAB IN PATIENTS WITH DIABETIC MACULAR EDEMA (BRDME) STUDY.

PURPOSE: To establish the predictive value of specific optical coherence tomography retinal features on visual outcomes and retinal thickness during anti-vascular endothelial growth factor treatment in patients with diabetic macular edema. METHODS: Post hoc analysis of compound data of a prospective, 6-month, multicenter, randomized controlled trial of 119 patients with diabetic macular edema receiving either intravitreal bevacizumab or ranibizumab were analyzed to assess the associations between baseline retinal morphologic parameters and change in best-corrected visual acuity and central subfield thickness. Based on the study protocol of the core study, best-corrected visual acuity and central subfield thickness were obtained before each mandatory monthly injection during 6 months. RESULTS: The presence of serous retinal detachment at baseline was associated with significant improvement in best-corrected visual acuity letter score at Month 3 and Month 6 (P < 0.001 and P = 0.01, respectively). In addition, the presence of disorganization of retinal inner layers was associated with lower best-corrected visual acuity letter score at Month 3 and Month 6 (P < 0.05 and P = 0.01, respectively). CONCLUSION: This study found that serous retinal detachment and disorganization of retinal inner layers were associated with different treatment responses to anti-vascular endothelial growth factor therapy in patients with diabetic macular edema.

Association of Circulating Markers With Outcome Parameters in the Bevacizumab and Ranibizumab in Diabetic Macular Edema Trial.

Purpose: The purpose of this study was to evaluate selected candidate biomarkers as potential markers for patients with diabetic macular edema (DME) who receive antivascular endothelial growth factor (VEGF) therapy. Methods: Selected biomarkers included blood levels of messenger RNA (mRNA) of retinoschisin, RPE65, rhodopsin, and endothelial progenitor cell markers CD34 and CD133. Blood samples were obtained from 89 patients with DME according to the study protocol of the Bevacizumab and Ranibizumab in Diabetic Macular Edema (BRDME) study. During each monthly visit, patients underwent optical coherence tomography scanning and visual acuity was measured. Anti-VEGF injections were administered at fixed monthly intervals over 6 months. Analyses of covariance using simplified and linear mixed models were used to examine the correlations between candidate markers and changes in visual acuity and central subfield thickness. Results: Plasma mRNA levels of retinoschisin were negatively associated with visual acuity, and plasma mRNA levels of rhodopsin were positively associated with visual acuity in patients with DME (P < 0.01 and P < 0.05, respectively). In addition, changes in central subfield thickness between baseline and months 1, 2, and 3 during anti-VEGF treatment were associated with mRNA levels of retinoschisin, rhodopsin, and the ratio of retinoschisin-to-rhodopsin (P < 0.01, all). Conclusions: This prospective, multicenter study found that circulating mRNA levels of retinoschisin and rhodopsin are associated with visual acuity and changes in central subfield thickness during anti-VEGF therapy in patients with DME. (ClinicalTrials.gov number: NCT01635790.).