br Discussion Arginase and Arginase

Discussion Arginase 1 and Arginase 2 are homologous enzymes that consume l-arginine to produce l-ornithine and urea [14], [15]. These enzymes compete with NO synthases for the same substrate [14], [15], [30], and therefore may reduce NO production [21], [24], [30]. The colocalization of arginases with NO synthases [19] and the fact that arginase inhibitors increase NO production [36] strengthen this possibility. Consistent with abnormal upregulation of arginases in the corpus cavernosum in ED [16], [24], [25], [29], arginase inhibitors were shown to improve erectile function in animal models of ED [24], [26]. In humans, upregulation of Arginase 2 in the corpus cavernosum from patients with ED [16] may exert similar effects to those found in human endothelial O-propargyl-puromycin submitted to hypoxic conditions, when reduced production of NO by eNOS is accompanied Arginase 2 upregulation [19]. However, there are no clinical studies that have evaluated whether the expression or activity of Arginases are increased in plasma from ED patients, neither whether genetic factors involving variations in the genes encoding Arginase 1 and 2 are associated with ED. Here, we were first to assess whether plasma Arginase 1 and 2 levels, and Arginase activity and genetic polymorphisms affect the risk for ED, and the degree of ED as reflected by the IIEF score. The main findings reported here are: 1) Plasma Arginase 2 levels are increased in patients with ED patients and may be a useful biomarker for ED; 2) Arginase 1 genotypes and haplotypes are associated with variations in the IIEF score in patients with ED, thus suggesting a role for Arginase 1 in the degree of ED. Arginase 1 and Arginase 2 are enzymes produced ubiquitously in human body [15]. While Arginase 1 is cytosolic and more abundantly expressed in the liver, Arginase 2 is mitochondrial and is more widely expressed in the kidney, prostate, gastrointestinal tract, and vasculature, although both forms were found in vascular endothelial and smooth muscle cells [15], [37]. However, our results suggest that Arginase 2 may be more relevant as a clinical biomarker for ED than Arginase 1. Indeed, Arginase 2 was increased in plasma of patients with ED (Fig. 1), which is consistent with previous reports on corpus cavernosum from ED patients [16]. Interestingly, we found lower plasma Arginase 1 than Arginase 2 concentrations, both in ED patients and in controls. Nevertheless, Arginase 1 may be relevant to disease conditions because it may be locally upregulated, as shown in animal models [24], and thus impair regional NO production without being detected at abnormal circulating concentrations. In agreement with this idea, we found that Arginase 1 and Arginase 2 correlated with plasma enzyme activity only under disease conditions (ED), which presumably leads to upregulation of both enzymes [16], [24]. To date there are no reports of arginase 1 and 2 levels in plasma as biomarkers for erectile dysfunction. Although there is evidence showing the expression of these enzymes in several tissues, there is a lack of evidence showing how these enzymes are present in plasma. We believe that these enzymes may be released from endothelial cells (which express both isoforms [19]) or from red blood cells (which express arginase 1 [36]) to plasma when they are destroyed naturally. In diseases that involve endothelial dysfunction, arginases were shown to be upregulated [16], [22], [23]. If this is true, it is very likely that the increase in Arginase 2 levels that we show here actually reflects a more general endothelial dysfunction and may not be specifically related to endothelial dysfunction occurring only in the corpus cavernosum. Nevertheless, this is consistent with the idea that ED would be an early manifestation of a more generic endothelial dysfunction, which over the years would end up presenting itself as coronary disease, or other cardiovascular diseases [4]. The effects of genetic polymorphisms of ARG1 and ARG2 genes on ED risk and on IIEF scores were also examined. We selected four polymorphisms of ARG1 gene (Table 3) with basis on previous functional or clinical implications. The rs2781659 G allele reduces the expression of ARG1 promoter [38], and the rs2791665 polymorphism (which is at high linkage disequilibrium with the rs2781667, evaluated here) also reduces the expression of ARG1 in vitro[38]. Moreover, the variant alleles of the four selected polymorphisms were associated conditions affected by NO signaling including asthma [38], [39], [40], [41], blood pressure [42], and myocardial infarction [43]. The selection of these four polymorphisms allowed the assessment of all most common ARG1 haplotype blocks in Caucasians, according to Seattle SNPS website (http://gvs.gs.washington.edu/GVS138/; using 10% as minor allele frequency cutoff). Regarding ARG2, while there is no clear functional evidence implicating the studied polymorphisms, it has been shown that both the rs3742879 and the rs10483801 polymorphisms affect exhaled NO concentrations significantly [44], and affect the susceptibility to asthma [41], [45], or the therapeutic responses of sickle cell disease patients to hydroxyurea, which depends on NO formation [46]. Although we found negative results with respect to the association of ARG1 and ARG2 polymorphisms with ED phenotype (Supplementary Tables 1 and 2), the rs2781659 AA and rs2781667 TT genotypes were found in association with worse (lower) IIEF scores among ED patients, whereas the rs2781659 GG and the rs2781667 CC genotypes were associated with better (higher) IIEF scores (Table 3, Table 4). Interestingly this was only observed in the Clinical ED group, which is consistent with our hypothesis that although Arginase 1 may not be upregulated in plasma, it may be locally upregulated under disease condition (as shown in animal models [24]) and may affect the severity of ED.