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-β (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 (RBP3), a retinol transport protein secreted mainly by photoreceptors, may inhibit progression of diabetic retinopathy (DR) by decreasing glucose uptake and inflammation in the retina. In people with type 1 diabetes with duration of ≥50yrs (Joslin Medalist Study), >40% of Medalists exhibited no to mild non-proliferative diabetic retinopathy (NPDR) and increased expression of RBP3 compared to those with severe DR. We examined 213 vitreous samples from a larger population of Medalists and patients with type 1 and type 2 diabetes with shorter diabetes duration (mean±SD 26.5±12.7 years) from the Joslin Beetham Eye Institute to determine whether vitreous RBP3 levels are correlated with DR severity and progression in a broad population with diabetes. RBP3 was increased in vitreous from people with no diabetes compared to subjects with type 1 and type 2 diabetes (p<0.0001). Type of diabetes (type 1 vs. type 2, p=0.44) and A1c (p=0.68) were not associated with RBP3. In patients with both type 1 and 2 diabetes, vitreous RBP3 levels gradually decreased from the highest concentration in no to mild NPDR (15.7 nM) to the lowest concentrations in advanced DR (moderate-severe: 8.2 nM, p=0.01 vs. no-mild NPDR; active proliferative DR: 8.4 nM, p=0.0003 vs. no-mild NPDR; quiescent proliferative DR: 3.5 nM, p<0.0001 vs. no-mild NPDR). RBP3 was higher in moderate-severe NPDR compared to quiescent PDR (p=0.03). RBP3 concentrations in surgical samples from living donors were lower than those from post-mortem Medalists (p<0.01), but were associated with DR severity in both groups (p=0.03 and p<0.0001, respectively). Increased RBP3 concentrations were associated with reduced risk of PDR development over time (n=34, p<0.001). These findings indicate that elevated vitreous RBP3 is associated with decreased DR severity and decreased risk of PDR development, supporting the postulate that RBP3 is a protective factor and therapeutic target for the progression of DR. Disclosure W. Fickweiler: None. G. L. King: Consultant; Self; Agios, Inc., Medtronic, Other Relationship; Self; Janssen Pharmaceuticals, Inc. H. Park: None. K. Park: None. T. Boumenna: None. J. Gauthier: None. I. Wu: None. J. D. Cavallerano: None. L. P. Aiello: Consultant; Self; KalVista Pharmaceuticals, Novo Nordisk, Regeneron Pharmaceuticals Inc., Stock/Shareholder; Self; KalVista Pharmaceuticals. J. Sun: Other Relationship; Self; Novo Nordisk, Roche Pharma, Research Support; Self; Adaptive Sensory Technology, Boehringer Ingelheim Pharmaceuticals, Inc., KalVista Pharmaceuticals, Optovue, Roche Pharma. Funding National Eye Institute (R01EY026080-01); National Institute of Diabetes and Digestive and Kidney Diseases (DP3DK094333-01); JDRF (17-2013-310); Beatson Foundation; Dianne Nunnally Hoppes Fund

Hyperglycemia has been evaluated extensively as risk factors for cardiovascular disease (CVD) in people with type 1 diabetes (T1DM). However, the effects of the autoantibody on atherosclerosis and immunogenic trigger are not well understood in T1D. Using the generated several mice models of atherosclerosis including NOD, congenic ApoE-/-/NOD (NDM and no- autoimmune), ApoE-/-/NOD (autoimmunity with insulitis, NDM) and ApoE-/-/NOD autoimmune DM mice, we analyzed immune cells composition of aorta and have observed the increasing numbers of CD3+(CD3+/CD25-), Th1 and Th17 subset of T cells and B cells and decreased Treg (CD3+/CD25+/Foxp3+) in the ApoE-/-/NOD and ApoE-/-/NOD-DM mice vs. control. In addition, we postulate that the identification of specific autoantibody(s) which induce dysregulaton of T-cells, resulting in enhancing its infiltration and accumulation at the plaques could aid new immunotherapies for atherosclerosis. Here we performed autoantigen microarrays to profile and identify specific autoantibodies, enhancing the severe atherosclerosis in subgroups of Medalist patients with chronic duration of T1D due to autoimmunity with positive HLA DR3/4 risk alleles and autoantibodies and compared to the people with monogenic diabetes lacking HLA DR3/4 risk alleles for T1D and autoantibodies. Autoantigen microarrays from the plasma of T1D patients with positive DR3/4 risk and CVD identified 8 autoantibodies changed in the context of atherosclerosis, compared to the people with monogenic diabetes. To confirm the autoantibody profiling data from the patients, we analyzed the autoantibody profiles in the T1D mice models of atherosclerosis described above. Autoantigen microarrays analysis of the plasma of T1D patients and mice showed several common findings that reached significance anti-Nucleolin Ab, p=0.005. These findings indicate autoimmunity may contribute to the progression of atherosclerosis in T1D. Further studies are in progress to replicate these findings in other cohorts of T1D. Disclosure K. Park: None. Q. Li: None. H. Park: None. J. Fu: None. S. Kissler: None. E. Maddaloni: Research Support; Self; European Foundation for the Study of Diabetes, Speaker's Bureau; Self; Abbott, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Merck KGaA. I. Wu: None. A. H. Lichtman: None. G. L. King: Consultant; Self; Agios, Inc., Medtronic, Other Relationship; Self; Janssen Pharmaceuticals, Inc. Funding DK036836, DK036836, EY026080

The Joslin Medalist Study, which characterizes people with type 1 diabetes duration of 50 years or more, reported that the levels of Retinol Binding Protein 3 (RBP3) are increased in the retina and vitreous of Medalists who are protected from the development of severe diabetic retinopathy (DR). This protective capacity of RBP3 is potentially mediated by its ability to delay the uptake of glucose into the cells of the retinal vasculature and neural retina in hyperglycemia. This study correlated the relationship between DR severity and neural retinal layer thickness with vitreous and serum RBP3 levels in a broad population of people with diabetes. Optical coherence tomography (OCT) was performed to obtain neural retinal layer thicknesses and structure. Specific ELISA assays were developed to measure vitreous and serum RBP3 concentrations in individuals with type 1 and type 2 diabetes recruited from the Joslin Beetham Eye Institute, Medalist Study, and the FinnDiane study. Vitreous RBP3 concentration was associated with DR severity (p<0.05), diabetes duration (p=0.02), age (p=0.01), and HDL (p<0.05) in the Medalists (n=68), individuals with shorter duration of type 1 and type 2 diabetes (mean±SD 28±14 years, n=24), and nondiabetic controls (n=20). OCT showed that thinning of the inner nuclear layer, outer nuclear layer, and the photoreceptor layer was associated with decreased vitreous RBP3 concentration (all p<0.0001). Serum RBP3 concentrations were more than 1000-fold less than those in vitreous, but correlated positively with vitreous RBP3 concentration (p<0.05). The presence of laser photocoagulation was associated with decreased serum RBP3 concentration (p=0.005). Repeated testing at 5 years showed that baseline and 5 year RBP3 serum concentrations were correlated in the Medalist Study (r=0.64, p=0.003) and the FinnDiane Study (r=0.77). These findings suggest that measurement of RBP3 systemically may be potentially useful in the clinical screening of DR. Disclosure W. Fickweiler: None. K. Park: None. E. Wolfson: None. H. Park: None. H. Yokomizo: None. I. Wu: None. D. Gordin: Consultant; Self; GE Healthcare. Speaker’s Bureau; Self; AstraZeneca, Fresenius Medical Care, Novo Nordisk A/S. Other Relationship; Self; CVRx, Sanofi-Aventis. V. Harjutsalo: None. P. Groop: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Sanofi, Sanofi. Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Medscape, Merck Sharp & Dohme Corp., Mundipharma International. L.P. Aiello: Advisory Panel; Self; Novo Nordisk Inc. Consultant; Self; KalVista Pharmaceuticals, Inc., Novo Nordisk Inc. Stock/Shareholder; Self; KalVista Pharmaceuticals, Inc. Other Relationship; Self; Optos. J. Sun: Research Support; Self; Adaptive Sensory Technology, Boehringer Ingelheim Pharmaceuticals, Inc., KalVista Pharmaceuticals, Inc., Novo Nordisk Inc., Optovue, Inc., Roche Pharma. Other Relationship; Self; Merck Foundation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Roche Pharma. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc. Funding National Eye Institute (R01EY026080-01); National Institute of Diabetes and Digestive and Kidney Diseases (DP3DK094333-01); JDRF (17-2013-310); Thomas J. Beatson, Jr. Foundation; Dianne Nunnally Hoppes Scholarship Fund

Hyperglycemia, hyperlipidemia and hypertension have been evaluated extensively as risk factors for cardiovascular disease (CVD) in people with type 1 diabetes (T1DM). However, the impact of autoimmunity in T1DM on CVD has not been determined. We have generated several mice models of atherosclerosis including ApoE-/-/NOD, ApoE-/-/NOD (congenic non diabetes and non-autoimmune), ApoE-/-/NOD (autoimmunity with insulitis, nondiabetes) and ApoE-/-/NOD autoimmune diabetic mice. All groups of mice have high plasma lipids but only ApoE-/-/NOD diabetic mice had hyperglycemia. However, ApoE-/-/NOD (insulitis and nondiabetes) and ApoE-/-/NOD (insulitis and diabetes) had high titers of autoantibodies to IA2 compared to control CongApoE-/-/NOD mice or nondiabetic ApoE-/-/NOD mice (P<0.0001). For the the severity of atherosclerosis, the presence of hyperglycemia in ApoE-/-/NOD-DM mice showed greater levels of lipid deposition in the descending aorta (22%) by en face assessment as compared to nondiabetic ApoE-/-/NOD mice (P<0.01). Similarly, we also observed in the macrophage content of the descending aorta, was greater in numbers in the ApoE-/-/NOD mice than in ApoE-/-/CongNOD mice (P<0.05) but less than in ApoE-/-/NOD-DM mice. FACS of the descending aorta showed the elevated CD3+/CD28- T-cells (CD4 and CD8) by 4.5 folds and decreased CD3+/CD28+ Treg cells by 46% at the atherosclerotic plaques of ApoE-/-/NOD mice, compared to those of CongApoE-/-/NOD mice. To further confirm the elevated infiltrate and accumulated CD3+ T-cells in vivo, we will investigate the levels of macrophages and CD3+ T-cells in the arterial wall of the coronary artery from the Medalists group T1D and autoimmunity with positive HLA DR3/4 risk alleles and autoantibodies and lacking monogenic diabetic variants, as compared to the group lacking HLA DR3/4 risk alleles and autoantibodies. These studies suggest that autoimmunity exacerbates atherosclerosis in T1D, independently and synergically hyperglycemia. Disclosure K. Park: None. Q. Li: None. H. Park: None. R. St-Louis: None. J. Fu: None. C. Rask-Madsen: None. E. Maddaloni: Consultant; Self; Merck KGaA. Speaker’s Bureau; Self; Abbott, AstraZeneca, Pikdare. H. Yokomizo: None. S. Kissler: None. A.H. Lichtman: None. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc. Funding National Institutes of Health (R01DK053105); Thomas J. Beatson, Jr. Foundation