**5. Gene polymorphisms influencing vasomotor endothelial function**

Nitric oxide (NO), is a key vasodilator. It is formed in the vascular endothelium by the oxidation of arginine through the catalytic activity of nitric oxide synthase (NOS). This reaction requires NADPH and O2 as co-substrate and yields NO and citrulline as end products. Importantly, the enzymatic activity of NOS is inhibited by methylated analogues of arginine, namely N-monomethylarginine (L-NMMA) and asymmetric dimethylarginine (ADMA) [63], which are synthesized in vivo by a family of enzymes known as protein arginine methyltransferases. Proteolysis of proteins containing L-NMMA and ADMA releases them into the endothelial cell cytosol, from where they are removed into the blood. Elevated serum ADMA levels are associated with atherosclerotic vascular disease [64].

More than 15 polymorphisms exist in the NOS3 promoter that might influence mRNA transcription and reduce gene expression. Two polymorphisms in NOS3, 786 T > C and 894G > T, are the most studied. 786 T > C resides in the promoter region of NOS3 and regulates transcriptional initiation [65]. However, the –786 T > C polymorphism has shown inconsistent associations with functional measures, and with clinical disease end points. The CC genotype at 786 T > C is associated with blunted forearm blood flow responses to Ach in hypertensive subjects [66] and no increases in NOS3 mRNA and endothelial nitric oxide synthase (eNOS) protein expression in response to laminar shear stress in endothelial cells from coronary heart patients [67]. Polymorphisms within the coding region of the NOS 3 gene could alter NOS enzymatic activity. The 894G > T polymorphism in exon 7 of NOS3 results in substitution of glutamate with aspartate at codon 298 (also denoted as Glu298Asp) [68]. There is currently a debate, with controversial studies on whether this polymorphism is indeed functional. Two studies have shown that eNOS Asp298 undergoes selective proteolytic cleavage in endothelial cells and vascular tissues, which may account for reduced vascular NO production [69]. However, other studies have suggested that this finding may be the result of an artifact [70].

ADMA is removed from the circulation by metabolism primarily by isoform two of the DDAH2 dimethylarginine dimethylaminohydrolase, which predominates in tissues that express eNOS, such as the endothelium. The main cause of elevated ADMA levels in patients at risk for vascular disease is not fully understood, but one potential explanation could be loss-of-function mutations in the DDAH enzyme gene that alter gene expression or enzyme activity. Six potentially pathological polymorphisms have been identified in the DDAH2 gene. Five of them are upstream of the translation start site and may affect gene transcription. An insertion-deletion polymorphism (6G/7G) at position 2871, which lies in the core promoter region, affected DDAH2 promoter activity in the promoter/reporter assay [71].

The realization that common gene variants can, at best, have little to moderate impact on physiology and disease susceptibility has led to the understanding that

future studies of susceptibility to complex diseases, whether they address clinical endpoints or intermediate phenotypes such as endothelial function, will need to be much larger and include more variables simultaneously. Because many GWAS identify SNPs outside protein coding regions or in non-coding intervals, the contribution of small non-coding RNA (e.g., lncRNA, microRNA) in modulating endothelial function should be addressed.
