Nuclear factor of activated T-cells (NFAT) regulation of IL-1ß-induced retinal vascular inflammation.

Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1ß has the reputation of being a major driver of cytokine-induced inflammation. IL-1ß and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1ß-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1ß-induced expression of IL-1ß and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1ß-induced increases of IL-1ß, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1ß-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1ß-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes.

A screen for deeply conserved non-coding GWAS SNPs uncovers a MIR-9-2 functional mutation associated to retinal vasculature defects in human.

Thousands of human disease-associated single nucleotide polymorphisms (SNPs) lie in the non-coding genome, but only a handful have been demonstrated to affect gene expression and human biology. We computationally identified risk-associated SNPs in deeply conserved non-exonic elements (CNEs) potentially contributing to 45 human diseases. We further demonstrated that human CNE1/rs17421627 associated with retinal vasculature defects showed transcriptional activity in the zebrafish retina, while introducing the risk-associated allele completely abolished CNE1 enhancer activity. Furthermore, deletion of CNE1 led to retinal vasculature defects and to a specific downregulation of microRNA-9, rather than MEF2C as predicted by the original genome-wide association studies. Consistent with these results, miR-9 depletion affects retinal vasculature formation, demonstrating MIR-9-2 as a critical gene underpinning the associated trait. Importantly, we validated that other CNEs act as transcriptional enhancers that can be disrupted by conserved non-coding SNPs. This study uncovers disease-associated non-coding mutations that are deeply conserved, providing a path for in vivo testing to reveal their cis-regulated genes and biological roles.

[Retinal vasculopathy with cerebral leukoencephalopathy carrying TREX1 mutation diagnosed by the intracranial calcification: a case report].

A 40-year-old woman with renal dysfunction for 2 years was admitted to our hospital suffering from a headache. Family history revealed that her mother had a headache, renal dysfunction, and brain infarction in younger age. She had a retinal hemorrhage, a retinal atrophy, pitting edema in her lower extremities. Her neurological findings were unremarkable. Brain imaging showed multiple white matter lesions accompanied with calcifications and slightly enhancement. Kidney biopsy showed the thrombotic microangiopathy, Gene analysis demonstrated a causative mutation in three-prime repair exonuclease-1 (TREX1) gene, c.703_704insG (p.Val235GlyfsX6), thereby we diagnosed her as retinal vasculopathy with cerebral leukoencephalopathy (RVCL). RVCL is an autosomal dominant condition caused by C-terminal frame-shift mutation in TREX1. TREX1 protein is a major 3' to 5' DNA exonuclease, which are important in DNA repair. While TREX1 mutations identified in Aicardi-Goutieres syndrome patients lead to a reduction of enzyme activity, it is suggested that mutations in RVCL alter an intracellular location of TREX1 protein. There are no treatments based evidences in RVCL. We administered cilostazol to protect endothelial function, and her brain lesions and renal function have not become worse for 10 months after. It is necessary to consider RVCL associated with TREX1 mutation if a patient has retinal lesions, white matter lesions accompanied with calcifications, and multiple organ dysfunction.

Circulating Endothelial Markers in Retinal Vasculopathy With Cerebral Leukoencephalopathy and Systemic Manifestations.

BACKGROUND AND PURPOSE: Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a monogenic small vessel disease, caused by C-terminal truncating TREX1 mutations, that can be considered a model for stroke and vascular dementia. The pathophysiology of RVCL-S is largely unknown, but systemic endothelial involvement has been suggested, leading to pathology in the brain and other highly vascularized organs. Here, we investigated circulating endothelial markers to confirm endothelial involvement and identify biomarkers for disease activity. METHODS: We measured circulating levels of von Willebrand factor (VWF) antigen, VWF propeptide, and angiopoietin-2 in members of 3 Dutch RVCL-S families and matched unrelated healthy controls. Stratified analyses based on symptomatology and age were performed. RESULTS: We found elevated levels of VWF antigen, VWF propeptide, and angiopoietin-2 in TREX1 mutation carriers (n=31) compared with family members without a TREX1 mutation (n=33) and unrelated healthy controls (n=31; Kruskal-Wallis test P<0.001 for all comparisons). Effects were most pronounced in mutation carriers with clinical manifestations aged ≥40 years (Mann-Whitney U test P<0.001 for all comparisons). Compared with healthy controls, levels of VWF antigen (P=0.02) and angiopoietin-2 (P=0.04) were also elevated in mutation carriers aged <40 years. All 3 markers showed moderate correlations with markers of kidney and liver disease and inflammation (ie, systemic symptoms of RVCL-S). CONCLUSIONS: Our results confirm an important role of the endothelium in RVCL-S pathophysiology. VWF antigen, VWF propeptide, and angiopoietin-2 might serve as early biomarkers of disease activity. Our findings might also help to understand the pathophysiology of common neurovascular disorders, such as stroke.

Papillophlebitis versus paediatric venous thrombosis. A case with 46C/T polymorphism in the F12 coagulation gene. / Papiloflebitis versus trombosis venosa pediátrica. Un caso con polimorfismo C46T del gen F12 de la coagulación.

CLINICAL CASE: An 8 year-old boy with no known diseases, with sudden loss of visual acuity (VA) in the left eye (LE). EXAMINATION: VA 1 in right eye, and 0.1 in LE, discrete left relative afferent pupil defect (RAPD). Normal biomicroscopy. Funduscopy: congestive papilla, venous tortuosity, peripapillary haemorrhages with macular oedema in LE. The systemic study only revealed A C46Tpolymorphism in the F12 coagulation gene. He had a VA of 1 and normal funduscopy 8 months later. DISCUSSION: Papillophlebitis is an inflammatory and non-ischaemic central retinal vein occlusion, ophthalmoscopically similar to central retinal vein thrombosis. The systemic study is essential to rule out underlying diseases.

VKORC1 C1173T and VKORC1 G-1639A gene polymorphisms in Turkish Behçet's patients with ocular and non-ocular involvement.

BACKGROUND: Behçet's disease (BD) is a multisystemic vasculitis with unknown etiology. Vitamin K epoxide reductase complex subunit 1 (VKORC1) is the key enzyme in the formation of active vitamin K that is a cofactor of various coagulation factors. Polymorphisms of the VKORC1 may affect the levels of active forms of vitamin K-dependent coagulation proteins and the tendency to thrombosis. The current study aimed to evaluate the role of VKORC1 gene polymorphisms in ocular and non-ocular Behçet's disease. METHODS: VKORC1 C1173T (rs 9934438) and G-1639A (rs 9923231) gene polymorphisms were evaluated by real-time polymerase chain reaction-based DNA analysis. The frequency of alleles and distribution of genotypes were assessed by the chi-squared test. Genotype distribution and Hardy-Weinberg equilibrium were tested with the χ(2) test for quality of fit. RESULTS: The distribution of GG, GA and AA and CC, CT, TT genotypes and the frequency of G,A and C,T alleles were not found to be different between patients and controls (p = 0.5651; p = 0.335 respectively), as well as patients with or without eye involvement (p = 0.9267; p = 0.384 respectively). CONCLUSION: VKORC1 polymorphisms seem not to be related with the thrombotic state of systemic and ocular Behçet's disease.

Profiling of human leukocyte antigens in Eales disease and tuberculosis.

To analyze the association between Eales disease, histocompatibility leukocyte antigen alleles (HLA-A/B, HLA-DRB/DQB) and tuberculosis infection, and to explore susceptible genes and protective genes associated with Eales disease and tuberculosis infection in a population of Han nationals from Zunyi City in Guizhou Province, China. The subjects were analyzed by a case-control study consisting of three groups--Eales disease group (47 cases), pulmonary tuberculosis group (36 cases) and normal control group (100 cases). The Eales disease group was divided into four parts. Part one consisted of 12 patients who had suffered from pulmonary tuberculosis. Part two consisted of 27 patients who had not suffered from pulmonary tuberculosis. Parts three and four consisted of 27 patients with a positive purified protein derivative test and 12 patients with a negative test, respectively. DNA samples from 47 patients with Eales disease, 36 patients with pulmonary tuberculosis and 100 healthy people were detected by polymerase chain reaction with sequence-specific primers. The 59 HLA-A/B and HLA-DRB/DQB alleles from Eales disease were compared with those from tuberculosis and normal control, and a correlativity test of common susceptible genes was performed to analyze the potential relationship between Eales disease and pulmonary tuberculosis. The frequency distribution of HLA-A*02 alleles (OR 9.719, OR 95 % CI 4.377-21.580, P = 0.000) and HLA-B*07 (OR 11.605, OR 95 % CI 2.397-56.191, P = 0.001) in the Eales disease group was higher than in the normal control group, but the HLA-A*11 alleles (OR 0.495, OR 95 % CI 0.245-1.000, P = 0.048) were lower than in the normal control group, showing a significant difference. Compared with parts two and four, the frequency distribution of HLA-A*02, HLA-A*11 and HLA-B*07 alleles in parts one and three showed no significant difference (P > 0.05). HLA-A*A02, HLA-A*24, HLA-B*07 and HLA-DRB*16 alleles between the Eales disease, pulmonary tuberculosis and normal control group showed statistical significance (P < 0.05). HLA-A*24 alleles in the pulmonary tuberculosis group were lower than the Eales disease group (χ(2) 7.289, P = 0.007), but HLA-A*02 alleles showed no significant difference (P > 0.05) between the two groups. The results show that HLA-A*02 and HLA-B*07 may be genetic predisposing genes, but HLA-A*11 alleles may be protective genes of Eales disease, the HLA-A*02 allele may be a common susceptible gene of Eales disease and pulmonary tuberculosis. HLA-A*11 and HLA-A24 alleles are protective genes of Eales disease and pulmonary tuberculosis, respectively. In summary, the frequency distribution of susceptible genes of Eales disease and pulmonary tuberculosis in a population of Han nationals from Zunyi City in Guizhou Province, China showed no significant difference.

Transthyretin Ala36Pro mutation in a Chinese pedigree of familial transthyretin amyloidosis with elevated vitreous and serum vascular endothelial growth factor.

The familial transthyretin (TTR) amyloidosis (FTA) demonstrates variable penetrance of clinical features associated with mutations in the plasma thyroid hormone-binding protein TTR gene. The purpose of this study was to assess the ocular features, to analyze vitreous and serum vascular endothelial growth factor (VEGF) levels, and to identify the genetic defect in a Chinese family with TTR FTA. The pedigree of interest was a three-generation family with eleven members. The primary ocular signs were vitreous opacities, beginning from the third or fourth decade, accompanied by retinal vasculitis, hemorrhages, and widespread pinpoint deposits in the peripheral retina. Two patients underwent vitrectomy with marked improvement of visual acuity postoperatively. Vitreous and serum samples for VEGF were analyzed with an enzyme-linked immunosorbent assay (ELISA). Forty-eight healthy adult volunteers were enrolled as a control group for the analysis of serum VEGF. Eight subjects who underwent vitrectomy for a macular epiretinal membrane or macular hole were enrolled as control for the analysis of vitreous VEGF. Both serum and vitreous VEGF levels of patients were raised compared to that of controls. Venous blood was collected from family members and the genomic DNA was extracted. All exons and exon-intron boundaries of the TTR gene were sequenced. A previously-described pathogenic transversion in exon 2 (c.G106C, p.Ala36Pro) was identified. Within this family eight individuals were confirmed as affected. In conclusion, a Chinese family with TTR Ala36Pro associated FTA is characterized by early ocular involvement. Widespread pinpoint lesions indicate RPE lesions caused by TTR deposition. FTA is associated with increased VEGF levels, both in serum and vitreous.

Familial amyloid polyneuropathy associated with the novel transthyretin variant Arg34Gly.

We report a 57-year-old man with pathognomonic bilateral vitreo-lenticular amyloid opacities (pseudopodia lentis) in whom a novel transthyretin (TTR) mutation was identified. The patient presented due to bilateral floaters. The vitreous cavities of both eyes showed course, fibrilar opacities attached to the posterior lens surface with pseudopodia. There was a history of bilateral carpal tunnel syndrome. Nerve conduction studies showed upper and lower limb axonal polyneuropathy. Magnetic resonance imaging of the brain and spinal cord, renal and cardiac function were normal. Vitreous and conjunctival biopsies confirmed the diagnosis of TTR-related amyloidosis. Genetic analysis of exon 2 of the TTR gene revealed that the patient was heterozygous for a single nucleotide substitution c.160 A>G, resulting in replacement of arginine with glycine at position 34 of the mature protein (Arg34Gly). Five years later the patient developed increasing sensory and motor neuropathy of both lower limbs, and neovascular glaucoma in one eye. We hypothesize that the reason for his neovascular glaucoma was retinal ischaemia secondary to amyloid retinal vasculopathy.

Intron F G79A polymorphism of the protein Z gene in Turkish Behçet patients.

PURPOSE: Vasculitis is one of the major findings of Behçet's disease (BD). Protein Z (PZ) is a glycoprotein that acts as a cofactor of PZ-dependent protease inhibitor and suppresses trombus formation by inhibiting activated factor Xa. Polymorphism of the PZ gene was mentioned as a genetic risk factor for various thrombotic events. The aim of this study is to investigate the intron F G79A polymorphism of the PZ gene in Behçet patients with and without ocular involvement. METHODS: Seventy-six patients and 70 controls were included in the study. Intron F G79A polymorphism of PZ gene was determined by polymerase chain reaction based DNA analysis. The frequency of A allele and the distribution of genotypes were assessed by χ(2) test and the genotype distribution and Hardy-Weinberg equilibrium were tested with the χ(2) test for quality of fit. RESULTS: The frequency of the A allele was significantly higher in overall Behçet patients than in controls (odds ratio [OR] = 6.8; 95% CI, 2.6 to 17.9; p = 0.0001). It was also significantly higher in patients with (OR = 5.3; 95% CI, 1.83 to 15.6; p = 0.0024) or without (OR = 8.2; 95% CI, 2.95 to 22.5; p = 0.0001) ocular involvement compared to controls. However, A allele frequency was not significantly different between patients with eye involvement versus patients without eye involvement (OR = 0.65; 95% CI, 0.3 to 1.4; p = 0.28). CONCLUSIONS: Although thrombosis in BD is multifactorial, intron F G79A polymorphism of PZ gene in BD may be one of the factors that contribute to this pathological process.

Impact of interleukin-6 promoter polymorphism and serum interleukin-6 level on the acute inflammation and neovascularization stages of patients with Eales' disease.

PURPOSE: To evaluate the role of interleukin-6 (IL-6) in the inflammatory and proliferative stages of Eales' disease (ED) and to determine the influence of IL-6-174G/C polymorphism in the IL-6 and IL-6-regulated protein expression, as well as the development of ED. METHODS: One hundred and twenty-one patients diagnosed with ED, 223 matched healthy controls, and 16 control patients with macular holes were recruited from the eastern Indian population. Serum and vitreous levels of IL-6 and vascular endothelial growth factors (VEGF) were measured by enzyme-linked immunosorbent assay. Serum levels of high-sensitivity C-reactive protein (hsCRP) were measured by enzyme immunoassay. Subjects were genotyped for the IL-6-174G/C polymorphism (rs1800795) by a custom TaqMan single-nucleotide polymorphism (SNP) Genotyping Assays system. RESULTS: Serum IL-6 (p<0.0001), hsCRP (p<0.0001), and VEGF (p=0.0031) levels were significantly higher in the inflammatory stage of ED than in healthy controls. Serum IL-6 also significantly correlated with hsCRP (Spearman's correlation coefficient; r=0.4992, p=0.0009), but not with VEGF in this stage in ED patients. At the proliferative stage of ED, significantly higher levels of vitreous IL-6 (p=<0.0001) and VEGF (p=<0.0001) were found compared with the vitreous of patients with macular holes. A significant correlation was observed between vitreous IL-6 and VEGF in ED patients (Spearman's correlation coefficient; r=0.5834, p=0.0087). A statistically significant association was found between the -174GG genotype (p=0.006) and occurrence of ED. Mean serum and vitreous concentrations of IL-6 were also higher in the subjects with the GG genotype than in those with the GC or CC genotype in this population. CONCLUSIONS: IL-6 expression, regulated by the allelic distribution of -174 loci and the enhanced level of IL-6, modulates CRP and VEGF concentration depending respectively on the acute inflammatory stimulation at the initial stage and angiogenic stimulation at the advanced stage of ED.

Association of interferon-gamma, interleukin-10, and tumor necrosis factor-alpha gene polymorphisms with occurrence and severity of Eales' disease.

PURPOSE: Eales' disease (ED) is an idiopathic retinal vasculitis characterized by capillary nonperfusion and neovascularization. Previous reports on ED demonstrated that T-cell-mediated immunoresponse and differential cytokine production in inflammatory and angiogenic stage seem to influence the extent and severity of this disease. Therefore, the purpose of this study is to investigate the influence of cytokine gene polymorphisms on occurrence and severity of ED. METHODS: One hundred twenty-one patients with ED were recruited from an Eastern Indian population and compared with 223 matched healthy control subjects. Genotyping of IFN-γ, IL-10, and TNF-α were performed by amplification refractory mutation system polymerase chain reaction (ARMS-PCR). RESULTS: A statistically significant association was found between the IL-10 -1082AA (P = 0.002), TNF-α -308AA (P = 0.0017) genotypes and the IL-10 ATA haplotype (P = 0.0123) and the occurrence of ED. In addition IL-10 -1082GG (P = 0.0005), TNF-α -308GG (P < 0.0001) genotype were found to be protective against disease occurrence. A synergistically low IL-10/high TNF-α genotype increased the risk of development (P < 0.0001) and the severity (P = 0.019) of ED. CONCLUSIONS: These data suggest that a low IL-10-expressing and high TNF-α-expressing genotype of the host can influence the occurrence and severity of outcome of ED.

Angiotensin-converting enzyme gene and endothelial nitric oxide synthase gene polymorphisms in Behçet's disease with or without ocular involvement.

PURPOSE: The association of known ACE gene and eNOS gene polymorphisms with BD in a group of Turkish patients with or without ocular involvement has been investigated. METHODS: The ACE and eNOS gene polymorphisms were investigated in 73 BD patients and 90 controls. RESULTS: The distrubition of "DD", "ID" and "II" genotypes of the ACE gene were 32 (43.8%), 29 (39.8%) and 12 (16.4%) for BD patients and 32 (35.5%), 35 (38.9%) and 23 (25.6%) for healthy controls. There was no significant difference between the groups (p = 0.140, OR 1.44, CI 0.90-2.30). When Behçet patients with ocular involvement were compared to the control group, statistical significance was found (p = 0.049, OR 2.18, CI 1.00-4.81). The "bb", "ba", and "aa" genotype frequencies of the eNOS gene were 48 (65.8%), 23 (31.5%), and 2 (2.7%) for patients with BD and 75 (83.3%), 15 (16.7%), and 0 (0%) for healthy controls, respectively. The significant difference found in allelic frequencies between the two groups (p = 0.011, OR 2.32, CI 1.11-4.87). When Behçet patients with ocular involvement were compared, sharper statistical significance was found (p = 0.001,OR 4.61,CI 1.85-11.52). DISCUSSION: The ACE gene polymorphism does not play a role in the pathogenesis of BD. The findings of the eNOS gene polymorphisms confirmed the significant association with BD and even more in patients with ocular involvement.

Ocular involvement in Adamantiades-Behçet's disease in Berlin, Germany.

BACKGROUND: Epidemiological studies demonstrate a prevalence of Adamantiades-Behçet's disease (ABD) in the range of 0.12-420 per 100,000 inhabitants, with the highest rates in Istanbul, Turkey, and the lowest rates in the USA. Ophthalmological data on the prevalence of ocular involvement are limited for ABD in Germany, because most epidemiological studies are based on rheumatological or dermatological data. Berlin is the city with the highest number of non-native German inhabitants, and its multi-ethnic character renders it uniquely appropriate for epidemiological studies on ABD. METHODS: We retrospectively analyzed all ABD patients seen in our department since 1982. All patients fulfilled the criteria of the International Study Group for Behçet's Disease. We found 140 patients (63 female, 77 male), with a mean follow-up of 6.4 years (0.5-22 years). RESULTS: The mean age was 23 at the first manifestation and 32 when the full-blown disease was noted. The mean age at onset of ocular involvement was 30. Most of the patients were of Turkish (n = 73) or German (n = 34) origin. Fifty-six percent developed ocular involvement, which was the first manifestation in 8.6% and the second manifestation in 19.3% of cases. CONCLUSIONS: More than half the patients developed ocular involvement. The calculated prevalence of ocular involvement in ABD is 1.77/100,000 inhabitants for the municipality of Berlin.

The association of the PTPN22 620W polymorphism with Behcet's disease.

OBJECTIVES: A single nucleotide polymorphism (SNP) of the gene encoding protein tyrosine phosphatase type 22 (PTPN22 620W) has recently been described as a strong common genetic risk factor for human autoimmune disease. We have analysed the association of PTPN22 620W in patients with Behçet's disease (BD). METHODS: Genomic DNA was obtained from 270 patients with BD from the UK and the Middle East. Normal controls (n = 203) were collected from the same populations. Patients with idiopathic retinal vasculitis from the UK (n = 136) were used as disease controls. PTPN22 620W was detected by SSP-PCR analysis and agarose gel electrophoresis. RESULTS: The results showed an inverse correlation between the presence of PTPN22 620W and Behçet's disease in either patient group tested. There was a greatly reduced prevalence in Middle Eastern compared to UK patients and controls. Finally, there was no association with either UK patients with retinal vasculitis compared with UK controls. CONCLUSIONS: The presence of PTPN22 620W was inversely associated with BD and the distribution of the SNP in the Middle East supports previous findings in the global prevalence.