Thoracic aortic aneurysm (TAA) is a life threatening condition, which is manifested by progressive enlargement of the thoracic aorta due to destructive changes in the aortic wall. Therapeutic agents that may influence the process are absent to date and the only therapeutic decision is a surgical intervention (Davis et al., 2014). Up to 20% of patients with TAA have a positive family history, confirming the autosomal-dominant nature of this disease. The genetic basis of TAA is heterogeneous and involves most importantly genes coding for components of the vascular smooth muscle contractile apparatus, the extracellular matrix of connective tissue and key molecules of the transforming growth factor-β signaling (Luyckx and Loeys, 2015).

TAAs are characterized by extensive loss of smooth muscle cells (SMC) and changes in their functionality (Della Corte et al., 2008; Forte et al., 2013; Phillippi et al., 2014; Malashicheva et al., 2016). At the same time, the role of SMC during development and progression of aortic aneurysms is not well defined. Vascular SMC are characterized by specific molecular markers and contractile functions. Unlike other terminally differentiated muscle cells, vascular SMC have a unique ability to reversibly modify their contractile phenotype to a dedifferentiated state in response to changes in local environmental cues (Owens, 1995; Owens et al., 2004). The molecular basis of SMC differentiation in response to pro-differentiation signals is the expression of several highly specific contractile proteins, including smooth muscle α-actin (SMA), SM22α, and calponin. Abnormal control of the SMC phenotype leads to progression of vascular pathologies, including aneurysms (Ailawadi et al., 2009).

Myocardin (MYOCD) is an essential factor for the SMC differentiated phenotype. This transcription cofactor is both necessary and sufficient for the development and differentiation of most SMC (Wang et al., 2003; Long et al., 2008; Miano, 2015). Myocardin is a cardiac and smooth muscle–specific coactivator of serum response factor (SRF) that is able to transcriptionally activate CArG box–containing cardiac and smooth muscle target genes, such as smooth muscle α-actin, or SMA (ACTA2), SM22α (TAGLN), and calponin (CNN1) (Wang et al., 2003; Qiu et al., 2005; Huang et al., 2008; Long et al., 2008).

TGF-beta has been for a long time considered as a key participant of driving SMC differentiation state in the aorta by activating the genetic program that includes a set of SMC differentiation marker genes (Guo and Chen, 2012).

TAA may occur in the presence of a tricuspid or a bicuspid aortic valve (TAV and BAV), respectively. Several lines of evidence suggest that the mechanism of aneurysm development is distinct between the two patient groups (Folkersen et al., 2011; Balistreri et al., 2013; Kjellqvist et al., 2013; Maleki et al., 2013; Phillippi et al., 2014; Paloschi et al., 2015). The genetic background underlying BAV/TAA is clearly not monogenic and complex. So far, a few genes such as NOTCH1 (Garg et al., 2005; Mohamed et al., 2006; Mckellar et al., 2007; Mcbride et al., 2008; Andreassi and Della Corte, 2016; Forte et al., 2016; Koenig et al., 2017) and GATA5 (Padang et al., 2012) have been associated with non-syndromic forms of BAV/TAA.

In the vascular system, Notch receptors (Notch1–4) and ligands (Jag1 and 2 and Dll1, 3, and 4) are expressed. Activation of Notch receptors requires binding to a transmembrane ligand presented by neighbor cells. This binding enables a series of cleavage events in the receptor, resulting in the release of the intracellular region of Notch protein (Notch intracellular domain, NICD). NICD, the transcriptionally active form of Notch, translocates to the nucleus where it regulates a broad range of target genes (Andersson et al., 2011). The outcome of Notch activation is cell type and context dependent with multiple combinations of receptors and ligands that transduce different biological effect (Mašek and Andersson, 2017).

Controversy exists regarding the effect of Notch signaling on vascular SMC phenotype. Notch signaling has been linked to smooth muscle differentiation both in vitro and in vivo (Doi et al., 2006; Noseda et al., 2006; Boucher et al., 2012). At the same time, some data are consistent with a model wherein Notch signaling represses SMC differentiation and maintenance of the contractile SMC phenotype (Sweeney et al., 2004; Morrow et al., 2005; Proweller et al., 2005).

Although, the role of Notch has been extensively studied in the context of development and cancer (Briot et al., 2016) recent experiments using in vitro assays and mouse models also showed that changes in Notch activity can impact organ homeostasis in adults (Rostama et al., 2014, 2015; Briot et al., 2015). A recent research established a molecular framework coupling angiogenesis, angiocrine signals and osteogenesis via Notch signaling (Ramasamy et al., 2014).

SMC have high plasticity and are able to convert from the differentiated contractile phenotype to a variety of synthetic dedifferentiated states exhibiting in some cases chondrogenesis and osteogenesis during the pathogenesis of vascular diseases (Hilaire et al., 2016). The mechanisms of bone and vascular calcification seem to be similar and are connected through Notch/BMP/TGF-β crosstalk (Hilaire et al., 2016; Towler, 2017). It is known also that aortic tissues of BAV-patients are predisposed to progressive calcification and thus proosteogenic mechanisms might be involved in the pathogenesis of BAV-associated aortopathies.

Thus, it is obvious that functionality of SMC associated with their differentiation state is attenuated in the cells deriving from the TAA patients of both BAV and TAV groups. Myocardin, TGF-β, Notch and BMP are the main pathways responsible for the functional state of SMC in the aortic wall. The objective of the present study was to elucidate more precisely the mechanisms that attenuate differentiation state of the diseased SMC and to reveal possible differences between BAV- and TAV-derived SMC. For this we stimulated TGF-β, osteogenic and Notch differentiation pathways and compared control and diseased cells from the BAV and TAV group. We show that myocardin, TGF-β, BMP and Notch–related mechanisms of SMC differentiation are attenuated in the smooth muscle cells of the patients with thoracic aortic aneurys; Notch-dependent and proosteogenic genes show distinct expression in smooth muscle cells of BAV- vs. TAV-related aortic aneurysms.

In this study we show that smooth muscle cell differentiation pathways that are dependent on myocardin and TGF-β are attenuated in the patients with thoracic aortic aneurysm associated with bicuspid or tricuspid aortic valve. BAV-derived SMC differ from TAV-derived SMC in sensitivity to Notch-activation and to proosteogenic signals.

Consistent with the previous study that smooth muscle cells from thoracic aneurysms are characterized by decreased expression of SMC contractile proteins (Malashicheva et al., 2016), both BAV- and TAV-derived smooth muscle cells also demonstrated reduced MYOCD expression level compared to the cells from healthy individuals. Moreover, in SMC from healthy aortas MYOCD level positively correlated with the levels of ACTA2 and TAGLN, which are early SMC markers, but not with later marker CNN1, which is activated in the middle stage of the SMC differentiation (Owens et al., 2004). At the same time, BAV-derived SMC demonstrated a strong negative correlation between MYOCD and CNN1 expression, while in TAV-derived SMC we did not observe any correlation between MYOCD and other transcripts.

Myocardin is important in maintaining the normal SMC contractile phenotype (Long et al., 2008; Miano, 2015). The exact relation of myocardin upregulation to the heart and vascular diseases remains unclear. Remarkably, mutations in the MYH11 (smooth muscle myosin heavy chain) and ACTA2 (smooth muscle actin) genes (Zhu et al., 2006; Guo et al., 2007) were shown to cause familial thoracic aneurysms and dissections. Each of these genes is regulated directly by myocardin in the postnatal vasculature confirming that dysregulated myocardin-dependent transcription can be directly involved in aneurysm formation in humans (Huang et al., 2008).

To our surprise, we revealed a direct correlation of myocardin mRNA with vimentin mRNA (VIM) in control and BAV-derived SMC (Figure 1), but not in TAV-derived cells. Vimentin is a cytoskeletal protein of the intermediate filaments. Increased expression of vimentin in the vascular SMC has been associated with vascular injury and used as a marker of SMC synthetic, dedifferentiated phenotype (Owens et al., 2004). However, it is apparent, that functions of vimentin are more complex, and vimentin is a necessary component of fully functional contractile apparatus in smooth muscle cells.

Our data suggest that the observed “imbalance” between myocardin and smooth muscle-specific genes expression in aortic SMC from BAV and TAV-associated aneurysms is likely to depend on additional still unknown signals that can modulate a role of myocardin in directing an endogenous SMC differentiation program in aneurysm and this requires further research.

TGF-β is among the most potent factors that promote and maintain the vascular SMC contractile phenotype by upregulating smooth muscle structural genes. We revealed that aortic SMC from BAV- and TAV-related aneurysms are more responsive to pro-differentiation effect of the exogenous TGF-β1 than SMC from healthy aortas. However, we did not observe any difference in the response to TGF-β1 between BAV- and TAV-derived SMC. TGF-β pathway is apparently involved in TAA progression. Accumulating evidences are associated with increased TGF-β signaling in aneurysms (Forte et al., 2013; Gomez et al., 2013; Nataatmadja et al., 2013). Recently increased TGF-β activity during TAA formation has been reported in patients with TAV, but not BAV, presumably as a result of an increased sequestration of TGF-β in the extracellular matrix in aortas of BAV patients (Paloschi et al., 2015). Evidently, TGF-β signaling in aneurysm progression is very complex and is also influenced by epigenetic mechanisms such as histone modifications, microRNA expression and possibly others (Forte et al., 2016; Albinsson et al., 2017).

The discriminant analysis of expression of genes responsible for SMC differentiation state and genes related to Notch and proosteogenic regulation (Figure 2) suggested that SMC from BAV and TAV patients have distinct gene expression profile of these groups of genes from the cells of healthy donors.

Notch is an important regulator of SMC (Boucher et al., 2012). Among all Notch receptors and ligands Notch2 and Notch3 appear to be the most important for SMC. These two receptors influence the phenotype and functions of SMC (Baeten and Lilly, 2015; Liu et al., 2015). Notch is a key signaling pathway in development, ensuring cross talk between different types of cells and their physiological differentiation (Andersson et al., 2011). Notch signaling in the endothelium of the vessel is positioned to mediate differentiation of underlying SMC ensuring integrity of the vessel wall (Pedrosa et al., 2015). We have shown recently that Notch signaling is attenuated in aortic endothelial cells of BAV patients (Kostina et al., 2016). In the present work we show alteration in Notch responsiveness in BAV-derived SMC. We suggest that the initial process of the vessel formation as well as further healing in response to constant shear stress in the aorta could be impaired in the BAV patients via Notch-dependent events in particular through inactive feedback loop connecting endothelial and smooth muscle cells and their mutual influence on cellular functional state.

Cardiovascular calcification is gaining an increased attention nowadays as it seems to be a “default” state of various pathologies (Hilaire et al., 2016). Osteogenic medium induced more elevated RUNX2 expression and more prominent calcification in BAV-derived SMC. These results suggest a dysregulation of proosteogenic pathways in the BAV-derived cells. Notch activation induced significant elevation of ACTA2 expression and corresponding SMA protein accumulation in BAV-derived cells. It has been shown previously that the cross-talk between BMP2 and Notch signaling pathways induces osteogenic differentiation of vascular SMC (Shimizu et al., 2011). At the same time increased expression of ACTA2 has been shown to directly reduce the clonal potential of human mesenchymal stem cells and to guide their differentiation toward osteoblasts. Hence, SMA not only identifies osteoprogenitors in mesenchymal populations as shown by others before (Kalajzic et al., 2008; Grcevic et al., 2012), but may be part of the mechanism driving differentiation (Talele et al., 2015). Our data are in line with this as we observed that only BAV-derived cells are able to elevate the level of ACTA2 in response to Notch stimulation. In addition we show that simultaneous action of osteogenic medium and Notch activation leads to elevated ACTA2 expression and to an increase in osteogenic differentiation in SMC. Thus, this Notch-dependent elevation of ACTA2 might be a direct prerequisite to calcification. Synergy between attenuated Notch and proosteogenic sensitivity could cause the final predisposition to osteogenic phenotype in BAV-derived SMC.

This study has some important limitations. First, the groups of the patients were not quite large. Second, we worked in vitro with isolated smooth muscle cells, but in aortic wall there is a community of SMC and endothelial cells and the communication between these different type of cells now is gaining more attention to understand proper function of the whole vessel (Lilly, 2014). Nevertheless, we suggest that our findings are relevant for searching potential targets to ameliorate aortic wall integrity.

EI and DK made experiments, analyzed data and wrote the manuscript. OI, VU, NG, and OM analyzed data of the patients and collected patient samples. AG and AK analyzed data. AM designed research, analyzed data, and wrote the manuscript.