Regulation of Keap1-Nrf2 axis in temporal lobe epilepsy-hippocampal sclerosis patients may limit the seizure outcomes.

BACKGROUND: Accumulation of reactive oxygen species (ROS) exacerbates neuronal loss during seizure-induced excitotoxicity. Keap1 (Kelch-like ECH-associated protein1)-nuclear factor erythroid 2-related factor 2 (Nrf2) axis is one of the known active antioxidant response mechanisms. Our study focused on finding the factors influencing Keap1-Nrf2 axis regulation in temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) patients. METHODS: Based on post-surgical follow-up data, patient samples (n = 26) were categorized into class 1 (completely seizure-free) and class 2 (only focal-aware seizures/auras), as suggested by International League Against Epilepsy (ILAE). For molecular analyses, double immunofluorescence assay and Western blot analysis were employed. RESULTS: A significant decrease in expression of Nrf2 (p < 0.005), HO-1; p < 0.02) and NADPH Quinone oxidoreductase1 (NQO1; p < 0.02) was observed in ILAE class 2. Keap1 (p < 0.02) and histone methyltransferases (HMTs) like SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) (p < 0.009) and enhancer of zeste homolog 2 (EZH2; p < 0.02) and methylated histones viz., H3K4me1 (p < 0.001), H3K9me3 (p < 0.001), and H3K27me3 (p < 0.001) was upregulated in ILAE class 2. Nrf2-interacting proteins viz., p21 (p < 0.001) and heat shock protein 90 (HSP90; p < 0.03) increased in class 1 compared to class 2 patients. CONCLUSION: Upregulation of HMTs and methylated histones can limit phase II antioxidant enzyme expression. Also, HSP90 and p21 that interfere with Keap1-Nrf2 interaction could contribute to a marginal increase in HO-1 and NQO1 expression despite histone methylation and Keap1. Based on our findings, we conclude that TLE-HS patients prone to seizure recurrence were found to have dysfunctional antioxidant response, in part, owing to Keap1-Nrf2 axis. The significance of Keap1-Nrf2 signaling mechanism in generation of phase II antioxidant response. Keap1-Nrf2 controls antioxidant response through regulation of phase II antioxidant enzymes like HO-1 (heme oxygenase-1), NQO1 (NADPH-Quinone Oxidoreductase1), and glutathione S-transferase (GST). Release of Nrf2 from negative regulation by Keap1 causes its translocation into nucleus, forming a complex with cAMP response-element binding protein (CBP) and small Maf proteins (sMaf). This complex subsequently binds antioxidant response element (ARE) and elicits and antioxidant response involving expression of phase II antioxidant enzymes. Reactive oxygen species (ROS) modify Cysteine 151 residue, p62 (sequsetosome-1), and interacts with Nrf2- binding site in Keap 1. p21 and HSP90 prevent Nrf2 interaction with Keap1. At transcriptional level, histone methyltransferases like EZH2 (enhancer of zeste homologue2), and SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) and corresponding histone targets viz., H3K27me3, H3K9me3, and H3K4me1 influence Nrf2 and Keap1 expression respectively.

Role of NAD+ and FAD in Ischemic Stroke Pathophysiology: An Epigenetic Nexus and Expanding Therapeutic Repertoire.

The redox coenzymes viz., oxidized ß-nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) by way of generation of optimal reducing power and cellular energy currency (ATP), control a staggering array of metabolic reactions. The prominent cellular contenders for NAD+ utilization, inter alia, are sirtuins (SIRTs) and poly(ADP-ribose) polymerase (PARP-1), which have been significantly implicated in ischemic stroke (IS) pathogenesis. NAD+ and FAD are also two crucial epigenetic enzyme-required metabolites mediating histone deacetylation and poly(ADP-ribosyl)ation through SIRTs and PARP-1 respectively, and demethylation through FAD-mediated lysine specific demethylase activity. These enzymes and post-translational modifications impinge on the components of neurovascular unit, primarily neurons, and elicit diverse functional upshots in an ischemic brain. These could be circumstantially linked with attendant cognitive deficits and behavioral outcomes in post-stroke epoch. Parsing out the contribution of NAD+/FAD-synthesizing and utilizing enzymes towards epigenetic remodeling in IS setting, together with their cognitive and behavioral associations, combined with possible therapeutic implications will form the crux of this review.

Impaired insulin exocytosis in chronic hepatitis C infection: contributory role of p38δ MAPK-protein kinase D-golgi complex axis.

Hepatitis C virus (HCV) infection and chronic hepatitis C (CHC) are associated with a measurable risk of insulin resistance (IR)/impaired glucose tolerance (IGT)/diabetes mellitus (DM). While loss of hepatic endocrine function contributes to liver cirrhosis in diabetic patients, onset and progression of IR/IGT to diabetes and exacerbation of incident hyperglycemia are ostensibly linked with chronic HCV infection. In this regard, the study by Chen J et al. appearing in Clinical Science (2020) (134(5) https://doi.org/10.1042/CS20190900) attempts to understand the mechanisms underlying the savaging effects of chronic HCV infection on insulin-producing pancreatic ß-cells and hence diabetic onset. The study investigated the role of mitogen-activated protein kinase (MAPK) p38δ-protein kinase D (PKD)-golgi complex axis in impacting insulin exocytosis. It was inferred that an insulin secretory defect of pancreatic ß-cells, owing to disrupted insulin exocytosis, to an extent explains ß-cell dysfunction in HCV-infected or CHC milieu. HCV infection negatively regulates first-phase and second-phase insulin secretion by impinging on PKD-dependent insulin secretory granule fission at trans-golgi network and insulin secretory vesicle membrane fusion events. This commentary highlights the study in question, that deciphered the contribution of p38δ MAPK-PKD-golgi complex axis to ß-cell dysfunction in CHC milieu. This pivotal axis proffers a formidable therapeutic opportunity for alleviation of double burden of glucose abnormalities/DM and CHC.

Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria.

Podocytes are specialized cells of the glomerulus and key component of the glomerular filtration apparatus (GFA). GFA regulates the permselectivity and ultrafiltration of blood. The mechanism by which the integrity of the GFA is compromised and manifest in proteinuria during ischemic stroke remains enigmatic. We investigated the mechanism of ischemic hypoxia-induced proteinuria in a middle cerebral artery occlusion (MCAO) model. Ischemic hypoxia resulted in the accumulation of HIF1α in the podocytes that resulted in the increased expression of ZEB2 (Zinc finger E-box-binding homeobox 2). ZEB2, in turn, induced TRPC6 (transient receptor potential cation channel, subfamily C, member 6), which has increased selectivity for calcium. Elevated expression of TRPC6 elicited increased calcium influx and aberrant activation of focal adhesion kinase (FAK) in podocytes. FAK activation resulted in the stress fibers reorganization and podocyte foot process effacement. Our study suggests overactive HIF1α/ZEB2 axis during ischemic-hypoxia raises intracellular calcium levels via TRPC6 and consequently altered podocyte structure and function thus contributes to proteinuria.

Roscovitine effectively enhances antitumor activity of temozolomide in vitro and in vivo mediated by increased autophagy and Caspase-3 dependent apoptosis.

Gliomas are incurable solid tumors with extremely high relapse rate and definite mortality. As gliomas readily acquire resistance to only approved drug, temozolomide (TMZ), there is increasing need to overcome drug resistance by novel therapeutics or by repurposing the existing therapy. In the current study, we investigated antitumor efficacy of roscovitine, a Cdk inhibitor, in combination with TMZ in vitro (U87, U373, LN 18 and C6 cell lines) and in vivo (orthotopic glioma model in Wistar rats) glioma models. We observed that TMZ treatment following a pre-treatment with RSV significantly enhanced chemo-sensitivity and suppressed the growth of glioma cells by reducing Cdk-5 activity and simultaneous induction of autophagy and Caspase-3 mediated apoptosis. Additionally, reduced expression of Ki67, GFAP and markers of angiogenesis (CD31, VEGF) was observed in case of TMZ + RSV treatments. Also, presence of reactive astrocytes in peri-tumoral areas and in areas around blood vessels was completely diminished in TMZ + RSV treated brain sections. Taken together, results in the current study provide evidence that RSV in conjunction with TMZ restricts glioma growth, reduces angiogenesis and also eliminates reactive astrocytes thereby preventing the spread of glioma to adjacent healthy brain tissues and thus might be more potent therapeutic option for glioma.

Association and gene-gene interaction analyses for polymorphic variants in CTLA-4 and FOXP3 genes: role in susceptibility to autoimmune thyroid disease.

PURPOSE: Polymorphic variants of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and forkhead box protein P3 (FOXP3) genes are implicated in dysregulated immune homeostasis and autoimmune disorders. We analyzed the association between CTLA-4 rs231775 and FOXP3 rs3761548, rs3761549 polymorphisms and predisposition to autoimmune thyroid disease (AITD), inclusive of Hashimoto's thyroiditis (HT) and Graves' disease (GD) in South-Indian population. METHODS: A total of 355 AITD subjects (comprising 275 HT and 80 GD) and 285 randomly selected age- and sex-matched control subjects were genotyped for the aforementioned polymorphisms by PCR-RFLP method. RESULTS: The rs231775 "G" allele was preponderant in HT and GD subjects when compared with controls and exerted a dominant influence on the susceptibility to HT (p = 0.009) and GD (p = 0.02), respectively. There was no allelic association of rs3761548 and rs3761549 polymorphisms with AITD susceptibility, albeit a significant difference in genotype distribution with respect to rs3761549. Haplotype analysis revealed an increased frequency of rs3761548 "C"-rs3761549 "T" in HT and GD subjects, thereby associating it with disease predisposition (p = 0.03). Epistatic interaction analysis by multifactor dimensionality reduction approach revealed redundancy between CTLA-4 and FOXP3 genes in influencing the susceptibility to AITD. CONCLUSIONS: The genetic variation in CTLA-4 gene with reference to rs231775 polymorphism contributes to an increased predisposition to HT and GD. Also, in conjunction with FOXP3 gene variants it seems to influence the susceptibility to HT and GD respectively. The significance of these findings in combination with antithyroid antibody screening could plausibly contribute towards meticulous case-finding for effective treatment of HT and GD.

Role of Nitric Oxide and Hydrogen Sulfide in Ischemic Stroke and the Emergent Epigenetic Underpinnings.

Nitric oxide (NO) and hydrogen sulfide (H2S) are the key gasotransmitters with an imperious role in the maintenance of cerebrovascular homeostasis. A decline in their levels contributes to endothelial dysfunction that portends ischemic stroke (IS) or cerebral ischemia/reperfusion (CI/R). Nevertheless, their exorbitant production during CI/R is associated with exacerbation of cerebrovascular injury in the post-stroke epoch. NO-producing nitric oxide synthases are implicated in IS pathology and their activity is regulated, inter alia, by various post-translational modifications and chromatin-based mechanisms. These account for heterogeneous alterations in NO production in a disease setting like IS. Interestingly, NO per se has been posited as an endogenous epigenetic modulator. Further, there is compelling evidence for an ingenious crosstalk between NO and H2S in effecting the canonical (direct) and non-canonical (off-target collateral) functions. In this regard, NO-mediated S-nitrosylation and H2S-mediated S-sulfhydration of specific reactive thiols in an expanding array of target proteins are the principal modalities mediating the all-pervasive influence of NO and H2S on cell fate in an ischemic brain. An integrated stress response subsuming unfolded protein response and autophagy to cellular stressors like endoplasmic reticulum stress, in part, is entrenched in such signaling modalities that substantiate the role of NO and H2S in priming the cells for stress response. The precis presented here provides a comprehension on the multifarious actions of NO and H2S and their epigenetic underpinnings, their crosstalk in maintenance of cerebrovascular homeostasis, and their "Janus bifrons" effect in IS milieu together with plausible therapeutic implications.

Interplay between mitochondrial metabolism and oxidative stress in ischemic stroke: An epigenetic connection.

The advent of epigenetics brought in a tectonic shift in the understanding of molecular basis of complex diseases like ischemic stroke (IS). Substantial scientific inquiry into the epigenetic basis of neurodegenerative diseases has bolstered the idea that altered carbon flux into central carbon metabolism and disturbed redox states govern the attendant transcriptional profiles through stochastic epigenetic changes. In view of an increasing understanding of the link between mitochondrial energy metabolism, oxidative stress and epigenetics in IS, the hitherto underappreciated 'neuroenergetics' is gaining sustained attention. Defined metabolic transitions during IS are necessarily a function of transiently altered abundance of critical metabolic substrates of Krebs cycle and other pathways viz., acetyl-CoA, citrate, 2-oxo-glutarate, succinate, fumarate, S-adenosyl methionine, ß-hydroxybutyrate and cofactors (NAD+, FAD, ATP, vitamin C) in neuronal mitochondria. These changes impinge on the cellular transcriptome by regulating the activity of several chromatin modifying enzymes that bring about epigenomic transition through alteration in DNA methylation and histone post translational modifications. This triggers downstream signaling cascades that circumstantially evoke adaptive and cell death responses during IS. Indeed, they also prevail on the functionality of neuronal network, brain plasticity and neurogenesis during post stroke recovery. Understanding the epigenetic underpinnings of IS that explicitly alter the brain transcriptomes could open new vistas of therapeutic opportunity. In the current review, we present an update on various aspects linking mitochondrial energy metabolism, oxidative stress and epigenetic modifications in the pathological setting of IS.

Poly(ADP-ribose)polymerase-1 hyperactivation in neurodegenerative diseases: The death knell tolls for neurons.

Neurodegeneration is a salient feature of chronic refractory brain disorders like Alzheimer's, Parkinson's, Huntington's, amyotropic lateral sclerosis and acute conditions like cerebral ischemia/reperfusion etc. The pathological protein aggregates, mitochondrial mutations or ischemic insults typifying these disease conditions collude with and intensify existing oxidative stress and attendant mitochondrial dysfunction. Interlocking these mechanisms is poly(ADP-ribose) polymerase (PARP-1) hyperactivation that invokes a distinct form of neuronal cell death viz., 'parthanatos'. PARP-1, a typical 'moonlighting protein' by virtue of its ability to poly(ADP-ribosyl)ate a plethora of cellular proteins exerts diverse functions that impinge significantly on cellular processes. In addition, its interactions with various nuclear proteins like transcription factors and chromatin modifiers elicit varied transcriptional outcomes that wield pathological cellular responses. Further, emerging leitmotifs like mitochondrial and nucleolar PARPs and the novel aspects of gene expression regulation by PARP-1 and poly(ADP-ribosyl)ation can provide a holistic view of PARP-1's influence on cell vitality. In this review, we discuss the pathological underpinnings of PARP-1, with a special emphasis on mitochondrial dysfunction and cell death subroutines, in the realm of neurodegeneration. This would provide a deeper insight into the functions of PARP-1 in neurodegenerative conditions that would enable the development of more effective therapeutic strategies.

Association Analysis of Polymorphisms in Genes Related to Oxidative Stress in South Indian Type 2 Diabetic Patients with Retinopathy.

BACKGROUND: Diabetic Retinopathy (DR) is one of the most common microvascular complications of type 2 diabetes mellitus (T2DM) and is polygenic with a multitude of genes contributing to disease susceptibility. The present study aimed at exploring the association between DR and seven polymorphisms in oxidative stress-related genes, i.e. ACE, eNOS, p22phox subunit of NAD(P)H oxidase, PARP-1 and XRCC1 in South Indian T2DM subjects. MATERIALS AND METHODS: The study included 149 T2DM subjects with DR (diagnosed through funduscopic examination) and 162 T2DM patients with no evidence of DR. The selected polymorphisms were genotyped by polymerase chain reaction (PCR) and Taqman allele discrimination assay. RESULTS: There was no significant difference in the genotype and allele distribution of ACE ins/del, eNOS-786T>C, 894G>T, 4a4b and p22phox 242C>T polymorphisms between T2DM groups with and without DR. Contrastingly, there appeared to be a significant association of PARP-1 Val762Ala and XRCC1 Arg399Gln polymorphisms with DR, wherein 762Ala allele seemed to confer significant protection against DR (p = 0.01; OR = 0.51 [0.3-0.86]), while the presence of 399Gln allele was associated with an enhanced risk for DR (p = 0.02; OR = 1.52 [1.07-2.15]). Multiple logistic regression analysis revealed a significant and independent association of Val762Ala and Arg399Gln polymorphisms and other putative risk factors with DR in T2DM individuals. CONCLUSIONS: The polymorphisms in the DNA repair genes PARP-1 and XRCC1 tended to associate significantly with DR. While Val762Ala polymorphism was associated with reduced susceptibility to DR, the Arg399Gln polymorphism contributed an elevated to risk for DR in South-Indian T2DM individuals.

Polymorphisms in oxidative stress pathway genes and risk of diabetic nephropathy in South Indian type 2 diabetic patients.

AIM: Diabetic nephropathy (DN), a common microvascular complication of type 2 diabetes mellitus (T2DM) is polygenic, with a vast array of genes contributing to disease susceptibility. Accordingly, we explored the association between DN and six polymorphisms in oxidative stress related genes, namely eNOS, p22phox subunit of NAD(P)H oxidase, PARP-1 and XRCC1 in South Indian T2DM subjects. METHODS: The study included 155 T2DM subjects with DN and 162 T2DM patients with no evidence of DN. The selected polymorphisms were genotyped by polymerase chain reaction and Taqman allele discrimination assay. RESULTS: No significant difference was observed in the genotype and allele distribution of eNOS -786T > C, intron 4a4b, p22phox 242C > T and XRCC1 Arg399Gln polymorphisms between T2DM groups with and without DN. Contrastingly, there appeared to be a significant association of eNOS 894G > T and PARP-1 Val762Ala polymorphisms with DN wherein, the presence of 894T allele was associated with an enhanced risk for DN [P = 0.005; OR = 1.78 (1.17-2.7)], while the 762Ala allele seemed to confer significant protection against DN [P = 0.02; OR = 0.59 (0.37-0.92)]. Multiple logistic regression analysis revealed a significant and independent association of eNOS 894G > T, PARP-1 Val762Ala polymorphisms and hypertension with DN in T2DM individuals. CONCLUSIONS: eNOS 894G > T and PARP-1 Val762Ala polymorphisms appeared to associate significantly with DN, with the former contributing to an enhanced risk and the latter to a reduced susceptibility to DN in South Indian T2DM individuals.

Arg399Gln polymorphism of X-ray repair cross-complementing group 1 gene is associated with angiographically documented coronary artery disease in South Indian type 2 diabetic patients.

AIMS: DNA damage resulting from oxidative stress contributes significantly to the development and progression of atherosclerosis in type 2 diabetic (T2DM) individuals, thereby implicating polymorphisms in DNA repair genes in the modulation of DNA repair efficiency. Based on this premise, we explored the association between X-ray repair cross-complementing group 1 (XRCC1) gene Arg399Gln polymorphism, coronary artery disease (CAD), and myocardial infarction (MI) in type 2 diabetic patients. We screened 283 T2DM patients, inclusive of 160 with angiographically defined CAD, 73 with MI, 89 without MI, and 121 T2DM individuals with no evidence of CAD for XRCC1 Arg399Gln polymorphism. RESULTS: There appeared to be a significant difference in the distribution of genotype and allele frequencies of XRCC1 Arg399Gln polymorphism between T2DM groups with and without CAD (p=0.03), albeit no significant association with MI was observed (p=0.055). A further analysis revealed that the frequencies of the Arg/Gln, Gln/Gln genotypes and 399Gln allele were considerably higher in patients with triple vessel disease (TVD) as compared with those with the single and double vessel disease (p=0.03), thereby associating this polymorphism with severity of CAD in T2DM individuals. Multiple logistic regression analysis revealed a significant and independent association of XRCC1 Arg399Gln polymorphism and other putative risk factors with CAD/TVD in T2DM individuals. CONCLUSIONS: These findings reveal a significant association between XRCC1 gene Arg399Gln polymorphism, CAD/TVD, and coincident putative risk factors in T2DM individuals in the South Indian population.

Association of the genetic variants of endothelial nitric oxide synthase gene with angiographically defined coronary artery disease and myocardial infarction in South Indian patients with type 2 diabetes mellitus.

INTRODUCTION: The polymorphic variants of endothelial nitric oxide synthase (eNOS) gene have been implicated in endothelial dysfunction and are highly relevant to macroangiopathies. We investigated the relationship between eNOS gene T-786C, G894T, intron 4a/b polymorphisms and coronary artery disease (CAD) in South Indian type 2 diabetic (T2DM) individuals. METHODS: We screened 283 T2DM patients, inclusive of 160 with angiographically defined CAD, 73 with myocardial infarction (MI), 89 without MI and 121 T2DM individuals with no evidence of CAD for eNOS gene polymorphisms. RESULTS: There appeared to be a significant difference in the genotype and allele distribution of eNOS T-786C polymorphism between T2DM groups with and without CAD (p=0.004), albeit no significant association with MI was observed. The frequencies of TC and CC genotypes and -786C allele were considerably higher in patients with triple vessel disease (TVD) as compared to those without CAD (p=0.003), thereby associating this polymorphism with severity of CAD. Genotype and allele distributions of G894T and intron 4a/b polymorphisms were not significantly different between T2DM subjects with and without CAD/MI. Significant linkage disequilibrium was observed between intron 4a/b and T-786C polymorphisms. Multiple logistic regression analysis revealed a significant and independent association of eNOS T-786C polymorphism and other putative risk factors with CAD/TVD in T2DM individuals. CONCLUSIONS: These findings reveal a significant association between eNOS T-786C polymorphism, CAD/TVD and coincident putative risk factors in T2DM individuals in South Indian population.

Relationship between NADPH oxidase p22phox C242T, PARP-1 Val762Ala polymorphisms, angiographically verified coronary artery disease and myocardial infarction in South Indian patients with type 2 diabetes mellitus.

INTRODUCTION: There has been compelling evidence for the role of oxidative stress in the pathogenesis of cardiovascular complications in type 2 diabetes mellitus (T2DM). We analyzed the association of C242T and Val762Ala polymorphisms of NADPH oxidase p22phox and poly (ADP-ribose) polymerase-1 (PARP-1) genes respectively with coronary artery disease (CAD) and its severity, myocardial infarction (MI) and cardiovascular risk factors in T2DM patients. MATERIALS AND METHODS: We screened 283 T2DM patients, inclusive of 160 with angiographically defined CAD, 73 with and 89 without MI and 121 T2DM individuals with no evidence of CAD for the two gene polymorphisms. RESULTS: The 242T and 762Ala alleles were significantly more frequent in T2DM subjects without CAD than those with CAD, thereby associating them with a significant protective effect against development of CAD [p=0.002 (C242T); 0.02 (Val762Ala)]. The association was further characterized by a relatively lower frequency of 242T and 762Ala alleles in T2DM patients with multi (MVD)/triple vessel disease respectively [p=0.003 (C242T); 0.02 (Val762Ala)]. Conversely, the genotype and allele frequencies of these polymorphisms were not significantly different in T2DM+CAD patients with or without MI. Stratification of risk by putative risk factors for CAD revealed a significant interaction with these polymorphisms. Multiple logistic regression analysis revealed a significant and independent association of C242T and Val762Ala polymorphisms and other putative risk factors with CAD/MVD in T2DM individuals. CONCLUSIONS: Our observations indicate a significant relationship between p22phox C242T and PARP-1 Val762Ala polymorphisms, CAD and its severity, but not with occurrence of MI in T2DM individuals with significant coronary stenoses.

Relationship between angiotensin-converting enzyme gene insertion/deletion polymorphism, angiographically defined coronary artery disease and myocardial infarction in patients with type 2 diabetes mellitus.

INTRODUCTION: The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been implicated in the pathogenesis of cardiovascular diseases. The objective of the present study was to investigate the influence of ACE gene I/D polymorphism on the development and progression of coronary artery disease (CAD) and myocardial infarction (MI) in type 2 diabetic (T2DM) patients. MATERIALS AND METHODS: We screened 283 T2DM patients, inclusive of 160 patients with angiographically defined CAD, 73 patients with MI, 89 patients without MI and 121 T2DM individuals with no evidence of CAD for ACE gene I /D polymorphism. RESULTS: There was no significant difference in the distribution of genotypes and alleles of ACE gene I/D polymorphism between T2DM+CAD and T2DM (non-CAD) groups. However, a significant association of this polymorphism with MI in T2DM+CAD patients (p=0.024) was observed. Further analysis revealed that the frequencies of the DD and ID genotypes increased with the number of stenosed coronary vessels (p=0.026). The DD genotype and the D allele were more frequent in the subgroup of T2DM patients with multivessel CAD (p=0.01) than in individuals with single vessel stenosis. CONCLUSIONS: These findings reveal a significant relationship between ACE gene I/D polymorphism, multivessel CAD and also the occurrence of MI in T2DM individuals with significant coronary stenoses in our population.