The most exploited circulating inflammatory marker in plasma is CRP. In addition to its role as a marker, it can exert modulatory effects through its well-demonstrated presence in atherosclerotic plaques [32]. CRP is an acute-phase protein produced in the liver and has a homopentameric structure, each with 206 amino acids that are combined in a three-dimensional structure, and Ca²⁺-binding specificity for phosphocholine [33]. Besides being a sensitive marker for the detection of subclinical inflammation, CRP has been suggested to participate in the pathophysiology of ATH. Moreover, it stimulates the secretion of proinflammatory cytokines and tissue factor and may contribute to vascular remodeling via its effects on eNOS [34].

A growing body of literature has demonstrated that CRP levels may be correlated with ED. Moreover, it may stimulate expression of various biomarkers, such as ICAM, vascular cell adhesion molecule 1 (VCAM-1) and E-selectin [35]. Moreover, according to experimental data CRP may also induce a significant impairment of endothelial-dependent vasorelaxation in various conditions which could lead to an increased cardiovascular risk profile [36]. In line with this evidence, Venugopal et al. [37] have demonstrated that CRP decreased eNOS mRNA, protein abundance, and enzyme activity in cultured human aortic ECs. Furthermore, increased CRP plasma levels across the coronary circulation have been associated with impairment in coronary endothelial-dependent function, even though its relationship with the severity and extent of coronary atherosclerosis is not clear [38].

Impaired endothelium dependent vasodilation along with alterations in the expression of adhesion molecules and proinflammatory molecules are present in state of ED. More specifically, increased IL-6, TNF-a, and especially ICAM-1, VCAM-1, and E-selectin are associated with ED. These molecules may also participate in ECs stimulation to promote an atherogenic phenotype [39]. In addition, other chemotactic factors could be expressed such as MCP-1, macrophage colony-stimulating factor, and TNF-β which contribute to the development of inflammation within the arterial wall related with the pathogenesis of atherosclerosis [40]. As aforementioned, vascular endothelium produces various other molecules that affect blood fluidity and thrombosis, such as plasminogen activator inhibitor-1 (PAI-1), tissue factor and vWF. These in turn, are regulated by the production of NO, prostacyclin, tissue plasminogen activator and thrombomodulin [41]. Moreover, CD40 ligand (CD40L) is expressed on monocyte/macrophages, ECs, smooth muscle cells and platelets, and as a consequence of CD40L binding to CD40, several inflammatory processes are initiated by the release of cytokines and the expression of adhesion molecules. In particular, the link between CD40/CD40L and CVDs has been established in numerous studies [41]. The pathophysiological substrate is still under investigation however, evidence suggests that initially, CD40L has an unfavorable effect on the vascular redox state [41].

Accordingly, it has been suggested that circulating markers of oxidative stress, including F2 isoprostanes and antibodies against oxLDL, are increased in humans with diabetes [42]. Notably, given that there is oxidative degradation of BH4 by ROS in ED, BH4 plasma levels along with levels in the vascular wall could reflect vascular endothelial health [43]. Moreover, clinical data have suggested that hyperhomocysteinemia may be associated with impaired renal function, potentially through affecting endothelial function. Thus, plasma homocysteine levels may be considered as an intermediate factor between ED and renal function [44]. Also, asymmetrical dimethylarginine (ADMA) as an endogenous eNOS inhibitor has been associated with ED in several CVDs, such as hypercholesterolemia and coronary artery disease [45]. It is worth mentioning, endothelial progenitor cells (EPCs) are currently considered to participate in vascular repair and maintain endothelial integrity thus, they may exhibit a role as prognostic biomarker in CVD. EPC number and function may be used as biomarkers and more specifically, reduced circulating numbers and functional capacity may predict future cardiovascular events, independently of other cardiovascular risk factors [46].

The eNOS gene includes 26 exons, 25 introns and it is located at 7q35–7q36 of chromosome 7, while the protein consists of 1,203 amino acids and has a molecular weight of 133 kDA [48]. It has been the focus of intensive research to identify potentially functional polymorphisms influencing ED, as more than 100 polymorphisms have been identified in the eNOS gene (Table 8.2). Some of them (e.g. T–786C promoter polymorphism) are situated in the eNOS promoter and might influence mRNA transcription reducing gene expression [53]. More specifically, Nakayama et al. [53] have demonstrated the existence of three linked mutations in the 5′-flanking region of the eNOS gene (T-786C, A-922G, and T-1468A) which are more prevalent in patients with coronary spasm than in the control group. Also, the T-786C mutation resulted in a significant reduction in eNOS gene promoter activity, whereas neither the A-922G nor the T-1468A mutation had any affect, suggesting that this single nucleotide polymorphism (SNP) may reduce endothelial NO synthesis and predispose to coronary spasm.

One of the most studied NOS polymorphisms is the polymorphism in exon 7 of NOS3, a G-T transition at position 894 that results in a Glu to Asp amino acid substitution for codon 298 (894GT) within exon 7. It is the only common polymorphism identified thus far that encodes an amino acid substitution. Significant data have shown that eNOS Asp298 is subjected to selective proteolytic cleavage in ECs and vascular tissues that might account for reduced vascular NO generation, even though it has been elsewhere suggested that eNOS Asp298 could be a marker for another possibly functional variant [51, 56, 57]. Moreover, given a novel method to accurately determine molar quantities of each variant of G894T polymorphism (expressing them as green fluorescent protein fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular ECs), the Asp substitution at 298 does not seem to have a major effect in modulating eNOS activity in vivo [51]. In turn, Guzik et al. [49] have investigated the relationships between NO-mediated endothelial function with the presence of the eNOS Glu298Asp variant via endothelium-dependent vasorelaxation to different agonists which were determined in human saphenous veins of coronary artery disease (CAD) patients. NO-mediated endothelial vasorelaxation was highly variable between patients, and reduced values were associated with increased number of clinical risk factors for ATH. This variant was not associated with any differences in contractions to phenylephrine, NO-mediated vasorelaxations to acetylcholine, bradykinin or calcium ionophore, nor relaxations to the NO donor sodium nitroprusside, suggesting that this polymorphism may not have a major direct functional effect on vascular eNOS activity in human ATH [49].

More recently however, a study by Antoniades et al. [52] has provided new evidence about its potential association with endothelial function and with markers of ECs injury and activation. The study population consisted of 56 patients with a history of premature myocardial infarction, while the forearm blood flow was measured using strain-gauge plethysmography. Of note, the presence of 894 T allele on eNOS gene is associated with impaired endothelial function and higher levels of von Willebrand factor in relatively young patients with myocardial infarction. Subsequently, the same investigators have attempted to explore the determinants of GCH1 gene expression, which codes for a rate-limiting enzyme in the biosynthesis of BH4, an eNOS cofactor important for maintaining enzymatic coupling. They have demonstrated that GCH1 gene expression modulated by a particular GCH1 haplotype, is a major determinant of BH4 bioavailability both in plasma and in the vascular wall thus, this genetic variation may be a determinant of eNOS coupling, vascular redox state, and endothelial function in human vascular disease [58].