Polymorphic markers of the NO synthase genes and genetic predisposition to diabetic polyneuropathy in patients with type 1 diabetes mellitus

The allele and genotype frequencies of polymorphic markers of NOS1, NOS2 and NOS3 genes, encoding three types of NO synthases, were compared in type 1 diabetes patients with and without diabetic polyneuropathty. 180 type 1 diabetes patients (T1DM) of Russian or Eastern Slavonic origin, living in Moscow city, were divided into two groups using non-overlapping (polar) phenotypes. 86 patients had overt DPN and T1DM duration in this group was less than 5 years (DPN+ group) and 94 patients had no clinical DPN and T1DM duration was more than 10 years (DPN- group). We have not found the significant differences of allele and genotype frequencies of polymorphic markers (CA)n of NOS1 gene, (CCTTT)n of NOS2 gene, ecNOS4a/4b and Glu298Asp of NOS3 gene that indicates that all these markers are not associated with diabetic polyneuropathty. Only in the case of (CCTTT)n marker of NOS2 gene we have found a tendency for the association of 14 allele with DPN development. The carriers of this allele have the lower risk of DPN in T1DM.     

Survey of the allelic frequency of a NOS2A promoter microsatellite in human populations: assessment of the NOS2A gene and predisposition to infectious disease.

Allelic frequencies of a (CCTTT)(n) pentanucleotide repeat in the NOS2A promoter region were determined in a total of 1393 unrelated individuals from five specific population groups in four continents: Africa, Europe, Asia, and the Caribbean. There were highly significant differences in allele frequencies between the ethnically diverse populations. The repeat variation may have implications for the selective pressure of malaria or other infectious diseases that may operate at the NOS2 locus.     

Chemotactic cytokine receptor 5 gene polymorphism: relevance to microvascular complications in type 2 diabetes

We investigated the involvement of chemotactic cytokine receptor 5 (CCR5) gene polymorphism in microvascular complications of T2DM. All subjects were genotyped with the 59029 SNP in the CCR5 gene. The genotype/allele frequencies did not differ between T2DM patients and controls. Genotype distribution was compared in patients with and without complications (nephropathy, retinopathy and neuropathy). The frequency of A allele was significantly higher in patients with complications (OR for A allele 3.07, 95% CI 2.49-3.77). The A allele carriage was associated with diabetic nephropathy (OR 6.17, 95% CI 3.28-11.6). An association was observed between 59029 polymorphism and age at T2DM onset. The A allele was more frequent in early onset than in late onset patients. For AA homozygotes OR was 2.38 (1.19-4.76) and 2.26 (1.12-4.58) in complicated and uncomplicated subgroups, respectively. These results suggest that CCR5 gene polymorphism is associated with diabetic nephropathy in T2DM.     

Polymorphism of gene encoding vascular angiotensin II receptor and microangiopathies in patients with insulin-dependent diabetes mellitus

Polymorphism A1166C of the AT1R gene encoding angiotensin vascular receptor [replacement of C (cytosine) for A (adenine)) at position 1166] was compared in patients with insulin-dependent diabetes mellitus (IDDM) complicated by diabetic nephropathy (DN) and in noncomplicated patients (n = 27 and n = 41, respectively) and also in patients with IDDM complicated by diabetic retinopathy (DR) and in correspondent noncomplicated individuals (n = 30 and n = 44, respectively). The frequency of AT1R gene alleles and genotypes in patients with IDDM complicated by DN did not differ significantly from that observed in patients with noncomplicated IDDM. In contrast, in patients with IDDM complicated by retinopathy, a significant decrease in the content of A allele (68.3% against 82.6%) and a significant increase in the content of C allele (31.7% against 17.4%) was found as compared with the control group. Thus, in the Moscow population, A1166C polymorphism of the AT1R gene is not associated with diabetic renal complications but indeed associated with diabetic retinal complications. C allele is a risk factor of DR (the relative risk, RR, is equal to 2.17), and A allele is, in contrast, a protective factor against early retinopathy development (RR is equal to 0.49).     

Lack of association between an ecNOS gene polymorphism and diabetic nephropathy in type 2 diabetic patients with proliferative diabetic retinopathy.

The development of diabetic nephropathy shows remarkable variation among individuals. Therefore, not only hyperglycemia but also genetic factors may contribute to the development of diabetic nephropathy. The aim of the present study was to examine the contribution of the 27-bp repeat polymorphism in intron 4 of the endothelial constitutive nitric oxide synthase gene (ecNOS4) to the development of diabetic nephropathy. For this purpose, we analyzed this polymorphism in 167 Japanese type 2 diabetic patients with proliferative diabetic retinopathy consisting of 102 patients with diabetic nephropathy (with macroalbuminuria) and 65 patients without diabetic nephropathy (with normoalbuminuria). The genotype and allele frequencies were not significantly different between patients with diabetic nephropathy and those without diabetic nephropathy (ecNOS4 "b/b" 79.4% vs. 84.6%, ecNOS4 "b/a" 20.6% vs. 15.4%, "b" allele 89.7% vs. 92.3%, "a" allele 10.3% vs. 7.7%). We conclude that the ecNOS4 polymorphism does not contribute to the development of diabetic nephropathy.     

Induction of MRP1 and gamma-glutamylcysteine synthetase gene expression by interleukin 1beta is mediated by nitric oxide-related signalings in human colorectal cancer cells

Treatment of human colorectal cancer cells HT29 with interleukin 1beta (IL-1beta) induces expression of the multidrug resistance protein (MRP1) gene encoding the ATP-dependent glutathione S-conjugate export (GS-X) pump and the gamma-glutamylcysteine synthetase (gamma-GCSh) gene encoding heavy (catalytic) subunit of gamma-glutamylcysteine synthetase, the rate-limiting enzyme for the biosynthesis of glutathione (GSH). The induction can be suppressed by N(G)-methyl-L-arginine, a specific inhibitor of nitric oxide synthase (NOS). These results suggest that IL-1beta-mediated MRP1 and gamma-GCSh induction involve nitric oxide (NO) -related signaling. Further supports to the involvement of NO in the induction of MRP1 and gamma-GCSh expression are made by the following observations. (i) Expression of MRP1 and gamma-GCSh genes were induced by treating the cells with NO donors, i.e., S-nitro-N-acetyl-D,L-penicillamide (SNAP) and S-nitroso-L-glutathione, in a concentration-dependent manner. (ii) Ectopic expression of inducible NOS (iNOS) activity by transfecting expressible recombinant iNOS cDNA encoding functional iNOS but not the nonfunctional version resulted in elevated expression of MRP1 and gamma-GCSh. We also demonstrated that HT-29 cells treated with either 1L-1beta or SNAP induced ceramide production, and addition of C2 or C6 ceramides into cultured HT-29 cells resulted in induction of gamma-GCSh but not MRP1 expression. Collectively, our results demonstrate that induction of MRP1 and gamma-GCSh by IL-1beta is regulated, at least in part, by an NO-related signaling, and induction of gamma-GCSh is by NO-related ceramide signaling.     

Effect of cyclosporin-A on the blood--retinal barrier permeability in streptozotocin-induced diabetes

BACKGROUND: Our previous results showed that in retinas from streptozotocin (STZ)-induced diabetic rats there is an increased level of interleukin-1beta (IL-1beta). This cytokine may be involved in the expression of the inducible isoform of the nitric oxide synthase (iNOS), with consequent synthesis of large amounts of NO and blood-retinal barrier (BRB) breakdown. AIMS: The aim of this work was to examine whether the administration of cyclosporin-A (Cs-A) to STZ-induced diabetic rats inhibits the synthesis of IL-1beta and the expression of the inducible proteins, iNOS and cyclo-oxygenase-2 (COX-2) in retinal cells, and whether the activity of these proteins contribute to BRB breakdown. METHODS: The level of IL-1beta was evaluated by ELISA and the NO production by L-[3H]-citrulline formation. Expression of iNOS and COX-2 proteins was determined by two methods, western blot and immunohistochemistry. The permeability of the BRB was assessed by quantification of the vitreous protein. RESULTS AND DISCUSSION: Our results indicated that the levels of IL-1beta and NO in retinas from Cs-A-treated diabetic rats are significantly reduced, as compared to that in non-treated diabetic rats. The treatment of diabetic rats with Cs-A also significantly inhibited the expression of the inducible proteins, iNOS and COX-2. The evaluation of the vitreous protein content revealed that Cs-A also reduces the BRB permeability. Taken together, these results suggest that the increased production of the inflammatory mediators, IL-1beta and NO, in diabetes may affect the BRB permeability and therefore contribute to the development of diabetic retinopathy.     

Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats

A nonselective inhibitor of cyclooxygenase (COX; high-dose aspirin) and a relatively selective inhibitor of inducible nitric oxide synthase (iNOS; aminoguanidine) have been found to inhibit development of diabetic retinopathy in animals, raising a possibility that NOS and COX play important roles in the development of retinopathy. In this study, the effects of hyperglycemia on retinal nitric oxide (NO) production and the COX-2 pathway, and the interrelationship of the NOS and COX-2 pathways in retina and retinal cells, were investigated using a general inhibitor of NOS [N(G)-nitro-l-arginine methyl ester (l-NAME)], specific inhibitors of iNOS [l-N(6)-(1-iminoethyl)lysine (l-NIL)] and COX-2 (NS-398), and aspirin and aminoguanidine. In vitro studies used a transformed retinal Müller (glial) cell line (rMC-1) and primary bovine retinal endothelial cells (BREC) incubated in 5 and 25 mM glucose with and without these inhibitors, and in vivo studies utilized retinas from experimentally diabetic rats (2 mo) treated or without aminoguanidine or aspirin. Retinal rMC-1 cells cultured in high glucose increased production of NO and prostaglandin E(2) (PGE(2)) and expression of iNOS and COX-2. Inhibition of NO production with l-NAME or l-NIL inhibited all of these abnormalities, as did aminoguanidine and aspirin. In contrast, inhibition of COX-2 with NS-398 blocked PGE(2) production but had no effect on NO or iNOS. In BREC, elevated glucose increased NO and PGE(2) significantly, whereas expression of iNOS and COX-2 was unchanged. Viability of rMC-1 cells or BREC in 25 mM glucose was significantly less than at 5 mM glucose, and this cell death was inhibited by l-NAME or NS-398 in both cell types and also by l-NIL in rMC-1 cells. Retinal homogenates from diabetic animals produced significantly greater than normal amounts of NO and PGE(2) and of iNOS and COX-2. Oral aminoguanidine and aspirin significantly inhibited all of these increases. The in vitro results suggest that the hyperglycemia-induced increase in NO in retinal Müller cells and endothelial cells increases production of cytotoxic prostaglandins via COX-2. iNOS seems to account for the increased production of NO in Müller cells but not in endothelial cells. We postulate that NOS and COX-2 act together to contribute to retinal cell death in diabetes and to the development of diabetic retinopathy and that inhibition of retinopathy by aminoguanidine or aspirin is due at least in part to inhibition of this NO/COX-2 axis.     

Cyclic AMP response element mediates dexamethasone induced suppression of prostaglandin H synthase-2 gene expression in human amnion derived WISH cells.

A human PGHS-2 promoter fragment (300 BP) linked to the luciferase reporter was used to study the regulation of PGHS-2 gene expression in human amnion-derived WISH cells. A cyclic AMP (cAMP) response element (CRE) was found to be important in the induction of PGHS-2 gene expression. This was demonstrated by showing that coexpression of CREB stimulated native but not CRE mutant promoter and that IL-1beta and PMA induced less activity with the mutant promoter as compared to the native promoter. The effect of dexamethasone on IL-1beta and PMA induced promoter activities was further examined. IL-1beta or PMA induced activity was blocked by dexamethasone, whereas IL-1beta or PMA induced mutant activity was not responsive to dexamethasone. Direct activation of CRE by a cAMP elevating agent, isoproterenol, was found to be inhibited significantly dexamethasone. These results suggest that CRE may mediate the induction of PGHS-2 by IL-1beta and PMA as well as the suppression of expression by dexamethasone in amnion-derived cells.     

The mitogen-activated protein kinase p38 is necesssary for interleukin 1beta-induced monocyte chemoattractant protein 1 expression by human mesangial cells

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1beta (IL-1beta)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1beta-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1beta treatment. p38 kinase immunoprecipitated from IL-1beta-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1beta-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-kappaB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1beta induction of NF-kappaB was measured. SB 203580 (up to 25 microM) had no effect on IL-1beta-induced NF-kappaB nuclear translocation or DNA binding activity. Our previous work showed that IL-1beta also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1beta-induced MCP-1 mRNA or protein levels, or on IL-1beta activation of NF-kappaB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1beta, but is not involved at the level of cytoplasmic activation of NF-kappaB. In contrast, ERK2 does not mediate IL-1beta induced MCP-1 gene expression.     

A new pentanucleotide STR-marker, located in the intron of the ING1 tumor suppressor gene and its allelic polymorphism

DNA samples of unrelated subjects from the Volga-Ural region of Russia were examined to study allele polymorphism of the pentanucleotide repeat (TTGTG)8 localized to an intron of the tumor suppressor gene ING1. STR marker was registered in the EMBL database with the accession number AJ277387. In a sample of 119 individuals, three pentanucleotide alleles consisting of seven, eight, and nine repeated monomers were revealed. The allele frequencies were 0.24, 0.74, and 0.02, respectively. Heterozygosity was 0.45. On the basis of these data, the repeat can be regarded as a polymorphic STR marker for the ING1 gene and used in population and clinical studies.     

Interleukin-1beta causes different levels of nitric oxide-mediated depression of contractility in different positions of rat thoracic aorta.

Interleukin-1beta (IL-1beta) can be synthesized by macrophages, endothelial cells and vascular smooth muscle cells when stimulated by bacterial lipopolysaccharide (endotoxin) during septic shock. The IL-1beta levels in the blood vessel wall are also elevated in atherosclerosis. IL-1beta can cause induction of inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cells and produce vasorelaxation, hypotension and ultimately tissue damage. We studied the depressions of vascular smooth muscle contractions at 3 hours after exposure to IL-1beta in different positions of rat thoracic aorta. The data show that the aortic rings from the cranial end of rat thoracic aorta had little response to IL-1beta (0.5 and 1.0 ng/ml) while those from the caudal end of thoracic aorta had larger depressant response. S-methylisothiourea sulfate (SMT), an iNOS inhibitor, completely blocked the depression of contraction caused by IL-1beta in intact aortic rings. If the endothelium was removed from the aortic rings before exposure to IL-1beta, all rings from different parts of the thoracic aorta showed an equal amount of vasodepression. Thus, the difference in the depressant response of IL-1beta in different portions of thoracic aorta is endothelium-dependent and involves induction of NOS.     

Lack of association between Gly82Ser, 1704G/T and 2184A/G of RAGE gene polymorphisms and retinopathy susceptibility in Malaysian diabetic patients

Diabetic retinopathy is the most common diabetic eye disease, occurring in about 60% of type 2 diabetic patients. Other than known clinical risk factors, the influence of genes has been suggested as part of the development of diabetic retinopathy. We investigated the association of Gly82Ser, 1704G/T and 2184A/G polymorphisms in the RAGE gene with retinopathy in type 2 diabetic patients in Malaysia. Ninety-eight unrelated retinopathy patients and 185 unrelated healthy controls from all over Malaysia were recruited in this study. The allele and genotype frequencies of the three gene polymorphisms were investigated using PCR-RFLP. The allele frequency of the three polymorphisms did not differ significantly between the control and the retinopathy group (P > 0.05). Analysis of the frequency of GA+AA, GT+TT and AG+GG in the retinopathy group did not reveal significant differences (P > 0.05) compared to the control group. We conclude that RAGE gene Gly82Ser, 1704G/T and 2184A/G polymorphisms are not associated with retinopathy development in the Malaysian population.     

Association between retinoid-X receptor-gamma genetic polymorphisms and diabetic retinopathy

Retinoid-X receptor (RXR) is one of the members of the nuclear hormone receptor superfamily. It forms heterodimers with many nuclear receptors, such as the peroxisome proliferative-activated receptor, which has been proposed to be involved in diabetic complications, including retinopathy. A recent study revealed that RXR-alpha has antioxidant properties and is associated with diabetic retinopathy. We found that the RXR-gamma gene is involved in the pathogenesis of diabetic nephropathy. We also hypothesized that the RXR-gamma gene has a role in the development of diabetic retinopathy. We examined 213 diabetic patients, who were divided into retinopathy or no retinopathy groups. Nine selected single nucleotide polymorphisms (SNPs) in the RXR-gamma gene were evaluated. The diabetic retinopathy group had longer diabetes duration, higher body mass indexes, and higher systolic blood pressure, as well as higher concentrations of fasting plasma glucose, blood urine nitrogen, and creatine. One SNP--rs3818569 of the RXR-gamma gene was found to be associated with increased risk for diabetic retinopathy in both allele and genotype frequencies (P = 0.0023 and 0.0057, respectively). Analysis with multivariate logistic regression revealed that the dominant RXR-gamma GG genotype is a risk factors for the development of diabetic retinopathy (odds ratio = 2.388; 95% confidence interval = 1.17-4.875). We conclude that the RXR-gamma rs3818569 SNP is associated with diabetic retinopathy development in the Taiwanese population.     

MyD88-Dependent Pathways in Leukocytes Affect the Retina in Diabetes

Background

Previous studies by us and other have provided evidence that leukocytes play a critical role in the development of diabetic retinopathy, suggesting a possible role of the innate immune system in development of the retinopathy. Since MyD88 is a convergence point for signaling pathways of the innate immune system (including Toll-Like Receptors (TLRs) and interleukin-1ß (IL-1ß)), the purpose of this study was to assess the role of MyD88 and its dependent pathways on abnormalities that develop in retina and white blood cells related to diabetic retinopathy.

Methods

C57BL/6J mice were made diabetic with streptozotocin. Chimeric mice were generated in which MyD88-dependent pathways were deleted from bone marrow-derived only. Mice were sacrificed at 2 mos of diabetes for assessment of, leukostasis, albumin accumulation in neural retina, leukocyte-mediated killing of retinal endothelial cells, and cytokine/chemokine generation by retinas of diabetic mice in response to TLR agonists,

Results

IL-6 and CXCL1 were generated in retinas from diabetic (but not nondiabetic mice) following incubation with Pam3CysK/TLR2, but incubation with other TLR ligands or IL-1ß did not induce cytokine production in retinas from nondiabetic or diabetic mice. Diabetes-induced abnormalities (leukostasis, ICAM-1 expression on the luminal surface of the vascular endothelium, retinal superoxide generation) were significantly inhibited by removing either MyD88 or the signaling pathways regulated by it (TLRs 2 and 4, and IL-1ß) from bone marrow-derived cells only. Leukocyte-mediated killing of endothelial cells tended to be decreased in the marrow-derived cells lacking TLR2/4, but the killing was significantly exacerbated if the marrow cells lacked MyD88 or the receptor for IL-1ß (IL-1ßr).

Conclusions

MyD88-dependent pathways play an important role in the development of diabetes-induced inflammation in the retina, and inhibition of MyD88 might be a novel target to inhibit early abnormalities of diabetic retinopathy and other complications of diabetes.     

Allele Polymorphism of a New Pentanucleotide STR Marker Located to an Intron of the Tumor Suppressor Gene ING1

Using polymerase chain reaction (PCR) DNA samples of unrelated subjects from the Volga-Ural region of Russia were examined to study allele polymorphism of the pentanucleotide repeat (TTGTG) _g localized to an intron of the tumor suppressor gene ING1. STR marker was registered in the EMBL database with the accession number AJ277387. In a sample of 119 individuals, three pentanucleotide alleles consisting of seven, eight, and nine repeated monomers were revealed. The allele frequencies were 0.24, 0.74, and 0.02, respectively. Heterozygosity was 0.45. On the basis of these data, the repeat can be regarded as a polymorphic STR marker for the ING1 gene and used in population and clinical studies.     

Association between interleukin-6 polymorphism and age-at-onset of type 1 diabetes. Epistatic influences of the tumor necrosis factor-alpha and interleukin-1beta polymorphisms

Multiple immune mediators have been mentioned as playing a role in the pathomechanism of type1 DM. Interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha play a central role in the autoimmune destruction of pancreatic beta-cells, whereas IL-6 inhibits TNF-alpha secretion, and may have some protecting effects. In our study, we aimed to investigate the association between these three cytokines' single nucleotide polymorphisms (IL-6 gene G(-174)C, TNF-alpha gene G(-308)A and IL-1beta gene C(3954)T polymorphisms) and age-at-onset of type 1 diabetes mellitus (T1DM) in 165 diabetic children (median age: 17 years). Polymorphisms were determined using the PCR-RFLP method. We found that the age-at-onset of T1DM was significantly different in patients with a different IL-6 genotype (median age-at-onset of T1DM was: 8, 6 and 4.5 years in children with the (-174)GG, GC and CC genotypes, respectively; p < 0.01). Adjusted for TNF-alpha and IL-1beta polymorphisms, patients with a IL-6 (-174)CC genotype have a 3.0-fold (95% CI: 1.2-7.1) increased risk of developing diabetes before the age of 6 years than (-174)G allele carrier patients. However, we found this association to be present only in patients who carried the TNF-alpha (-308)A or IL-1beta (3954)T allele, i.e. in patients with high TNF-alpha and high IL-1beta producer genotypes. We suppose that in the case of high TNF-alpha and IL-1beta producer genotypes, elevated proinflammatory cytokine levels result in a higher production of IL-6 in (-174)G allele carrier patients. This elevated IL-6 level may have a protective effect against the development of T1DM and may delay the destruction of pancreatic beta-cells.