Modulation of diabetes-related retinal pathophysiology by PTX3.

Diabetic retinopathy (DR) is a common complication of diabetes characterized by vascular pathology and neuroinflammation. Pentraxin 3 (PTX3) is a soluble pattern recognition molecule that functions at the crossroads between innate immunity, inflammation, and tissue remodeling. DR is known to involve inflammatory pathways, although the potential relevance of PTX3 has not been explored. We found that PTX3 protein levels increased in the retina of diabetic mice. Similarly, evaluation of a publicly available transcriptomic human dataset revealed increased PTX3 expression in DR with diabetic macular edema and proliferative retinopathy, when compared to nondiabetic retinas or diabetic retinas without complications. To further understand the role of PTX3 within DR, we employed the streptozotocin-induced diabetes model in PTX3 knockout mice (PTX3KO), which were followed up for 9 mo to evaluate hallmarks of disease progression. In diabetic PTX3KO mice, we observed decreased reactive gliosis, diminished microglia activation, and reduced vasodegeneration, when compared to diabetic PTX3 wild-type littermates. The decrease in DR-associated pathological features in PTX3KO retinas translated into preserved visual function, as evidenced by improved optokinetic response, restored b-wave amplitude in electroretinograms, and attenuated neurodegeneration. We showed that PTX3 induced an inflammatory phenotype in human retinal macroglia, characterized by GFAP upregulation and increased secretion of IL6 and PAI-1. We confirmed that PTX3 was required for TNF-α-induced reactive gliosis, as PTX3KO retinal explants did not up-regulate GFAP in response to TNF-α. This study reveals a unique role for PTX3 as an enhancer of sterile inflammation in DR, which drives pathogenesis and ultimately visual impairment.

Multi-ancestry GWAS analysis identifies two novel loci associated with diabetic eye disease and highlights APOL1 as a high risk locus in patients with diabetic macular edema.

Diabetic retinopathy (DR) is a common complication of diabetes. Approximately 20% of DR patients have diabetic macular edema (DME) characterized by fluid leakage into the retina. There is a genetic component to DR and DME risk, but few replicable loci. Because not all DR cases have DME, we focused on DME to increase power, and conducted a multi-ancestry GWAS to assess DME risk in a total of 1,502 DME patients and 5,603 non-DME controls in discovery and replication datasets. Two loci reached GWAS significance (p<5x10-8). The strongest association was rs2239785, (K150E) in APOL1. The second finding was rs10402468, which co-localized to PLVAP and ANKLE1 in vascular / endothelium tissues. We conducted multiple sensitivity analyses to establish that the associations were specific to DME status and did not reflect diabetes status or other diabetic complications. Here we report two novel loci for risk of DME which replicated in multiple clinical trial and biobank derived datasets. One of these loci, containing the gene APOL1, is a risk factor in African American DME and DKD patients, indicating that this locus plays a broader role in diabetic complications for multiple ancestries. Trial Registration: NCT00473330, NCT00473382, NCT03622580, NCT03622593, NCT04108156.

Association of SDF-1-3' Gene A Variant with Diabetic Retinopathy in the Hungarian Population.

We investigated the association between the SDF-1-3' (c801G > A) variant and the development of diabetic macular edema (DME) or proliferative diabetic retinopathy (PDR) in a Hungarian cohort. SDF-1-3' (c801G > A) was genotyped in 103 patients with diabetic retinopathy and 31 age- and sex-matched non-diabetic controls. Central retinal and choroidal thickness was measured by swept-source optical coherence tomography. The distribution of heterozygous and homozygous SDF-1-3' (c801G > A) genotypes was similar in diabetic and control subjects. The SDF-3'(c801AA) genotype was associated with DME (n = 94 eyes, allele distribution p = 0.006, genotype distribution p = 0.01 OR: 2.48, 95% CL: 1.21-5.08) in both univariable and multivariable modelling, independent of duration and type of diabetes, HbA1C, hypertension and microalbuminuria (p = 0.03). DME occurred earlier in patients carrying the SDF-1 (c801A) allele (Kaplan-Meier analysis, log-rank test p = 0.02). A marginally significant association was found between the presence of the SDF-1 (c801A) allele and the development of PDR (n = 89 eyes, p = 0.06). The SDF-1-3' (c801A) allele also showed a correlation with central retinal (p = 0.006) and choroidal (p = 0.08) thickness. SDF-1-3' (c801G > A) is involved in the development of macular complications in DM independent of critical clinical factors, suggesting that SDF-1 may be a future therapeutic target for high-risk patients, especially those carrying the SDF-1 (c801A) allele.

Identifying Genetic Biomarkers Predicting Response to Anti-Vascular Endothelial Growth Factor Injections in Diabetic Macular Edema.

Intraocular anti-vascular endothelial growth factor (VEGF) therapies are the front-line treatment for diabetic macular edema (DME); however, treatment response varies widely. This study aimed to identify genetic determinants associated with anti-VEGF treatment response in DME. We performed a genome-wide association study on 220 Australian patients with DME treated with anti-VEGF therapy, genotyped on the Illumina Global Screening Array, and imputed to the Haplotype Reference Consortium panel. The primary outcome measures were changes in central macular thickness (CMT in microns) and best-corrected visual acuity (BCVA in ETDRS letters) after 12 months. Association between single nucleotide polymorphism (SNP) genotypes and DME outcomes were evaluated by linear regression, adjusting for the first three principal components, age, baseline CMT/BCVA, duration of diabetic retinopathy, and HbA1c. Two loci reached genome-wide significance (p < 5 × 10−8) for association with increased CMT: a single SNP on chromosome 6 near CASC15 (rs78466540, p = 1.16 × 10−9) and a locus on chromosome 12 near RP11-116D17.1 (top SNP rs11614480, p = 2.69 × 10−8). Four loci were significantly associated with reduction in BCVA: two loci on chromosome 11, downstream of NTM (top SNP rs148980760, p = 5.30 × 10−9) and intronic in RP11-744N12.3 (top SNP rs57801753, p = 1.71 × 10−8); one near PGAM1P1 on chromosome 5 (rs187876551, p = 1.52 × 10−8); and one near TBC1D32 on chromosome 6 (rs118074968, p = 4.94 × 10−8). In silico investigations of each locus identified multiple expression quantitative trait loci and potentially relevant candidate genes warranting further analysis. Thus, we identified multiple genetic loci predicting treatment outcomes for anti-VEGF therapies in DME. This work may potentially lead to managing DME using personalized treatment approaches.

Association of VEGF Gene Family Variants with Central Macular Thickness and Visual Acuity after Aflibercept Short-Term Treatment in Diabetic Patients: A Pilot Study.

INTRODUCTION: Diabetic retinopathy (DR) is one of the major vision-threatening causes worldwide. Searching for an individualized therapeutic strategy to prevent its progress is challenging. OBJECTIVE: This work aimed to investigate the association of angiogenesis-inducer vascular endothelial growth factor (VEGF) gene family and related receptor variants (rs833069, rs12366035, rs7664413, rs7993418, and rs2305948) with susceptibility of DR and the response to 1 dose of aflibercept treatment in type 2 diabetes mellitus (T2DM). METHODS: Consecutive eligible patients with T2DM (n = 125) and 110 unrelated controls were enrolled in this preliminary prospective case-controlled study. Genotyping was identified using TaqMan real-time PCR. Adjusted odds ratio (OR) with 95% confidence interval (CI) was applied to assess the strength of the association with the clinical/ophthalmological characteristics and early response to intravitreal aflibercept treatment in terms of improved visual acuity (BCVA) and central macular thickness (CMT). RESULTS: We found that both VEGFB rs12366035 and VEGFC rs7664413 conferred higher risk for DR progression under allelic (OR [95% CI]: 1.71 [1.07-2.74]), homozygote comparison (3.55 [1.32-9.57]), and recessive (3.77 [1.43-9.93]) models for the former and under allelic (2.09 [1.25-3.490, homozygote comparison (2.76 [1.02-7.45]), and recessive (2.62 [0.98-6.98] models for the latter. In contrast, VEGFR1 rs7993418 conferred protection against DR under heterozygote comparison and dominant models. The rs12366035*T/T genotype showed the worst pretreatment BCVA score (0.35 ± 0.24) compared to other corresponding genotypes (0.66 ± 0.26 in C/T and 0.54 ± 0.25 in C/C carriers) (p = 0.008). Meanwhile, patients with rs7993418*G/G of VEGFR1 exhibited a significant reduction in CMT after aflibercept injection (12.26 ± 35.43 µ in G/G vs. 3.57 ± 8.74 µ in A/A) (p = 0.037). CONCLUSIONS: Polymorphisms of the studied VEGF/receptors could be considered as genetic risk factors of DM/DR development and could play an important role in aflibercept early response for DR patients in the study population.

Micro-RNAs in the aqueous humour of patients with diabetic macular oedema.

IMPORTANCE: Micro-RNAs (miRNAs) have been studied as new biomarkers or mediators in various diseases, but the value of aqueous humour (AH) miRNAs in diabetic macular oedema (DMO) is still not known. BACKGROUND: To compare AH miRNAs and related cytokine expression in DMO patients and healthy controls. DESIGN: Prospective cross-sectional study. PARTICIPANTS: Twenty naïve DMO patients and 13 control subjects, who were scheduled for intravitreal injection and cataract surgery, respectively. METHODS: AH samples were collected at the beginning of each procedure and analysed using a miRNA polymerase chain reaction (PCR) array composed of 84 miRNAs, reverse transcripase-quantitative PCR (qPCR) for verifying selected differentially expressed miRNAs, and a cytokine assay, the results of which were compared with bioinformatics conducted to find out genes associated with DMO-related miRNAs. MAIN OUTCOMES MEASURES: AH expression of miRNAs and cytokines and the bioinformatics results. RESULTS: Five miRNAs (hsa-miR-185-5p, hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-15b-5p and hsa-miR-15a-5p) showing a fold change greater than -50 in log2 values in the miRNA PCR array were selected, all significantly down-regulated in the DMO group compared to the control group (P < .05), and showed a direct relationship with tumour necrosis factor, nuclear factor kappa B subunit 1 and interleukin-6 (IL-6) in bioinformatics analysis, all of which were related to vascular endothelial growth factor (VEGF). In the cytokine assay, the aqueous concentrations of VEGF, placental growth factor, IL-6 and IL-8 were significantly higher in the DMO group compared to the control group. CONCLUSIONS AND RELEVANCE: This study is the first to perform miRNA profiling of the AH of DMO patients. We identified differentially expressed miRNAs in DMO AH, which may be used as potential biomarkers or novel therapeutic targets for DMO.

MicroRNA Expression in the Aqueous Humor of Patients with Diabetic Macular Edema.

We identified and compared secreted microRNA (miRNA) expression in aqueous humor (AH) and plasma samples among patients with: type 2 diabetes mellitus (T2D) complicated by non-proliferative diabetic retinopathy (DR) associated with diabetic macular edema (DME) (DME group: 12 patients); T2D patients without DR (D group: 8 patients); and non-diabetic patients (CTR group: 10 patients). Individual patient AH samples from five subjects in each group were profiled on TaqMan Low Density MicroRNA Array Cards. Differentially expressed miRNAs identified from profiling were then validated in single assay for all subjects. The miRNAs validated in AH were then evaluated in single assay in plasma. Gene Ontology (GO) analysis was conducted. From AH profiling, 119 mature miRNAs were detected: 86 in the DME group, 113 in the D group and 107 in the CTR group. miRNA underexpression in the DME group was confirmed in single assay for let-7c-5p, miR-200b-3p, miR-199a-3p and miR-365-3p. Of these four, miR-199a-3p and miR-365-3p were downregulated also in the plasma of the DME group. GO highlighted 54 validated target genes of miR-199a-3p, miR-200b-3p and miR-365-3p potentially implied in DME pathogenesis. Although more studies are needed, miR-200b-3p, let-7c-5p, miR-365-3p and miR-199a-3p represent interesting molecules in the study of DME pathogenesis.

Imaging and Biomarkers in Diabetic Macular Edema and Diabetic Retinopathy.

PURPOSE OF REVIEW: Diabetic retinopathy (DR) is the leading cause of acquired vision loss in adults across the globe. Early identification and treatment of patients with DR is paramount for vision preservation. The aim of this review paper is to outline current and new imaging techniques and biomarkers that are valuable for clinical diagnosis and management of DR. RECENT FINDINGS: Ultrawide field imaging and automated deep learning algorithms are recent advancements on traditional fundus photography and fluorescein angiography. Optical coherence tomography (OCT) and OCT angiography are techniques that image retinal anatomy and vasculature and OCT is routinely used to monitor response to treatment. Many circulating, vitreous, and genetic biomarkers have been studied to facilitate disease detection and development of new treatments. Recent advancements in retinal imaging and identification of promising new biomarkers for DR have the potential to increase detection, risk stratification, and treatment for patients with DR.

Endothelial Cell-Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature.

Objective- Blood-CNS (central nervous system) barrier defects are implicated in retinopathies, neurodegenerative diseases, stroke, and epilepsy, yet, the pathological mechanisms downstream of barrier defects remain incompletely understood. Blood-retina barrier (BRB) formation and retinal angiogenesis require ß-catenin signaling induced by the ligand norrin (NDP [Norrie disease protein]), the receptor FZD4 (frizzled 4), coreceptor LRP5 (low-density lipoprotein receptor-like protein 5), and the tetraspanin TSPAN12 (tetraspanin 12). Impaired NDP/FZD4 signaling causes familial exudative vitreoretinopathy, which may lead to blindness. This study seeked to define cell type-specific functions of TSPAN12 in the retina. Approach and Results- A loxP-flanked Tspan12 allele was generated and recombined in endothelial cells using a tamoxifen-inducible Cdh5-CreERT2 driver. Resulting phenotypes were documented using confocal microscopy. RNA-Seq, histopathologic analysis, and electroretinogram were performed on retinas of aged mice. We show that TSPAN12 functions in endothelial cells to promote vascular morphogenesis and BRB formation in developing mice and BRB maintenance in adult mice. Early loss of TSPAN12 in endothelial cells causes lack of intraretinal capillaries and increased VE-cadherin (CDH5 [cadherin5 aka VE-cadherin]) expression, consistent with premature vascular quiescence. Late loss of TSPAN12 strongly impairs BRB maintenance without affecting vascular morphogenesis, pericyte coverage, or perfusion. Long-term BRB defects are associated with immunoglobulin extravasation, complement deposition, cystoid edema, and impaired b-wave in electroretinograms. RNA-sequencing reveals transcriptional responses to the perturbation of the BRB, including genes involved in vascular basement membrane alterations in diabetic retinopathy. Conclusions- This study establishes mice with late endothelial cell-specific loss of Tspan12 as a model to study pathological consequences of BRB impairment in an otherwise intact vasculature.

BMP9 (Bone Morphogenetic Protein-9)/Alk1 (Activin-Like Kinase Receptor Type I) Signaling Prevents Hyperglycemia-Induced Vascular Permeability.

Objective- Diabetic macular edema is a major cause of visual impairment. It is caused by blood-retinal barrier breakdown that leads to vascular hyperpermeability. Current therapeutic approaches consist of retinal photocoagulation or targeting VEGF (vascular endothelial growth factor) to limit vascular leakage. However, long-term intravitreal use of anti-VEGFs is associated with potential safety issues, and the identification of alternative regulators of vascular permeability may provide safer therapeutic options. The vascular specific BMP (bone morphogenetic protein) receptor ALK1 (activin-like kinase receptor type I) and its circulating ligand BMP9 have been shown to be potent vascular quiescence factors, but their role in the context of microvascular permeability associated with hyperglycemia has not been evaluated. Approach and Results- We investigated Alk1 signaling in hyperglycemic endothelial cells and assessed whether BMP9/Alk1 signaling could modulate vascular permeability. We show that high glucose concentrations impair Alk1 signaling, both in cultured endothelial cells and in a streptozotocin model of mouse diabetes mellitus. We observed that Alk1 signaling participates in the maintenance of vascular barrier function, as Alk1 haploinsufficiency worsens the vascular leakage observed in diabetic mice. Conversely, sustained delivery of BMP9 by adenoviral vectors significantly decreased the loss of retinal barrier function in diabetic mice. Mechanistically, we demonstrate that Alk1 signaling prevents VEGF-induced phosphorylation of VE-cadherin and induces the expression of occludin, thus strengthening vascular barrier functions. Conclusions- From these data, we suggest that by preventing retinal vascular permeability, BMP9 could serve as a novel therapeutic agent for diabetic macular edema.

Genome-wide association studies for diabetic macular edema and proliferative diabetic retinopathy.

BACKGROUND: Diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR) are sight-threatening complications of diabetes mellitus and leading causes of adult-onset blindness worldwide. Genetic risk factors for diabetic retinopathy (DR) have been described previously, but have been difficult to replicate between studies, which have often used composite phenotypes and been conducted in different populations. This study aims to identify genetic risk factors for DME and PDR as separate complications in Australians of European descent with type 2 diabetes. METHODS: Caucasian Australians with type 2 diabetes were evaluated in a genome-wide association study (GWAS) to compare 270 DME cases and 176 PDR cases with 435 non-retinopathy controls. All participants were genotyped by SNP array and after data cleaning, cases were compared to controls using logistic regression adjusting for relevant covariates. RESULTS: The top ranked SNP for DME was rs1990145 (p = 4.10 × 10- 6, OR = 2.02 95%CI [1.50, 2.72]) on chromosome 2. The top-ranked SNP for PDR was rs918519 (p = 3.87 × 10- 6, OR = 0.35 95%CI [0.22, 0.54]) on chromosome 5. A trend towards association was also detected at two SNPs reported in the only other reported GWAS of DR in Caucasians; rs12267418 near MALRD1 (p = 0.008) in the DME cohort and rs16999051 in the diabetes gene PCSK2 (p = 0.007) in the PDR cohort. CONCLUSION: This study has identified loci of interest for DME and PDR, two common ocular complications of diabetes. These findings require replication in other Caucasian cohorts with type 2 diabetes and larger cohorts will be required to identify genetic loci with statistical confidence. There is considerable overlap in the patient cohorts with each retinopathy subtype, complicating the search for genes that contribute to PDR and DME biology.

VEGF-A gene polymorphisms and responses to intravitreal ranibizumab treatment in patients with diabetic macular edema.

PURPOSE: To investigate the association between VEGF gene polymorphisms and the responses to treatment with intravitreal ranibizumab (IVR) in patients with diabetic macular edema (DME). METHODS: This prospective study, conducted at the Kutahya Dumlupinar University Faculty of Medicine, included 95 patients with DME that were treated with IVR and 32 patients without DME despite proliferative diabetic retinopathy (PDR). The participants were divided into three groups: DME with non-proliferative diabetic retinopathy, DME with PDR, and PDR without DME; patients with DME who were treated with IVR were further divided into two groups based on their response to the treatment. Each patient was genotyped for five single nucleotide variations (SNVs) in VEGF-A: rs2010963, rs2146323, rs10434, rs833069, and rs6921438. RESULT: There was a statistically significant difference in allelic distribution of VEGF-A rs833069 polymorphism in relation to the severity of diabetic retinopathy (DRP) (p = 0.031). The allelic distribution of VEGF-A rs2146323 polymorphism tended to be associated with the severity of DRP (p = 0.069). There were no statistically significant differences in the allelic distribution of the studied five SNVs in DME patients regarding the patients' responses to IVR therapy. CONCLUSIONS: There is no association between the studied VEGF-A SNVs and the responses to IVR therapy in DME. However, the VEGF-A rs833069 gene polymorphism has a clear association with the severity of diabetic retinopathy.

Retinopathy with central oedema in an INS C94Y transgenic pig model of long-term diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is a severe complication of diabetes mellitus that often leads to blindness. Because the pathophysiology of diabetic retinopathy is not fully understood and novel therapeutic interventions require testing, there is a need for reliable animal models that mimic all the complications of diabetic retinopathy. Pig eyes share important anatomical and physiological similarities with human eyes. Previous studies have demonstrated that INS C94Y transgenic pigs develop a stable diabetic phenotype and ocular alterations such as cataracts. The aim of this study was to conduct an in-depth analysis of pathological changes in retinas from INS C94Y pigs exposed to hyperglycaemia for more than 2 years, representing a chronic diabetic condition. METHODS: Eyes from six INS C94Ypigs and six age-matched control littermates were analysed via histology and immunohistochemistry. For histological analyses of retinal (layer) thickness, sections were stained with H&E or Mallory's trichrome. For comparison of protein expression patterns and vessel courses, sections were stained with different antibodies in immunohistochemistry. Observed lesions were compared with reported pathologies in human diabetic retinopathy. RESULTS: INS C94Ypigs developed several signs of diabetic retinopathy similar to those seen in humans, such as intraretinal microvascular abnormalities, symptoms of proliferative diabetic retinopathy and central retinal oedema in a region that is cone rich, like the human macula. CONCLUSIONS/INTERPRETATION: The INS C94Ypig is an interesting model for studying the pathophysiology of diabetic retinopathy and for testing novel therapeutic strategies.

Dominant cystoid macular dystrophy.

OBJECTIVE: To describe the clinical characteristics and long-term follow-up in patients with autosomal dominant cystoid macular dystrophy (DCMD). DESIGN: Retrospective case series. PARTICIPANTS: Ninety-seven patients with DCMD. METHODS: Extensive ophthalmic examination, including visual acuity (VA), fundus photography, fluorescein angiography (FA), fundus autofluorescence (FAF) imaging, optical coherence tomography (OCT), color vision testing, dark adaptation testing, full-field electroretinography (ERG), and electro-oculography (EOG). Blood samples were obtained for DNA extraction and subsequent haplotype analysis. MAIN OUTCOME MEASURES: Age at onset, VA, fundus appearance, and characteristics on FA, FAF, OCT, ERG, and EOG. RESULTS: Cystoid fluid collections (CFCs) were the first retinal abnormalities detectable in DCMD, developing during childhood. At long-term follow-up, the CFCs decreased in size and number, and eventually disappeared with concurrent development of progressive chorioretinal atrophy and hyperpigmented deposits in the posterior pole. Dominant cystoid macular dystrophy could be classified into 3 stages, based on characteristics on ophthalmoscopy, FAF, FA, and OCT, as well as on results of electrophysiologic analysis. The staging system correlated with age and VA. In stage 1 DCMD (20 patients; 22%), patients generally were younger than 20 years and had CFCs with fine folding of the internal limiting membrane and mild pigment changes. In stage 2 DCMD (48 patients; 52%), the CFCs tended to decrease in size, and moderate macular chorioretinal atrophy developed. Patients with stage 3 DCMD (24 patients; 26%) generally were older than 50 years and showed profound chorioretinal atrophy, as well as coarse hyperpigmented deposits in the posterior pole. Most patients were (highly) hyperopic (72 patients; 92%). All DCMD patients shared the disease haplotype at the DCMD locus at 7p15.3. CONCLUSIONS: Dominant cystoid macular dystrophy is a progressive retinal dystrophy, characterized primarily by early-onset cystoid fluid collections in the neuroretina, which distinguishes this disorder from other retinal dystrophies. The phenotypic range of DCMD can be classified into 3 stages. The genetic locus for this retinal dystrophy has been mapped to 7p15.3, but the involved gene is currently unknown.

Common Sequence Variation in the VEGFC Gene Is Associated with Diabetic Retinopathy and Diabetic Macular Edema.

PURPOSE: To investigate associations between single nucleotide polymorphisms (SNPs) in the VEGFC gene and the development of diabetic retinopathy (DR) in white patients with type 1 diabetes mellitus (T1DM) or type 2 diabetes mellitus (T2DM). DESIGN: Cross-sectional, case control study. PARTICIPANTS: White patients with T1DM or T2DM (n = 2899) were recruited from ophthalmology and endocrine clinics in Australia and the United Kingdom. Patients with T2DM were required to have diabetes mellitus (DM) for at least 5 years and be receiving oral hypoglycemic treatment or insulin. METHODS: Participants were categorized according to their worst-ever DR grading, as having "no DR" (no history of nonproliferative DR [NPDR], proliferative DR [PDR], or diabetic macular edema [DME]) or "any DR" (further subclassified as NPDR or PDR, without or with DME). Clinical characteristics, glycemic control (hemoglobin A1c [HbA1c]), and presence of diabetic complications were determined at recruitment. Genotyping was performed for 13 VEGFC tag SNPs. MAIN OUTCOME MEASURES: Odds ratios (ORs) were determined for associations with DR of VEGFC tag SNPs, individually and within haplotypes. Logistic regression was used to adjust for clinical covariates, including DM type, age, sex, DM duration, hypertension, nephropathy, HbA1c, and smoking. RESULTS: Participants with DM but "no DR" (n = 980) were compared with 1919 participants with DM and "any DR." Three VEGFC SNPs were associated with DR after logistic regression: rs17697419 (P = 0.001; OR, 0.67; confidence interval [CI], 0.52-0.85), rs17697515 (P = 0.001; OR, 0.62; CI, 0.47-0.81), and rs2333526 (P = 0.005; OR, 0.69; CI, 0.54-0.90). rs17697515 Was also specifically associated with DME in those with T2DM (P = 0.004; OR, 0.53; CI, 0.35-0.82). Haplotype analysis revealed 2 significantly associated haplotypes, both protective against DR development. CONCLUSIONS: Significant associations were found between VEGFC tag SNPs (individually and within haplotypes) and the presence of any DR or DME in white participants with T1DM and T2DM.

Molecular analysis of blood-retinal barrier loss in the Akimba mouse, a model of advanced diabetic retinopathy.

The molecular mechanisms of vascular leakage in diabetic macular edema and proliferative retinopathy are poorly understood, mainly due to the lack of reliable in vivo models. The Akimba (Ins2(Akita)VEGF(+/-)) mouse model combines retinal neovascularization with hyperglycemia, and in contrast to other models, displays the majority of signs of advanced clinical diabetic retinopathy (DR). To study the molecular mechanism that underlies the breakdown of the blood-retinal barrier (BRB) in diabetic macular edema and proliferative diabetic retinopathy, we investigated the retinal vasculature of Akimba and its parental mice Kimba (trVEGF029) and Akita (Ins2(Akita)). Quantitative PCR, immunohistochemistry and fluorescein angiography were used to characterize the retinal vasculature with special reference to the inner BRB. Correlations between the degree of fluorescein leakage and retinal gene expression were tested by calculating the Spearman's correlation coefficient. Fluorescein leakage demonstrating BRB loss was observed in Kimba and Akimba, but not in Akita or wild type mice. In Kimba and Akimba mice fluorescein leakage was associated with focal angiogenesis and correlated significantly with Plvap gene expression. PLVAP is an endothelial cell-specific protein that is absent in intact blood-retinal barrier, but its expression significantly increases in pathological conditions such as DR. Furthermore, in Akimba mice BRB disruption was linked to decreased expression of endothelial junction proteins, pericyte dropout and vessel loss. Despite fluorescein leakage, no alteration in BRB protein levels or pericyte coverage was detected in retinas of Kimba mice. In summary, our data not only demonstrate that hyperglycemia sensitizes retinal vasculature to the effects of VEGF, leading to more severe microvascular changes, but also confirm an important role of PLVAP in the regulation of BRB permeability.

Association of monocyte chemoattractant protein-1 (MCP-1)2518A/G polymorphism with proliferative diabetic retinopathy in northern Chinese type 2 diabetes.

BACKGROUND: The pathogenesis of proliferative diabetic retinopathy (PDR) remains poorly understood. Recent studies have implicated that monocyte chemoattractant protein-1 (MCP-1) is associated with diabetic microvascular or macrovascular complications. However, the relationship between single nucleotide polymorphism(SNP)c.2518A/G -rs1024611 in the MCP-1 gene with diabetic retinopathy remains controversial. In the present study, we evaluated the association of SNP in the MCP-1 gene with diabetic retinopathy (DR) and diabetic macular edema (DME) in a Chinese population from Northern China with type 2 diabetes. METHODS: We conducted a case-control study, which enrolled 1,043 subjects with type 2 diabetes (528 with DR, including 277PDR; 515 without DR), and SNP genotyping of c.2518A/G in the MCP-1 gene was performed using the polymerase chain reaction. Genomic DNA was isolated from 3 ml samples of whole blood using a modified conventional DNA extraction method. The genotype and allele frequencies of 2518A/G were studied by using an automated DNA sequencer (ABI PRISM 3730 DNA Sequencer). RESULTS: The demographic and clinical characteristics did not differ among genotype subgroups. The MCP-1(-2518) GG genotype was significantly associated with DR susceptibility with OR of 1.481 (95 % CI, 1.019-2.153) (P = 0.046). There were no significant differences in the MCP-1(-2518) G allele frequencies in DR compared to non-diabetic retinopathy (DNR) (P > 0.05, OR = 0.841, 95 % CI, 0.705-1.002). The MCP-1(-2518) GG genotype was significantly associated with high-risk PDR susceptibility with OR of 2.656 (95 % CI, 1.222-5.775) (P = 0.014). The MCP-1(-2518) G allele was significantly increased in high-risk PDR patients (P = 0.020, OR = 1.481, 95 % CI, 1.070-2.051) compared with A allele. Genotype and allele frequencies of various DME of the DR patients were compared, but there were no significant associations established (P > 0.05). CONCLUSIONS: It is likely that the MCP-1 c.2518G/G genotype is a susceptibility gene for DR in Chinese type 2 diabetic patients, especially the high-risk PDR. There is no association with DME and c.2518G/G.

Genetic study of diabetic retinopathy: recruitment methodology and analysis of baseline characteristics.

BACKGROUND: Diabetic retinopathy (DR) is a blinding disease of increasing prevalence that is caused by a complex interplay of genetic and environmental factors. Here we describe the patient recruitment methodology, case and control definitions, and clinical characteristics of a study sample to be used for genome-wide association analysis to detect genetic risk variants of DR. METHODS: One thousand six hundred sixty-nine participants with either type 1 (T1) or type 2 (T2) diabetes mellitus (DM) aged 18 to 95 years were recruited in Australian hospital clinics. Individuals with T2DM had disease duration of at least 5 years and were taking oral hypoglycaemic medication, and/or insulin therapy. Participants underwent ophthalmic examination. Medical history and biochemistry results were collected. Venous blood was obtained for genetic analysis. RESULTS: Six hundred eighty-three diabetic cases (178 T1DM and 505 T2DM participants) with sight-threatening DR, defined as severe non-proliferative DR, proliferative DR or diabetic macular oedema were included in this analysis. Eight hundred twelve individuals with DM but no DR or minimal non-proliferative DR were recruited as controls (191 with T1DM and 621 with T2DM). The presence of sight-threatening DR was significantly correlated with DM duration, hypertension, nephropathy, neuropathy, HbA1C and body mass index. Diabetic macular oedema was associated with T2DM (P < 0.001), whereas proliferative DR was associated with T1DM (P < 0.001). CONCLUSIONS: Adoption of a case-control study design involving extremes of the DR phenotype makes this a suitable cohort, for a well-powered genome-wide association study to detect genetic risk variants for DR.

Vascular endothelial growth factor gene polymorphism prevalence in patients with diabetic macular oedema and its correlation with anti-vascular endothelial growth factor treatment outcomes.

BACKGROUND: To study the possible association between vascular endothelial growth factor gene polymorphism and diabetic macular oedema, and its correlation to the outcomes of anti-vascular endothelial growth factor treatment. DESIGN: Prospective study. PARTICIPANTS: 392 diabetic patients were included; 180 patients of them had no retinopathy, 212 patients had diabetic retinopathy. Diabetic retinopathy patients were classified into four groups as defined by the absence or presence of macular oedema or proliferative retinopathy. METHODS: In all subjects, polymerase chain reaction-restriction fragment length polymorphism was conducted to detect the vascular endothelial growth factor gene C-634G polymorphism. Serum levels of vascular endothelial growth factor were estimated. Changes of visual acuity and central macular thickness after bevacizumab treatment in diabetic macular oedema patients of different genotypes were monitored for 9-12 months. MAIN OUTCOME MEASURES: Vascular endothelial growth factor C-634G genotypes distribution in different groups; correlation between genotypes, and changes in visual acuity and central macular thickness after intravitreal bevacizumab treatment. RESULTS: CC genotype was significantly prevalent among diabetic macular oedema patients (P = 0.019). Significant higher serum levels of vascular endothelial growth factor were detected in diabetic retinopathy and diabetic macular oedema patients with CC genotype (P = 0.02, 0.016). After bevacizumab treatment, individuals with genotypes CG and GG have a decreased chance of positive treatment outcomes compared t with CC genotype (P < 0.001). CONCLUSIONS: Vascular endothelial growth factor C-634G polymorphism (CC genotype) is a genetic risk factor for diabetic macular oedema, and its presence provides significantly better visual outcome following bevacizumab treatment.

Genetic association of TIE2 with diabetic retinopathy and diabetic macular edema.

PURPOSE: To evaluate the associations of the TIE2 gene with diabetic retinopathy (DR) and diabetic macular edema (DME). METHODS: This study included a Chinese cohort of 285 non-proliferative DR patients and 433 healthy controls. The DR patients were classified further into those with or without DME. Thirty haplotype-tagging single-nucleotide polymorphisms (SNPs) in TIE2 were genotyped using TaqMan technology. Associations of DR and subtypes were analyzed by logistic regression adjusted for age and sex. Stratification association analysis by sex was performed. RESULTS: TIE2 rs625767 showed a nominal but consistent association with DR [odds ratio (OR) = 0.71, P = 0.005] and subtypes (DR without DME: OR = 0.69, P = 0.016; DME: OR = 0.73, P = 0.045). SNP rs652010 was consistently associated with overall DR (OR = 0.74, P = 0.011) and DR without DME (OR = 0.70, P = 0.016), but not with DME. Moreover, SNPs rs669441, rs10967760, rs549099 and rs639225 showed associations with overall DR, whilst rs17761403, rs664461 and rs1413825 with DR without DME. In stratification analysis, three SNPs, rs625767 (OR = 0.62, P = 0.005), rs669441 (OR = 0.63, P = 0.006) and rs652010 (OR = 0.64, P = 0.007), were associated with DR in females, but not in males. Moreover, one haplotype T-T defined by rs625767 and rs669441 was significantly associated with DR in females only. CONCLUSIONS: This study revealed TIE2 as a susceptibility gene for DR and DME in Chinese, with a sex-specific association in females. Further validation should be warranted.