BAV, with an incidence of 1–2% in the population, represents the most common congenital heart defect [1]. The heritability of BAV, estimated to be between 0.75 and 0.89, is high. Family-based genomewide analyses have revealed a linkage between BAV and chromosomal regions 5q, 13q, and 18q [2], with an autosomal dominant mode of inheritance, a reduced penetrance and a non-Mendelian inheritance pattern [3]. Mutations were detected in the transmembrane receptor NOTCH1 (gene map locus 9q) [4]. Moreover, mutations in the vascular smooth muscle cell alpha actin gene (ACTA2, mapped to chromosome 10q) have been identified in patients with BAV and aortic aneurysms [5]. The ubiquitin fusion degradation 1-like gene (UFD1L), which is highly expressed in the outflow tract during embryogenesis, is downregulated in the cusps of patients with BAV compared with those of control patients [6]. Furthermore, BAV is associated with left ventricular outflow tract abnormalities, including aortic coarctation, arch hypoplasia and supravalvular and mitral valve stenosis [7]. Tissue-specific deletion of the gene encoding activin receptor type 1 (Alk2/Acvr1) in the cushion mesenchyme results in the formation of aortic valve abnormalities, including BAV [8]. A male predominance of more than 3:1 has been reported for BAV, and this anomaly is very frequent in X0 Turner’s syndrome (incidence rate: 22–34%), suggesting an X-linked etiology [9]. Analysis of a subpopulation with Andersen syndrome described four members with BAV. In Andersen syndrome, a mutation in the potassium inwardly-rectifying channel, subfamily J, member 2 gene (KCNJ2) is observed [10]. Some studies have reported the involvement of miRNAs in BAV. MiRNAs are small (approximately 22 nucleotides in length), noncoding nucleotide RNAs that have been shown to modulate mRNA stability and translation. Nigam et al. demonstrated that miR-26a was downregulated in fused aortic valves, and miR-26a was also found to be downregulated in aneurysms [11,12]. Recent studies have also reported a significant decrease in the amount of eNOS protein present in BAV-associated aneurysmal aortic tissue compared with that of tricuspid aortic valve (TAV) tissue [13]. Mohamed et al. detected decreased levels of eNOS protein in different areas of ascending aortic aneurysms. In conjunction with BAV, this can influence the cytosolic protein levels of pro-apoptotic HTRA2/Omi. It is speculated that the local differences in eNOS protein expression observed in BAV compared with TAV aortic aneurysms are probably caused by variations in aortic wall shear stresses [14]. There are several known polymorphisms in the eNOS gene (NOS3), some of which have biological relevance [15,16]. This study aimed to investigate the frequency of NOS3 mutations in patients with BAV and ascending aneurysms. Whole genomic 23.5 kb eNOS gene, including 28 exons, was analyzed. Subsequently, SNP analysis was performed using the MassARRAY® method and MALDI-TOF mass spectrometry for analytic detection. A set of 18 variants of the eNOS gene was generated from direct sequencing, whereas 16 variants represent SNPs and two are insertion/deletions and STR variants, respectively. The distribution of alleles was compared in 90 patients with BAV (mean age 52.3 ± 12.8 years, aortic diameter 48.1 ± 8.9 mm) and 11 patients with TAV (mean age 53.7 ± 18.2 years, aortic diameter 51.2 ± 13.3 mm). Statistical analysis revealed that three SNPs (rs1549758, rs1799983 and rs2566514) featured a significantly different allele distribution between BAV and controls. However, the association between these SNPs and BAV aortopathy requires further replications.

Many studies have observed similarities between the histology of the aneurysmal tissue of the aorta in MFS and the histology of the corresponding tissue in BAV. However, the current grading of aortic abnormalities during histopathological evaluation of BAV is based on conflicting reports [17]. De Sa et al. graded aneurysmatic tissue according to the severity of seven histopathological features: fibrosis, atherosclerosis, medionecrosis, cystic medial necrosis, smooth muscle cell orientation, elastic fiber fragmentation, and inflammation [18]. They found more pathological changes in aneurysmatic tissue obtained from patients with BAV compared with those of TAV patients. Other studies by Bechtel et al., found that, the ascending aorta abnormalities were more severe in TAV than in BAV [19]. These latter results correlated with recently observed results demonstrated here in Figure 1. Thus, the issue of whether the mechanisms underlying the aneurysmal histological alterations in BAV are caused by genetic heterotopy, wall stress caused by flow turbulence, or both, requires further investigation. In MFS, a mutation of the gene encoding the extracellular matrix protein fibrillin-1 has been detected. This mutation leads to dysregulation of transforming growth factor-beta (TGF-β) signaling. The discovery of fibrillins by Dietz et al. (1991) contributed to our understanding of molecular genetic causes of MFS. Dietz and his group found that a single mutation led to structural changes and caused complex diseases within various organs of the body [20]. According to Mendelian inheritance patterns, MFS is an autosomal dominant inherited disease of the extracellular connective tissue that can occur also spontaneously. Regardless of gender, MFS is diagnosed with a frequency of 1/3000 to 1/5000 [21,22]. Based on the severity of aortic wall abnormalities, further studies are needed to evaluate fibrosis, atherosclerosis, medionecrosis, cystic medial necrosis, smooth muscle cell orientation, elastic fragmentation and inflammation in aneurysmal tissue obtained from patients with MFS and BAV.

Figure 1: Comparison of histological scores. In terms of fibrosis, atherosclerosis, medionecrosis, cystic medial necrosis, smooth muscle cell orientation, elastic fiber fragmentation, and inflammation in intra-operative obtained biopsies of the sinus section of ascending aorta aneurysms: Aneurysms in the BAV-group demonstrate mild changes in the media with up to incipient moderate ones. There was a significant difference in the histological scores in terms of elastic fragmentation between the groups of TAV and MFS by p=0.031, but a highly significant difference was between the groups of BAV and MFS by p<0.001.

Historically, the development of aneurysms in patients with BAV was attributed to altered hemodynamic forces [23]. However, family members of patients with BAV and ascending aneurysm can show aortic dilatation without accompanying valve abnormality. It is also reported that aneurysms do progress after valve replacement in BAV patients [24]. BAV and ascending aneurysms may be variably penetrant manifestations of a common underlying gene defect. Moreover, the aortic valve and ascending aorta share a common embryonic origin; BAV may result from abnormal neural crest cell migration leading to the fusion of endocardial cushions, the precursors of the semilunar valves. Typical histopathological findings in the media of BAVassociated ascending aneurysms include elastic fiber fragmentation, increased ground substance, metalloproteinase activity, and apoptosis of vascular smooth muscle cells (VSMCs). In contrast to known connective tissue disorders, BAV is associated with dilatation above the sinotubular junction of the right antero-lateral aspect (convexity). Recent studies have indicated that aortic wall shear stress differs locally between BAV and control patients, when examined by magnetic resonance imaging [25,26]. Furthermore, two independent studies have provided evidence that VSMCs show different apoptotic behavior in the convex and opposite concave portions of the dilated aorta [27]. Inhibition of caspase-3 protected cultured cells derived from the tunica media of the concavity to a greater extent than those derived from the convexity of the aorta [28]. The convex aortic site is more prone to acute aortic dissection, which is a life-threatening condition with high morbidity and mortality rates and is generally an unpredictable event. By using a simultaneous detection system on the profiles of six MMPs and their four inhibitors at four different aortic sites (concave, convex, distal and proximal) of ascending aortic aneurysms. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2, TIMP-3 and TIMP-4 were all detected and quantified. This method is as accurate as older methods and minimizes the errors that can occur at the mRNA level using PCR techniques [29]. In a recent publication, Jackson and colleagues presented interesting results on the elevation of MMP-14 and -19 associated with TAVassociated thoracic aortic aneurysms, demonstrating upregulation of MMP-14 and -19 in the media of dilated aortas [30]. Of note, MMP-19 was the only MMP that was found to be upregulated in acute aortic dissection (AAD) [31]. Patients with BAV are at an increased risk of developing AAD (5 to 9 times higher than normal and12.5 to 20.5 times higher than that of patients with MFS) [32].

In the subsequent molecular biological investigations of AAD, further genes were described, and their proteins were altered in the media layer of patients with aortic dissection. The High-mobilitygroup- AT-hook-2 (HMGA2) protein was detected along with SNAI1 and vimentin, a marker for the epithelial-mesenchymal-transition, in the vasa vasorum of dissection patients. HMGA2 appears so to be involved in the transformation of vasa-vasorum-endothelial cells into mesenchymal cells during AAD [33].

These studies provide new fundamental knowledge about the pathogenesis of BAV, the ascending aorta and AAD. However, present knowledge of ascending aortic aneurysms in patients with BAV reflects only a part of the complex pathogenesis process [34].

BAV presents a technical challenge in heart valve operations, with regard to how this malformation and associated diseases can be optimally corrected in a way that preserves or imitates specific physiological properties. Because approximately >50% BAV patients present with an aneurysm of the ascending aorta, it makes sense to consider potential complications associated with the replacement. Untreated aortic aneurysm can constitute a life-threatening emergency situation and can be fatal if rupture occurs. While the pathogenesis of aneurysm in patients with connective tissue disorders, such as Marfan’s syndrome, is fairly well understood, the emergence of thoracic aneurysm in patients with BAV warrants further investigation. Figure 2 depicts the theoretical future direction of research on BAV.

Figure 2: Genetic basis and pathogenesis of BAV and BAV associated aortopathy. The etiology of bicuspid aortic valve/of the thoracic aortic aneurysm appears to be multiple. At the onset of valvulogenesis a number of mechanisms (e.g. Genes, epigenetic factors, fluid forces) may be involved, either alone or combined, in the pathogenesis. The growth of the embryonic outflow tract (OFT, descendant of the second heart field) shortens at specific stages according to programmed cell death. During cardiac valve formation, when the heart is a simple tube, it invades the extracellular matrix to build the endocardial cushions in the OFT. Migratory cells from pharyngeal arches, i.e., neural crest cells, participate and differentiate into VSMCs that populate the interior of semilunar valves, wall of the ascending aorta, aortic arch, and head vessels. Whether mechanisms related to existing aneurysms in BAV are due to genetic heterotopy and/or wall stress induced by flow turbulence late in adulthood requires further investigation.

The authors would like to acknowledge and thank members of the Department of Thoracic and Cardiac Vascular Surgery in Luebeck. Dr. Hoeppner and his team provided support for the MassARRAY® method and MALDI-TOF mass spectrometry for analytic detection of mutations of NOS3. The authors would also like to acknowledge and thank the Leducq Fondation for supporting our work and giving us the opportunity to study BAV aortopathy over the coming years. We are about to commence a significant study in this area.