In the present investigation, we observed associations of a variant in the promoter region of the SOD3 gene with cardiovascular morbidity and mortality in prospective cohorts of type 1 and type 2 diabetic patients. The minor T-allele of rs2284659 was inversely associated with the prevalence of myocardial infarction at baseline and with the incidence during follow-up of myocardial infarction, cardiovascular death and all-cause mortality in subjects with type 1 diabetes from GENESIS and GENEDIAB studies. The same allele was also inversely associated with the incidence of myocardial infarction and all-cause mortality during follow-up in the DIABHYCAR cohort of type 2 diabetic subjects. The protective T-allele of rs2284659 was associated with higher plasma EC-SOD and lower plasma AOPP concentrations in GENEDIAB.
AOPP concentration reflects the oxidation of plasma proteins, especially albumin, and is a reliable marker of oxidant-mediated protein damage . Isoprostanes are a biologically active product of arachidonic acid metabolism formed as the result of oxygenation of polyunsaturated fatty acids. Circulating isoprostane concentration reflects lipid peroxidation associated with oxidative stress . We observed higher plasma AOPP and isoprostane concentrations at baseline in type 1 diabetic subjects who died during the follow-up than in subjects who were alive at the end of the study. Isoprostane concentration at baseline was also higher in incident case of myocardial infarction. Previous studies have shown plasma AOPP and isoprostane to be an independent risk factors for coronary artery disease [10,11,19,20].
SOD3 gene and risk of cardiovascular morbidity and mortality
Data from the literature on the association of the genes encoding the SOD enzymes with cardiovascular complications are scare. We have previously observed in the DIABHYCAR cohort associations of SOD1 variants with cardiovascular mortality . Mollsten and coworkers observed an association of a missense variant of SOD2 with increased cardiovascular risk in a Danish cohort of type 1 diabetic patients . Regarding SOD3, investigations dealt mostly with the rare functional variant rs1799895 (Arg213Gly) in the heparin-binding domain of EC-SOD. The Gly allele is associated with decreased EC-SOD affinity for heparin, decreased tissue binding, and a 6-to-10-fold increase in circulating EC-SOD concentration [17,23,24]. Despite higher circulating levels of the enzyme, the 213Gly isoform was shown to present only minimal anti-oxidant effects in the vascular wall , the loss of function being attributed to decreased tissue binding. Associations of the Gly allele with cardiovascular risk factors, morbidity or mortality were reported in a few studies [25,26].
Genetic analysis of rs1799895 in our study was inconclusive due to the very low frequency of the Gly allele in our cohorts. It is therefore unlikely that the associations we have observed with the promoter rs2284659 could be explained by a rs1799895 Gly allele effect. Moreover, linkage disequilibrium between the two variants was not strong (D’ = 0.687 and r2 = 0.009). The genetic mechanism behind the allelic associations that we have observed remains unclear, as rs2284659 does not seem to modify known transcription factor binding sites. However, it is noteworthy that rs2284659 is in complete linkage disequilibrium with two other SNPs in the promoter of SOD3 (rs2159757 and rs13435617) that might affect transcription. The region surrounding and including rs2159757 matches the binding site for the PBX (pre-B-cell leukemia) family of transcription factors, and that surrounding and including rs13435617 matches the binding sites for HLF (hepatic leukemia factor) and E4BP4 (adenovirus E4 promoter-binding protein) transcription factors (http://consite.genereg.net). Interestingly, PBX transcription factors play a significant role in the morphogenesis, patterning and formation of the ventricular outflow tract (OFT). Mice lacking PBX1 gene have a range of OFT malformations, including failure of cardiac septation . A missense variant of PBX3 (p.A136V) predicted to be deleterious for transcriptional function was four times more frequent in patients with congenital heart defects as compared to controls . E4BP4 is implicated in the regulation of mammalian circadian oscillatory mechanism, anti-inflammatory response and cell survival. E4BP4 is expressed in the human heart and was shown to inhibit apoptotic proteins and to be overexpressed in cardiomyocytes following myocardial infarction . The implication of these transcription factors on SOD3 expression needs to be evaluated.
Oxidative stress and vascular complications of diabetes
Oxidative stress is involved in the pathogenesis of atherosclerosis . Increased production of reactive oxygen species (ROS) impairs endothelial function and endothelium-dependent vasodilation in humans, notably by inactivation of NO [31,32]. Oxidative stress also induces cell proliferation, hypertrophy, cardiac remodeling, apoptosis and inflammation in endothelial and smooth cells of the vascular wall and in myocardial cells [31,33,34]. Moreover, oxidative stress is associated with the metabolic syndrome and with many risk factors for atherosclerosis such as hypertension, dyslipidemia, obesity and diabetes . Biomarkers of oxidative stress predict the risk of mortality in patients with heart disease . Oxidative stress was shown to play a role in the premature vascular morbidity and mortality in people with diabetes . Studies in animal models corroborate the implication of oxidative stress in cardiovascular morbidity and mortality related to diabetes [38,39].
Antioxidant enzymes such as the SODs scavenge ROS and inhibit NO degradation in the vascular wall . The vascular wall contains large amounts of EC-SOD, which is produced and secreted to the extracellular space by smooth muscle cells [7,40]. The expression of SOD3 was shown to be significantly reduced, and endothelium-bound EC-SOD activity to be correlated to flow-dependent endothelium-mediated dilation in subjects with coronary artery disease, suggesting that reduced EC-SOD activity might contribute to endothelial dysfunction in these patients . Overexpression of SOD3 in human vascular endothelial cells in vitro was shown to decrease endothelial-cell-derived superoxide production and LDL oxidation . The implication of EC-SOD in cardiovascular protection is supported by studies in animal models. The overexpression of SOD3 in transgenic mice was associated with protection of myocardial function after global ischemia/reperfusion . Increased EC-SOD levels by adenovirus-mediated transfection of human SOD3 cDNA was associated with protection against myocardial stunning and with decreased reperfusion infarct size in a rabbit model of ischemia/reperfusion injury . Atherosclerosis was not increased in SOD3 knockout mice given an atherogenic diet . However, in an experimental model of focal cerebral ischemia, total infarct volume was 81% greater and hemiparesis more severe in SOD3 knockout mice as compared to wild-type animals , while mice overexpressing SOD3 had increased resistance to ischemia .
Strengths and limitations
The main strengths of our study are the assessment of clinical phenotypes (myocardial infarction, cardiovascular and all-cause mortality) in three prospective cohorts of type 1 and type 2 diabetes and the genotyping of tagSNPs covering the haplotype block containing SOD3. We also investigated associations of the clinical phenotypes and SOD3 genotypes with three markers of redox status. Overproduction of ROS and oxidative stress induced by chronic hyperglycemia are features of type 1 and type 2 diabetes . Nevertheless, despite the observation in our study of similar genetic associations in both types of diabetes, we acknowledge that proper replication studies are needed in type 1 and type 2 diabetes cohorts. Our study has limitations, notably in issues related to the measurement of the redox biomarkers. AOPP and isoprostane were assayed in plasma-EDTA samples collected at baseline and kept frozen for ~20 years, and only in a subset of participants. These issues might have affected, at least in part, comparisons of plasma AOPP and isoprostane concentrations between groups. Moreover, measurements of redox biomarkers were performed only in GENEDIAB participants for whom plasma samples were available, and thus were not replicated in the present investigation. Another limitation of the study is the relatively small sample size of our type 1 diabetes cohorts and the small number of new cases of myocardial infarction during follow-up. The statistical power was adequate to detect SNP effects with HR ≥1.5, but might have been insufficient to detect effects of smaller magnitude.