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.
Cardiovascular disease is a major cause of morbidity and mortality worldwide, prompting the need for a better understanding of the underlying pathogenic mechanisms.
Non-canonical WNT signalling involves an evolutionarily conserved and ubiquitous range of pathways affecting fundamental processes such as inflammation, metabolism, cell motility, oxidative stress and homeostasis.
Non-canonical WNT signalling is a promising target in vascular disease, influencing vascular oxidative stress, endothelial dysfunction, inflammation, vascular smooth muscle cell phenotypes and cellular insulin resistance, all of which can affect atherosclerosis progression and plaque stability.
Non-canonical WNT signalling has putative links to cardiac disease by influencing myocardial oxidative stress, inflammation, repair capacity, energetics and remodelling, including fibrotic or adipose infiltration of the myocardium, all of which can generate the substrate for contractile dysfunction and arrhythmogenic potential.
Non-canonical WNT ligands are secreted by adipose tissue and are upregulated in obesity, acting as an endocrine and paracrine link between obesity and cardiovascular complications via adipose tissue–cardiovascular system crosstalk.
Non-canonical WNT signalling can be therapeutically targeted at multiple levels and could involve several state-of-the-art technologies; however, more research in this area is required.
Cardiovascular diseases, including atherosclerosis and myocardial disease, remain the leading cause of mortality worldwide1. Atherosclerosis is an inflammatory disease2,3,4 influenced by genetic and demographic risk factors5,6,7 and is associated with complex phenotypic changes in endothelial cells, vascular smooth muscle cells (VSMCs) and macrophages8,9. Myocardial disease is often associated with the presence of coronary atherosclerosis and involves processes such as myocardial oxidative stress10,11, fibrosis and remodelling12, which can lead to diseases such as heart failure and arrhythmias11,13. Despite substantial advances over the past decade, there is an unmet need to describe cardiovascular disease pathogenesis more accurately.
The WNT ligand family includes several secreted glycoproteins (19 in mammals) that activate an evolutionarily conserved spectrum of signalling pathways14,15. WNT signalling is implicated in embryogenesis and development via the regulation of cell motility and cell polarization. WNT signalling also regulates cell survival, growth and motility and has therefore been pathophysiologically linked with tumorigenesis14,16 and cellular metabolism17. WNT signalling can be classified into two main modes of signalling: the canonical pathway, which was described first and involves a reduction in β-catenin degradation followed by the induction of the expression of β-catenin target genes, and the non-canonical pathway, which involves β-catenin-independent mechanisms such as Ca2+ signalling and activation of small GTPases18. WNT ligands have varying degrees of selectivity towards the two pathways and can activate either of the two pathways depending on spatiotemporal parameters and receptor availability19. Certain WNT ligands, such as WNT5A and WNT11, seem to predominantly activate non-canonical WNT signalling15. Non-canonical WNT ligands have been implicated in multiple biological processes such as inflammation, cell motility and metabolism14. WNT5A and WNT11 have even been proposed as markers of respiratory distress syndrome severity in patients with coronavirus disease 2019 (COVID-19)20, which might be related to the capacity of these ligands to regulate cell migration, survival and apoptosis in lung injury. Additionally, non-canonical WNT signalling has emerged as a potential regulator of vascular disease pathogenesis via mechanistic links with inflammation, cell motility and oxidative stress21,22,23. Moreover, activation of non-canonical WNT signalling might be increased in obesity via the increased secretion of non-canonical WNT ligands, such as WNT5A, by adipose tissue15.
In this Review, we explore the complex roles of non-canonical WNT signalling in cardiovascular disease. First, we provide an overview of WNT signalling, followed by a comprehensive description of the role of non-canonical WNT signalling in cardiovascular disease pathogenesis. Finally, we explore the clinical potential of targeting non-canonical WNT signalling to treat atherosclerosis.
WNT pathways and molecular targets
The prototype WNT signalling, currently referred to as canonical WNT signalling, was first described in Drosophila, where it was shown to be involved in wing formation by signalling mediated via the glycoprotein product of the Wg gene24. This gene was later found to be homologous to the mammalian gene INT1; therefore, the nomenclature for this family of Wg/INT1 genes fused and became WNT1, and more WNT ligands have been described since14,25. In mammals, the WNT family of ligands consists of 19 glycoproteins (Table 1) that undergo a variety of post-translational modifications, including glycosylation and palmitoleic acid modifications15,25. As a result of these modifications, WNT ligands have moderate water solubility, which facilitates rapid protein–protein interactions and the propagation of paracrine signalling25.
WNT ligands are ubiquitously secreted molecules without exclusive tissue sources. Pathophysiologically important sources include adipose tissue15 and immune cells23. WNT ligands are also secreted by cardiovascular cells, including cardiomyocytes and the endothelium, although the baseline expression of WNT ligands in vascular cells is lower than in adipose tissue15,26. Nonetheless, the relative contribution of each tissue to the systemic WNT ligand pool has been poorly described. For example, WNT ligand release is a complex process involving post-translational modifications and vesicle secretion, and the upstream stimuli are unclear27. WNT ligands are often constitutively expressed and certain processes, such as inflammation and obesity, can upregulate their expression via mediators such as adiponectin28.
WNT signalling is initiated after the binding of extracellular, secreted WNT ligands to various membrane receptors, mainly the Frizzled (FZD) family of G protein-coupled, seven-transmembrane receptors14. The tyrosine-protein kinase transmembrane receptors ROR1 and ROR2 and the tyrosine-protein kinase RYK have also been shown to interact with WNT ligands but these interactions are less well characterized than WNT–FZD interactions29. The WNT ligand–receptor interaction triggers an incompletely characterized chain of molecular events involving downstream protein interactions that lead to transcriptional regulation14,25. To date, two main pathways downstream of the WNT–receptor interaction have been described: the canonical WNT pathway and the non-canonical WNT pathway29. The canonical pathway prevents the degradation of β-catenin, thereby allowing β-catenin-dependent transcriptional regulation to occur, which affects cell proliferation and survival30. The non-canonical pathway involves β-catenin-independent downstream signalling and can be broadly divided into the planar cell polarity (PCP) pathway and the Ca2+-dependent pathway29. WNT signalling is negatively regulated by the interaction of WNT ligands with secreted FZD-related proteins (SFRP1–SFRP5 in humans), which are structurally similar to FZD receptors and thus have affinity to WNT ligands and act as decoy receptors29.
Of note, WNT ligands and receptors comprise an extremely complex canvas of interrelated pathways, and classification into canonical and non-canonical pathways and ligands is virtual and schematic. In reality, most WNT ligands might be able to activate both canonical or non-canonical WNT signalling, with the primary effects on one of the pathways depending on context, spatiotemporal parameters and receptor availability19. Moreover, a negative interaction between the canonical and non-canonical pathways has been described, suggesting continuous crosstalk between the various pathways and ligands31,32.
Non-canonical WNT signalling
After the description of the canonical, β-catenin-dependent WNT signalling pathway, it became evident that certain WNT ligands exerted a wide range of biological effects in β-catenin-independent ways, namely the PCP and the Ca2+-dependent pathway33. These non-canonical pathways are not fully characterized; however, certain relevant downstream mediators and phenotypic consequences have been identified33 (Fig. 1).
The PCP pathway involves binding of a WNT ligand to FZD, ROR or RYK receptors33. This interaction leads to Dishevelled (DVL)-mediated GTP-dependent activation of small GTPases, such as RHOA and RAC1, although the exact intermediate events are not clear29. Activated RHOA and RAC1, in turn, stimulate the activation of JUN N-terminal kinase (JNK) via phosphorylation34. Given the involvement of RHOA and RAC1 in regulating cytoskeletal dynamics, changes in cell polarization and motility are the main phenotypes associated with activation of the PCP pathway33,35. In addition, WNT-mediated JNK activation has been linked with several other important pathophysiological processes such as inflammation and insulin resistance34,36. RAC1 can also influence oxidative stress via activation of NADPH oxidases15, indicating that oxidative stress can be regulated by the PCP pathway.
WNT ligands can also trigger β-catenin-independent effects via regulation of intracellular Ca2+ concentration33. Indeed, WNT signalling acts in synergy with phospholipase C to increase intracellular Ca2+ levels37, prompting activation of the Ca2+-sensitive kinases protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII)33. Ca2+-mediated WNT signalling activates the Ca2+-sensitive nuclear factor of activated T cells (NFAT) pathway33, which is important in immune response regulation38. Ca2+-dependent WNT signalling has mainly been explored in the context of embryonic development, and its clinical relevance in adults is less clear. However, considering the wide-ranging effects of its downstream targets PKC, CaMKII and NFAT, this mode of WNT signalling might contribute to several important biological processes35.
Importantly, non-canonical WNT signalling is negatively regulated by canonical WNT signalling. The extracellular domain of LDL receptor-related protein 6 (LRP6), a membrane co-receptor of the canonical WNT signalling pathway, has been shown to interact physically with WNT5A, acting as a decoy receptor for non-canonical WNT signals39. In vivo, Lrp6–/– mice have congenital defects, which are rescued by Wnt5a deletion39, suggesting that this LRP6-related phenotype is mediated by non-canonical WNT5A signalling. Similar defects were described in Xenopus embryos with knockdown of lrp5 or lrp6, which were reversed by knockdown of the non-canonical WNT ligands wnt5a and wnt11 (ref.39). WNT5A can activate or inhibit canonical WNT signalling in a spatiotemporal, tissue-specific manner40, which could be a result of spatially different receptor and related regulatory co-receptor profiles, similar to LRP5 and LRP6. Non-canonical WNT effects could therefore occur indirectly via changes in canonical WNT dynamics.
Non-canonical WNT signalling in vascular diseases
Non-canonical WNT signalling has been implicated in key contributory factors to atherosclerosis (Box 1), including oxidative stress, endothelial dysfunction, VSMC phenotypic switching and migration, and inflammation, as implied by observational association studies29 and mechanistic evidence15,23. Of note, WNT5A is the best-studied non-canonical WNT ligand in cardiovascular disease to date, followed by other ligands such as WNT11 (refs23,29). Therefore, most of the relevant studies used WNT5A as a representative ligand that primarily activates non-canonical WNT signalling in the cardiovascular system. Nevertheless, the downstream pathways described in these studies could be activated by any WNT ligand in the appropriate circumstances, and WNT5A itself can also activate canonical WNT signalling in vascular cells41,42.
One of the first pieces of conclusive evidence for a causal link between WNT signalling and atherosclerosis came from a study published in 2007 on the genetic basis of the risk of early coronary artery disease (CAD) in a family with a high prevalence of CAD43. Genome-wide linkage analysis followed by direct DNA sequencing of the annotated genes and in vitro functional assessments demonstrated a link between the missense mutation R611C in LRP6 and the early CAD phenotype43, thereby causally linking WNT signalling with vascular disease. Further research has explored the mechanisms underlying this link, particularly regarding non-canonical WNT signalling.
Observational data indicate that high levels of circulating WNT5A are associated with the presence of atherosclerosis and related diseases such as obesity and diabetes mellitus15,23,44. Work from our group has demonstrated that, in humans, CAD is associated with elevated WNT5A bioavailability in the plasma independently of demographic risk factors15. In addition, in patients who underwent two CT scans for coronary artery calcium score quantification, plasma WNT5A levels were positively associated with calcified plaque burden and new-onset calcification, independent of traditional risk factors15. Furthermore, WNT5A is locally expressed in mouse and human atherosclerotic plaques21. Levels of the non-canonical ligands WNT5A, WNT5B and WNT11 are also upregulated in human aortic calcified valves compared with non-calcified valves45. Given the link between these ligands and non-canonical activation of osteogenesis-related pathways in a variety of in vitro models46, non-canonical WNT might be hypothesized to contribute to valve or vascular wall calcification.
The aforementioned observational findings provide strong proof of concept for a link between non-canonical WNT signalling (mainly WNT5A) and atherosclerosis47 but observational results do not confirm causality. However, consistent with the observational findings, several experimental studies have revealed underlying cellular hubs through which non-canonical WNT signalling causally interacts with key mechanisms of atherosclerosis (Fig. 2).
WNT5A signalling increases the production of reactive oxygen species (ROS) in the human granulosa-like tumour cell line KGN, which was speculated to be related to the induction of inflammation considering that lipopolysaccharide stimulation had the same effect on ROS production in these cells36. Despite the incomplete mechanistic data, this observation supports the notion that WNT5A might directly increase ROS production. By contrast, WNT5A has been shown to protect against oxidative stress-induced VSMC apoptosis, although this effect was achieved with a supraphysiological concentration of WNT5A that cross-activated the canonical pathway42. Therefore, this finding should be regarded as being mediated by canonical WNT signalling rather than a specific WNT5A-mediated effect.
Work from our group was the first to directly explore the role of WNT5A in the regulation of vascular oxidative stress in the context of atherosclerosis15. After demonstrating the specificity of physiological WNT5A concentrations towards non-canonical signalling, we showed that WNT5A directly increased NADPH oxidase activity in vitro in arteries from patients with CAD15. This effect was mediated via increased GTP-dependent activation and membrane translocation of RAC1 (which is both a downstream target of the PCP pathway and a key subunit of the NADPH oxidase isoforms NOX1 and NOX2)15. This finding was further validated in vitro in WNT5A-treated primary VSMCs from patients with CAD as well as in aortic segments from mice overexpressing WNT5A15. Importantly, transcriptome analysis of WNT5A-treated human primary VSMCs revealed that WNT5A induces the differential expression of a large number of genes, and the expression profile of several of these genes was restored by administration of pegylated superoxide dismutase, suggesting that WNT5A is involved in redox-dependent transcriptional regulation in human vascular tissue15. Finally, we identified the ubiquitinating enzyme USP17 as a redox-sensitive downstream target of WNT5A that increases the GTP-dependent activation of RAC1 (ref.15). This study was the first to establish a causal role of WNT5A in increasing oxidative stress in human atherosclerosis and identifies USP17 and RAC1 as the key mediators of WNT5A-induced oxidative stress.
In contrast to our findings, other work suggests that WNT5A inhibits hydrogen peroxide (H2O2)-induced apoptosis in VSMCs via CCN family member 4 (also known as WISP1), an effect mediated through canonical WNT signalling42. This finding could suggest that WNT5A has an antioxidant function; however, WNT5A was used at a supraphysiological concentration in this study, which might mean that this canonical WNT signalling effect on H2O2-induced apoptosis might not be physiological. Conversely, H2O2 is a more stable type of ROS than superoxide and has subtler signalling roles42. As such, WNT5A-mediated signalling might theoretically stimulate the production of both superoxide (which could be detrimental when in excess) and H2O2 (which could provide a more prolonged signalling effect), hinting towards a delicate WNT5A-regulated redox balance, which warrants further investigation.
Given that the activation of endothelial nitric oxide synthase (eNOS) is partly mediated by CaMKII48 and that both PKC and CaMKII interact with NADPH oxidases49,50,51, it is highly plausible that non-canonical WNT Ca2+ signalling also regulates vascular oxidative stress and thereby contributes to atherosclerosis. However, this hypothesis remains to be studied.
Although canonical WNT signalling has been linked to endothelial cell survival and angiogenesis52, the role of non-canonical WNT signalling in endothelial cells has been explored only during the past decade. A study in primary endothelial cells from patients with diabetes revealed that WNT5A signalling impairs AKT phosphorylation, eNOS activity, nitric oxide (NO) bioavailability and, ultimately, endothelial function in vitro, mediated by non-canonical JNK-mediated signalling53. Consistent with this finding, our group demonstrated that WNT5A-mediated signalling directly impairs NO bioavailability and endothelial function, evidenced by ex vivo endothelium-dependent vasorelaxation of WNT5A-incubated artery samples from patients with CAD15. This reduced NO bioavailability was secondary to NADPH oxidase activation by WNT5A, as explained in previous sections, which led to oxidative depletion of tetrahydrobiopterin15. The absence of tetrahydrobiopterin resulted in eNOS uncoupling and production of superoxide instead of NO15.
These findings provide proof of the causal role of WNT5A in propagating a dysfunctional endothelial phenotype in humans. However, the underlying mechanisms for this role have not been fully explored. In addition, as mentioned in the previous section, Ca2+-dependent WNT signalling potentially affects both eNOS coupling and activity via CaMKII and PKC48,54,55,56 and could, therefore, be hypothesized to contribute to endothelial dysfunction, which warrants further investigation.
VSMC function and phenotype
Cell motility, migration and polarity are the best-studied phenotypes regulated by non-canonical WNT signalling, which has been demonstrated in a variety of embryonic development models and studies of tumours or cell types such as erythrocytes23,57,58,59. Canonical WNT signalling has been linked to VSMC biology, particularly cell migration42,60,61. However, evidence also suggests roles for non-canonical signalling in VSMCs.
After the demonstration of a link between the LPR6 R611C variant and CAD, further research has shed light on the underlying mechanisms with the use of VSMC in vitro assays and transgenic mouse models62. Indeed, loss of normal LRP6 activity as a result of the R611C variant is associated with increased non-canonical WNT signalling and is evidenced by increased RHOA and JNK activity62. The activation of non-canonical WNT signalling resulted in the activation of serine/threonine-protein kinase NLK followed by phosphorylation and subsequent ubiquitylation and degradation of transcription factor 7-like 2, a transcription factor that is associated with canonical WNT signalling62. These signalling events were associated with a phenotypic switch in VSMCs from a contractile to a synthetic phenotype, arterial media thickening and CAD promotion in mice62. Exogenous treatment with the canonical ligand WNT3A reversed the effects of the LRP6-R611C variant62. This finding is an elegant example of the crosstalk between the canonical and non-canonical pathways, with reciprocal effects on VSMC phenotypes.
Our group has shown that WNT5A induces redox-sensitive migration of human primary VSMCs without affecting their proliferation15. This effect could be partly mediated by RAC1 activation, but transcriptome analysis showed that WNT5A-mediated signalling also affected the expression of multiple genes related to migration in primary human VSMCs15. In addition, WNT5A can inhibit oxidative stress-induced apoptosis of VSMCs via the induction of WISP1 (ref.42). However, this effect was shown to be mediated by β-catenin signalling42 and might not be relevant in vivo given that WNT5A, at physiological concentrations, is a selective ligand for non-canonical WNT signalling. Another non-canonical WNT ligand, WNT4, has been shown to stimulate VSMC proliferation, whereas its downregulation attenuates intima–media thickening in mice63.
Beyond its effects on VSMC migration, non-canonical WNT signalling has been shown to induce a phenotypic switch in human primary VSMCs characterized by the loss of expression of the contractile markers aortic smooth muscle actin (also known as α2-SMA) and transgrelin, and an increased expression of matrix metalloproteinase 9 (MMP9)15. MMP9 has been shown to induce destabilization of atherosclerotic plaques in mouse models of atherosclerosis and in human genetics studies64,65,66,67. Taken together, these findings suggest that non-canonical WNT signalling might promote an ‘aggressive’ phenotype in VSMCs, characterized by an increased propensity for migration, the switch to a less differentiated phenotype and the upregulation of potentially plaque-destabilizing MMPs. WNT5A has been reported to be among the WNT ligands expressed by VSMCs isolated from artery samples from patients undergoing coronary artery bypass graft surgery, which might be related to neointima formation68.
Non-canonical WNT signalling can also influence atherosclerotic plaque calcification by inducing changes in VSMC biology. WNT5A was found to stimulate the expression of genes related to chondrogenesis in both mouse and human VSMCs69. WNT5A-mediated gene expression was inhibited by peroxisome proliferator-activated receptor-γ (PPARγ) signalling via SFRP2, suggesting that WNT5A promotes vascular calcification69. In addition, a correlation has been observed between the WNT5A–ROR2 pathway and the degree of VSMC calcification in vitro70. Canonical WNT signalling has also been shown to regulate vascular calcification in mouse aorta via bone morphogenetic protein 2 signalling46. Interestingly, co-expression of the non-canonical WNT receptor FZD10 and the canonical co-receptors LRP5 or LRP6 in VSMCs leads to cross-inhibitory signals between the two pathways71. By contrast, LPR5 or LRP6 loss of function increased non-canonical WNT signalling and inhibited canonical β-catenin signalling, which was associated with a shift towards osteochondrogenic programming and calcification in VSMCs71. These findings suggest a competition between the canonical and non-canonical pathways with regard to VSMC calcification, with LRP5 and LRP6 at the core.
Non-canonical WNT signalling can affect intracellular cholesterol accumulation. In an Apoe–/– mouse model of atherosclerosis, in vivo adenovirus-mediated delivery to aortic tissues of small interfering RNA targeting Wnt5a reduced atherosclerotic plaque lipid content without affecting blood lipid levels36. WNT5A has also been suggested to facilitate foam cell formation23. By contrast, another in vitro study indicated that WNT5A reduces intracellular cholesterol in VSMCs treated with oxidized LDL mediated by stimulating the expression of ATP-binding cassette transporter 1 (ABCA1)72, which is involved in reverse cholesterol transport. Overall, non-canonical WNT signalling seems to be involved in lipid handling in atherosclerotic plaque cells, but the exact effects and the mechanisms involved are unclear.
Non-canonical WNT signalling might also affect neoangiogenesis. In a rat model of ischaemic myocardial injury, peri-infarct injection of conditioned medium from WNT11-overexpressing mesenchymal stem cells improved cardiac function and reduced infarct size, which was shown to involve non-canonical JNK–PKC signalling73. By contrast, mutations in Wnt5a or Wnt11 in myeloid cells were associated with increased angiogenesis in mouse retinas mediated by downstream suppression of Flt1 expression74, which encodes an inhibitor of vascular endothelial growth factor (VEGF). These findings suggest potentially complex roles for non-canonical WNT signalling in the regulation of angiogenesis.
Extensive evidence links non-canonical WNT signalling to inflammation and inflammatory conditions such as rheumatoid arthritis23,75, psoriasis75,76,77 and atherosclerosis23. Indeed, non-canonical WNT signalling influences key mechanisms of vascular inflammation via multiple effects on vascular wall cells, thereby contributing to atherosclerosis23.
WNT5A (but not WNT3A, a canonical WNT ligand) was shown to increase the expression of genes encoding pro-inflammatory factors, including cyclooxygenase 2, IL-6, IL-1α, Toll-like receptor 4, granulocyte colony-stimulating factor, granulocyte–macrophage colony-stimulating factor (GM-CSF), CC-chemokine ligand 2 (CCL2) and CCL8, in human aortic endothelial cells in vitro via non-canonical Ca2+–PKC signalling22. These expression changes were associated with increased permeability of the endothelial cell monolayer22. WNT5A also activates pro-inflammatory nuclear factor-κB (NF-κB) signalling in endothelial cells22,36. Interestingly, activation of the Ca2+–NFAT pathway, a downstream target of non-canonical WNT signalling, has been shown to induce a pro-inflammatory phenotype in human coronary artery endothelial cells in vitro that is characterized by an increased expression of pro-inflammatory molecules78. However, a direct link between non-canonical WNT signalling and NFAT signalling in endothelial cells has not yet been documented. By contrast, cardiac-specific overexpression of WNT11 in mice attenuated the inflammatory response to myocardial infarction and facilitated recovery after myocardial infarction in vivo79. This finding suggests an anti-inflammatory role for non-canonical WNT signalling in this context.
WNT5A can be secreted by circulating monocytes59 and is involved in innate responses in monocytes and macrophages as evidenced by the upregulation of WNT5A gene expression in pathogen-activated macrophages and during monocyte differentiation into macrophages in response to GM-CSF and IL-4 treatment in vitro80,81,82. Importantly, pro-inflammatory and pro-oxidant signals, such as oxidized LDL, which are present in atherosclerotic plaques, upregulate the production of macrophage-derived WNT5A83. WNT5A in turn stimulates the secretion of pro-inflammatory cytokines (such as IL-1β, IL-6 and IL-8) from macrophages via Ca2+–CaMKII signalling and facilitates foam cell formation21,84,85. Evidence suggests that WNT5A promotes transforming growth factor-β (TGFβ)-mediated macrophage polarization, as demonstrated in the context of kidney fibrosis86. Extensive research in oncology suggests that WNT5A has a wide range of effects on macrophage activation such as in establishing an NF-κB autocrine loop87,88. However, these WNT5A effects have not been fully investigated in atherosclerosis.
Several observational and mechanistic studies in in vivo models of atherosclerosis have further supported a role for non-canonical WNT signalling in promoting a vascular pro-inflammatory phenotype. In an Apoe–/– mouse model of atherosclerosis, Wnt5a knockdown inhibited lipid accumulation and inflammation in atherosclerotic plaques, which was suggested to involve ROR2 non-canonical WNT signalling and downstream nuclear translocation of NF-κB89. Furthermore, circulating WNT5A levels are increased in a variety of inflammatory processes, such as sepsis, as shown in experimental models, further supporting a connection between non-canonical WNT signalling and inflammation23,75. Despite the available strong evidence, further research is required to elucidate the full spectrum of the effects of non-canonical WNT ligands on cytokine production, inflammatory cell recruitment and activation, and overall lipid-driven and cytokine-driven vascular inflammatory responses.
Cellular insulin resistance
Observational data show that increased bioavailability of WNT5A in the circulation and high WNT5A expression in adipose tissue are both associated with systemic insulin resistance (defined as glucose intolerance) as first demonstrated by a team led by Walsh, who were pioneers in the study of the metabolic and cardiovascular implications of non-canonical WNT signalling44,90,91,92. This association could be indirectly linked to atherosclerosis via the detrimental effects of hyperglycaemia on vascular function6. However, evidence suggests that WNT5A is associated with molecular insulin resistance in the vasculature, defined as abnormal vascular insulin signalling34,53. Indeed, ex vivo insulin-mediated vasorelaxation of arterioles from visceral adipose tissue isolated from individuals with obesity was impaired compared with that of arterioles from subcutaneous adipose tissue from the same individuals34. JNK activation and Wnt5a expression was increased in visceral adipose tissue compared with subcutaneous fat34. Furthermore, in endothelial cells isolated from adipose tissue, treatment with recombinant WNT5A stimulated JNK activation and induced insulin resistance, demonstrated by a reduction in downstream AKT and eNOS phosphorylation34. Consistently, the capacity of WNT5A to abolish insulin-induced AKT and eNOS phosphorylation and NO production has been replicated in studies using primary endothelial cells from patients with diabetes53.
Given the well-described link between JNK activity and molecular insulin resistance93, the link between non-canonical WNT signalling, particularly the PCP pathway, and induction of vascular insulin resistance is not surprising. Inflammatory factors, such as NF-κB and tumour necrosis factor, which are induced by non-canonical WNT signalling, can also interfere with molecular insulin signalling94,95,96,97, providing indirect links between non-canonical WNT signalling and vascular insulin resistance. Finally, Ca2+–PKC non-canonical WNT signalling might also contribute to vascular insulin resistance given that certain PKC isoforms directly induce cellular insulin resistance6.
Adipose tissue secretome
Adipose tissue is a dynamic organ that interacts with the vascular wall in paracrine and endocrine manners via the secretion of biologically active molecules5,98,99. Metabolic diseases, such as visceral obesity and diabetes, are associated with a pro-inflammatory phenotype of visceral adipose tissue, which secretes molecules that can directly reach the vascular wall via the bloodstream and exert biological effects100,101. Epicardial adipose tissue is a multifaceted, dynamic marker of cardiometabolic disease that is affected by pleiotropic pharmacological therapies and influences the heart in multiple paracrine ways98,102,103,104,105. Perivascular adipose tissue (PVAT) can exert paracrine effects on the vasculature owing to its close proximity to the vascular wall and can also act as a receiver of biological signals from the vascular wall, thereby establishing bidirectional crosstalk with the vasculature106. PVAT senses signals of adjacent coronary inflammation, changing its phenotype and lipid content107. This phenomenon can be captured by an imaging biomarker of coronary inflammation derived from coronary CT angiography, which has been standardized and is used in clinical practice107,108,109,110.
Our group has shown that WNT5A is the predominant WNT ligand expressed by PVAT surrounding internal mammary arteries in patients undergoing coronary artery bypass graft surgery15. WNT5A expression in PVAT is upregulated in the context of obesity and is independently associated with the activity of NADPH oxidases in the underlying vessels15. Furthermore, human primary VSMCs co-cultured with human adipocytes with WNT5A knockdown produced less NADPH oxidase-derived superoxide than VSMCs co-cultured with control adipocytes15. This finding confirms the concept that adipocyte-derived WNT5A might have paracrine pro-oxidant effects on vascular cells in humans. These findings are in agreement with a study reporting elevated levels of WNT5A and reduced levels of SFRP5 in the plasma of patients with peripheral occlusive arterial disease than in the plasma of healthy individuals111. Therefore, non-canonical WNT signalling, based on findings from the paradigm ligand WNT5A, is a novel paracrine link between obesity and vascular disease pathogenesis (Fig. 3).
Obesity and diabetes mellitus
Non-canonical WNT signalling has multiple broad roles related to the pathogenesis of metabolic diseases such as obesity and diabetes. These roles include multifaceted effects on adipose tissue, liver and pancreas, ranging from the regulation of energy storage and handling to adipogenesis and apoptosis91,112,113. Work by Fuster, Walsh and colleagues strongly suggests a causal association between obesity and increased bioavailability of WNT5A in the circulation and visceral adipose tissue in animal models and humans15,90,91,114. Furthermore, WNT5A overexpression in mouse myeloid cells augmented adipose tissue inflammation in vitro, and WNT5A directly induced JNK signalling and molecular insulin resistance in adipocytes of visceral adipose tissue in obese mice91. All these effects might influence obesity-related adipose tissue function and, indirectly, cardiovascular biology via regulation of the adipose tissue secretome.
Non-canonical WNT signalling influences adipose tissue biology in the context of obesity and diabetes in terms of adipose tissue volume, distribution, and secretome and, therefore, also influences the interactions between adipose tissue and the cardiovascular system. Non-canonical WNT signalling, mediated by WNT10B and WNT5A, is believed to have an anti-adipogenic effect via FZD and LRP receptors leading to reduced adipocyte size and also regulates the adipose tissue secretome and overall insulin sensitivity115. Non-canonical WNT signalling also induces adipose tissue inflammation independently of adipose tissue expansion91. In summary, non-canonical WNT signalling can contribute to the formation of small adipocytes, with low lipid content and increased inflammation, which are all hallmarks of visceral adipose tissue. We must note that WNT ligands create a complicated, cross-interacting canvas in adipose tissue, which makes deciphering the integrated effects extremely challenging and prone to inaccuracies115.
Non-canonical WNT signalling in cardiac diseases
Cardiac diseases, such as heart failure and arrhythmias, are caused by various pathogenic mechanisms such as arrhythmogenesis116, dysregulated cardiac biomechanics117, structural remodelling118, abnormal energetics119,120 and oxidative stress121. Observational evidence suggests that non-canonical WNT signalling is linked to cardiac diseases. For example, circulating levels of WNT5A were elevated in patients with heart failure compared with individuals without heart failure and were associated with haemodynamic markers of heart failure such as ejection fraction and filling pressures122. In a cohort of patients with dilated cardiomyopathy, higher plasma WNT5A levels were associated with increased right ventricular filling pressures and decreased right ventricular ejection fraction, and WNT5A expression was elevated in the right ventricle compared with the left ventricle123. Importantly, several experimental studies suggest that non-canonical WNT signalling is a causal regulator of cardiac disease (Fig. 4).
Arrhythmias are caused by abnormal electrical impulse generation or conduction116,124. Abnormal impulses arise from increased automaticity, early afterdepolarizations causing slow action potential repolarization, or late diastolic depolarizations caused by sarcoplasmic reticulum Ca2+ leakage124. Abnormal conduction includes accessory pathways and re-entry circuits (often caused by a structural substrate such as regional fibrosis)124. Triggers include myocardial ischaemia, electrolyte disorders, medications and genetic channelopathies116.
A large number of experimental studies have linked canonical WNT signalling to arrhythmogenic conditions such as arrhythmogenic cardiomyopathy, which is not surprising given the crucial role of β-catenin in the regulation of desmosomal intercellular junctions125. Furthermore, canonical WNT signalling regulates myocardial fibrosis, as shown in mouse models, thereby interfering with the formation of re-entry substrates such as in atrial fibrillation126,127. Both canonical β-catenin WNT signalling and non-canonical RHO-mediated WNT signalling have been associated with the pathophysiology of arrhythmogenic right ventricular cardiomyopathy (ARVC) in in silico models, which showed that the inactivation of the aforementioned pathways is linked with increased PPARγ expression and ARVC pathogenesis128.
By contrast, the mechanistic role of non-canonical WNT signalling in human arrhythmogenesis has not been adequately explored. In a study in patients with rheumatic valve disease undergoing valve surgery, the presence of atrial fibrillation was associated with elevated expression of the transcription factor SNAIL1 and several WNT ligands, including the non-canonical WNT ligands WNT5A and WNT11, in the right atrium129. WNT5A has been associated with the upregulation of SNAIL1 protein levels in melanoma cells via non-canonical PKC activation, which was associated with epithelial-to-mesenchymal transition (EMT) and metastasis potential130. Furthermore, WNT5A has been linked to TGFβ-dependent fibrosis and EMT in a variety of organs such as the liver126,131. Atrial fibrillation is tightly linked to processes such as EMT and fibrosis132. Therefore, these findings might imply a causal role for non-canonical WNT signalling in arrhythmogenesis by facilitating the development of re-entrant circuits.
Cardiac remodelling involves structural changes caused by myocardial wall stress (as a result of, for example, hypertension or valvular disease) and inflammation (such as after ischaemic myocardial injury)118. At the cellular level, remodelling is caused by cardiomyocyte hypertrophy and collagen deposition in the extracellular matrix caused by pro-inflammatory and redox signalling and metabolic signals118,121,133. These changes can induce arrhythmogenic substrates or impair contractile efficiency120.
Non-canonical WNT signalling might contribute to cardiac fibrosis. For example, WNT5A was shown to stimulate human primary cardiac fibroblasts in vitro, inducing the production of IL-6 and tissue inhibitor of metalloproteinase 1 (TIMP1) and the activation of ERK1/ERK2 signalling122. This finding suggests that WNT5A can potentially promote inflammation and fibrosis in vivo. WNT5A also induces a reduction in glycogen synthase kinase 3β (GSK3β) levels in human cardiac fibroblasts in vitro, which promoted fibrosis partly via transactivation of TGFβ, although this effect seemed to be predominantly mediated by canonical WNT signalling134. In human ventricular cardiomyocytes, activation of non-canonical, Ca2+-dependent WNT signalling stimulates the activation of the NFAT–calcineurin pathway123, which has been shown to contribute to cardiac fibrosis in experimental models.
Non-canonical WNT signalling has been shown to stimulate cardiomyocyte hypertrophy126. More specifically, WNT5A activated PCP signalling mediated by Dapper 1 and led to downstream activation of JNK to promote hypertrophy in human cardiomyocytes in vitro, as assessed by microscopy and cardiomyocyte surface area135. Interestingly, in a mouse model of left ventricular hypertrophy induced by aortic constriction, both neutrophil depletion and myeloid-specific knockdown of Wnt5a reduced neutrophil infiltration in the myocardium and cardiac hypertrophy136. This finding suggests that WNT5A might also regulate cardiac hypertrophy in indirect ways involving neutrophils and local inflammatory responses in the heart.
Fatty infiltration of the myocardium is found in a spectrum of myocardial disease phenotypes ranging from arrhythmogenesis to contractile dysfunction137. Although suppression of canonical WNT signalling has been linked to fatty infiltration of the myocardium, for example, in animal models of ARVC138, limited evidence exists on non-canonical WNT signalling and fatty remodelling of the myocardium. Non-canonical WNT signalling has been implicated in fatty infiltration in other organs139,140 such as in a mouse model of non-alcoholic fatty liver disease, which was rescued by canonical WNT3A signalling141.
Under physiological conditions, fatty acid oxidation is the major source of ATP in the heart, followed by glycolysis and lactate metabolism120. Different myocardial diseases are associated with different metabolic profiles but most eventually lead to ATP depletion, reducing contractile efficiency and altering a wide range of downstream signalling events142. Several types of myocardial dysfunction are characterized by a shift towards glycolysis and ketone body oxidation to meet the increasing energy demands of the failing heart under stress143. Medications such as sodium–glucose cotransporter 2 inhibitors might exert some of their beneficial effects by regulating ketone body metabolism, glycolysis substrate inflow and fatty acid oxidation144, which highlights the concept that metabolic pathway balance and fuel shifts might be crucial for modifying myocardial performance143.
Evidence suggests that WNT signalling mediates metabolic pathway reprogramming in a variety of cell types although this action mainly involves canonical signalling145,146. Non-canonical WNT signalling attenuates mitochondrial aggregation in the HEK93 cell line147 and protects mitochondria from fission–fusion alterations and prevents mitochondrial loss in neurons148, both via PKC and regulation of intracellular Ca2+ dynamics. Overexpression of the non-canonical WNT ligand WNT11 preserves mitochondrial membrane potential and protects cardiomyocytes against hypoxia in vitro via mechanisms involving insulin-like growth factor 1 and VEGF149. Non-canonical WNT signalling also accelerates glucose oxidative metabolism in the liver, which leads to steatosis via de novo lipogenesis150. Although these findings provide putative links to myocardial metabolism, the role of non-canonical WNT signalling in myocardial energetics has not been directly explored.
Oxidative stress has a key pathophysiological role in myocardial diseases such as in ischaemia–reperfusion injury after myocardial infarction151 and through redox signalling in heart failure and arrhythmia152. Oxidative stress results from ischaemia–reperfusion injury153, inflammation-mediated stimulation of NADPH oxidases121,154 and disturbed mitochondrial energetics120,155. Myocardial ROS, in turn, can induce hypertrophy, apoptosis, autophagy, metabolic enzyme dysregulation and impaired contractility, thereby drastically contributing to myocardial disease11,121,153,155.
Ischaemia–reperfusion injury is characterized by sudden oxygen abundance in a stunned myocardium, in which free radicals are produced because of an imbalance between pro-oxidant and antioxidant enzymes151,153. The excess free radicals induces cardiomyocyte death via multiple intracellular pathways, including proteolysis, caspase activation and mitochondrial regulation151,153. Co-expression of AKT1 and the non-canonical ligand WNT11 stimulates the proliferation and differentiation of mesenchymal stem cells into cardiomyocytes and attenuates hypoxia–reperfusion injury156. By contrast, a study in mice showed that blockade of non-canonical WNT signalling attenuates myocardial ischaemia–reperfusion injury157. NADPH oxidases have been implicated in atrial fibrillation and heart failure via a multitude of redox-sensitive intracellular transcription pathways154,158. Non-canonical WNT signalling is linked to NADPH oxidases via RAC1, as shown in VSMCs in vitro15. Whether these findings are relevant to the human myocardium remains to be proven.
Targeting non-canonical WNT signalling
The studies discussed above indicate a strong mechanistic link between non-canonical WNT signalling and cardiovascular disease. Therefore, successful targeting of this pathway could have multiple beneficial effects in patients, especially in the context of obesity, in which the bioavailability of the principal non-canonical WNT ligand, WNT5A, is increased15,23. Interestingly, the downstream mediators of non-canonical WNT signalling (PLC–CaMKII, RAC1, RHOA and JNK) are convergence points for multiple non-WNT pathways such as the catecholamine and renin–angiotensin–aldosterone pathways159,160,161. The exact contribution of WNT ligands to cardiovascular pathophysiology in this context is unknown. However, targeting WNT signalling might offer an advantage over targeting downstream molecules, which would also influence the outputs of multiple other, non-WNT pathways.
Non-canonical WNT signalling is a conserved pathway affecting virtually all cell types and governing fundamental biological processes18,162. Consequently, its targeting is prone to non-specific, off-target adverse effects (with tumorigenesis being the most important) and is challenging when embryonic development is relevant (for example, in pregnant women)18,162. The interconnected network of WNT ligands and receptors further complicates targeting a single ligand18,162.
So far, early attempts have mainly focused on targeting the canonical WNT signalling pathway, especially in the context of cancer18,162,163. By contrast, targeting of non-canonical WNT signalling has not been explored. Advances in biotechnology, gene editing and drug delivery, coupled with a better understanding of the downstream mediators of non-canonical WNT signalling, might help towards this end.
Targeting non-canonical WNT signalling might be theoretically achieved with the use of chemical inhibitors of, for example, SFRPs or RAC1 (refs15,18). The use of monoclonal antibodies, antisense oligonucleotides (ASOs) and gene-editing methods (such as CRISPR–Cas9) can allow more efficient targeting of non-canonical WNT signalling than previously thought164,165,166,167. More efficient drug delivery to target tissues might also be achieved with the use of nanoparticles164,168,169. The main challenge of using monoclonal antibodies is their systemic, whole-body delivery, posing the risk of substantial adverse effects resulting from systemic WNT inhibition in tissues outside of the cardiovascular system. This issue might be bypassed by using tissue-specific targeting of WNT elements with tools such as ASOs and CRISPR–Cas9 editing. However, these strategies are not clinically feasible at present and require extensive validation and optimization. Nanoparticle technology might be used to guide drugs and vectors to sites of interest via systemic or local administration of nanoparticles with affinity to particular molecules such as endothelial markers (for example, vascular cell adhesion molecules). This approach, in combination with molecular techniques (CRISPR–Cas9 or ASOs), could increase local efficacy and tissue specificity of WNT targeting, as demonstrated with the use of macrophage-specific, promoter-driven plasmids contained in lipid nanoparticles to target inflammatory cells such as macrophages170. The characteristics of these strategies are summarized in Fig. 5.
Non-canonical WNT signalling comprises a complex network of receptors and downstream molecules. Therefore, one could, in theory, interfere with non-canonical WNT signalling at different levels by using the techniques mentioned in the previous section. Importantly, the canonical and non-canonical pathways cross-regulate each other; therefore, any intervention on non-canonical WNT signalling is expected to also have effects on canonical WNT signalling162,171.
WNT ligands and SFRP inhibitors
Targeting non-canonical WNT ligands would be the most obvious way to inhibit all downstream signalling. However, simultaneous targeting of all non-canonical WNT ligands would not be feasible. WNT5A is a paradigm non-canonical WNT ligand, having been the focus of most research on non-canonical WNT signalling; therefore, WNT5A would seem to be an appropriate therapeutic target in this context15,63. Systemic WNT5A targeting (for example, with monoclonal antibodies or exogenously administered recombinant forms of SFRPs) might be the most obvious way forward, but this approach would pose the aforementioned risks of global inhibition. Considering the pathophysiological mechanisms evaluated in the previous sections, tissue-specific (for example, adipose tissue-specific) targeting would be more efficient, although still not feasible at present.
Downregulation of WNT receptors, such as FZD2 and FZD5, could markedly attenuate non-canonical WNT signalling. This strategy could be important given that FZD2 and FZD5 have been shown to be upregulated in internal mammary arteries from individuals with obesity, suggesting a higher sensitivity to WNT ligands15. Downregulation of WNT receptors could be achieved by ASO-mediated silencing or CRISPR–Cas9 gene editing, in which the use of tissue-specific promoters would be ideal. However, as mentioned, these strategies remain hypothetical at present.
Downstream signal transduction molecules
Non-canonical WNT signalling converges on a number of downstream molecules, including RAC1, JNK, CaMKII and PKC as well as USP17, a newly described WNT5A target15,52. Targeting of these downstream targets might reverse the detrimental effects of non-canonical WNT signalling. However, many non-WNT pathways also converge on these downstream molecules, which would make their targeting prone to non-specific effects.
Studies in mouse models of RAC1 depletion have reported beneficial effects, such as reduced endoplasmic reticulum stress and reduced cardiac oxidative stress172. In addition, several allosteric inhibitors of RAC1 have been developed. Several JNK inhibitors have been used in animal models of diseases such as neurodegeneration173 and in a phase Ib clinical trial in patients with pulmonary fibrosis174, supporting the clinical relevance of targeting JNK. Similarly, CaMKII inhibition in vivo is associated with decreased atherosclerotic plaque burden in Apoe–/– mice175. Administration of the CaMKII inhibitor KN93 in a mouse model of heart failure induced by pressure overload had beneficial effects176. PKC inhibition in vivo is more challenging given the large number of PKC isoforms6. Finally, USP17 is a newly identified link between WNT signalling, RAC1 activation and downstream redox signalling, which might warrant further investigation as a potential therapeutic target15.
Non-canonical WNT signalling, acting via receptor-mediated pathways and through activation of second messengers, such as RAC1, JNK, Ca2+-mediated CaMKII and PKC, is causally linked to vascular and myocardial disease in animal and human experimental models. In particular, non-canonical WNT signalling induces vascular oxidative stress via NADPH oxidase activation, promotes endothelial dysfunction and insulin resistance via JNK signalling and oxidative eNOS uncoupling, increases vascular inflammation in endothelial cells and macrophages, and triggers a VSMC contractile-to-synthetic phenotypic switch that might promote atherosclerotic plaque instability. Importantly, non-canonical WNT signalling is upregulated in adipose tissue (including PVAT) in obesity, exerting paracrine and endocrine vascular effects. Non-canonical WNT signalling has putative links to a wide spectrum of cardiac disease phenotypes via regulation of myocardial metabolism, fibrosis, and adipogenesis pathways and of redox signalling mediators such as NADPH oxidases and NF-κΒ, potentially contributing to myocardial remodelling, arrhythmogenic substrate formation and contractile dysfunction.
Non-canonical WNT signalling is therefore a multifaceted causal mediator of atherosclerosis and a link between obesity and vascular disease. Advances in biotechnology have opened up new potential approaches to modulate non-canonical WNT signalling through targeting specific tissues, proteins or genes related to WNT ligands, receptors or the downstream signalling network, all of which warrant further investigation.
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C.A. acknowledges support from the British Heart Foundation (CH/F/21/90009, TG/19/2/34831 and RG/F/21/110040), theOxford BHF Centre of Research Excellence (RE/18/3/34214) and the Oxford NIHR Biomedical Research Centre.
C.A. is founder, shareholder and director of Caristo Diagnostics, a CT image analysis company. The other authors declare no competing interests.
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Akoumianakis, I., Polkinghorne, M. & Antoniades, C. Non-canonical WNT signalling in cardiovascular disease: mechanisms and therapeutic implications. Nat Rev Cardiol (2022). https://doi.org/10.1038/s41569-022-00718-5