Non-canonical WNT signalling in cardiovascular disease: mechanisms and therapeutic implications

Abstract

WNT signalling comprises a diverse spectrum of receptor-mediated pathways activated by a large family of WNT ligands and influencing fundamental biological processes. WNT signalling includes the β-catenin canonical pathway and the non-canonical pathways, namely the planar cell polarity and the calcium-dependent pathways. Advances over the past decade have linked non-canonical WNT signalling with key mechanisms of atherosclerosis, including oxidative stress, endothelial dysfunction, macrophage activation and vascular smooth muscle cell phenotype regulation. In addition, non-canonical WNT signalling is involved in crucial aspects of myocardial biology, from fibrosis to hypertrophy and oxidative stress. Importantly, non-canonical WNT signalling activation has complex effects in adipose tissue in the context of obesity, thereby potentially linking metabolic and vascular diseases. Tissue-specific targeting of non-canonical WNT signalling might be associated with substantial risks of off-target tumorigenesis, challenging its therapeutic potential. However, novel technologies, such as monoclonal antibodies, recombinant decoy receptors, tissue-specific gene silencing with small interfering RNAs and gene editing with CRISPR-Cas9, might enable more efficient therapeutic targeting of WNT signalling in the cardiovascular system. In this Review, we summarize the components of non-canonical WNT signalling, their links with the main mechanisms of atherosclerosis, heart failure and arrhythmias, and the rationale for targeting individual components of non-canonical WNT signalling for the treatment of cardiovascular disease.

Fig. 1. Overview of non-canonical WNT signalling pathways.

Non-canonical WNT signalling pathways involve the planar cell polarity pathway (part a) and the Ca²⁺-dependent pathway (part b). Both pathways are initiated by binding of a WNT ligand to WNT receptors, such as Frizzled (FZD) receptors, the tyrosine-protein kinase transmembrane receptors ROR1 and ROR2, and tyrosine-protein kinase RYK, which belong to the family of G protein-coupled receptors (also known as seven-transmembrane receptors), with the potential contribution of various co-receptors. Binding of WNT ligands to secreted FZD-related proteins (SFRPs) blocks the WNT–receptor interaction. The downstream events involved in the planar cell polarity pathway are not well defined, but include Dishevelled (DVL) and lead to GTP-dependent activation of small GTPases, such as RAC1 and RHOA, which in turn activate JUN N-terminal kinase (JNK), and ultimately regulate cell polarity and motility and gene transcription. The Ca²⁺-dependent pathway involves activation of PLC and protein kinase C (PKC), which leads to increased intracellular Ca²⁺ concentration, triggering the activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and the nuclear factor of activated T cells (NFAT) pathway, leading to transcriptional regulation.

Fig. 2. Non-canonical WNT signalling in atherosclerosis.

Non-canonical WNT ligands, such as WNT5A, can enter the vascular wall from the circulation and are also released by vascular macrophages and the perivascular adipose tissue (PVAT). In endothelial cells, binding of non-canonical WNT ligands to their receptors leads to the upregulation of the expression of intracellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), which promotes monocyte recruitment to the arterial intima. Non-canonical WNT signalling in endothelial cells also induces the activation of NADPH oxidases (NOX), which leads to increased production of reactive oxygen species (ROS), activation of pro-inflammatory redox signalling, reduced nitric oxide (NO) bioavailability and endothelial dysfunction. In monocytes and macrophages, non-canonical WNT signalling promotes pro-inflammatory activation, uptake of oxidized LDL (oxLDL) and foam cell formation, and production of pro-inflammatory cytokines such as C-C motif ligand 2 (CCL2), IL-1β and IL-6. In vascular smooth muscle cells (VSMCs), non-canonical WNT signalling induces a switch from a contractile to a synthetic phenotype and increases cell migration via ROS signalling, thereby promoting the migration of synthetic VSMCs into the atherosclerotic plaque, where they produce extracellular matrix (ECM) components and matrix metalloproteinases (MMPs). These mechanisms interact in complex ways to establish a vicious cycle, directly promoting atherogenesis and plaque instability.

Fig. 3. Non-canonical WNT signalling and bidirectional interactions between adipose tissue depots and the cardiovascular system.

Obesity, systemic insulin resistance and systemic inflammation induce systemic upregulation of non-canonical WNT ligands, partly via upregulation of WNT secretion from adipose tissue depots. WNT ligands can reach the heart and blood vessels via the systemic circulation and from adjacent adipose tissue depots such as epicardial adipose tissue and perivascular adipose tissue, respectively. Regardless of the source, WNT ligands exert multiple effects on the cardiovascular system, including the induction of reactive oxygen species (ROS), myocardial remodelling, endothelial dysfunction, vascular smooth muscle cell (VSMC) migration and phenotypic switch, and inflammation. These changes in turn stimulate the release of signals that influence WNT expression (red arrows) in epicardial adipose tissue and perivascular adipose tissue, thereby establishing potential paracrine interaction loops between the cardiovascular system and adipose tissues.

Fig. 4. Non-canonical WNT signalling in myocardial disease.

Summary of established and putative mechanisms linking non-canonical WNT signalling and myocardial disease. In the myocardium, sources of non-canonical WNT ligands, such as WNT5A, include cardiomyocytes, the microcirculation, blood in the cardiac cavities and the adjacent epicardial adipose tissue (red arrows). Non-canonical WNT signalling can stimulate cardiac fibroblasts, inducing the upregulation of expression of IL-6, transforming growth factor-β (TGFβ), ERK1 and ERK2, and tissue inhibitor of metalloproteinase 1 (TIMP1), thereby potentially promoting fibrosis and inflammation. Non-canonical WNT signalling can induce cardiac hypertrophy via JUN N-terminal kinase (JNK) activation. Non-canonical WNT signalling can promote oxidative stress through activation of NADPH oxidases mediated by the small GTPase RAC1. WNT might be linked to mitochondrial biology, regulating mitochondrial aggregation and the production of mitochondrial reactive oxygen species (ROS), although the direction of this interaction is unclear. WNT can also regulate desmosome and ion channel function, potentially promoting arrhythmogenesis. WNT secreted by epicardial adipose tissue can cause adipose tissue expansion and myocardial fatty infiltration, facilitating the development of re-entrant circuits and arrhythmias such as atrial fibrillation. ECM, extracellular matrix.

Fig. 5. Potential strategies for therapeutic targeting of non-canonical WNT signalling.

Modulating WNT effects can be achieved by targeting the main sources of WNT ligands, such as adipose tissue, with the use of tissue-specific knockdown technologies, for example, antisense oligonucleotides (ASOs) and CRISPR–Cas9 methods. However, these approaches require considerable research before their eventual translation into the clinic. The balance between different circulating WNT ligands can be targeted by using recombinant decoy receptors of WNT ligands (such as secreted frizzled-related proteins (SFRPs)) or monoclonal antibodies against WNT ligands. Overall, targeting of WNT signalling systemically might be associated with off-target adverse effects. WNT signalling can also be targeted by blocking or decreasing WNT receptors, with the added theoretical benefit of tissue specificity when using knockdown methods. Finally, downstream signalling could be targeted by developing chemical inhibitors or knockdown technologies. Targeting individual downstream molecules instead of the WNT ligands would provide a theoretical benefit of more specifically targeting individual subphenotypes caused by WNT signalling activation. Given that obesity is associated with increased WNT bioavailability, behavioural interventions, such as interventions to induce weight loss, and the use of medications with known metabolic effects might have pleiotropic effects on WNT signalling although the effect of these approaches on WNT signalling is unclear. CaMKII, Calcium/calmodulin-dependent protein kinase II; DVL, Dishevelled; FZD, Frizzled; JNK, JUN N-terminal kinase; NFAT, nuclear factor of activated T cells; PKC, protein kinase C; ROR, tyrosine-protein kinase transmembrane receptor ROR; RYK, tyrosine-protein kinase RYK.