VC Pathogenesis at the Molecular Level

There are numerous molecular components involved in the pathogenesis of calcification, described in detail by Sage and colleagues (2010), and a number of proteins relating to osteogenesis/chondrogenesis have been identified in arterial vascular cells (Papadopouli et al., 2008). VC mechanisms are mediated by transcription factors that also control osteogenesis/chondrogenesis, such as Runx2, sex-determining region Y box-9 (Sox9) and osterix, and their downstream matrix components ALP, bone sialoprotein (BSP) and osteocalcin (OCN) (Tintut and Demer, 2006). Furthermore, BMPs, central regulators of osteogenic differentiation, are also known to have potent pro-calcific effects in the vasculature, as are a number of pro-inflammatory cytokines (e.g. TNF, interleukin (IL)-6) (Al-Aly, 2008;

Davenport et al., 2016; Hénaut and Massy, 2018). The most relevant of these factors to the

1.2.4.2.1 Pro-calcific Transcription Factors

There are several pro-calcific transcription factors involved in the regulation of bone morphogenesis that are also known to regulate VC. Runx2, for example, a “master regulator”

of osteoblast differentiation and bone development, has been implicated as one of the central osteoblastic transcription factors involved in VC pathogenesis (Lin et al., 2015). Runx2 is responsible for inducing the expression of a wide range of osteoblastic genes (Byon et al., 2011; Pratap et al., 2003; Weng and Su, 2013), and also trans-activates another osteoblastic transcription factor, osterix (Nishio et al., 2006). Indeed, Runx2 has been shown to be elevated in calcified arteries (Moe et al., 2003), and Runx2 deletion has been shown to attenuate osteochondrogenic differentiation and subsequent calcification in VSMCs (Lin et al., 2015).

Another transcription factor, msh homeobox 2 (Msx2) also plays a crucial role in osteoblastic differentiation of VSMCs, but is not believed to be necessary for subsequent calcification (Andrade et al., 2017).

Sox9, a chondrocytic transcription factor with a primary role in chondrocyte differentiation, is also responsible for driving a number of pro-calcific genes (Akiyama et al., 2002). Unlike Runx2, however, Sox9 has only recently been implicated in the VC process (Kauffenstein et al., 2014) and is of particular interest given that the calcification process is now thought to closely resemble chondrocytic rather than osteoblastic differentiation (Speer et al., 2009). Sox9 has also been detected in calcified vessels (Kauffenstein et al., 2014) and is known to be expressed in cultured VSMCs (Tyson et al., 2003). In a vascular context, the particular stimuli that induce Sox9 expression are relatively unclear, but it has been shown to potentiate BMP-2-induced cartilage formation (Liao et al., 2014). Interestingly, Sox9 prevents Runx2-mediated osteoblastic gene expression in bone (Cheng and Genever, 2010), however, the expression of osteoblastic targets have been identified alongside Sox9 expression in calcified vascular cells (Tyson et al., 2003). Thus, the role of this transcription factor in a vascular setting may vary considerably from their traditional roles in osteo/chondrogenesis and remain to be delineated.

1.2.4.2.2 Downstream Protein Mediators

There are a number of proteins downstream of these transcription factors that regulate the VC process. ALP, for example, is a metalloenzyme regulated by Runx2 that is actively involved in the mineralisation process, and is known to be highly expressed in calcified vascular tissue (Sheen et al., 2015). ALP is localised to the extracellular side of the plasma membrane, but can

also be identified intracellularly (attached to matrix vesicles) and in secretory form (Davenport et al., 2016; Golub and Boesze-Battaglia, 2007). Extracellular ALP is the most relevant to VC, given that it promotes mineralisation by reducing inorganic pyrophosphate (PPi) levels (an inhibitor of mineralisation) and increasing inorganic phosphate (Pi) levels (a promoter of mineralisation) in the extracellular space (Schoppet and Shanahan, 2008). Budding matrix vesicles containing hydroxyapatite crystals (a calcium- and phosphate-containing mineral), which are then deposited into the extracellular matrix if the PPi/Pi balance is correct (Orimo, 2010) (Figure 1.4). In addition to ALP, BSP and OCN, early and late markers of VC, respectively (Huang et al., 2007), also promote the mineralisation process in VSMCs by promoting nucleation and binding of hydroxyapatite crystals (Hunter and Goldberg, 1994). As these osteoblastic markers are upregulated, natural VSMC markers (e.g., SMα-actin, SM22α) are downregulated, resulting in a “calcifying” VSMC phenotype. A range of proteins are also involved in the negative regulation of VC; for example, MGP inhibits VC in its role as a BMP inhibitor (Boström et al., 2001), while OPN binds strongly to calcium atoms to prevent mineralisation (Sodek et al., 2000). However, upregulation of the aforementioned pro-calcific transcription factors have also been associated with a decrease in the expression of these proteins (Schinke et al., 2000; Zhang et al., 2012).

1.2.4.2.3 BMPs

BMPs are known to play a central role in the regulation of VC, and vascular cells have been shown to express BMP receptors (Kim et al., 2013; Yu et al., 2007). There are multiple BMPs involved in the regulation of osteoblastogenesis, some of which are particularly relevant in a vascular setting. BMP-2, for example, is known to induce the calcification process, and exerts its osteoblastic function by increasing the expression of the transcription factors Runx2 (Sage et al., 2010) and osterix (Lee et al., 2003), which follow on to induce ALP activity. BMPs exert their function via Smad phosphorylation and translocation, promoting osteoblastic gene expression (Hruska et al., 2005). Of significant interest is the role for BMP-2 identified in endothelial:smooth muscle cell communication; in this respect, endothelial cells directly exposed to pro-calcific stimuli in circulation secrete BMP-2 as a pro-calcific paracrine signal to the underlying VSMCs (the location of medial VC in vivo) (Davenport et al., 2016; Osako et al., 2010). BMP-4 has also been implicated in the promotion of VC in smooth muscle (Panizo et al., 2009), whilst BMP-7 has been shown to inhibit calcification (Mathew et al., 2006).

1.2.4.2.4 Pro-inflammatory and Pro-oxidant Mediators

Inflammation is closely related to calcification, as immune/vascular cells at the site of atherosclerotic plaque release inflammatory cytokines that contribute to VC regulation (Johnson et al., 2006; Mody et al., 2001). In this respect, TNFα and IL-6 have both been shown to induce VC (Al-Aly, 2008; Hénaut and Massy, 2018) and can activate many of the aforementioned pro-calcific mediators in vascular cells (e.g. Runx2, ALP, BMP-2) (Illiandri et al., 2016; Kurozumi et al., 2016). Of further note, oxidative stress (induced by ROS) is a well-known inducer of pro-calcific and pro-inflammatory proteins in the vasculature (Al-Aly et al., 2007; Byon et al., 2008), and ROS are also known to be secreted by vascular cells under pro-calcific conditions (Clempus and Griendling, 2006; Farrar et al., 2015). Of particular relevance to T2DM, hyperglycemia (a pro-oxidant, pro-inflammatory stimulus) has also been shown to stimulate chondrocytic trans-differentiation of human VSMCs, alongside increased levels of Runx2 and Sox9 expression, increased ALP activity and mineralisation (Bessueille et al., 2015).

Figure 1.4. The regulation of calcific genes and proteins in calcifying vascular cells. Pro-calcific stimuli, pro-inflammatory stimuli and pro-oxidant stimuli can induce a number of osteoblastic/chondrocytic transcription factors in vascular cells (Runx2, Sox9, osterix, Msx2). These transcription factors drive the expression of pro-calcific proteins (ALP, BMPs, BSP, OCN) and downregulate anti-calcific proteins (MGP, OPN) and smooth muscle markers (SMα-actin, SM22α). ALP is secreted alongside hydroxyapatite (in matrix vesicles), and converts PPi to Pi to promote mineral deposition. These osteoblastic vascular cells also secrete BMPs, pro-inflammatory cytokines and reactive oxygen species to further promote the calcification process. ALP, alkaline phosphatase; BMP, bone morphogenetic protein; BSP, bone sialoprotein; MGP, matrix gla protein; Msx2, msh homeobox 2; OPN, osteopontin; (P)Pi, inorganic (pyro)phosphate;

Runx2, runt-related transcription factor-2; SM, smooth muscle; Sox9, sex determining region Y box-9.