Prevalence and risk factors for diabetic retinopathy at diagnosis (DRAD) in patients recently diagnosed with type 2 diabetes (T2D) or latent autoimmune diabetes in the adult (LADA).

PURPOSE: To study prevalence of diabetic retinopathy (DR) at diagnosis (DRAD) and to estimate contributing risk by sociodemographic, cardiovascular and metabolic characteristics present in patients recently diagnosed with type 2 diabetes (T2D) or latent autoimmune diabetes in the adult (LADA). METHODS: Patients (n=2174) recently diagnosed T2D (93%) or LADA (7%) were included upon arrival for their baseline DR screening. Fundus photographs of 4902 eyes were graded by a senior ophthalmologist according to the International Diabetic Retinopathy Disease Severity Scale. Official registers held by Statistics Sweden provided sociodemographic variables. The National Patient Register and Swedish Prescribed Drug Register were used to assess cardiovascular risk. Beta cell function (HOMA2%b) and insulin sensitivity (HOMA2%s) were estimated from fasting (f) C-Peptide using the homeostasis model assessment (HOMA) 2 calculator. Odds ratios (OR) for DRAD were estimated using generalized estimating equation models. RESULTS: The prevalence of DRAD was 12% (7% mild and 5% moderate) and of diabetic macular edema it was 11% (all within vascular arch). The prevalence did not significantly differ between T2D and LADA. Due to sample size, the regression analysis of LADA patients did not yield any significant estimates. In T2D low educational level (≤9years) increased risk for DRAD by 44% (OR 1.44; 95% CI 1.07-1.93) and <50% beta-cell function adjusted for HbA1c and insulin sensitivity at diagnosis increased the risk by 77% (OR 1.77; 95% CI 1.28-2.44). For every unit increase in BMI, risk for DRAD decreased by 3% (OR 0.97; 95% CI 0.95-0.99). CONCLUSIONS: DRAD prevalence in patients recently diagnosed with T2D or is 12%. Low educational level and low beta cell function at diagnosis are risk factors for DRAD. Estimation of beta cell function from (f)C-Peptide and (f)P-Glucose may be a valuable tool in identifying patients at risk for DRAD.

Profile of intraocular tumour necrosis factor-α and interleukin-6 in diabetic subjects with different degrees of diabetic retinopathy.

PURPOSE: To assess and correlate the levels of inflammatory mediators in the eyes from non-diabetic and diabetic subjects without retinopathy (NDR), with non-proliferative diabetic retinopathy (NPDR) or with proliferative diabetic retinopathy (PDR) to corresponding erum levels. METHODS: The levels of interleukin 1ß, interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were analysed by an ELISA-mimicking technique in the vitreous from 26 diabetic subjects with active PDR and 27 non-diabetic subjects, or by a multiplex bead assay in the aqueous humour from 35 diabetic subjects with NDR/NPDR and 40 non-diabetic subjects. Intraocular protein production was estimated in vitreous specimens by calculating a vitreous/serum ratio. RESULTS: In the vitreous, IL-6 was higher in diabetic [157.5 (25.0-1401.0) pg/ml; median (min-max)] than in non-diabetic subjects [44.0 (5.0-4425) pg/ml; p = 0.021]. The vitreous/serum ratio was high (55.5:1 and 16:1, respectively), suggesting intraocular production. TNF-α was lower in diabetic [18.0 (8.0-46.0) pg/ml] than in non-diabetic subjects [22.0 (13.0-47.0) pg/ml; p = 0.034], but the vitreous/serum ratio was elevated in both groups (2:1 and 3.4:1, respectively). TNF-α levels were higher in serum from diabetic subjects [9.0 (5.0-53.0) pg/ml versus 6.7 (3.0-11.0) pg/ml; p < 0.001]. Aqueous levels of inflammatory mediators did not differ between diabetic subjects with NDR/NPDR and non-diabetic subjects despite elevated TNF-α in serum [27.8 (6.8-153.7) pg/ml versus 16.4 (4.1-42.4) pg/ml; p = 0.021]. CONCLUSION: Intraocular inflammation seems to be involved in PDR but does not seem to be prominent in early retinopathy stages, i.e. NDR or NPDR. Diabetic subjects have an overall increased inflammatory activity compared to non-diabetic subjects, as demonstrated by increased serum levels of TNF-α.

Vascular cellular adhesion molecule-1 (VCAM-1) expression in mice retinal vessels is affected by both hyperglycemia and hyperlipidemia.

BACKGROUND: Inflammation has been proposed to be important in the pathogenesis of diabetic retinopathy. An early feature of inflammation is the release of cytokines leading to increased expression of endothelial activation markers such as vascular cellular adhesion molecule-1 (VCAM-1). Here we investigated the impact of diabetes and dyslipidemia on VCAM-1 expression in mouse retinal vessels, as well as the potential role of tumor necrosis factor-α (TNFα). METHODOLOGY/PRINCIPAL FINDINGS: Expression of VCAM-1 was examined by confocal immunofluorescence microscopy in vessels of wild type (wt), hyperlipidemic (ApoE(-/-)) and TNFα deficient (TNFα(-/-), ApoE(-/-)/TNFα(-/-)) mice. Eight weeks of streptozotocin-induced diabetes resulted in increased VCAM-1 in wt mice, predominantly in small vessels (<10 µm). Diabetic wt mice had higher total retinal TNFα, IL-6 and IL-1ß mRNA than controls; as well as higher soluble VCAM-1 (sVCAM-1) in plasma. Lack of TNFα increased higher basal VCAM-1 protein and sVCAM-1, but failed to up-regulate IL-6 and IL-1ß mRNA and VCAM-1 protein in response to diabetes. Basal VCAM-1 expression was higher in ApoE(-/-) than in wt mice and both VCAM-1 mRNA and protein levels were further increased by high fat diet. These changes correlated to plasma cholesterol, LDL- and HDL-cholesterol, but not to triglycerides levels. Diabetes, despite further increasing plasma cholesterol in ApoE(-/-) mice, had no effects on VCAM-1 protein expression or on sVCAM-1. However, it increased ICAM-1 mRNA expression in retinal vessels, which correlated to plasma triglycerides. CONCLUSIONS/SIGNIFICANCE: Hyperglycemia triggers an inflammatory response in the retina of normolipidemic mice and up-regulation of VCAM-1 in retinal vessels. Hypercholesterolemia effectively promotes VCAM-1 expression without evident stimulation of inflammation. Diabetes-induced endothelial activation in ApoE(-/-) mice seems driven by elevated plasma triglycerides but not by cholesterol. Results also suggest a complex role for TNFα in the regulation of VCAM-1 expression, being protective under basal conditions but pro-inflammatory in response to diabetes.

Tumor necrosis factor-alpha does not mediate diabetes-induced vascular inflammation in mice.

OBJECTIVE: Vascular inflammation is a key feature of both micro- and macrovascular complications in diabetes. Several lines of evidence have implicated the cytokine tumor necrosis factor (TNF) alpha as an important mediator of inflammation in diabetes. In the present study we evaluated the role of TNF alpha in streptozotocin (STZ)-induced diabetes on vascular inflammation in C57BL/6 wild-type and apoE-/- mice. METHODS AND RESULTS: Diabetes increased the expression of vascular cell adhesion molecule (VCAM)-1 in cerebral arteries 150 m in diameter as well as the macrophage accumulation in aortic root atherosclerotic plaques in apoE-/- mice. A more pronounced vascular inflammatory response was observed in diabetic TNF alpha-deficient apoE-/- mice. These mice were also characterized by increased accumulation of IgG and IgM autoantibodies in atherosclerotic lesions. Diabetes also increased VCAM-1 expression and plaque formation in apoE-competent TNF alpha -/- mice, whereas no such effects were observed in C57BL/6 wild-type mice. CONCLUSIONS: The present findings suggest that TNF alpha does not mediate diabetic-induced vascular inflammation in mice and reveal an unexpected protective role for TNF alpha. These effects are partly attributable to a direct antiinflammatory role of TNF alpha, but may also reflect a defective development of the immune system in these mice.

Inflammatory markers in nondiabetic and diabetic rat retinas exposed to ischemia followed by reperfusion.

PURPOSE: To examine the retinal inflammatory response to ischemia-reperfusion in nondiabetic and diabetic rats injected with either an omega-3-polyunsaturated fatty acid (docosahexaenoic acid [DHA]) or a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (pravastatin). METHODS: Diabetes was induced by an intraperitoneal injection of streptozocin, and retinal ischemia was induced by ligation of the optic nerve and vessels, followed by reperfusion for 1 hour or 24 hours. Five minutes before surgery, an intravenous injection of DHA, pravastatin, or vehicle (ethanol) was administered. The mRNA expressions of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, caspase-1, IL-1beta, P-selectin, vascular cellular adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1 were compared between ischemic and nonischemic retinas as well as diabetic and nondiabetic nonischemic retinas. RESULTS: Ischemia induced increased expressions of TNF-alpha (P

TNF-alpha is an independent serum marker for proliferative retinopathy in type 1 diabetic patients.

PURPOSE: This study aimed to determine if there are any associations between serum levels of inflammatory markers and proliferative retinopathy (PDR) in type 1 diabetic patients. DESIGN: A cross-sectional design was utilized for this study. METHODS: One hundred twenty-eight type 1 diabetic patients underwent stereo fundus photography according to the Early Treatment Diabetic Retinopathy Study and were divided into two retinopathy groups: no or nonproliferative retinopathy (NDR/NPDR; n=62) and PDR (n=66). Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, IL-6, soluble vascular cellular adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), P-selectin, and high-sensitivity C-reactive protein (hsCRP) were analyzed. Statistical analysis was performed using nonparametric Mann-Whitney U test and multivariate logistic regression analysis. RESULTS: Patients with PDR had higher levels of TNF-alpha [7.0 pg/ml (<4-17) vs. 6.0 pg/ml (<4-25); P=.009], sVCAM-1 [860 ng/ml (360-2120) vs. 700 ng/ml (310-1820); P<.001], and P-selectin [180 ng/ml (39-400) vs. 150 ng/ml (42-440); P=.017; figures are expressed as median (range)]. There were no differences in serum levels of sICAM-1 or hsCRP. IL-1 beta was not detectable in any patient, and IL-6 was detectable in only 22.7% of the patients. In multivariate logistic regression analysis, TNF-alpha was the single, persistent, independent determinant inflammatory marker for PDR. CONCLUSION: The association between TNF-alpha and PDR in type 1 diabetic patients suggests that inflammation might play a role in the pathogenesis of proliferative diabetic retinopathy.

Circulating antipericyte autoantibodies: a novel modifier of risk of progression of diabetic retinopathy?

BACKGROUND: Antipericyte autoantibodies (APAAs) are present in high frequency among diabetic subjects with and without nonproliferative retinopathy. This study aimed to determine whether progression of retinopathy in type 2 diabetes was associated with the same medical risk factors in APAA-positive subjects as in APAA-negative subjects. METHODS: Type 2 diabetic patients with nonproliferative diabetic retinopathy at baseline were followed prospectively for 2 years monitoring progression of retinopathy. Thirty-eight (21.7%) of 175 patients had progression in Early Treatment Diabetic Retinopathy Study grade by > or =2 steps in at least 1 eye. Serum APAAs were detected by immunofluorescence on tissue-cultured bovine retinal pericytes. RESULTS: Progression of retinopathy was associated with HbA(1c) level (P = 0.002), diabetes duration (P = 0.03), and albumin/creatinine ratio (P = 0.02) in APAA-negative subjects but not in APAA-positive subjects. The association between progression and APAAs was strongest in the upper quartile for HbA(1c) level (>8.0%), where 71.4% of patients negative for APAAs had progression of retinopathy while only 24.1% of patients positive for APAAs had progression (P = 0.007). CONCLUSION: The results suggest that APAA presence is a modifier of risk of progression of retinopathy due to hyperglycemia and that it could be useful as a biochemical marker of risk of progression of diabetic retinopathy in type 2 diabetic patients with poor metabolic control.

Expression of antioxidant enzymes in rat retinal ischemia followed by reperfusion.

To evaluate the expression and protein levels of antioxidant enzymes in the rat retina exposed to oxidative stress induced by ischemia-reperfusion injury. Retinal ischemia was induced in female Wistar rats by ligation of the optic nerve and vessels behind the left eye bulb, and was followed by reperfusion for 0, 3, 6, or 24 hours. The right eye served as control. RNA and protein were extracted simultaneously from each retina. Expressions of the endogenous antioxidant enzymes glutathione peroxidase (GPx1), catalase (CAT), copper/zinc superoxide dismutase, manganese superoxide dismutase, and the catalytic subunit of glutamylcysteine ligase (GCLc) were analyzed with real-time reverse transcription polymerase chain reaction and related to the endogenous control cyclophilin B. Protein levels were measured with Western blot analysis. During the early phase (0 or 3 hours) of reperfusion, no changes were seen in enzyme expression. After 6 hours, GCLc expression increased by a factor of 1.14 (P = .034), followed by a decline of 0.80 after 24 hours (P = .00004), according to the comparative Ct method. After 24 hours of reperfusion, GPx1 expression increased by a factor of 1.14 (P = .028), and CAT had decreased by 0.82 (P = .022). Expressions of copper/zinc superoxide dismutase and manganese superoxide dismutase showed a tendency toward a decrease by factors of 0.86 (P = .055) and 0.88 (P = .053), respectively, after 24 hours. Protein levels did not differ for any of the antioxidants, regardless of reperfusion time. The slightly increased messenger RNA expression of GPx1 after 24 hours of reperfusion with a concomitant very modest decrease in CAT and GCLc expression and no change in protein levels indicate a very modest, if any, response to oxidative stress generated by ischemia followed by reperfusion in rat retina.

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

Transpupillary thermotherapy for occult subfoveal choroidal neovascularization: a 1-year, prospective randomized pilot study.

PURPOSE: To evaluate the 1-year effect of transpupillary thermotherapy (TTT) on occult choroidal neovascularization membranes (CNV). METHODS: In this prospective, randomized, controlled pilot study of 28 patients with occult or minimally classic (< 10%) neovascularization membranes with a diameter less than 4500 microm, 19 patients were treated with TTT, while nine received sham treatment. Outcome measures were membrane diameter, visual acuity and reading ability. RESULTS: The median age of patients randomized to TTT was 78 years (range 24 years); that of patients randomized to sham was 79 years (range 9 years). There was no difference regarding membrane diameter at baseline between the two groups; the median membrane diameters were 3400 microm (range 2400 microm) in the TTT group and 3200 microm (range 2300 microm) in the sham group (p = 0.639). Visual acuity (VA) was similar, with a median of 0.2 (minimum-maximum 0.08-0.5) in the TTT group and a median of 0.16 (min-max 0.10-0.32) in the sham group. A total of 21 patients were followed for 1 year, 13 in the TTT group (2.7 treatments/patient) and eight in the sham group. Membrane diameter increased in both groups, by a median of 350 microm (range 1600 microm) in the TTT group and 800 microm (range 1700 microm) in the sham group (p = 0.414), respectively, and there was a loss in VA of > or = 15 letters in 5/13 patients (38%) in the TTT group compared with 2/8 patients (25%) in the sham group (p = 0.266). Reading ability deteriorated equally over time in both groups. Seven patients were lost to follow-up due to reluctance to continue in the study (n = 4) or development of a classical component > 50% (n = 3) requiring photodynamic therapy (PDT). CONCLUSION: The results from this randomized, prospective pilot study of TTT for occult CNV did not indicate that TTT has a beneficial effect on visual outcome 1 year after treatment compared with the visual outcome that results from the natural course of the disease. The small study size limits statistical power and results from large control studies are needed.