VC Pathogenesis – a Role for TRAIL

In recent years, a novel role has emerged for an additional ligand in the vasculature: namely, TRAIL. This ligand has been shown to interact with OPG and RANKL in the modulation of the calcification process (Schoppet et al., 2004), and although its precise functions in this context are poorly defined, mounting evidence now points to a vasoprotective role for TRAIL in the prevention of VC. Prior to discussing the effects of TRAIL in a vascular context, some background will first be provided on the function and regulation of this complex ligand in vivo.

1.2.4.4.1 TRAIL – Location, Function and Regulation

As its name suggests, the primary function for TRAIL is as an inducer of apoptosis, and although originally believed to exert this function on malignant or transformed hematopoietic cells, it is now known to have diverse pleiotropic roles in multiple cell types (Forde et al., 2016;

Wiley et al., 1995). TRAIL has been identified in multiple tissue locations including spleen, prostate and lung (Wiley et al., 1995), but its mRNA and protein expression has thus far only been demonstrated in immune, vascular, hepatocytic and myocytic cells (Falschlehner et al., 2009; Forde et al., unpublished observations; Gochuico et al., 2000; Sato et al., 2006; Spierings et al., 2004). TRAIL is also present in the circulation (albeit in the picomolar range), and interestingly, serum TRAIL has been shown to be inversely correlated with a number of pathologies including CVD (Volpato et al., 2011).

At a molecular level, TRAIL, a member of the TNF ligand superfamily, is a type-II transmembrane protein with the ability to bind five different receptors found on numerous cell types, as well as a C-terminal domain that can also be cleaved from the cell surface (or secreted in vesicles) to release a soluble form (Ehrlich et al., 2003). Both membrane-bound and soluble TRAIL can induce apoptosis, although soluble TRAIL is thought to exert a more potent apoptotic effect (Kim et al., 2004). Two TRAIL receptors (DR4 and DR5) have a cytoplasmic death domain, whilst two decoy receptors (DcR1 and DcR2) lack a functional death domain;

thus TRAIL-induced apoptosis via DR4 and DR5 is antagonised by the competitive inhibitory effect of DcR1 and DcR2 (Ravi et al., 2001). OPG acts as an additional soluble decoy receptor for TRAIL (and vice-versa); therefore, OPG has a second protective function in addition to its ability to block RANKL-induced calcification, by virtue of its ability to block TRAIL-dependent apoptotic signalling (Emery et al., 1998).

The regulation of TRAIL in vivo is mediated by a number of factors including the NF-κB family of transcription factors (Wurzer et al., 2004) and interferons (Kayagaki et al., 1999;

Miura et al., 2006), described in detail by Forde et al. (2016). TRAIL receptor regulation, although not yet fully defined, appears to be mediated by many of the same pathways that regulate TRAIL (Kamohara et al., 2004; Ravi et al., 2001). TRAIL receptors are expressed at varying levels in a wide range of cells and tissues in vivo (hepatocytes, neurons, cardiac myocytes, colon, kidney, lung, vasculature, and pancreas among others) (Daniels et al., 2005;

Pan et al., 1998; Zauli et al., 2006), and as such, it is no surprise that TRAIL exerts complex pleiotropic functions dependent on the location-specific expression of death and decoy receptors (and the pathways activated by TRAIL:TRAIL receptor binding).

The mechanisms associated with TRAIL signalling are complex, but, as noted, TRAIL’s primary function as an apoptosis-inducing ligand is best defined. Indeed, TRAIL’s apoptotic function is initiated by DR4 and DR5 binding, resulting in the formation of the death-inducing signalling complex (Almasan et al., 2003) and downstream caspase activation (Suliman et al., 2001). There are also a number of signalling pathways activated by TRAIL, however, that result in anti-apoptotic functions, but these pathways are less understood. As a brief summary, TRAIL has the ability to induce NF-κB activation, which can function to either promote cell survival or apoptosis depending on the cellular context (Degli-Esposti, 1999; Ravi et al., 2001).

In this respect, high concentrations of TRAIL (1 µg/mL) cause the NF-κB-dependent upregulation of anti-apoptotic proteins which may reduce cellular sensitivity to TRAIL-induced apoptosis (Almasan et al., 2003). Secondly, TRAIL can initiate the mitogen activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) signalling cascade (Falschlehner et al., 2007), which results in an anti-apoptotic effect in vascular cells (Secchiero et al., 2003). A third non-apoptotic signalling cascade activated by TRAIL is the protein kinase B/Akt pathway (Secchiero et al., 2003); Akt activation also indirectly protects from apoptosis through P53 degradation and NF-κB activation (Hausenloy et al., 2004). While these pathways can be activated by DR4 and DR5 under the correct conditions, the decoy receptor DcR2 (while incapable of inducing apoptosis) is capable of activating other signalling pathways (e.g. NF-κB) which may accentuate the anti-apoptotic role of TRAIL (Delgi-Esposti et al., 1997).

For a comprehensive review on the pleiotropic roles of TRAIL in vivo, and an in-depth summary of the pathways that activate, and are activated by TRAIL, see Forde et al. (2016).

For the purpose of the current review, however, a focus on the specific role for TRAIL in the

1.2.4.4.2 TRAIL in the Vasculature

The pro-survival role of TRAIL has led to the hypothesis that this ligand may exert pleiotropic roles in many cell types, including the circulatory system (Forde et al., 2016). Although there is still much debate over whether or not TRAIL is expressed in the vascular endothelium (Secchiero and Zauli, 2008), TRAIL receptors are expressed by endothelial cells (Zauli et al., 2006), and VSMCs express both TRAIL and TRAIL receptors (Cheng et al., 2014; Harith et al., 2016; Li et al., 2016). Additionally, immune cells present at sites of atherosclerotic plaque and intimal calcification are sources of surface and soluble TRAIL (Ehrlich et al., 2003;

Kamohara et al., 2004) while the TRAIL receptors on these cells are widely expressed (Guicciardi and Gores, 2009). As noted, TRAIL is also present in circulation (Volpato et al., 2011), further supporting a physiological role for TRAIL in the vasculature.

An emerging hypothesis within the field of vascular research has proposed a vasoprotective role for TRAIL, possibly via the mediation of atherogenic, inflammatory and anti-oxidant effects. In this respect, TRAIL delivery has been shown to promote anti-atherosclerotic activity in diabetic mice, while TRAIL-deficient mice also developed accelerated medial VC (di Bartolo et al., 2013). Furthermore, TRAIL deficiency has also been associated with increased atherosclerotic plaque size in Apolipoprotein E(ApoE)^-/- mice (Watt et al., 2011), and Secchiero et al. (2006) also found that TRAIL reduced plaque mass and increased macrophage apoptosis within atherosclerotic lesions. Furthermore, TRAIL has been shown to exert anti-oxidant and anti-inflammatory effects on the endothelium via the modulation of endothelial nitric oxide synthase (eNOS) (Zauli et al., 2003), and endothelial survival and proliferation has also been observed in response to TRAIL treatment (Pritzker et al., 2004;

Secchiero et al., 2003). From a clinical perspective, decreased serum TRAIL levels correlate with acute cardiovascular events (including myocardial infarction, heart failure) and resulting mortality (Niessner et al., 2009; Secchiero et al., 2009; Volpato et al., 2011), potentially implicating lower circulating TRAIL with poorer prognoses.

Contrastingly, however, some competing theories point to a potential role for TRAIL as an inducer of calcification. In this respect, elevated levels of TRAIL have been identified in vulnerable atherosclerotic plaque (Michowitz et al., 2005; Schoppet et al., 2004) and both TRAIL and its decoy receptor OPG have been identified at elevated levels alongside medial VC (Schoppet et al., 2004). There is also evidence that TRAIL induces apoptosis in endothelial cells (Li et al., 2003) and VSMCs (Sato et al., 2006) in vitro, yet TRAIL has also been shown

to exhibit anti-apoptotic activity in these cells under certain conditions (Kavurma et al., 2008;

Secchiero et al., 2003; Secchiero et al., 2004). As such, the precise role(s) for TRAIL within the vasculature are as of yet unclear, and remain the focus of a complex debate within the field of vascular research.

As mentioned, OPG can also serve as a decoy receptor for TRAIL. Importantly, both TRAIL and RANKL compete for OPG with similar binding affinities, with OPG favouring the protein of greatest concentration, resulting in a competitive effect between the two ligands (Vitovski et al., 2007). With excessive TRAIL levels, OPG:RANKL binding is reduced, allowing unopposed RANKL activity to ensue (and vice versa) (Figure 1.6). Therefore, the concentration of all three ligands are relevant to the ultimate biological effects within the vascular system, a fact which may be relevant in explaining the apparent contradictions in TRAIL function.

Overall, there is evidence to suggest that TRAIL has substantive yet diverse functional roles within the vasculature (which may be dependent on or independent of OPG/RANKL), but further research is necessary to confirm if these effects are beneficial in the context of VC.

Figure 1.6. Interactions between OPG, RANKL and TRAIL in the vasculature. (i) OPG is in excess of RANKL, preventing RANKL:RANK binding. (ii) OPG is in excess of TRAIL, preventing TRAIL binding to its receptors. (iii) RANKL is in excess of OPG, thus excess RANKL can bind to RANK receptors. There is no OPG available to prevent TRAIL binding to its receptors. (iv) TRAIL is in excess of OPG, thus excess TRAIL can bind to receptors. There is no OPG available to prevent RANKL binding to its receptors. OPG, osteoprotegerin, RANK, receptor activator of nuclear factor kappa-B; RANKL, receptor activator of nuclear factor kappa-B ligand; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.