macro- and microvascular complications are common in long - standing type 2 diabetes ( t2d ) and diminish the quality of life and life - time expectancy of patients [ 1 , 2 ] . in addition to genetic and lifestyle factors , poor glycemic control , arterial hypertension , and/or hyperlipidemia are predisposing factors for the development of these complications [ 3 , 4 ] . high blood glucose levels and dyslipidemia may stimulate the production of reactive oxygen species ( ros ) , leading to increased oxidative stress and chronic inflammation that appears to be the major driver of molecular processes leading to late diabetic complications [ 58 ] . recent studies suggested the involvement of inflammatory pathways that may be triggered by inflammasome , a crucial complex that senses and is activated by metabolic damage signals [ 9 , 10 ] . inflammasomes are a large family of multiprotein complexes that can be activated by pathogen - associated or damage - associated molecular patterns ( pamps or damps , resp . ) , among others also such as ros , cholesterol crystals , and possibly high glucose levels . nlrp3 inflammasome is composed of the nlrp3 scaffold protein , card containing adaptor protein , and caspase-1 . activation of inflammasome leads to assembly of the multiprotein complex that cleaves and activates caspase-1 , resulting in cleavage of pro - interleukin-1 ( pro - il-1 ) and release of il-1 that triggers downstream inflammatory response [ 9 , 10 ] . nlrp3 inflammasome has been suggested as a link between insulin resistance , obesity , circulating immune markers , immunogenetic susceptibility , macrophage function , and chronic inflammation . genetic variations leading to the altered production or function of inflammasome and inflammatory cytokines were linked to various inflammatory diseases , including obesity , insulin resistance , and t2d [ 13 , 14 ] . two inflammasome related single nucleotide polymorphisms ( snps ) nlrp3 rs35829419 ( p.gln705lys ) is a gain - of - function polymorphism associated with increased production of il-1 . on the other hand , card8 rs2043211 ( p.cys10ter ) results in nonfunctional protein and leads to loss of card-8 inhibition of caspase-1 . another common snp associated with mrna stability , nlrp3 rs10754558 , was linked to insulin resistance and increased risk for t2d in chinese han population . the aim of our study was to investigate if nlrp3 and card8 polymorphisms play a role in the risk for macro- and microvascular complications in t2d . we performed a retrospective study that included t2d patients aged between 18 and 75 years . all the patients were treated and followed up at the outpatient clinic at the general hospital trbovlje , slovenia . patients with diabetes type 1 , gestational diabetes , other types of diabetes , active cancer , addiction to alcohol or illegal drugs , and dementia or severe psychiatric disorders were not eligible for the study . patients were included as they were coming for their regular follow - up visits and were receiving treatment according to the established clinical guidelines . blood pressure , body height , and body weight were measured at every follow - up visit and the body mass index ( bmi ) was calculated . information on the history of diabetes , presence of arterial hypertension , hyperlipidemia and chronic macrovascular and microvascular diabetic complications , smoking status , and other medications was obtained from the medical records and from the interview at the inclusion in the study . plasma lipid levels , urea and creatinine , and urine albumin and albumin / creatinine ratio were determined at least once per year . all patients were referred to consulting ophthalmologist for screening for diabetic retinopathy at least once a year . echosonography and exercise stress test ( cycloergometry ) were performed at the first visit and also at any complaints suggestive for ischemic heart disease . the study was approved by the national ethics committee and conducted in accordance with the declaration of helsinki . written informed consent was obtained from all subjects . all the laboratory parameters were measured using standard laboratory procedures at the biochemistry laboratory of the general hospital trbovlje , slovenia . genomic dna was isolated from peripheral blood leukocytes using qiagen flexigene kit ( qiagen , hilden , germany ) . dna extraction and genotyping analyses were performed at the pharmacogenetics laboratory , institute of biochemistry , faculty of medicine , university of ljubljana . genotyping of nlrp3 rs35829419 ( c.2113c > a , p.gln705lys ) and card8 rs2043211 ( c.304a > t , p.phe102ile , p.cys10ter ) was carried out using a fluorescence - based competitive allele - specific ( kaspar ) assay according to the manufacturer 's instructions ( kbiosciences , herts , uk ) . genotyping was performed blindly to any clinical data and was randomly repeated in 20% of samples to check for genotyping reliability . median and interquartile range were used to describe central tendency and variability of continuous variables , while frequencies were used to describe the distribution of categorical variables . standard chi square test or mann - whitney test was used to compare clinical characteristics between different patient groups . all statistical analyses were performed using ibm spss statistics 19.0 ( ibm corporation , armonk , ny , usa ) . dominant genetic model was used in all analyses and the level of statistical significance was set at 0.05 . in total 181 patients ( 105 males and 76 females ) with t2d were included in the study . the median ( 2575% range ) age of the patients was 64 ( 58.570.5 ) years and duration of t2d was 11 ( 617 ) years . on the prescribed treatment regimen most of the patients achieved relatively good blood glucose control with the average hba1c 6.9% ( 6.37.6 ) , well controlled blood pressure , and plasma lipid levels ( table 1 ) . in total 46 ( 25.4% ) patients suffered from macrovascular complications : 10 ( 5.5% ) had peripheral arterial occlusive disease ( paod ) , 16 ( 8.85% ) ischemic cerebrovascular disease ( icd ) , and 32 ( 17.7% ) myocardial infarction ( mi ) . only 34 ( 18.8% ) patients had microvascular complications : 25 ( 13.8% ) suffered from end - stage kidney failure due to diabetic nephropathy , 13 ( 7.2% ) from neuropathy , and 15 ( 8.3% ) from retinopathy . duration of t2d and plasma cholesterol levels were the most important clinical risk factors for macrovascular complications in logistic regression analysis ( or = 1.06 ; 95% ci = 1.021.11 ; p = 0.007 and or = 0.67 ; 95% ci = 0.460.96 ; p = 0.031 , resp . ) . the most important clinical risk factor for microvascular complications was duration of t2d ( or = 1.12 ; 95% ci = 1.071.17 ; p < 0.001 ) . all the patients were genotyped for card8 rs2043211 and nlrp3 rs35829419 polymorphisms ; however , card8 genotype data could not be obtained for one patient . in total 89 ( 49.4% ) patients were homozygous for the wild type card8 aa allele , 70 ( 38.9% ) carried one , and 21 ( 11.7% ) carried two polymorphic t alleles . nlrp3 polymorphism was less common : 160 ( 88.4% ) were homozygous for the wild type cc genotype , while 21 ( 11.6% ) patients carried one of the polymorphic a alleles : 20 ( 11.0% ) carried one and 1 ( 0.6% ) carried two a alleles . all genotype frequencies were in agreement with hwe ( p = 0.213 and 0.666 for card8 and nlrp3 , resp . ) . we observed an association between nlrp3 rs35829419 polymorphism and the risk for macrovascular complications ( or = 3.93 ; 95% ci = 1.5410.0 ; p = 0.004 ) ( table 2 ) . this association remained significant after adjustment for duration of t2d and plasma cholesterol levels ( or = 4.36 ; 95% ci = 1.5812.01 ; p = 0.004 ) . although carriers of one or two polymorphic nlrp3 rs35829419 alleles had higher risk for mi , paod , and icd , these associations were statistically nonsignificant ( p = 0.052 , p = 0.078 , and p = 0.092 , resp . , table 2 ) . when stratified by sex , a significant association of nlrp3 rs35829419 with macrovascular complications was observed only in males ( or = 4.44 ; 95% ci = 1.3914.25 ; p = 0.012 ) ( table 2 ) . additionally , significant association with paod was observed among males ( or = 5.93 ; 95% ci = 1.1730.06 ; p = 0.032 ) . this association could not be tested in females as only 3 female patients had paod and all were nlrp3 cc homozygous . no association was observed between the investigated card8 rs2043211 and the risk for macrovascular complications in univariable ( or = 0.97 ; 95% ci = 0.501.90 ; p = 0.930 ) or multivariable analysis ( or = 1.02 ; 95% ci = 0.502.08 ; p = 0.951 ) . we also observed no associations of card8 rs2043211 with individual macrovascular complications ( data not shown ) . the presence of individual microvascular complications was also investigated , but no association was observed between card8 or nlrp3 polymorphisms and these complications ( table 2 ) . however , male carriers of at least one polymorphic nlrp3 rs35829419 allele tended to have more microvascular complications ( p = 0.050 ) and had increased risk for end - stage kidney failure ( p = 0.030 , table 2 ) . to our knowledge , this is the first study that investigated the influence of genetic variability in inflammasome on t2d long - term complications . we observed that polymorphic nlrp3 rs35829419 a allele is associated with increased risk for development of any macrovascular complications in long - term t2d patients . despite the small numbers of individuals with a particular complication , nlrp3 polymorphism showed statistically nonsignificant association with mi , paod , and icd . these preliminary data are concordant with previous reports on association of nlrp3 and card8 polymorphism with the proinflammatory phenotype . in a large cohort of healthy blood donors higher il-1 levels were observed in carriers of polymorphic alleles of these genes than in noncarriers . our results are also in agreement with reports on potential role of nlrp3 inflammasome in mi . recent genome - wide association study   as well as a large cardiovascular candidate gene study   observed association of nlrp3 locus with higher fibrinogen levels that may lead to higher atherothrombotic risk . inflammasome proteins were indeed shown to be expressed not only in neutrophils and macrophages , but also in vascular endothelia and cardiac fibroblasts . knockout mice showed declined inflammatory responses and were more protected against myocardial damage after mi . contrary to our findings , a large first - ever myocardial infarction study in ac - county ( fia ) showed no association between nlrp3 rs35829419 and mi . however , they observed a gender - specific effect of nlrp3 rs35829419 : polymorphic a allele conferred protection from development of mi in females but was associated with increased crp levels as a marker of inflammation in males . in our study group , the risk was highest for paod , while a tendency of increased risk of mi was also observed . it was shown that nlrp3 may play a role in vascular lipid deposition and atherosclerosis . we observed no associations between any complications and nlrp3 polymorphisms among females ; however , in our study group fewer females than males presented with complications . the discrepancy between our results and the fia study may be due to the differences in the study design between the two studies : while fia was a large population based study with cases and controls carefully matched for age and gender , our study included patients with t2d as they were coming for their regular follow - up visits and patients with and without complications were not selected for age or gender . as the number of patients with complications was limited , any protective effect of nlrp3 rs35829419 polymorphism in females may have been missed in our study due to the small numbers . we also have to note that both studies recruited patients from a small geographical area and other factors contributing to mi , such as lifestyle and physical activity , may differ between the two countries . the authors of the fia study acknowledge that differences in lifestyle factors , including type of food intake and physical activity , between males and females of northern sweden may contribute to the gender - specific genetic association and mi observed in their study . in our study males also showed some tendency for increased risk of microvascular complications among polymorphic nlrp3 rs35829419 a allele carriers , in particular in end - stage kidney failure . inflammasome was also implicated in inflammatory , autoimmune , and obstructive kidney disease and in ischemia - reperfusion type kidney injury , mostly in rat or mouse models , but the role of nlrp3 inflammasome in diabetic nephropathy remains to be elucidated . serum il-18 levels were elevated in patients with diabetic nephropathy , but not in other diabetic complications . we observed no association between card8 rs2043211 and any long - term t2d complications . to our knowledge so far , card8 polymorphism was associated with psoriasis   and rheumatoid arthritis susceptibility in some , but not all , studies , but was not associated with juvenile idiopathic arthritis . recently , card8 rs2043211 t allele was found to be significantly associated with a protective effect of ischemic stroke , but not with coronary artery disease . due to its exploratory nature the present study the study was retrospective in nature and limited to a single regional diabetic outpatient clinic ; therefore the number of patients with a particular long - term t2d complication was relatively small . nevertheless all the patients were clinically well characterized and were followed up by the same diabetologist ; therefore , the discrepancies in clinical data collection procedures were minimalized . furthermore all the patients were recruited in a small geographic area with ethnically homogeneous population ; therefore , there was no bias due to genetic heterogeneity . as diabetic complications are multifactorial , we carefully checked for known clinical parameters as possible covariates that could influence our findings . glycemia was well controlled in all patients , while blood pressure and dyslipidemia were even more tightly controlled in patients with complications to reduce the risk of diabetes - related mortality [ 33 , 34 ] . nlrp3 polymorphism could be used as a molecular marker to identify patients in which pharmacological approach could be used to counteract the proinflammatory phenotype . furthermore components of nlrp3 inflammasome and downstream signalling pathways are potential novel targets for pharmacological prevention of late complications of t2d , as clinical trials targeting other inflammatory cytokines such as il-6 and tnfa have not been successful [ 37 , 38 ] . in conclusion , our preliminary data suggest the role of nlrp3 polymorphism in diabetic macrovascular complications , especially in mi .
background . 
 it is generally accepted that poor glycemic control , arterial hypertension and/or hyperlipidemia , and the associated oxidative stress may contribute to the development of macro- and microvascular complications in type 2 diabetes ( t2d ) . 
 such metabolic damage signals may activate inflammasome and trigger chronic inflammation . 
 we investigated common polymorphisms in inflammasome coding genes and the risk for macro- and microvascular complications in t2d . 
 methods . in total 181 clinically well - characterised t2d patients 
 were genotyped for nlrp3 rs35829419 and card8 rs2043211 . 
 risk for diabetic complications was assessed using logistic regression . 
 results . 
 patients with median duration of t2d 11 ( 617 ) years had relatively well controlled blood glucose and lipid levels and blood pressure on the prescribed treatment regimen . 
 duration of t2d and plasma cholesterol levels were the most important clinical risk factors for macrovascular complications ( p = 0.007 and p = 0.031 ) . 
 nlrp3 rs35829419 was associated with increased risk for macrovascular complications ( p = 0.004 ) , with myocardial infarction in particular ( p = 0.052 ) . 
 no association was observed between card8 polymorphism and any of t2d complications . 
 conclusions . 
 our preliminary data suggest the role of nlrp3 polymorphism in diabetic macrovascular complications , especially in myocardial infarction .