WNT Signaling in Cardiac and Vascular Disease

A. Synthesis and Posttranslational Modifications of WNT

WNTs are secreted proteins for which homologs have been detected in multicellular organisms ranging from sponges to vertebrates. They contain 22–24 conserved cysteine residues, responsible for the maintenance of the spatial structure by the formation of disulfide bonds. The 19 WNT genes identified in mammals range in molecular weight from 39–46 kDa and belong to 12 subfamilies: many WNT genes (including WNT2, -3, -5, -7, -8, -9, and -10) have shown duplications, giving rise to two closely related family members (e.g., WNT 3a and WNT 3b). Wnts can also be classified according to their function. A large group of Wnts is capable of inducing oncogenic transformation and duplication of the body axis in developing Xenopus embryos (WNT1, -3a, -7a, -7b, -8), whereas another group does not (e.g., WNT4, -5a, -6, and -11). This distinction is related to different signal transduction pathways that are activated by these WNTs, as will be further discussed in section V of this review (Croce and McClay, 2008). However, it has to be noted that not only the subtype of WNT protein, but also the FZD family member and probably also the coreceptors, are important in determining the choice of the signaling pathway (Schulte, 2015).

WNT proteins are subjected to extensive posttranslational modification to become secreted in a biologically active form. All WNT proteins carry a signal sequence that is required for their secretion (Langton et al., 2016). All WNT proteins except Drosophila WntD carry a palmitate group at a conserved cysteine residue located near the N terminus (Cys⁷⁷ in WNT3A) and a palmitoleate group at a conserved serine residue (Ser²⁰⁹ in WNT3A). Originally, the palmitate was thought to be necessary for receptor binding, internalization, and signaling, whereas the palmitoleate group was thought to be required for intracellular transport (Willert et al., 2003; Schulte, 2010; Harterink and Korswagen, 2012). However, after the resolution of the crystal structure of the xWnt8/Fzd₈ complex, WNT was shown to have a three-dimensional structure resembling a hand, with two extension resembling the thumb and index finger grasping the CRD of FZD. The palmitoleated Ser²⁰⁹ residue was found to be located at the tip of the so-called “thumb” region, which, together with the index finger, has been shown to be involved in the interaction of WNT proteins with receptors from the FZD family (Janda et al., 2012; Langton et al., 2016). Since the palmitated Cys⁷⁷ residue is not located at a distinctive region involved in receptor interaction, the role of its lipidation has become less clear. However, the addition of a hydrophobic group may help to concentrate active WNT proteins at the cell membrane, which may assist in activation of signaling (Kaemmerer and Gassler, 2016). The addition of the palmitoleate group to WNT proteins takes place in the endoplasmic reticulum by an enzyme called porcupine (PORCN). PORCN is a member of the membrane-associated O-acyl transferase enzymes, which promote the coupling between a palmitoleate and the conserved serine residue in the thumb region. Next to lipidations, WNT proteins can also be glycosylated at two specific sites (Lorenowicz and Korswagen, 2009). For WNT3A, this glycosylation was found to precede the palmitoyleation (Komekado et al., 2007). The glycosylation appears to be critical in controlling WNT folding and secretion, but is not essential for WNT signaling (Willert and Nusse, 2012).

Apart from its importance in binding to FZD, attaching a palmitoleate to the WNT protein also plays a crucial role in the secretion of WNT proteins because it stimulates the binding of WNTs to Wntless (WLS), a chaperone protein that escorts the palmitoleated WNT to the plasma membrane (Lorenowicz and Korswagen, 2009; Langton et al., 2016). As illustrated in Fig. 1, the binding of WNT to WLS occurs in the endoplasmic reticulum and is essential for the progression through the Golgi network. When reaching the plasma membrane, the WNT/WLS complex dissociates and WLS is recycled back to the Golgi apparatus by the retromer complex, which is responsible for endosome to Golgi trafficking (MacDonald et al., 2009; Harterink and Korswagen, 2012). Interestingly, WNT proteins have been shown to be released in a polarized fashion in epithelial cells, with different secretion profiles on the basal and apical sides of the cell (Lorenowicz and Korswagen, 2009; Langton et al., 2016). However, there is still debate whether secretion of WNT proteins from cells is actually necessary for their biologic activity, because WNTs have been found attached to cytonemes, actin-based cellular extensions that extend distances of several hundreds of micrometers (Stanganello and Scholpp, 2016).

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Fig. 1.

Synthesis, posttranslational modification, and exporting of WNT proteins. After translation of WNT proteins, a palmitoleate group is attached by Porcupine in the endoplasmic reticulum (ER). This promotes the binding to Wntless (WLS), a chaperone protein that facilitates the migration of WNT through the Golgi complex. When WNT has reached the plasma membrane, it can either be secreted in exosomes or be attached to lipoproteins or Swim proteins; this is required to shield the large lipophilic moiety and increase the water solubility of the WNT protein. WNT can also attach to the plasma membrane of the producing cell by forming a complex with heparin sulfate proteoglycan (HSPG). Upon delivery of WNT to the plasma membrane, WLS is recycled via the retromer complex to the Golgi apparatus.

Due to the attachment of the lipophilic groups, the solubility of WNT proteins in an aqueous environment is poor. Therefore, four mechanisms have been proposed for the secretion of WNT proteins into the extracellular compartment: 1) association with a lipoprotein particle such as lipophorin in Drosophila (Panakova et al., 2005); 2) binding to the secreted WNT-interacting molecule Swim, a member of the lipocalin family (Mulligan et al., 2012); 3) spreading of WNT over the plasma membrane with the lipid group inserted in the membrane (Stanganello et al., 2015); and 4) secretion of WNT proteins in exosomes (Gross et al., 2012). The first two mechanisms are thought to increase the water solubility by shielding the palmitoleate group, whereas in the latter two mechanisms this group is inserted in the hydrophobic membrane environment (Langton et al., 2016).

B. Frizzled Proteins

The name “frizzled” is derived from a Drosophila homolog of this gene, the mutants of which give rise to a distinctive derangement of the orientation of cutical wing hairs and bristles (Gubb and Garcia-Bellido, 1982). Up until now, 10 FZD homologs have been identified in mammals. On the basis of structural homology, they can be clustered in five subfamilies: FZD_1/2/7, FZD_5/8, FZD_9/10, FZD₄, and FZD_3/6; smoothened is a protein structurally related to the FZDs but no member of the FZD family. Major differences exist in the genetic structure of the different FZD genes with FZD₃ and FZD₆ having five introns in their coding region and FZD₄ having a single exon, whereas the other FZDs are intronless.

FZD proteins are a family of seven transmembrane (TM) receptors, meaning that there are seven helices of hydrophobic amino acids present in the protein allowing its embedding in a membranous structure. Although this feature is similar to many other members of the class of G protein-coupled receptors, the International Union of Basic and Clinical Pharmacology has categorized FZD proteins into a separate class, together with the smoothened protein, a member of the Hedgehog family (Foord et al., 2005). The reason for this classification is that FZDs have a unique extracellular domain at the N-terminal side, in which five disulfide bonds formed by 10 highly conserved Cys residues determine the three-dimensional shape. This so-called cysteine-rich domain (CRD) is thought to be the site of interaction with WNT proteins. Interestingly, similar CRDs are present in other non-seven transmembrane (7TM) proteins such as soluble frizzled-related proteins (sFRPs), the receptor tyrosine kinase-like orphan receptor (ROR), carboxypeptidase Z and a splice variant of collagen XVIII. For several of these proteins, interaction with WNT has actually been demonstrated (Xu and Nusse, 1998). Upstream of the CRD, the N-terminal part of the FZD proteins contains a signal sequence, necessary for transport to the plasma membrane, which is cleaved off in the active receptor protein. The CRD of the receptor is connected to the 7TM domain by means of a spacer of variable length (Schulte, 2010). On the intracellular side of the receptor, three loops and the free C-terminal end of the protein are present as in all G protein-coupled receptors. Although several domains important for G protein coupling still need to be defined (Gammons et al., 2016), there is increasing evidence that, among coupling to other signal transduction pathways, G protein signaling can actually be activated by FZD proteins, as discussed in more detail in section V.E of this review (Dijksterhuis et al., 2014). A remarkable feature of the C-terminal part of the FZDs is the fully conserved KTxxxW domain, located at the eighth helix (Gammons et al., 2016). Mutation experiments have shown that this domain is indispensable for interaction with the PDZ-domain of disheveled (DVL), a protein involved in most WNT/FZD signal transduction pathways (Schulte, 2010).

Targeted mutagenesis experiments have revealed that FZDs can show significant functional redundancy within their subfamily, so mice lacking a single FZD gene quite frequently show no clear phenotype. In the context of this review, we focus on the (combinations of) deletions that give rise to a cardiovascular phenotype; for a comprehensive overview of the phenotypes of all FZD knockouts, we refer the reader to the recent review by Wang et al. (2016a). When focusing on the FZD_1/2/7 subfamily, Fzd₁^−/− mice show no clear phenotype, whereas about half of the Fzd₂^−/− mice show a cleft palate and ±15% of the Fzd₇^−/− have a ventricular septal defect. Combining the different targeted mutants aggravates the frequency of the phenotypes: all Fzd₁^−/−;Fzd₂^−/− double knockouts were found to have a cleft palate and the majority of them had cardiac defects as well, ranging from a ventricular septal defect to a double outlet right ventricle (Yu et al., 2010). The combination of Fzd₂^−/−;Fzd₇^−/− was found to be embryonically lethal due to failure of closing its neural tube and failure to develop cardiac asymmetry, impairing cardiac function (Yu et al., 2012). These results demonstrate that adequate signaling via the FZD_1/2/7 subfamily is a prerequisite for a correct development of the heart.

Loss of FZD₄ has been shown to reduce vascular cell proliferation and migration. Moreover, tube formation by endothelial cells (ECs) is impaired, resulting in a diminished arterial network in heart and kidney (Descamps et al., 2012). A severe defect in retinal vascularization was observed in mice lacking Fzd₄ (Wang et al., 2012). This is associated with the progressive development of hearing loss and degeneration of the cerebellum, the latter caused by leakage of the blood-brain barrier in these mice (Wang et al., 2001). The combined findings stress the importance of proper FZD₄ signaling for the development and maintenance of vascular function, which will be further elaborated in section VII.C.

C. Lipoprotein-Related Receptor Protein 5/6

After the identification of FZD proteins as receptors for WNT, further research revealed that additional binding partners are required to activate WNT/β-catenin signaling. This led to the simultaneous publication by three independent research groups of two members of the low-density lipoprotein receptor family. These proteins are called low-density lipoprotein-related receptor (LRP) 5 and 6 and are homologs of Drosophila Arrow (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000). These receptor proteins are over 1600 amino acids in size and have a single TM domain. The bulky extracellular part of LRP5 and 6 has a modular structure consisting of four six-bladed β-propellers connected by EGF-like domains. A flexible hinge region between the two pairs of β-propellers allows for some bending of the protein, giving rise to a concave top surface of the extracellular domain (Chen et al., 2011; Cheng et al., 2011b). The β-propeller part is connected to the transmembrane region via three LDL type A repeats. Overexpression of the LRP6 intracellular domain can activate WNT signaling in a constitutive fashion, confirming the regulatory role of the extracellular domain in the control of the signal transduction (Niehrs and Shen, 2010).

The intracellular part of LRP5/6 contains multiple phosphorylation sites, the phosphorylation of which is a key step in the initiation of the signal transduction via WNT/β-catenin signaling. Several kinases have been found to phosphorylate LRP6, either in a WNT-inducible (G protein receptor kinase 5/6) or WNT-independent (protein kinase A, PFTAIRE protein kinase 1) way. For an overview, we refer to a review article on this topic (Niehrs and Shen, 2010).

WNT proteins can bind to LRP5/6 at two different sites, albeit with lower affinity than to FZD proteins (Tamai et al., 2000). The most N-terminal β-propeller can bind most WNT members, with the exception of WNT3 and WNT3A that bind to the third β-propeller. Several endogenous inhibitors of LRP5 and 6, including members of the dickkopf (DKK) family, SOST and WISE, were shown to interact with the first β-propeller, whereas DKK proteins also bind to the third β-propeller. Interestingly, antibodies specific for each of the two binding sites have been generated, allowing the specific blocking of each site (Joiner et al., 2013).

Mice lacking either the Lrp5 or -6 gene show distinct phenotypes. Inactivating mutations of Lrp5 led to low bone mass and abnormal retinal vascularization, combined with defects in cholesterol and glucose metabolism. Delays in mammary development and resistance to WNT1-induced mammary tumorigenesis have also been reported. Global deletion of Lrp6 led to postnatal lethality with skeletal, neurologic, and mammary gland defects and cardiac abnormalities including a double outlet right ventricle. Double mutants show abnormal posterior patterning of the epiblast. Several mutations in LRP5 and -6 are linked to human diseases, including osteoporosis pseudoglioma, Alzheimer’s disease and degenerative joint disease (Joiner et al., 2013). Similar to the phenotype of the FZD₄ mutant described above, mutations in LRP5 can lead to familial exudative vitreoretinopathy (Toomes et al., 2004). The R611C mutation in LRP6 was found to be associated with elevated serum lipids and glucose, hypertension, and premature coronary artery disease (Mani et al., 2007). Moreover, increased LRP5 levels have been associated with calcified aortic valves, a finding closely linked to the importance of WNT signaling in bone formation, as described in more detail in section VIII.B (Rajamannan, 2011).

D. Receptor Tyrosine Kinase-like Orphan Receptor 1/2 and Ryk

ROR1 and -2 were initially identified because of their layout showing similarity to tyrosine kinase receptors. These receptor proteins contain a single TM domain and the intracellular parts display kinase activity. Unlike other tyrosine kinase receptors, however, these receptor proteins were found to have an extracellular CRD domain that closely resembles the CRDs of FZD proteins (Saldanha et al., 1998). This suggests that WNT proteins serve as ligands for ROR1 and -2, which was indeed shown to be the case (Oishi et al., 2003). In the meantime, it has become clear that, in contrast to LRP5/6, ROR1 and -2 are mostly involved in β-catenin-independent WNT signaling (Green et al., 2014).

Ror2 null mice show skeletal defects, abnormal orientation of inner ear hairs, and abnormalities in lungs and ventricular septal defects (Takeuchi et al., 2000). The phenotype of Ror1-deficient mice is less severe, but deletion of Ror1 augments the phenotype of Ror2 deficiency and introduces additional defects such as transposition of the great arteries. This suggests a genetic interaction between Ror1 and -2 in cardiac and skeletal development (Nomi et al., 2001).

Ryk proteins also have a single membrane-spanning domain, like the ROR proteins, but contain an extracellular domain with homology to WNT inhibitory factor (WIF) rather than the CRD found in FZD and ROR proteins. Moreover, Ryk also deviates from RORs in that its intracellular domain has no enzymatic activity. This suggests that Ryk most likely functions as a coreceptor for WNTs together with FZD proteins (Green et al., 2014).

E. Interaction between WNT, FZD, and Their Coreceptors

A. WNT/Notum/Tiki

In addition to a complex intracellular machinery for the secretion of WNT proteins, there are several extracellular mechanisms controlling the activity of secreted WNT proteins. Membrane-associated glypicans (named Dally in Drosophila) can bind WNT proteins to the cell surface, regulating their extracellular distribution and signaling activity. Adenomatous polyposis coli downregulated 1 (APCDD1) has a similar mode of action and has been shown to inhibit WNT signaling in cell culture assays (Cruciat and Niehrs, 2013). More recently, WNT-specific metalloproteinases from the Tiki family have been described that can cleave the first 9–20 amino acids from amino-terminal side of WNT2B, -3, -3A, -5A, -5B, -6, -8A, -8B, and -16, thereby rendering them inactive (Zhang et al., 2016b). Finally, a carboxylesterase named Notum has been shown to enzymatically remove the palmitoleate from WNT proteins, which results in a loss of biologic activity (Kakugawa et al., 2015). The many mechanisms involved in the secretion and (in)activation of WNT proteins highlight the complexity and tight control of WNT activity.

B. Secreted Frizzled-Related Proteins

As the name implies, sFRPs resemble FZD proteins in that they contain a CRD at the N-terminal side of the protein that shows 30%–50% sequence similarity with the FZD CRD. The first sFRP, originally named SFRB, which later turned out to be sFRP3, was in fact discovered based on its homology to FZD proteins (Hoang et al., 1996). In humans, five different sFRPs have been identified varying in size from 295 to 346 amino acids (Cruciat and Niehrs, 2013). The CRD contains 10 conserved Cys residues; the disulfide bridges formed by these residues contribute to the tertiary structure of the CRD. At the C-terminal side of the protein, a Netrin-related motif is located containing six conserved Cys-residues that form three disulfide bridges (Chong et al., 2002). Netrin-related motifs are present in many unrelated proteins, including the axon-guidance molecule Netrin, tissue inhibitors of metalloproteinases (TIMPs), type-1 procollagen C-protein enhancer proteins, and complement proteins C3, C4, and C5 (Cruciat and Niehrs, 2013). The sFRPs can be classified into two subgroups based on phylogenetic analysis: the sFRP1, -2, and -5 subgroup and the sFRP3 and -4 subgroup (Jones and Jomary, 2002). Posttranslational modifications have been observed in sFRP1 and -5 (N-glycosylation and sulphation of two Tyr residues), but these are absent in sFRP2, -3, and -4 (Bovolenta et al., 2008).

The main mechanism of action of sFRPs is the inhibition of WNT signaling by scavenging extracellular WNT proteins, thereby preventing their interaction with the receptor complex. At first sight, the CRD region of the sFRPs appears to be responsible for this scavenging effect, as it can be anticipated to bind WNT proteins in a fashion similar to FZD proteins (Cruciat and Niehrs, 2013). However, structure-function analyses of sFRP1 has shown that the Netrin-related motif in the C-terminal region contributes to the inhibition of WNT function (Bhat et al., 2007; Bovolenta et al., 2008). The binding affinity for the interaction of sFRPs and WNT proteins has been reported to be in the nanomolar range, similar to the affinities of the WNT-FZD interaction. However, some specificity has been reported for the sFRP-WNT interaction: WNT3A binds to all sFRPs except sFRP5, whereas WNT5 only binds to SFRP1 and -2. As for the WNT-FZD interaction, so far no comprehensive studies have been published where all possible combinations were tested in a systematic way (Bovolenta et al., 2008).

In the meantime, several studies on sFRPs have offered conflicting results. Different sFRPs were shown to have opposing effects on apoptosis in breast tumors, and sFRP2 was shown to block the inhibitory effects of sFRP1 on kidney tubule formation (Bovolenta et al., 2008). Moreover, structural analysis of the CRDs of sFRPs has suggested that these CRDs might be able to form homo- or heterodimers with CRDs or either FZD or other sFRPs (Dann et al., 2001). Based on these observations, four mechanisms have been proposed by which sFRPs can modulate WNT signaling, two of which are inhibitory and two of which are stimulatory: 1) by scavenging WNT protein, which inhibits signaling because WNT is unavailable for interaction with its receptors; 2) by forming homo- or heteromeric sFRP complexes, which would favor WNT signaling; 3) by direct interaction with the CRDs of FZD, which would inhibit WNT signaling; and 4) by binding WNT to the Netrin-related motif, presenting WNT to the CRD of FZD, which would stimulate WNT signaling (Bovolenta et al., 2008). It is at present not clear how this control mechanism is directed in either of these four directions.

C. WNT-inhibitory Factor

WIF1 is a 379-amino acid protein that has been shown to inhibit WNT signaling by binding WNT proteins (Hsieh et al., 1999). The protein contains a WIF domain, five EGF repeats, and a hydrophilic tail. In fact, the WIF domain is similar to that present at the extracellular end of the RYK tyrosine kinase receptor. WIF1 has been shown to interact with multiple WNTs from both the WNT1 (WNT/β-catenin) and WNT5A (WNT/β-catenin independent) subclasses. However, it is not clear what the mechanism of action is for the regulation of WNT signaling (Cruciat and Niehrs, 2013).

D. Leucine-rich Repeat-containing G Protein-Coupled Receptor 4/5/R-Spondin

Regulation of WNT signaling does not only depend on the amount of WNT available for receptor activation, but also on the receptor density on the cell membrane. A very effective mechanism for the regulation of the density of FZD proteins has been identified by analysis of WNT target gene expression (Fig. 3). Two highly homologous single transmembrane proteins, RNF43 and ZNRF3, were found to be upregulated as result of activation of WNT signaling. Both RNF43 and ZNRF3 are E3 ligases, capable of transferring ubiquitin groups to multiple lysine residues located in the intracellular loops of FZD proteins via the RING domain that is present in their cytoplasmic tail. This ubiquitination induces the endocytosis of the FZD proteins, making them inaccessible for ligand stimulation and causing an effective downregulation of WNT signaling (Hao et al., 2012; Koo et al., 2012).

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Fig. 3.

Regulation of the amount of FZD protein on the plasma membrane. (A) Association of the transmembrane E3 ligases RNF43 and/or ZNRF3 with the FZD receptor complex induces its ubiquitination and leads to internalization of the receptor complex, making it unavailable for stimulation with WNT. (B) R-spondin is capable of redirecting the E3 ligases toward LGR4 or -5, inducing the internalization of this transmembrane protein rather than the FZD/LRP5/6 complex. This effectively leaves more FZD receptors at the plasma membrane, available for stimulation with WNT proteins.

Interestingly, the localization at the plasma membrane of the E3 ligases RNF43 and ZNRF3 is under the control of another group of WNT target genes, LGR4, -5, and -6. LGR5 was originally identified as a marker for intestinal stem cells (Barker et al., 2007). Together with LGR4 and -6, these receptors form a subgroup of the leucine-rich repeat-containing G protein-coupled receptor (LGR) family. This seven-transmembrane receptor family is characterized by a large extracellular domain consisting of leucine-rich repeats, which is connected to the seven-transmembrane domain via a hinge region. Other members of this family are the receptors for follicle-stimulating hormone (LGR1), luteinizing hormone (LGR2), and thyroid-stimulating hormone (LGR3) (de Lau et al., 2014). The ligands for LGR4 and 5, members of the R-spondin family, are known as secreted enhancers of WNT signaling (de Lau et al., 2011). Binding of an R-Spondin to the leucine-rich repeat of LGR4/5 initiates the formation of a complex with RNF43 or ZNRF3, which is rapidly internalized. The loss of RNF43 and ZNRF3 from the plasma membrane leads to an attenuation of the ubiquitination of FZD, so more FZD remains at the plasma membrane to interact with WNT and activate the signal transduction (de Lau et al., 2014). It has to be noted, however, that an alternative mechanism has been described for the internalization of FZD4, i.e., via a DVL2-mediated recruitment of β-arrestin-2 (Chen et al., 2003).

E. Dickkopf

The first DKK protein was originally identified as an inducer of the Spemann organizer, a tissue with strong head-inducing capacity, which explains the name of the protein (dickkopf is German for big head) (Glinka et al., 1998). The DKK family consists of four members in vertebrates, named DKK1-4, varying in size between 255 and 350 amino acids. DKK1, -2, and -4 have a high structural similarity with an N-terminal cysteine-rich domain (DKK_N) and a C-terminal collipase fold (DKK_C). DKK3 deviates from the other DKK family members in that this protein contains an additional soggy domain that is also found in dickkopf-like protein-1 (Cruciat and Niehrs, 2013).

DKK proteins -1, -2, and -4 can bind to LRP5/6 coreceptors with nanomolar affinity, thereby acting as functional antagonists of WNT/β-catenin signaling (Joiner et al., 2013). As described above, the extracellular part of LRP5/6 consists of two pairs of β-propellers connected by EGF-like domains. Crystallization studies revealed that the DKK1_C domain binds to LRP6 β-propeller pair located closest to the plasma membrane. Unexpectedly, however, the complex appeared to consist of an equimolar mixture of a bound LRP6 inner propeller pair-DKK1_C complex and an unbound LRP6 inner propeller pair, a finding that has not yet been explained. The DKK1_N binds to the outer LRP6 β-propeller pair at the site where interaction with most of the WNT proteins takes place (Ahn et al., 2011).

Apart from binding to LRP5/6, DKK1 and -2 have been shown to bind to Kremen receptors 1 and 2 with high affinity. The interaction between LRP6, DKK, and Kremen results in the rapid endocytosis of the complex from the plasma membrane, thereby providing an additional mechanism for the inhibition of WNT signaling (Mao et al., 2002).

E. Micro-RNAs

Micro-RNAs (miRNAs) are short noncoding RNA molecules that can regulate the translation of many genes by controlling mRNA stability. Most miRNAs are encoded by genomic regions distant from annotated genes but some are located in introns of annotated genes. The primary transcripts are subsequently processed by Drosha and Dicer to form the typical double-stranded RNA strands with a length of 20–25 nucleotides. One of these strands associates with the RNA-induced silencing complex to interact with the target mRNA. This interaction is initiated by the seed sequence, a stretch of 6–8 nucleotides located at the 5′ terminus of the miRNA and stabilized by the anchor sequence at the 3′-side of the miRNA. It is now increasingly clear that the translation of multiple genes can be regulated by a single miRNA, but on the other hand, single target mRNA can have multiple miRNA binding sites (Song et al., 2015). Over the last years an overwhelming number of studies have been published where the role of miRNA regulation in a variety of diseases and signaling pathways, including WNT signaling, is reported (Anton et al., 2011).

The expression of virtually all components of the WNT/β-catenin signal transduction pathway are under the control of at least one, but usually many more, miRNAs. This is also true for many of the inhibitors, including sFRPs, DKK proteins, and WIF. The result is a highly dynamic posttranscriptional regulation of the pathway, which may contribute to diseases such as cancer. A comprehensive overview of the many miRNAs regulating the different components of WNT signaling is beyond the scope of this review so we refer to a recent publication by Peng et al. (2017), where the crosstalk between miRNAs WNT/β-catenin signaling is extensively discussed. Here we will focus on the miRNAs that have been reported to regulate WNT components in a cardiovascular context.

Regulation of WNT signaling by miRNAs has been shown to affect cardiac physiology in several studies. As an example, overexpression of miR-19b targets the expression of ctnnb1, the gene encoding β-catenin, thereby disturbing the left-right symmetry and the development of the heart in zebrafish embryos (Li et al., 2014a). Both miR-218 and miR-499 have been shown to affect cardiomyocyte differentiation of cardiac and mesenchymal stem cells via modulation of the expression of sFRP2 and β-catenin, respectively (Zhang et al., 2012; Wang et al., 2016b). miRNAs can also modulate the activation of cardiac fibroblasts. This was recently shown for miR-154, which, by suppressing the DKK2 expression, activated WNT signaling (Sun et al., 2016). Moreover, the stability of atherosclerotic lesions was found to be enhanced by miR-210, targeting the expression of the APC gene and thereby regulating smooth muscle cell differentiation and survival (Eken et al., 2017). It is highly likely that these studies just form the starting point for a multitude of investigations where the modulation of WNT signaling via miRNAs will be further explored in a cardiovascular context.

A. Disheveled

The DVL proteins form an essential component in the transduction of WNT signaling from the receptor level to the intracellular compartment. DVL acts as a scaffold for the formation of the WNT/FZD receptor complex. The protein was originally described as a segment polarity gene required for wingless signaling in Drosophila (Klingensmith et al., 1994; Noordermeer et al., 1994) where disheveled mutants show a phenotype similar to the wingless mutant (Nusslein-Volhard and Wieschaus, 1980). Three DVL homologs have been identified in vertebrates, but the domain structure of the protein is highly conserved. At the N-terminal side of the protein, a DIX (disheveled-axin) domain that, as the name implies, is involved in the binding of the axin protein. The DIX domain is involved in the transduction of WNT/β-catenin signals, where a complex of DVL, FZD, axin, and LRP5/6 is formed. In the central part of the DVL protein, there is a PDZ domain where the interaction with the KTxxxW motif of FZD proteins takes place. At the C-terminal side of the protein the DEP (disheveled-EGL10-plekstrin) domain is located. This domain can interact with a bipartite motif in the third intracellular loop of FZD and forms electrostatic interactions with membrane lipids. Recently, the DEP domain was shown to undergo a conformational change, followed by its dimerization, to trigger the formation of the WNT/FZD signalosome (Gammons et al., 2016). All DVL proteins are heavily phosphorylated and, although the mechanisms leading to this modification are still obscure, some phosphorylation sites have been shown to be necessary for DVL function (Dijksterhuis et al., 2014; Mlodzik, 2016).

Targeted mutations of the Dvl genes in mice give rise to relatively mild phenotypes. The Dvl1 knockout mouse shows neuronal and social behavioral abnormalities (Lijam et al., 1997). Dvl2 and Dvl3 null mutants show cardiac outflow tract defects, including double outlet right ventricle, transposition of the great arteries, or persistent truncus arteriosus (Hamblet et al., 2002; Etheridge et al., 2008). All these abnormalities can be attributed to defects in PCP signaling, whereas DVL proteins show a high level of redundancy for WNT/β-catenin signaling: the presence of only a single allele of one of the three Dvl genes is sufficient for β-catenin-mediated signaling in mice (Wynshaw-Boris, 2012).

Next to their role in WNT/β-catenin and WNT-PCP signaling, several other cellular functions have been described for DVL proteins, including nuclear functions, localization and/or anchoring of ciliary basal bodies in multiciliated cells, inhibition of Notch signaling, and a WNT-independent role in cell viability (Mlodzik, 2016). The relevance of these mechanisms, compared with the role of DVL in WNT signaling, still needs to be elucidated.

B. WNT/β-Catenin Signaling

1. Characteristics of β-Catenin.

β-Catenin is a central molecule in the WNT/β-catenin signaling pathway. β-Catenin is protein of ∼80 kDa that is present throughout the animal kingdom. The characteristics of this multifunctional protein have been extensively described in a review by Valenta et al. (2012), so we will only provide a brief summary here. β-Catenin has two rather diverse functions in the cell: on the one hand, it has a structural role as part of the cadherin-catenin cell adhesion complex, whereas on the other hand it acts as a modulator of gene transcription. It has a highly conserved structure with 12 so-called Armadillo repeats (Armadillo is the Drosophila homolog of β-catenin) in the middle, flanked by N-terminal and C-terminal domains. The Armadillo repeats form a positively charged groove where many partners, including cadherins, APC protein, and transcription factors from the TCF/LEF family, can bind. Extensive mutation analyses have demonstrated that the N-terminal side of the Armadillo domain supports hetrodimerization with α-catenin and subsequent formation of the cadherin-catenin complex, whereas the region C-terminal from the Armadillo domain is involved in the binding of transcription coactivators. There are multiple phosphorylation sites present in β-catenin, allowing the fine tuning of its function and degradation. (Valenta et al., 2012).

The β-catenin-dependent signal transduction pathway is unusual in that activation of WNT/FZD signaling inhibits the degradation of the intracellular signaling protein β-catenin, leading to a higher concentration of β-catenin inside the cell. This is in contrast to most GPCRs, the activation of which stimulates the intracellular production of a second messenger rather than its degradation. This β-catenin subsequently can either move to the cell adhesion complex or migrate to the nucleus where it can activate gene transcription (Fig. 4). At present it is not clear how β-catenins distribute over the nucleus and cell adhesion complexes. In contrast to the nuclear β-catenin pool, which has a half-life of minutes, β-catenin that is incorporated in cell adhesion complexes appears to be very stable (Clevers and Nusse, 2012). This rapid turnover of β-catenin in the nucleus may create a concentration gradient with the cytoplasm, favoring the transport to the nucleus, where β-catenin can be bound to a transcription complex, as will be discussed in detail below.

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Fig. 4.

Schematic representation of the WNT/β-catenin signal transduction pathway. (Left) In the “Off” state, β-catenin is bound in a so-called β-catenin destruction complex containing glycogen synthase kinase 3β (GSK3β), axin, adenomatous polyposis coli (APC) and casein kinase-1 (CK-1). The kinases in this complex phosphorylate β-catenin, thereby targeting it for degradation by the ubiquitin proteasome system. (Right) In the “On” state, the receptor complex consisting of frizzled and LRP5/6 bind WNT, which recruits the disheveled (DVL) protein to the plasma membrane. Subsequently, several components of the β-catenin destruction complex are recruited to the membrane, which prevents the phosphorylation of β-catenin. Therefore, this protein can now accumulate in the cytoplasm and translocate to the nucleus to associate with transcription factors and stimulate the transcription of WNT target genes such as cycline-D1, c-myc, and axin2.

C. WNT/Ca²⁺ Signaling.

One of the most abundantly used second messengers is Ca²⁺, leading to processes as diverse as muscle contraction, activation of enzymes such as PKC, and gene transcription. Ca²⁺-mediated signal transduction of FZD proteins was already reported in the 90s to involve PKC and phosphatidyl inositol signaling (Cook et al., 1996; Slusarski et al., 1997). The current view on this topic is that two pathways can be employed in WNT/Ca²⁺ signaling, one involving phospholipase-C-mediated production of inositol triphosphate and diacylglycerol and the other via the cyclic GMP-selective phosphodiesterase and p38-MAPK (Ma and Wang, 2007). It is generally accepted that for both pathways the interaction between FZD proteins and heterotrimeric G proteins is needed, more specifically the Gα_i/o and the Gα_t subfamilies; for further details, refer to section V.E of this review. At present it is not conclusively established whether DVL is essential for the activation of WNT/Ca²⁺ signaling (Schulte, 2010).

D. WNT/Planar Cell Polarity Signaling

During development, cell proliferation and differentiation are required to make the embryo grow and develop different organs and tissues. However, another essential mechanism is the transfer of directional information to form specific structures and to allow organs and/or tissues to grow in their final shape. Planar cell polarity (PCP) was identified as a mechanism that is involved in the generation of both global and local directional information. Examples are the morphologic changes in mesenchymal cells but also the apical/basal polarization of epithelial cells (Yang and Mlodzik, 2015). PCP was first demonstrated in Drosophila where cuticular structures such as bristles and wing hairs show a clear polarization (Adler, 2012). In vertebrates, PCP signaling regulates anterior-posterior body axis elongation during gastrulation through convergent extension and is also involved in neural tube formation. PCP signaling is also necessary for the orientation of sensory hair cells in the inner ear, as well as the orientation of hair follicles in mouse skin and cilia in airway epithelium (Yang and Mlodzik, 2015). Although less well understood than in Drosophila, the importance of PCP in vertebrate development is stressed by defects in cardiac outflow tracts and incomplete closure of the neural tube as observed in spina bifida, which are caused by inadequate PCP signaling. Mutations in WNT/PCP signaling components were reported for both conditions (Phillips et al., 2005; Kibar et al., 2007).

Six core factors have been identified that interact with each other in Drosophila PCP signaling: the 7TM receptor protein FZD, the 4TM protein Van Gogh/striabismus, the cadherin Flamingo, and the cytoplasmic factors DVL, Prickle, and Diego. The vertebrate homologs of these proteins are the transmembrane proteins FZD, Van Gogh-like 1 and -2 (Vang1 and -2) and Celsr, and the cytoplasmic proteins DVL, Prickle, and Inversin/Diversin. As illustrated in Fig. 5, these core PCP proteins form two complexes on the opposite sides of each cell: on one side, FZD forms a complex with DVL, Inversin/Diversin, and Celsr, whereas at the opposing side the complex consists of Vangl1/2, Prickle, and Celsr. The intracellular components of both complexes are mutually inhibitory, supporting their spatial separation at two sides of the cell. However, interaction of the complexes from two neighboring cells appears to have a stabilizing effect of the complexes where the extracellular structures of Vang1 interact with the extracellular structures of FZD. Gene dosage effects have learned that either too much or too little of a PCP core factor has detrimental effects on tissue polarity (Yang and Mlodzik, 2015).

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Fig. 5.

Schematic representation of the planar cell polarity pathway. In this pathways two receptor complexes are formed at the opposite sides of a cell: On one side, frizzled forms a complex with Flamingo/Celsr, disheveled (Dvl), and Diego/Diversin, whereas the other side the complex consists of Flamingo/Celsr, Van Gogh/Vang, and Prickle. The extracellular parts of these complexes interact, thereby controlling the cellular polarization via activation of JNK/p38 MAPK, small GTPase and Rho-associated kinase (ROCK) signaling. Although the role of Wnt in this signal transduction cascade is only partially understood, this protein may interfere with the Vangl1-FZD interaction. This could subsequently disturb the balance between the signaling of the Vang1/Prickle/Celsr complex and the FZD/DVL/Diego/Celsr complex.

The interaction of these core PCP components can activate several signaling pathways. The FZD/DVL complex can activate the FZD/DVL PCP pathway via the PDZ and DIX domains of DVL, which can induce cytoskeletal reorganization but can also modulate gene transcription. The cytoskeletal reorganization is linked to the activation of small GTPases (such as Rho, Rac, and Cdc42) and Rho-associated kinase. Rho-associated kinase can mediate the cytoskeletal rearrangements, which are characteristic for planar polarity signaling. On the other hand, PCP signaling can also induce a transcriptional response via JNK/p38-type MAP kinase signaling (Yang and Mlodzik, 2015).

The role for WNT in the PCP pathway is only beginning to be elucidated. In Drosophila, PCP axes are directed toward the prospective margins that are a source of WNT. In this model WNT proteins are thought to modulate the extracellular Vangl1-FZD interactions, thereby disturbing the balance between the signaling of the Vang1/Prickle/Celsr complex on the one hand and the FZD/DVL/Diego/Celsr complex on the other hand. In this model, WNTs are instructive regulators of the FZD/Vang1 polarization (Wu et al., 2013). However, it has to be noted that most of these studies have been carried out in Drosophila; for vertebrates additional research will be needed to fully elucidate the exact role of WNT in PCP signaling. Complicating factors are the multiple vertebrate WNTs, only two of which (WNT5A and -11) show selectivity for PCP signaling. Moreover, PCP signaling does not make use of LRP5/6; several PCP-specific coreceptors have been identified in vertebrates such as ROR1/2 and Ryk, tyrosine kinase receptors that may serve in the relay of the signal by phosphorylating DVL.

E. Involvement of Heterotrimeric G Proteins in WNT/FZD Signaling

Despite their seven-transmembrane spanning structure, FZD proteins deviate sufficiently from regular G protein-coupled receptors (GPCRs) to form a distinct class of receptors (Schulte, 2010). At present, it is not entirely clear what these structural differences mean for the interaction with components of different signal transduction pathways. Although the most prominent differences between FZD proteins and regular GPCRs are located at the extracellular side of the receptor, where FZD proteins show a bulky and highly conserved CRD, intracellular structures, particularly the third intracellular loop and the C-terminal end, also deviate. Because these intracellular domains form the point of interaction between regular GPCRs and G proteins, there is an ongoing debate whether FZD proteins can actually interact with G proteins and what the consequences of this interaction would be (Dijksterhuis et al., 2014). As mentioned before, the strongest evidence for FZD-G protein interaction is available for the WNT/Ca²⁺ signaling route, where interactions with Gα_i/o and the Gα_t subfamilies are well established (Schulte, 2010). However, there is accumulating evidence for G protein involvement in WNT/β-catenin signaling, e.g., in mouse primary microglia cells where pertussis toxin (an inhibitor of Gα_i/o) was shown to block specific events such as phosphorylation of LRP6 and stabilization of β-catenin (Halleskog and Schulte, 2013). Moreover, interactions of the Gα_o subunit with axin and its βγ-subunit with DVL have been demonstrated (Egger-Adam and Katanaev, 2010). An unresolved issue in this context is that there is overlap between the predicted binding sites for DVL and the G protein at the intracellular side of the FZD protein, and because interaction with DVL is generally accepted to be essential for WNT/β-catenin signaling, it is not quite clear how the interaction with the G protein actually will take place (Schulte, 2015).

A. β-Catenin-Mediated

Early during development, the mesodermal cell layer is the source of cardiac progenitor cells. The formation of the mesoderm appears to be dependent on WNT/β-catenin signaling. Mice devoid of β-catenin, a key player in the WNT/β-catenin pathway, lack mesoderm (Huelsken et al., 2000), and WNT3A deficiency is associated with attenuated expression of mesodermal markers (Liu et al., 1999), making it challenging to analyze their function during cardiogenesis. However, these findings suggest a necessary requirement of WNT/β-catenin signaling during formation of mesoderm, a source of cardiac progenitor cells. The initial data on the role of WNT signaling during cardiac development in vertebrates has been seemingly confusing and demonstrated contrasting results. Because of the known role of WNT signaling in promoting cardiogenesis in Drosophila, it was assumed that similar results would apply to vertebrates (Park et al., 1996). However, the activation of the WNT/β-catenin signaling in chicken and Xenopus embryos suppressed cardiac differentiation, and the opposite effect was seen by applying WNT inhibitors (Marvin et al., 2001; Schneider and Mercola, 2001). Also, similar results were observed in mice, where multiple ectopic hearts were seen by knocking out β-catenin in the endoderm (Lickert et al., 2002). Other studies with cell-based experiments suggest a positive role for WNT/β-catenin signaling on cardiac development (Nakamura et al., 2003). However, these seemingly contradicting results can be explained by the findings that the specific effects of WNT signaling are time and concentration dependent (Naito et al., 2006; Ueno et al., 2007; Wu, 2008). Once the heart tube is formed, second heart field (SHF) cells, a group of Isl1 and Nkx2.5 expressing cells, migrate to the developing heart from the dorsal pericardial wall (Buckingham et al., 2005). These highly proliferative cells form the second heart field and are tightly regulated by Wnt2 via the β-catenin pathway (Tian et al., 2010; Norden and Kispert, 2012; Ruiz-Villalba et al., 2016). Removal of the β-catenin resulted in a reduction of cells within SHF with subsequent aberrant development of the right ventricle and outflow tract (Klaus et al., 2007; Pahnke et al., 2016).

B. Involvement of Non-β-Catenin-Mediated WNT Signaling

Non-β-catenin-mediated WNT signaling, although less well studied, is also known to play an important role in cardiac development. WNT11 appears to be required for proper heart development by displaying cardiac promoting activity, and these effects are mainly though to be mediated by non-β-catenin-mediated signaling branches (Pandur et al., 2002). Overexpression of Wnt11 in Xenopus explants induces the formation of cells with a contractile phenotype. This Wnt11 promoting activity has been shown in other models as well, such as murine embryonic stem cells, bone marrow-derived stem cells, and endothelial progenitor cells (Terami et al., 2004; Belema Bedada et al., 2005; Koyanagi et al., 2005). Furthermore, these analyses have shown that the Wnt11 procardiogenic effects are impaired by inhibiting JNK or PKC and stimulated by their activation.

WNT5A, another member of the WNT family, promotes troponin-positive cells through non-β-catenin-mediated signaling in synergy with BMP6 and sFRP1 (Chen et al., 2008). However, treatment with either factor did not result in cardiomyocyte formation, which suggests that WNT5A acts in a supportive role but is not required for cardiogenesis. In contrast to WNT5A, WNT2B is required for cardiogenesis. (Wang et al., 2007). WNT2B-deficient embryonic stem cells have a significantly impaired ability to form cardiogenic progenitor cells but not hematopoietic progenitor cells. The loss of WNT2B leads to an accelerated expression of Bry and Flk1. Interestingly, WNT/β-catenin signaling also plays a positive role in Bry expression. In addition, activation of WNT/β-catenin signaling during differentiation of embryonic stem cells leads to enhanced differentiation of these cells (Anton et al., 2007). Furthermore, the loss of WNT2B function is very similar to the WNT3A function; it suggests that WNT2B might activate non-β-catenin-mediated WNT signaling. Terminal differentiation of cardiomyocytes in mouse embryonic cells is associated with a significant increase of Wnt11 expression and adding WNT11 to cultured embryonic stem cells causes an increase of beating cells (Naito et al., 2006). In Xenopus, two Wnt11 homologs have been identified, and one of them, called Wnt11A (also called Wnt11R), is expressed later (Garriock et al., 2005). Reduction of Wnt11R leads to a decrease of marker genes for cardiac terminal differentiation, and it is a cell adhesion molecule DM-GRASP that could be a non-β-catenin-mediated WNT pathway target gene (Gessert et al., 2008).

Based on the data on the role of WNT signaling during cardiac specification, the β-catenin-dependent WNT signaling pathway is required for mesoderm specification, and subsequently this pathway needs to be downregulated for formation of multipotent cardiovascular progenitor cells. This critical step is supported by the activation of the non-β-catenin-mediated WNT pathway. In the next step, the β-catenin-mediated WNT pathway is necessary for the proliferation of these cardiac progenitor cells, and finally, for the terminal differentiation, it is the non-β-catenin-mediated WNT signaling that is activated and the β-catenin-dependent WNT signaling that is downregulated (Gessert and Kuhl, 2010). After heart tube formation, the noncanonical pathway of WNT5A and Wnt11 appears to be critical in the SHF cells migration to the arterial pole of the heart (Cohen et al., 2012).

C. Formation of Outflow Tract, Valves, and Conduction System

Outflow tract formation and septation is intrinsically linked to cardiac neural crest cells, which are a subpopulation of cranial neural crest cells. These cells originate at the dorsal neural tube and migrate along the aortic arch arteries through the pharyngeal arches 3, 4, and 6. The development of cardiac neural crest cells as well at outflow tract appears to be significantly dependent on the WNT signaling pathways. These crest cells express WNT1 and WNT3A and loss of β-catenin on WNT1-positive cells leads to reduction of cardiac neural crest cells as well as outflow hypoplasia in mice (Brault et al., 2001). In addition, WNT5A and WNT 11 appear to be expressed in the outflow tract myocardium. Loss of WNT5A leads to septation defects of the outflow tract, resulting in congenital heart defects such as double outlet right ventricle or persistent truncus arteriosus (Schleiffarth et al., 2007). In addition, WNT11 activates a non-β-catenin-mediated signaling pathway in the outflow tract and regulates the expression of TGFβ-2 (Zhou et al., 2007). However, the underlying mechanisms leading to double outlet right ventricle formation could be due aberrant second heart field components or abnormal contribution of cardiac neural crest cells to the outlfow tract (van den Hoff and Moorman, 2000). Outflow tract formation and septation is intrinsically associated with valve formation. Hence it stands to reason that WNTs participate in valve formation.

Valve formation is a delicate and complex process where cells from the atrioventricular canal, the endocardium located between the atrial and ventricular chambers, undergo an epithelial-mesenchymal transition (EMT), migrate in the space between endocardium and myocardium, proliferate, and form the so-called cardiac cushions. Subsequently, these cardiac cushions are the precursors of the tricuspid valve and mitral valve, which separate the right and the left chambers, respectively. WNT/β-catenin signaling is active during endocardial cushion development (Gitler et al., 2003). Furthermore, loss of APC function in zebrafish, which in turn leads to an abundance of active β-catenin signaling, results in excessive endocardial cushion formation. Overexpression of APC leads to inhibition of cardiac cushion formation (Liebner et al., 2004), demonstrating that WNTs by its inhibitory effect likely exert an inhibition on cushion formation.

WNTs likely also contribute to the development of the conduction system. This system originates from the Isl1-positive progenitor cells (Sun et al., 2007). This process starts early in development, and around embryonic day 8 in mice the cardiac pacemaking region starts taking shape in the atrium, giving rise later to the sinoatrial node where the impulses originate. These impulses are transmitted to the rest of the conduction system, which is composed of the atrioventricular node, the His bundle, bundle branches, and as Purkinje fibers (Sun et al., 2007). WNT7A and WNT11 are upregulated in the developing conduction system, as early as embryonic day 8 (HH 34) in chicken embryo; however, there is no functional data to delineate the precise role of WNT signaling in the formation of the conduction system (Bond et al., 2003). Also, the proper patterning of the atrioventricular canal is important on timely delay the electrical impulses from the atria to the ventricles. Recent data suggests that β-catenin-dependent WNT signaling is an important regulator of this maturation process (Gillers et al., 2015). Concurrently, coronary artery formation is a critical part of the proper cardiac development and studies suggest that WNT9b plays an important role of this process via the β-catenin pathway (Zamora et al., 2007). A summary of the WNTs in cardiovascular development is shown in Table 1.

D. Vessels: WNT Signaling and the Control of Angiogenesis

The formation of blood vessels is under control of two physiological processes: vasculogenesis and angiogenesis. Vasculogenesis is the formation of blood vessels from the de novo production of ECs in embryos, after which angiogenesis takes place to provide new blood vessels from pre-existing ones to all organs and tissues in both physiological and pathologic conditions.

Vasculogenesis, the initial step of vasculature development, requires the differentiation of ECs from mesodermal precursors. This first step is under the control of many signaling cues, including WNT signaling. Previous studies have demonstrated a role of WNT2, WNT3A, and WNT5A in the differentiation of embryonic stem cells toward ECs (Wang et al., 2006, 2007; Yang et al., 2009). The latter seems to involve both the β-catenin and PKCα pathways (Yang et al., 2009). This will be covered in more detail in section IX of this review. The contribution of R-spondin-3 also seems to be important for vasculogenesis, as well as angiogenesis. A common role for R-spondin-3 has been identified in Xenopus embryos and in mice and human ECs, thus highlighting the highly conserved nature of this signaling (Kazanskaya et al., 2008). R-Spondin-3 promotes the differentiation process of ESC toward angioblasts and activates the β-catenin pathway, resulting in an increased vascular endothelial growth factor (VEGF) expression (crosstalk with VEGF pathway, see section X.C) (Kazanskaya et al., 2008).

Angiogenesis is a complex process following sequential steps including the induction of tip cells, sprout elongation, vascular branching, and stabilization of the newly formed vascular network. After the initial differentiation of precursors cells to ECs, the initiation of sprouting is mediated by ECs that harbor different phenotypes: tip and stalk cells. The vascular sprout is under the guidance of tip cells at the vascular front, which have a migratory behavior. They are followed by stalk cells, which are proliferative ECs important for the extension of the sprout and involved in the creation of the vascular lumen.

2. Influence of WNT Signaling on Tip Cells.

Evidence for a role of WNT-signaling on tip cell induction and function is scarce. WLS, a key component of the WNT secretion machinery, is a cargo receptor that mobilizes WNT proteins from the Golgi to the cell surface (Bartscherer et al., 2006). The postnatal deletion of WLS in mouse ECs (Evi-ECKO) was associated with a decreased microvascular density in postnatal retina as well as in tumor tissues (Korn et al., 2014). Although the tip cell density at the vascular front was reduced in this transgenic model, no differences in the number of filopodia per tip cell were observed. This suggests that tip cell function is not impacted by WNT proteins (Korn et al., 2014). More recently, the MMP (matrix metalloproteinase) inhibitor Reck (reversion-inducing-cysteine-rich protein with Kazal motifs) and the adhesion G protein-coupled receptor Gpr124/ADGRA2 have been identified as key players of a WNT7A/WNT7B signaling complex for brain angiogenesis and blood-brain barrier (BBB) formation (see section VI.E) (Vanhollebeke et al., 2015; Noda et al., 2016). This atypical WNT/β-catenin signaling was shown to selectively control the function of tip cells in central nervous system (CNS) angiogenesis (Vanhollebeke et al., 2015). Furthermore, cell migration, an important feature for tip cells, has been shown to be impacted by a WNT/β-catenin-independent pathway, via phosphorylation of APC (Dejana, 2010; Harris and Nelson, 2010). Phosphorylated APC is present in cellular extensions during cell migration and is mandatory for cell migration and the organization of the cytoskeleton (Dejana, 2010; Harris and Nelson, 2010). Indirectly, WNT signaling could also regulate the selection of endothelial tip cells via interfering with Jagged-1. Jagged-1 is a Notch ligand that regulates the selection of tip cells by interfering with the Dll4/Notch signaling from the stalk to the tip cell (Fig. 6). Jagged-1 was significantly upregulated in ECs upon β-catenin-dependent WNT signaling (Reis et al., 2012). The study, however, supported that Jagged-1 is not directly regulated by β-catenin, because this upregulation was observed only after 48 hours of WNT3A stimulation while other genes were already regulated after 18 hours (Reis et al., 2012). Moreover, upon WNT/β-catenin stimulation, the expression of tip cell receptors (e.g., VEGFR2) was downregulated, whereas those expressed on stalk cells (e.g., VEGFR1) were upregulated, suggesting that WNT signaling rather promotes the induction of a stalk cell-like phenotype (Reis et al., 2012).

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Fig. 6.

The role of WNT signaling in sprouting angiogenesis. In this process, tip cells provide directional cues to the sprout cells that can proliferate and initiate the formation of a new vessel. WNT signaling can contribute to the proliferation and tube formation of the stalk cells. The effect of WNT signaling on the tip cells is likely to be indirect via the expression of delta-like ligand-4 (Dll4) in the stalk cells, which activates Notch signaling in the tip cells.

E. WNT Signaling in the Formation of the Blood-Brain Barrier.

The perfusion of the cerebral tissue is a critical process that needs to be strictly controlled to avoid the entrance of nondesired blood components into the CNS. This is achieved due to the unique features of the CNS ECs, which are fundamental for the establishment and maintenance of the blood-brain barrier (BBB) (Abbott et al., 2010). The BBB comprises tight junction proteins that allow a very low permeability to non-CNS components, but also specific transporters that enable the transport of nutrients (e.g., glucose, amino acids).

Supporting evidence for the contribution of WNT signaling in the development of cerebral vasculature and BBB has been found less than a decade ago (Liebner et al., 2008; Stenman et al., 2008; Daneman et al., 2009). The invasion of the CNS by the vasculature and the concomitant BBB development requires the presence of Wnt7A and -B in the developing neuroepithelium (Stenman et al., 2008). By using the Cre-recombinase technique, the deletion of Wnt7A and -B was feasible at the embryonic stage (to avoid early lethality as Wnt7B is required for placental development), and the embryos can develop up to E12.5. Subsequently these Wnt7A/B mutants die from a severe hemorrhage specifically localized to the CNS (Stenman et al., 2008). In addition, the expression of the glucose transporter (Glut1), an early BBB marker, was strongly downregulated in the Wnt7A/B mutants, suggesting the absence of a proper barrier when Wnt7A and -B are lacking (Stenman et al., 2008). The study suggested that Wnt7A and -B produced by the neuroepithelium can directly activate the ECs in the perineural vascular plexus surrounding the neural tube to promote angiogenesis and to acquire a BBB phenotype (Stenman et al., 2008). This was further confirmed in another study in which in situ hybridization experiments were performed. This study revealed that several WNT ligands are expressed by neural progenitor cells in the developing mouse brain, which could be responsible for the WNT signaling activation in ECs. Wnt7A and -B expression in particular was temporally associated with activation of WNT/β-catenin pathway in several areas of the developing CNS vasculature. The impact of Wnt7A on BBB was further explored by studying the transcriptional profiles of primary brain endothelial cells using microarrays. In accordance with the findings described above, Wnt7A upregulated several BBB influx transporters, including Glut1. However, the expression of tight junction molecules was not modulated (Daneman et al., 2009). Recent studies have revealed the existence of functional interactions between WNT7A/B and Reck and Gpr124/ADGRA2, an orphan G protein-coupled receptor, in genetically engineered mice and zebrafish and luciferase reporter assays (Noda et al., 2016). Although many questions remain on the exact molecular interactions taking place between those partners, this newly discovered WNT7A/B-Gpr124-Reck complex seems important for the WNT/β-catenin signaling, which is of relevance for cerebrovasculature development and BBB formation.

Further in vivo evidence was collected to confirm the major contribution of WNT/β-catenin signaling for CNS vasculature development and BBB formation. First, WNT inhibition in developing embryos, using the injection of adenoviruses encoding a soluble FZD₈-FC fusion protein, led to many vascular defects that were limited to the CNS (Daneman et al., 2009). Second, studies using reporter mice for WNT/β-catenin signaling, have identified active WNT/β-catenin signaling in ECs during brain angiogenesis, which progressively declined with the maturation of the BBB (Liebner et al., 2008; Daneman et al., 2009). In adult mice, most cerebral vessels did not exhibit WNT/β-catenin activity, suggesting that a fully mature BBB does not require a permanent activation of this pathway. Furthermore, conditional activation of endothelial β-catenin in vivo was associated with an increased claudin-3 expression (a tight junction protein) and a decreased plasmalemma vesicle-associated protein expression (PLVAP, a component of endothelial fenestrations) (Liebner et al., 2008). On the contrary, the conditional inactivation of β-catenin in endothelial cells in vivo was associated with a strong upregulation of PLVAP as well as an increased BBB permeability. This WNT3A/β-catenin/claudin-3 signaling was confirmed in vitro using primary endothelial cells isolated from mouse brains. Treatment of the cells with a WNT3A-conditioned medium led to a selective increase of claudin-3 expression in association with an activation of the β-catenin pathway. WNT5A conditioned medium had no impact on the expression of claudin-3 or other tight junction proteins (Liebner et al., 2008). In addition, the upregulation of claudin-3 was dependent on the transcriptionally active form of β-catenin rather than the structural association of β-catenin with cadherins at junctional levels, thus confirming that claudin-3 is a major key effector of WNT/β-catenin signaling at the BBB level (Liebner et al., 2008).

Although the contribution of β-catenin in this study was linked to an increased transcription of a tight junction protein, it is of importance to highlight the role of β-catenin (together with α-catenin) in the composition of adherens junctions. The lack of association of β-catenin with cadherins would certainly be detrimental for BBB integrity. Unfortunately, the relative contribution of β-catenin to the adherens junctions compared with β-catenin-mediated WNT signaling in the context of BBB formation is poorly understood and should be subject to further study.

Apart from the WNT/β-catenin pathway, a contribution of WNT signaling for BBB function through the activation of the planar cell polarity complex (PCP) has been identified in immortalized human brain ECs (Artus et al., 2014). The PCP complex, which includes three interacting molecules, Par-3, Par-6, and an atypical PKC, is expressed in human brain ECs and colocalizes with tight junction proteins (zona occludens-1) (Artus et al., 2014). Stimulation of ECs with WNT5A was able to activate the Par/PKCα/PCP complex in this cellular model. Subsequently the cellular polarization increased and the cellular permeability decreased, thus highlighting the involvement of the Par/PKCα/PCP pathway to the WNT-mediated control of the BBB function.

WNT coreceptors are also crucial to control CNS vasculature. Deletion of both LRP5 and -6 in ECs severely impairs the CNS vasculature and is associated with cerebral bleeding (Zhou et al., 2014). In Lrp5^−/− mice, there is a decreased capillary regression in the retinas, resulting especially in the retention of the hyaloid vasculature throughout life (Kato et al., 2002).

The absence of norrin (NDP, Norrie disease protein, or X-linked exudative vitreoretinopathy 2 protein) is detrimental for the regulation of angiogenesis as well as for BBB formation. Norrin can activate the WNT/β-catenin signaling pathway by binding to FZD₄ and LRP5/6. This complex has been shown to be of major importance for both blood retinal and blood-brain barriers (Wang et al., 2012). In the absence of norrin/FZD₄ signaling, vascular growth in retinas is retarded and the capillary network does not form (Wang et al., 2012). In the brains of Fzd₄^−/− mice, BBB integrity is altered in the cerebellum, olfactory bulb, spinal cord, and retina (blood-retinal barrier or BRB), but not in the cerebral cortex, striatum, or thalamus (Wang et al., 2012). Furthermore, when Fzd₄ deletion was induced during adulthood, the vasculature exhibited an increased expression of PLVAP and a reduced expression of claudin-5 and this was associated with loss of BBB and BRB integrity. This highlights that FZD₄ is crucial for the maintenance of mature BBB/BRB function. This conclusion has been supported by another study, in which the injection of an anti-FZD₄ monoclonal antibody, blocking its function, induced a BRB dysfunction (Paes et al., 2011).

In addition to its role in BBB/BRB, norrin/FZD₄ signaling plays a central role in retinal vascular growth. Norrie disease, due to loss-of-function mutations in the Norrie disease gene (NDP, the gene coding for norrin), is associated with a severely decreased vascularization of the retina (see section VII.C) (Warden et al., 2007). Mutations downstream of the norrin pathway, in FZD₄, LRP5, or TSPAN12 genes, are also associated with several retinopathies such as familial exudative vitreoretinopathy (see section VII.C) (Warden et al., 2007).

Although the important role played by norrin has been recognized for BBB formation, less is known regarding its impact in pathologic conditions. Its role was recently investigated in a rat model of subarachnoid hemorrhage (Chen et al., 2015). First, norrin expression increased over time after induction of subarachnoid hemorrhage, thus highlighting the importance of norrin signaling in this pathologic condition. Second, intracerebroventricular administration of norrin led to β-catenin nuclear translocation, increased expression levels of occludin, VE-cadherin, and zona occludens 1, as well as a reduced BBB permeability shown by a reduced brain water content, a reduced Evans blue extravasation, and improved neurologic functions (Chen et al., 2015). These effects were abolished when a pretreatment with a FZD₄ siRNA was performed. Thus, norrin protects BBB integrity in the context of subarachnoid hemorrhage by activating the FZD₄/β-catenin pathway (Chen et al., 2015).

Taken together, these data imply that multiple WNT signaling components are important for BBB (and BRB) formation but can also play crucial roles for BBB maintenance in both physiologic and pathologic conditions. Although the development of drugs targeting the WNT signaling could be of major importance to seal and injured BBB, WNT-targeting compounds inducing a transient opening of the BBB on the contrary could also be of interest to allow the passage of non-BBB permeable drugs into the CNS.

A. Atherosclerosis

Atherosclerosis is a main cause of cardiovascular diseases. It is a chronic inflammatory condition that can lead to an acute clinical event due to plaque rupture and thrombosis. Although the incidence of atherosclerosis-related diseases and the associated mortality in Westernized countries have declined in the last decades, it remains a therapeutic priority (Lusis, 2000; Herrington et al., 2016). Clinical and preclinical studies have identified WNT signaling as being a key player in the development of this pathology. A schematic overview of the pathways described in this section is provided in Fig. 7.

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Fig. 7.

WNT signaling in atherosclerosis. The contribution of WNT signaling to the development and progression of atherosclerosis is described at the intima and media levels with a central role for the contribution of monocytes/macrophages (section VII.A). ABCA1, ATP-binding cassette transporter 1; agLDL, aggregated LDL; ox-LDL, oxidized LDL; MCP-1, monocyte chemotactic protein-1.

1. Clinical Evidence for WNT Signaling in Atherosclerosis.

A first clinical evidence linking WNT signaling to atherosclerosis was obtained after identifying a mutation in the LRP6 gene in a family with autosomal dominant early coronary disease, hypertension, hyperlipidemia, and osteoporosis. The functional significance of the corresponding mutation, an amino acid substitution (Cys-Arg) that affects an epidermal growth factor-like domain, was further confirmed in vitro in cells transfected with the mutant LRP6 and WNT reporter genes (Mani et al., 2007). A few years later, this 1062V LRP6 variant was demonstrated to be an independent risk factor for carotid artery stenosis in hypertensive patients. (Sarzani et al., 2011).

Next to LRP6, WNT5A has been shown to be also of importance in atherosclerotic lesions. First, WNT5A is highly expressed in the macrophage-rich regions of both human and murine atherosclerotic lesions (Christman et al., 2008). Second, WNT5A mRNA and proteins were more elevated in advanced than in less advanced arterial lesions (Bhatt and Malgor, 2014; Malgor et al., 2014). Circulating plasma WNT5A was also higher in atherosclerotic patients than in healthy controls (Malgor et al., 2014).

Furthermore, the LRP5/6 inhibitor DKK1 was also found to be increased in plasma and lesions of patients with coronary artery disease and patients with carotid plaques (Ueland et al., 2009). Similarly to the findings described above on WNT5A, DKK1 was more elevated in advanced and unstable disease. This result was confirmed in ApoE^−/− mice, an experimental model for atherosclerosis (Ueland et al., 2009). Immunostaining of human material as well as in vitro experiments revealed platelets as being an important cellular source of DKK1 (Ueland et al., 2009). Further mechanistic demonstrations of the role of DKK1 in the context of atherosclerosis are discussed below.

B. Vascular Calcification

Vascular calcification is a key process involved in the development of atherosclerosis as well as other cardiovascular pathologies such as congestive heart failure and chronic kidney disease (Demer and Tintut, 2008; Vervloet and Cozzolino, 2017). Vascular calcification has similarities with embryonic bone formation, in which WNT signaling is playing an important role (Wang et al., 2014b).

Early studies have identified a link between Msx2, a homeodomain transcription factor involved in bone anabolism, and WNT signaling (Shao et al., 2005, 2007; Cheng et al., 2008b). Indeed, Msx2 has been found to upregulate WNT3A and WNT7A, while downregulating the expression of the β-catenin-dependent inhibitor DKK1 in primary aortic myofibroblasts (Shao et al., 2005). Further in vivo analyses with mice overexpressing Msx2 in the aorta, confirmed the suppression of aortic DKK1 and the upregulation of vascular WNT expression (Shao et al., 2005). It was thus hypothesized that Msx2 activation was acting on the calcification process in a paracrine manner by signaling via the WNT pathway. In addition, mice overexpressing Msx2 exhibited an increased bone volume compared with their wild-type littermates (Cheng et al., 2008b). In vitro investigations revealed WNT7-LRP6 signaling as being required for the induction of osteogenic differentiation of mesenchymal cells by Msx2 (Cheng et al., 2008b). The signaling cascade was further investigated at the vascular level and bone morphogenetic protein 2 (BMP2) was identified as an upstream regulator of medial artery calcification in vivo (Shao et al., 2006, 2007). Injection of BMP2 in LDLR ^−/− mice was, as expected, able to increase aortic Msx2 expression, aortic WNT signaling and aortic calcium content (Shao et al., 2006). The relevance of BMP2 for vascular calcification is supported by clinical data showing an elevation of serum BMP2 levels in chronic kidney disease patients (Rong et al., 2014). Mechanistic explanations were provided by in vitro experiments using rat vascular smooth muscle cells. Stimulation of these cells with BMP2 induced cell apoptosis and upregulation of β-catenin, Msx2, and Runx2. This effect was dependent on β-catenin signaling (Rong et al., 2014). It was suggested that TNF-α, a known activator of BMP2, could constitute a relevant stimulus of the BMP2-Msx2-WNT signaling in vascular pathologies (Shao et al., 2007).

Other pathologic triggers should also be considered for the study of WNT signaling in vascular calcification. The advanced glycation end products (AGEs) and their receptors RAGEs are of particular relevance for diabetic disorders as well as other inflammatory diseases. A recent study performed in human arterial smooth muscle cells has shown that AGEs/RAGEs can promote calcification via WNT/β-catenin signaling (Liu et al., 2016). It is hypothesized that RAGEs may activate genes, promoting the differentiation of arterial smooth muscle cells into osteogenic cells through the WNT/β-catenin pathway. The contribution of WNT/β-catenin signaling for the osteogenic transdifferentiation of vascular smooth muscle cells and calcification seems to be under the control of Runx2, a transcription factor of osteoblasts (Cai et al., 2016). Activation of the WNT/β-catenin pathway (e.g., with WNT3A) induces the expression of Runx2, which in turn, controls key processes for osteoblast differentiation. On the contrary, blockade of WNT/β-catenin using DKK1 inhibits Runx2 induction and calcium depositions in vascular smooth muscle cells (Cai et al., 2016). Moreover, when bone marrow-derived mesenchymal stem cells (MSCs) are in contact with calcified vascular smooth muscle cells, they display an osteoblast phenotype. This differentiation seems to be due to an activation of the WNT5A/ROR2 pathway (Xin et al., 2013; Guan et al., 2014).

Matrix metalloproteinases (MMPs) are remodeling the extracellular matrix and are therefore suggested to play a role during vascular calcification. Their contribution has been evidenced recently in uremic vascular calcifications (Hecht et al., 2016). Furthermore, the calcification of vascular smooth muscle cells by WNT activation seemed to involve an enhanced expression of MMP-2 and MMP-9 (Freise et al., 2016). Increased expression and activity of MMPs is suggested to further promote the process of vascular calcification.

Although most findings seem to support the deleterious effect of WNT signaling on vascular calcification, a recent study identified WNT16 as being able to attenuate TGFβ-induced chondrogenic transformation of vascular smooth muscle cells (Beazley et al., 2015). In this study, WNT16 was found to sustain Notch activity, which in turn maintained the contractile phenotype of vascular smooth muscle cells (Beazley et al., 2015). LRP6 was also found to limit the process of vascular calcification. Indeed, the conditional deletion of LRP6 in the vascular smooth muscle cell lineage increased the aortic calcium content and decreased aortic distensibility in diabetic LDLR ^−/− mice (Cheng et al., 2015). The protective vascular function of LRP6 may be due to a downregulation of β-catenin-independent FZD signaling (Cheng et al., 2015).

C. Norrie Disease

Norrin is a retinal growth factor with angiogenic and neuroprotective properties (see section IX) (Xu et al., 2004). Norrin binds to FZD₄ receptors and subsequently activates the WNT/β-catenin pathway. This activity is furthermore increased in the presence of Tspan12 within the norrin/FZD₄ receptor complex. A defect in norrin or FZD₄ has a detrimental impact on retinal vasculature during developmental angiogenesis (Ohlmann and Tamm, 2012). The so-called Norrie disease, associated with mutations in the NDP gene, causes blindness as well as progressive deafness and mental retardation (Berger et al., 1992). This also applies for familial exudative vitreoretinopathy, retinopathy of prematurity, and other retinal hypovascularization diseases (Ye et al., 2010; Ohlmann and Tamm, 2012). Next to norrin and FZD₄, LRP5 and TSPAN12 have been found to be also associated with familial exudative vitreoretinopathy, thus highlighting the importance of the norrin/FZD₄ signaling for proper retinal vasculature development (Wang et al., 2012; Gilmour, 2015). The cysteine-rich domain (CRD) of FZD₄ has been shown to play a critical role in norrin-FZD₄ binding (Smallwood et al., 2007). Specific mutations of the FZD₄ in the cysteine-rich domain led to a defective norrin/β-catenin signaling in Xenopus embryos (Zhang et al., 2011).

The retinal vasculature in mice lacking norrin, FZD₄ and Tspan12 is massively impaired. The superficial retinal vascular plexus is delayed and incomplete compared with WT and they all lack the intermediate and deep vascular plexi (Zuercher et al., 2012). Further analyses of the retinal vasculature development revealed that, in a norrin knockout model, the vascular density and number of branching points were decreased, whereas the number of filopodia was significantly increased (Zuercher et al., 2012). However, the morphology of the filopodial protrusions was different compared with WT with more narrow angles and a reduced amount of long tip cell protrusions (Zuercher et al., 2012). In addition, an excessive recruitment of mural cells, required to stabilize the nascent vasculature, was observed in the retinal vessels from the norrin knockout mice, suggesting a potential interaction with PDGF-β/PDGFR-β signaling. The overall decreased vascular density and impaired vascular growth is suggested to be related to a decreased proliferation of the retinal ECs in the norrin knockout mice. This was evidenced directly in the retina after systemic injection of bromodeoxyuridine, as well as during an in vitro assay in which norrin was identified as a mitogenic stimulus for engineered HEK293 cells (Zuercher et al., 2012).

By using mouse genetic and cell culture models, it was demonstrated that Muller glia cells are responsible for norrin production, which then stimulates FZD₄ receptors located on retinal ECs (Ye et al., 2009). Subsequently, the norrin/FZD₄/LRP5/6 signaling is responsible for a transcriptional program in ECs to promote vascular growth and the endothelial-mural cell interactions. This program is at least partly under the control of the transcription factor Sox17. It was shown that the transfection of Sox17 was able to rescue the ability of Fzd₄ knockout retinal ECs to form capillary-like structures in a Matrigel assay (Ye et al., 2009). In addition, although FZD₄ is also expressed in photoreceptors and some other retinal neurons, it was shown that the impaired visual function in Fzd₄ knockout mice is linked to a defect in retinal vasculature rather than an impaired signaling in retinal neurons (Ye et al., 2009).

Finally, norrin/FZD₄/LRP5/6 signaling is also involved in other retinopathies, opening thus new avenues for therapeutic interventions. In addition, this signaling complex seems to be also of importance for other nonretinal disorders, such as female infertility (Hsieh et al., 2005; Luhmann et al., 2005). Its contribution in this field needs to be further investigated.

D. Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a life-threatening disease associated with increased pulmonary pressures, subsequently followed by development of right-sided heart failure. The pathology is characterized by a microvascular rarefaction and an increased pulmonary vascular resistance resulting from wall thickening due to smooth muscle cell proliferation (Rabinovitch, 2012).

The relevance of WNT signaling within this context was revealed by the expression profile of laser-microdissected pulmonary arterial resistance vessels obtained from idiopathic PAH and donor lung tissue (Laumanns et al., 2009). Pathway analysis demonstrated a significant upregulation of the WNT/PCP pathway. Moreover, β-catenin expression was also upregulated in ECs of pulmonary arteries from PAH patients compared with control subjects (Laumanns et al., 2009).

Thickening of the pulmonary arterial wall is observed in PAH due to an increased proliferation of vascular smooth muscle cells in the media. This has been found to be associated with higher levels of active β-catenin within pulmonary artery smooth muscle cells (PASMCs) in PAH patients (Takahashi et al., 2016). In addition, PASMCs from PAH patients also harbor higher expression levels of the platelet derived growth factor (PDGF)-BB and its receptors. PDGF-induced growth of PASMCs that turned out to be dependent of β-catenin activation and GSK3β inhibition (Takahashi et al., 2016). WNT5A was able to repress PDGF-BB-induced proliferation in PASMCs from healthy patients but not from PAH patients (Takahashi et al., 2016), highlighting a potential defective signaling in PASMCs during PAH.

Idiopathic PAH can also result from mutations in bone morphogenetic protein (BMP) receptor II (BMPRII). Such mutations are associated with apoptosis of pulmonary artery ECs as well as microvascular rarefaction during idiopathic PAH (de Jesus Perez et al., 2009). This is of particular interest as the proliferation, survival, and migration of pulmonary artery ECs induced by BMP2 is dependent of β-catenin to mediate GSK3β inactivation, but also dependent of Smad1 phosphorylation to recruit DVL and activate the RhoA/Rac1 pathway (de Jesus Perez et al., 2009). The relationship between the BMPRII and WNT signaling pathways has been further studied using primary cells from patients (induced pluripotent stem cell-derived mesenchymal and ECs) to identify molecular signatures of PAH (West et al., 2014). The results confirmed the increased WNT signaling in the cells of PAH patients and established a link with decreased BMPR2 signaling (West et al., 2014). In particular, expression analyses from PAH patient lung tissue and BMPRII mutant mice revealed an increased expression of the WNT inhibitor sFRP2 (West et al., 2014).

Finally, the progression and severity of PAH pathology is associated with a dysregulation of miRNA expression (Courboulin et al., 2011; Rhodes et al., 2013). Recently, a systematic investigation has been carried out via miRNA profiling of lung tissue from end-stage idiopathic PAH (Wu et al., 2016a). A pathway enrichment analysis identified WNT/β-catenin signaling as being the most affected. Five miRNAs (let-7a-5p, miR-26b-5p, miR-27b-3p, miR-199a-3p, and miR-656) relevant for PAH pathogenesis were significantly increased and correlated with hemodynamics and histopathological parameters of PAH (Wu et al., 2016a). Moreover, many WNT family genes (FZD₄, FZD₅, CTNNB1) and downstream targets (CCDN1, VEGFA, axin2) were upregulated in end-stage idiopathic PAH lung tissue (Wu et al., 2016a).

In conclusion, WNT signaling pathways appear to exert a key role of the preservation of pulmonary vascular homeostasis. Future studies are required to investigate the therapeutic potential of WNT signaling for PAH (de Jesus Perez et al., 2014).

E. Conclusions and Suggestions for Further Research

Wnt signaling is not only an important key pathway for atherosclerosis development but seems to be also of importance for its regression. The activation of Wnt signaling in atherosclerosis has been firmly established, not only in experimental models but also in patients by measuring pathway components in the circulation. This activation is likely to contribute to endothelial dysfunction, the inflammatory response, and VSMC proliferation and appears to involve both β-catenin-mediated and non-β-catenin-mediated WNT signaling. Most of the studies published to date on this subject made use of genetic interventions, so a suggestion for further research would be to include pharmacological interventions. Because of the involvement of multiple signaling pathways, intervening in the WNT production and/or at the level of the receptor complex appears to be a good starting point for these experiments. In the case of vascular calcification, most of the experimental data presented so far point to activation of the β-catenin-mediated signaling. Therefore, it would be interesting to study interventions in this branch of the Wnt signaling pathway. There is a rapidly growing number of drugs specifically developed for targeting β-catenin-mediated WNT signaling (please refer to section XI), and these drugs appear to be interesting candidates to be tested in vitro and in animal models of vascular calcification. PAH is a condition for which new therapeutic approaches are urgently needed. The field has been hampered for a long time by the lack of animal models that adequately represent the pathologic aspects of this condition. Fortunately, new animal models for PAH have been proposed (Colvin and Yeager, 2014) that can be used to test the effects of therapeutic interventions in WNT signaling on the pulmonary vascular resistance.

A. WNT Signaling in Myocardial Infarction

Myocardial infarction (MI) is one of the most frequent acute cardiovascular events. It usually occurs as a consequence of the progression of an atherosclerotic plaque toward an unstable phenotype, where the fibrous cap ruptures and releases the content of the plaque into the circulation. However, myocardial infarction can also be the result of plaque erosion with distal embolization or coronary vasospasm (Regitz-Zagrosek et al., 2016). In any case, due to the interrupted blood flow the affected region of the heart is deprived of oxygen and nutrients, resulting in cell loss in that area.

Despite the fact that stem cells have been identified in the heart, the repair of the infarct area does not result in the significant regeneration of the lost cardiac muscle (Laflamme and Murry, 2011). Instead, it shows many similarities to the wound healing process in the skin: The initial response consists of an inflammatory reaction involving polymorphonuclear neutrophils and macrophages. The function of these cells is to clear the infarct area from the necrotic debris, which is an essential first step in the repair of the infarct. The inflammatory phase is followed by the formation of granulation tissue, which typically is rich in (myo)fibroblasts and newly formed blood vessels, embedded in a loosely organized extracellular matrix. The myofibroblasts in the granulation tissue can be derived from different sources such as resident fibroblasts, circulating fibrocytes, or epicardial cells undergoing epithelial-to-mesenchymal transition (EMT) (Daskalopoulos et al., 2012). Because of their unique combination of contractile and matrix synthesizing properties, the myofibroblasts play an important role in the formation of a compact and strong replacement of the lost cardiac cells (van den Borne et al., 2010) This granulation tissue gradually develops into scar tissue by losing cells and increasing the amount and the cross-linking of extracellular matrix (Hermans et al., 2016).

B. WNT Signaling in Valvular Disease

Heart valves have a typical building plan that is highly conserved in evolution and makes them optimally suited to control the direction of blood flow within the heart. Heart valves consist of three layers: the spongiosa is situated in the middle, flanked by the fibrosa layer on the non-flow side, and the atrialis or ventricularis on the flow side of the valve. The valves are covered by a layer of valvular ECs. The atrialis/ventricularis is rich in radially oriented elastin fibers, whereas the fibrosa contains high amounts of densely packed collagen fibers that maintain the structural integrity of the closed valve. The spongiosa contains proteoglycans and glycosaminoglycans, allowing some movement of the atrialis/ventricularis and the fibrosa (Orton et al., 2012). The most abundant cell type in the different layers of the valve is the valvular interstitial cell, involved in maintenance of the valve by synthesis and degradation of matrix molecules. Under normal circumstances, valvular interstitial cells resemble quiescent fibroblasts, but upon injury they differentiate into myofibroblasts.

Valvular defects are a common cause of inadequate pump function of the heart. Generally, a distinction is made between valvular stenosis, common in the aortic valve and leading to obstructed outflow, and valvular degeneration, frequently found in the atrioventricular valve and leading to regurgitation of blood from the ventricle into the atrium in the systolic phase. The prevalence of aortic valve stenosis is particularly high in the elderly, with 15%–25% of subjects over 65 year of age being affected (Mathieu et al., 2014). Clinically significant mitral regurgitation due to a myxomatus (degenerated) mitral valve has been reported in 1.7% of the general population, but this rises to 10% in the population over 75 year of age (Rajamannan, 2014). The most prominent histologic abnormalities appear to be the formation of bone tissue in the calcified aortic valves (Liu and Xu, 2016), whereas cartilage formation can be detected in myxomatous mitral valves (Rajamannan, 2014).

The driving force behind valvular stenosis is calcification of the valve leaflets, the resulting stiffening of the valve leading to an obstruction in the outflow. In many ways this calcification resembles the formation of bone (Rajamannan, 2010; Liu and Xu, 2016). The control of bone formation by WNT/β-catenin signaling has been firmly established. Genetic intervention studies have shown that β-catenin is essential for the differentiation of mature osteoblasts and multiple WNT genes are expressed in either osteoblast precursors or adjacent cells during embryonic development (Hartmann, 2006). Moreover, mutations in LRP5 are associated with either high bone density (Boyden et al., 2002) or a genetic form of bone loss called osteoporosis pseudoglyoma syndrome (Gong et al., 2001), underscoring the central role of WNT/β-catenin signaling in the regulation of bone density. These observations have induced extensive studies of involvement of WNT signaling in valvular defects. The expression of osteoblastic genes is controlled by the balance between Notch and WNT/β-catenin signaling (Mathieu et al., 2014), and indeed elevated levels of LRP5 were reported in calcified aortic valves and, to a lesser extent, degenerative mitral valves (Caira et al., 2006). Moreover, a positive association between circulating DKK1 levels and calcified aortic stenosis was recently reported in patient with angiographically normal coronary arteries, but this association was less clear in the presence of concomitant atherosclerosis of the coronaries (Motovska et al., 2015).

Involvement of WNT/β-catenin signaling is not restricted to valvular calcification but was recently implicated in myxomatous valve disease as well. Myxomatous valve diseases is characterized by a thickening of the valvular leaflets and alterations in the extracellular matrix. It is the result of altered growth factor signaling, among which Wnt, BMP, and Notch are important participants. Excessive deposition of proteoglycans and a concomitant degradation of collagen and elastin are characteristics of myxomatous valve disease, where the phenotype of the spongiosa resembles that of cartilage (Orton et al., 2012). Loss of β-catenin signaling in valvular interstitial cells activated the transcription factor Sox9 and subsequent expression of chondrogenic matrix genes (Fang et al., 2014). Inactivation of axin2, a negative regulator of WNT/β-catenin signaling, showed immature heart valves and a progressive myxomatous valve degeneration in mice at 4 months of age, underscoring the importance of this signaling pathway in valve homeostasis (Hulin et al., 2017).

C. WNT Signaling in Cardiac Arrhythmias

Cardiomyocytes are attached to each other by specialized structures called intercalated disks. These intercalated disks not only provide strong mechanical connections between cardiomyocytes but also allow the fast propagation of electrical signals through the myocardial tissue, which is an essential feature for their simultaneous and coordinated contraction. Three main structures can be identified in the intercalated disk: desmosomes, anchoring the adjacent cells; adherens junctions, connecting actin filaments to the cell adhesion complex; and gap junctions, involved in the electric and metabolic coupling of neighboring cells. Although these structures are often studied separately, it is becoming increasingly clear that all intercalated disk components closely cooperate and should be seen as an integrated functional unit of the cardiomyocyte (Vermij et al., 2017). Therefore the term area composita was introduced to emphasize the integrated nature of desmosomes and adherens junctions (Patel and Green, 2014).

Apart from their role in WNT signaling, members of the catenin family are an important component of both desmosomes and adherens junctions. In the latter, N-cadherins are single membrane-spanning proteins which form intercellular connections via interaction of the extracellular domains extending from two neighboring cells. On the cytoplasmic side, N-cadherin is attached to the actin cytoskeleton via a complex consisting of α- and β-catenin and plakoglobin, also known as γ-catenin. Desmosomes are sturdy, symmetrical anchoring structures that provide structural support to the cell-cell junction. They are not involved in the connection with the actin cytoskeleton but they connect to intermediate filaments. Desmosomes consist of the single membrane spanning cadherins desmoglein and desmocollin. Desmin is bound to these cadherins via a complex formed by plakoglobin, plakophilin, and desmoplakin. Gap junctions consist of complexes of gap junction proteins from the connexin (Cx) family, forming a tubular structure. The typical ventricular gap junction consists of a complex of six Cx43 proteins in the plasma membrane, connected to a similar complex in the neighboring cell. These structures allow the rapid propagation of action potentials and transport of small molecules between cells (Patel and Green, 2014). Other members of the connexin family, including Cx40 and Cx45, have been identified in fast-conducting structures such as the His/Purkinje system (Lo, 2000).

There are several lines of evidence that the expression of Cx43 is under the control of WNT signaling. First, activation of WNT/β-catenin signaling in neonatal rat cardiomyocytes by coculturing with WNT1-secreting cells or with the GSK3-inhibitor lithium chloride (LiCl) resulted in an increase in Cx43 expression (Ai et al., 2000). Rapid electrical stimulation of neonatal rat cardiomyocytes resulted in nuclear translocation of β-catenin within 10 minutes and an increase in Cx43 expression within 60 minutes, suggesting a WNT/β-catenin-mediated regulation. This was accompanied by a significant increase in conduction velocity in these cells. Moreover, in a cardiomyopathic mouse model showing arrhythmias and remodeling of gap junctions, decreased levels of β-catenin and Cx43 were observed (Nakashima et al., 2014). Recently, mutations in the lamin A/C gene (LMNA) causing cardiomyopathy with conduction abnormalities were shown to decrease WNT/β-catenin signaling, which was accompanied by an increase in sFRP expression and reduced expression of Cx43 (Le Dour et al., 2017).

The involvement of aberrant WNT signaling in arrhythmogenic cardiomyopathy (AC) has been extensively studied and documented. AC is an inheritable cardiac disease characterized by life-threatening ventricular arrhythmias and heart failure and is a major cause of death in young athletes. Originally the condition was thought to be concentrated in the right ventricle, giving rise to the name arrhythmogenic right ventricular cardiomyopathy, which is now replaced by AC. A typical pathologic finding in these hearts is the replacement of cardiomyocytes by adipose and fibrous tissue (Pilichou et al., 2016). In most families, AC is inherited in a autosomal dominant fashion with incomplete penetrance. Mutations in five genes encoding the desmosomal proteins plakoglobin, desmoplakin, plakophilin-2, desmoglein-2, and desmocollin-2 have been reported in about half of the affected patients, therefore AC is generally considered a desmosomal disease (Sen-Chowdhry et al., 2005; Basso et al., 2011). The link between AC and WNT/β-catenin signaling can be found in desmosomal crosstalk with the nucleus. Decreasing desmoplakin expression via siRNA in cultured atrial myocytes resulted in nuclear translocation of plakoglobin, which competes with β-catenin for interaction with the TCF/LEF transcription factors. This induced a twofold reduction in WNT/β-catenin signaling and induced the expression of adipogenic and fibrogenic genes as observed in AC patients. An AC phenotype was also found in mice by the heterozygous deletion of the desmoplakin (Dsp) gene (Garcia-Gras et al., 2006) and in mice with a cardiomyocyte-specific silencing of β-catenin and plakoglobin (MerCreMer under control of the αMHC promoter) (Swope et al., 2012). Genetic fate mapping experiments have shown that the adipocytes in AC hearts originate from second heart field progenitor cells, in which the attenuated WNT/β-catenin signaling resulting from nuclear plakoglobin induced an adipogenic fate (Lombardi et al., 2009). The importance of WNT/β-catenin signaling in AC was recently illustrated in two mouse models with mutated desmosomal proteins (Chelko et al., 2016). The mice showed GSK3 accumulation in the intercalated disks, similar to those observed in AC patients. Treatment with SB216763, a GSK3 inhibitor, normalized the protein distribution in the intercalated disks and improved ventricular fibrosis and inflammation, pointing to GSK3 as a therapeutic target in AC.

D. WNT Signaling in Cardiac Hypertrophy

Cardiac hypertrophy is an adaptive response of the heart to a wide range of external factors and neurohormonal/mechanical stimuli, such as pressure overload, β-adrenergic stimulation, MI, etc. These factors activate different signaling pathways and key molecules, including, but not restricted to, NFAT, PI3K/Akt, GATA4, CaMKII, PKC, ERK, JNK, calcineurin (Hunter and Chien, 1999), and WNT (Blankesteijn et al., 2008). Hypertrophy can have profound effects on pathologic cardiac remodeling, which progresses to impairment of cardiac function and eventually leads, when left untreated, to lethal congestive heart failure. In various studies, cardiac hypertrophy has been linked with changes in WNT signaling and here we will discuss the involvement of various WNT components in the mediation of pro- and antihypertrophic stimuli.

E. WNT Signaling and Heart Failure

Heart failure (HF) is a progressively disabling chronic disease, which comes as an aftermath of a variety of pathologies, most importantly MI and hypertrophy (covered above). Initially, following injury, endogenous compensatory mechanisms prevent cardiac failure; however, eventually, cardiac remodeling produces a detrimental effect on cardiac architecture and function and leads to lethal HF. HF is a major public health issue, and the total number of HF patients around the globe is estimated to be more than 26 million (Ambrosy et al., 2014). According to the latest statistics from the American Heart Association (Benjamin et al., 2017), about 20% of people suffering an MI after 65 years of age will develop HF, and approximately one in two patients diagnosed with HF will die within 5 years. The prognosis for the United States alone is even worse because the HF prevalence is expected to rise 46% by 2030. It is obvious that the financial impact of HF is enormous: in 2012 the total cost associated with HF in the United States was more than $30 billion, and the estimations are expected to increase to $69 billion by 2030. Currently, available anti-HF pharmacotherapy is not curative but mainly directed at the symptoms and it only attenuates the deterioration to end-stage HF, without completely preventing it. WNT signaling has been shown to be involved in mediating adverse cardiac remodeling and the progression to HF, thus interventions in the pathway may provide new targets for HF therapy.

F. Conclusions and Directions for Future Research

The effect of interventions in Wnt signaling in models of cardiac disease has been the subject of extensive investigation in the last decade. From these studies it can be concluded that the expression of components of the WNT signaling pathway is modulated in a variety of cardiac pathologies, suggesting an activation of the pathway. Unfortunately, the results are often ambiguous when it comes to the question whether WNT signaling should be activated or inhibited. This is particularly the case in studies in which genetic interventions were applied. Interventions in a specific member of the large family of genes which act in WNT signaling may be compensated by the expression of other family members, hampering the interpretation of the results.

In contrast, from the studies in which pharmacological inhibitors of the pathway have been applied in models of myocardial infarction a rather clear picture is emerging: intervention at the level of either PORCPN (Bastakoty et al., 2016; Moon et al., 2017), FZD (Laeremans et al., 2011; Uitterdijk et al., 2016), the β-catenin destruction complex (Murakoshi et al., 2013), or β-catenin-mediated gene transcription (Sasaki et al., 2013) all have shown beneficial effects on infarct size and, in most cases, on cardiac function. Unfortunately, the numbers of studies in which pharmacological interventions have been applied in other models of cardiac disease are too small to draw firm conclusions, so it would be desirable to test the available compounds in models of cardiac hypertrophy and arrhythmias. Moreover, little is known about the optimal level where the pathway should be targeted: Upstream targeting (at the level of WNT production or interaction with the receptor) can target both β-catenin-dependent and -independent signaling, whereas downstream targeting (at the level of signal transduction) could introduce selectivity for a specific signaling pathway. To clarify this issue, future research should concentrate on interventions at different levels of the pathway in a single experiment to compare the effects on infarct composition and cardiac function. These studies should pay specific attention to the induction of cardiac renewal as a potential mechanism for the beneficial effects of WNT inhibition.

A. WNT Signaling Controls Stem Cell Differentiation

In the adult mammal, stem cells or progenitors are thought to reside in specialized microenvironments called niches. Signaling within a niche determines the fate of a stem cell to self-renew or differentiate into target parenchymal cells of that tissue. An understanding of signaling pathways thus provides critical insight into mechanisms of stem cell maintenance or exhaustion. WNTs are an important family of proteins that regulate the biology of stem cells and determine cell fate decisions. WNTs can regulate differentiation of progenitors to various directions, including neuronal, hepatic, myogenic, chondrongenic, osteogenic, adipogenic, and cardiogenic cell fate (Visweswaran et al., 2015). It has been shown that WNT signaling promotes neurogenic differentiation. More specifically, in cortical mouse neural precursor cells, WNT7A promoted neuronal differentiation (Hirabayashi et al., 2004). Furthermore, WNT5A appears to drive neuronal differentiation via FZD₃ and _-5, and signaling through the WNT5A-JNK pathway (Jang et al., 2015). WNT activation can also enhance myogenic differentiation. Various WNT molecules have been shown to promote myogenic markers, with WNT11 being the strongest (Belema Bedada et al., 2005). Hepatic differentiation appears to be influenced by WNT signaling as well. In humans, depending on the stem cells source, the WNT signaling pathways can have different effects. They are activated in hepatic differentiation of embryonic stem cells, but downregulated in hepatic differentiation of adipose tissue-derived stem cells (Heo et al., 2013). Additionally, WNT signaling regulates stem cell differentiation in osteogenic and chondrogenic lineages as well. The data on the WNT pathways on bone development have been contradictory, with reports suggesting enhancing as well as inhibitory effects (Visweswaran et al., 2015).

With regard to WNTs in stem cell differentiation into cardiac lineage, the data suggest that they play a critical role as mentioned in detail in section IX.C. It is important to highlight that the WNT/β-catenin pathway regulates the early differentiation of murine embryonic stem cells (Anton et al., 2007). Furthermore, regulation of WNT3A transcription serves as a turning point for cardiomyogenic differentiation (Deb et al., 2008).

B. WNT Signaling and Cardiovascular Regeneration in Lower Vertebrates

The remarkable cardiac regeneration ability in the lower vertebrates, including frogs, newts, and axolotls, was initially studied more than 50 years ago (Rumyantsev, 1966; Rumyantsev and Marakjan, 1968). More recent studies provided evidence of full regeneration of the adult hearts in newts over a period of 60 days (Witman et al., 2011). Although earlier studies showed only limited regenerative capacity, those studies were likely prematurely terminated for analysis (Porrello and Olson, 2014). In addition, the location of the injury could suggest a differential regenerative capacity of the progenitor cells or cardiomyocytes. However, zebrafish show an exceptional regenerative capacity following different modalities of cardiac injury (Kikuchi and Poss, 2012). The different modalities include surgical removal of 20% of the apex of the ventricle, cryoablation-induced injury of 25% of the apex of the ventricle, or ablation of up to 70% of cardiomyocytes by transgenic expression of diphtheria toxin (Poss et al., 2002; Chablais et al., 2011; Wang et al., 2011a). Regardless of the type of injury, the cellular mechanisms involved in heart regeneration appear to be closely similar, regardless of the injury models. The epicardium appears to be essential for these cellular changes to occur. Interestingly, genes that are normally expressed during development, such as Wilms’ tumor 1b, appear to be upregulated during the regeneration process. Although there is ample evidence of the role of WNT signaling pathways participating in mammalian heart regeneration and remodeling, little is known regarding their role heart regeneration in lower vertebrates. WNT/β-catenin signaling appears to be upregulated following ventricular regeneration after resection (Stoick-Cooper et al., 2007). In addition, a WNT target gene is also activated in the zebrafish after injury (Kizil et al., 2009). However, further studies are needed to elucidate the role of WNT signaling in lower vertebrate heart regeneration.

C. WNT Signaling in Cardiomyocyte Differentiation of Stem/Progenitor Cells

As mentioned in section VI, the studies on WNT signaling during cardiac development in vertebrates have yielded mixed results. In chicken and frog embryo experiments, the activation of β-catenin-dependent WNT signaling inhibited cardiac differentiation and WNT inhibitors had the opposite effect (Marvin et al., 2001; Schneider and Mercola, 2001). In addition, application of LiCl, an activator of β-catenin-dependent WNT signaling, increased the myogenic differentiation of mesenchymal stem cells derived from cardiac patients (Brunt et al., 2012). Non-β-catenin-mediated signaling plays also a role in promoting cardiomyocyte differentiation. Mesenchymal stem cells transduced with WNT11 appeared to upregulate GATA-4, likely through signaling via PKC or JNK pathway (He et al., 2011). From a developmental standpoint, these insights have been applied in other projects with a direct potential clinical impact. It has been proposed that human embryonic stem cells could serve as a source of cardiomyocytes. Subsequently, cultured embryonic stem cells form clusters called embryonic bodies that appear to differentiate in random fashion, even to a few beating cardiomyocytes. However, addition of WNT3A to these cultures significantly increased the onset of beating activity and expression of cardiac genes (Kwon et al., 2007). Furthermore, addition of a WNT signaling inhibitor resulted in complete abolition of the beating embryonic bodies (Kwon et al., 2007). These findings suggest that β-catenin-dependent WNT signaling not only drives the cardiomyocyte formation in embryonic stem cells but also is a necessary pathway for such events.

D. WNT Signaling in Cardiac Fibrosis and Repair

The importance of WNT signaling in development cannot be fully appreciated without elaborating on their role in cardiac repair and fibrosis (Deb, 2014). As discussed in detail in section VIII.A, expression of several WNTs, such as WNT1, -2, -4, -7A, -10B, and -11 increases significantly shortly after an ischemic cardiac injury (Kobayashi et al., 2009; Aisagbonhi et al., 2011; Duan et al., 2012). The effects of WNT/β-catenin signaling appears to be profibrotic and antagonism of this pathway with sFRPs appears to have beneficial effects on fibrosis, cardiac function, and left ventricular remodeling (Kobayashi et al., 2009). However, the specific mechanisms of beneficial effects of WNT antagonism following injury remains unclear, although it is tempting to speculate that this induces cardiac regeneration. Which cell populations express different WNTs in the injured hearts and how WNT/FZD signaling changes in different cell populations after cardiac injury will need to be further studied, especially to develop therapeutic strategies in the ischemic heart. Cardiac hypertrophy, in contrast to myocardial infarction, is characterized by an increase in cardiomyocyte cell size and increase fibrosis as well. Gene expression of WNTs does not appear to be altered after cardiac hypertrophy (ter Horst et al., 2012), but when patients with a hypertrophic heart develop heart failure, WNT signaling appears to be depressed (Kobayashi et al., 2009). In addition, β-catenin loss-of-function mutations in the myocytes have been associated with enhanced cardiac function, and gain-of-function mutations have been associated with reduced cardiac function resembling dilated cardiomyopathy (Baurand et al., 2007; Zelarayan et al., 2008). These findings suggest that WNTs signaling in cardiac myocytes promotes cardiac hypertrophy and suggests that inhibition of the β-catenin-dependent WNT pathway signaling might be a pharmacological target for cardiac hypertrophy.

E. Conclusions and Future Perspectives

Although there is ample evidence for the involvement of WNT signaling in stem cell homeostasis, e.g., in the intestine, its role in cardiovascular stem cell regulation is only beginning to be explored. Because regeneration of heart tissue would be a major step forward in the treatment of damage, e.g., caused by cardiac ischemia, further exploration of interventions in Wnt signaling aiming at the regeneration of heart tissue will be an exciting area of research for the near future. However, it will be challenging to specifically address the regeneration process in the heart, without affecting stem cells in other organs. A detailed molecular analysis of the signaling pathways involved in cardiac regeneration is needed to identify targeting points that are sufficiently specific to avoid unacceptable side effects in other stem cell populations.

A. Transforming Growth Factor β

Rather than operating in isolation, WNT signaling appears to be strongly intertwined with other signaling pathways, including the TGFβ/BMP and YAP/TAZ signaling. Multiple examples of crosstalk between these pathways can be found, and this crosstalk appears to be taking place at different levels in the signaling cascade (Guo and Wang, 2009). Activation of this signaling network is particularly relevant for the control of fibrosis of different organs, where differentiation of effector cells from different origins into myofibroblasts plays a key role (Piersma et al., 2015). Here we will discuss this crosstalk at multiple levels: the extracellular level, at the intracellular signal transduction, and in the nucleus.

B. Renin Angiotensin System

Besides its central role for cardiovascular homeostasis, the renin angiotensin system (RAS) is also involved in other organ systems and is interconnected to many signaling systems both in physiologic and pathologic conditions (de Gasparo et al., 2000; Bader and Ganten, 2008).

AngII, the main effector of the renin angiotensin system, is formed by the enzymatic degradation of angiotensinogen by renin and angiotensin converting enzyme, respectively. AngII has been shown to modulate the mRNA expression of FZD₂ in rat vascular smooth muscle cells in a time-dependent manner (Castoldi et al., 2005). The initial rapid upregulation of FZD₂ after AngII stimulation was accompanied by a rapid upregulation of TGFβ-1, suggesting a potential crosstalk between AngII and FZD₂, most probably indirectly via the contribution of TGFβ-1 signaling (see section X-A) (Castoldi et al., 2005). Following this early finding, more direct evidence linking the RAS to WNT signaling was identified more recently.

The (pro)renin receptor (PRR), one of the latest identified RAS family members, is able to bind renin and its precursor prorenin. Shortly after its discovery, PRR was recognized as the full-length form of a smaller protein described earlier, associated with the V-ATPase (vacuolar H⁺-ATPase), a multiprotein complex involved in the acidification of vesicles (Nguyen, 2011). More recently PRR was proposed to be a component of the WNT receptor complex independently of its function for renin. PRR seems to act as an adaptor between WNT receptors and the V-ATPase complex (Buechling et al., 2010; Cruciat et al., 2010). Coimmunoprecipitation experiments showed that PRR could bind FZD₂, FZD₈, and LRP6. In addition, loss of function and pharmacological inhibition of V-ATPase in vitro and in vivo revealed its necessity for WNT/β-catenin signaling (Cruciat et al., 2010). Results from this study have thus shed light on an unsuspected role for PRR/V-ATPase and acidification during WNT/β-catenin signaling. The authors hypothesized that the WNT/PRR complex is internalized upon stimulation of the FZD receptor, followed by an acidification of the endocytosed complex that allows the phosphorylation of LRP6 and the subsequent β-catenin activation (Cruciat et al., 2010). In addition to its involvement in the WNT/β-catenin pathway, the PRR has been identified to be also part of the WNT/PCP pathway in Drosophila (Buechling et al., 2010; Hermle et al., 2010).

Recent in vitro and in vivo results have identified that multiple RAS genes encoding angiotensinogen, renin, angiotensin-converting enzyme, and the receptors for AngII are downstream targets of WNT/β-catenin signaling. The overexpression of β-catenin or different WNT ligands upregulates the expression of RAS genes, whereas blockade of WNT/β-catenin signaling with a small molecule inhibitor effectively downregulates RAS genes. In vivo, the inhibition of the WNT/β-catenin/RAS axis was able to reduce renal inflammation and fibrosis, thus opening new therapeutic avenues (Zhou et al., 2015).

C. Vascular Endothelial Growth Factor

Vascular endothelial growth factors (VEGF-A, VEGF-B, VEGF-C, and VEGF-D) are key players influencing angiogenesis through VEGF receptors (VEGFR1, VEGFR2, and VEGFR3). This VEGF system has many sites of interaction with other signaling systems such as TGFβ, Notch, and FGF (Holderfield and Hughes, 2008; Dejana, 2010; Lieu et al., 2011). Studies performed in the last decade have also revealed synergistic or antagonistic interactions with WNT signaling.

The interaction between VEGF and WNT signaling was first identified in colonic neoplasia (Zhang et al., 2001). Similarly, the importance of the WNT/β-catenin signaling pathway in VEGF signaling has also been evidenced in human retinal pigment epithelial cells subjected to hypoxia. In this model, angiogenesis induced by tissue factor was decreased in presence of the WNT/β-catenin signaling pathway inhibitor IWR-1-endo (Wang et al., 2016c).

Later, researchers performed a high-throughput RNA interference screen to identify novel modulators of the WNT/β-catenin pathway and thus potential new therapeutic targets (Naik et al., 2009). A library of siRNAs was tested in a luciferase assay for activation of WNT/β-catenin transcription with WNT-stimulated human cells. Follow up studies with the best hits identified VEGFR1 (but not VEGFR2 or VEGFR3) as a positive regulator of WNT signaling (Naik et al., 2009). The study showed that this VEGFR1-WNT synergy was independent of GSK3β. A VEGFR1-tyrosine phosphorylation of β-catenin itself was proposed by the authors as the mechanism for this synergistic effect (Naik et al., 2009).

Further evidence for a crosstalk between WNT and VEGF signaling was obtained in the field of developmental angiogenesis. Zebrafish embryos deficient for Rspo1 or its receptor Kremen 1 exhibited primary vessels resulting from vasculogenesis, but did not demonstrate further vascular development (Gore et al., 2011). This was found in association with a decreased expression of VEGFC. Additional experiments revealed that the expression of VEGFC was dependent of Rspo1 and WNT and that the proangiogenic effects of Rspo1/WNT signaling are mediated by VEGFC/VEGFR3 signaling (Gore et al., 2011). At an earlier developmental stage, it has been shown that VEGF signals through the WNT/β-catenin pathway to favor the vasculogenic differentiation of mesenchymal stem cells (Zhang et al., 2016c).

Although WNT signaling is well known for its contribution to vascular development, its interplay with VEGF signaling remains poorly investigated. Additional studies are required to confirm the previous observations and to further decipher the complex WNT-VEGF interactions.

D. Conclusions and Future Perspectives

It is becoming increasingly clear that signaling pathways do not operate in isolation but are in fact strongly intertwined. The WNT signaling pathway appears to be no exception, as illustrated by its interactions with TGFβ signaling, the RAS and VEGF described in this section. It is quite conceivable that some of the effects that we observe by a therapeutic intervention in, e.g., the RAS are in fact the result of inhibition of an interacting signaling pathway. Although these interactions add significantly to the complexity of the study of WNT signaling, they are important because they can be a source of adverse effects of interventions. This calls for a more holistic approach in the preclinical phase of drug discovery in general to reduce the number of drugs that fail in clinical trials.

A. WNT Synthesis–Secretion

The first level of the WNT signaling cascade is undoubtedly the production and secretion of the WNT ligand. Following its production, WNT is not released as is, but undergoes glycosylation and palmitoylation, two processes which modify WNT and are indispensable for the secretion of the active WNT ligand (Komekado et al., 2007; Kurayoshi et al., 2007). PORCN, residing in the endoplasmic reticulum of WNT-producing cells, is a critical factor in this transformation and secretion of WNTs (Komekado et al., 2007). A wide range of PORCN inhibitors has been developed with demonstrated effects on WNT secretion, thus mediating the prevention of WNT signaling activation. Additionally, as shown by in vivo studies with transgenic mice, genetic loss of Porcn leads to dramatic inhibition of endogenous WNT production, but mice can still respond to exogenous WNTs, indicating that the receptor compartment is not affected (Barrott et al., 2011). Studies with human astrocytes showed that involvement of PORCN in the WNT production might be cell type and WNT subtype dependent (Richards et al., 2014). Chen et al. (2009a) were the first to show that four compounds, named IWP-1 to -4 (inhibitors of WNT production), can attenuate the secretion of the WNT ligands. These four IWP molecules induce PORCN-mediated inhibition of WNT 1, 2, and 3a production. A few years later they reported another 13 IWP Porcn inhibitors and attention was drawn toward IWP-L6, which was shown to be 100 times more potent than the originally designed IWP-2 (Wang et al., 2013b). In addition, LGK974, a potent and specific PORCN-inhibitor, was first discovered by the research group of Harris; the compound is also referred to as WNT974. The orally bioavailable molecule inhibits PORCN, as well as operating more downstream too by blocking WNT-mediated LRP5/6 phosphorylation, and is very effective against various types of cancer both in vitro and in vivo (Liu et al., 2013). Interestingly, LKG974 can work in synergy with the PI3K inhibitor GDC-0941 and produce antitumor effects in MDA-MB-231 breast cancer cells (Tzeng et al., 2015). Furthermore, LGK974 has also entered a phase I clinical trial (https://clinicaltrials.gov/ct2/show/NCT01351103), including patients with colorectal cancer, pancreatic adenocarcinoma, and other cancer types with well-established association with the WNT signaling cascade. The beneficial effects of this compound, as well as the PORCN inhibitor GNF-6231, on infarct healing was previously discussed (section VIII.A.1). Quite recently, ETC-159 was demonstrated to strongly inhibit PORCN, block WNT secretion, attenuate the expression of the β-catenin target genes TCF7, c-myc and axin2, and to possess robust antitumor capabilities versus teratocarcinomas and colon cancers, as well as preventing remissions (Madan et al., 2016a). Proffitt et al. (2013) propose the compound WNT-C59 as a robust and safe inhibitor of PORCN. All PORCN isoforms and all WNT members that can activate the β-catenin-dependent pathway were reported to be substantially inhibited by WNT-C59 and this is reflected by its excellent antitumor effects in mice, where it prevents MMTV-WNT1 growth (Proffitt et al., 2013). WNT-C59 has the additional advantage of being orally bioavailable. Treatment with WNT-C59 leads to the robust attenuation of renal fibrosis in a unilateral ureteral obstruction mouse model. This is effectuated by a complex crosstalking mechanism and since the disruption of porcupine leads to blunted WNT secretion, this has the inhibition of inflammatory cytokine activation and thus a suppression of ECM-depositing fibroblasts expression as a consequence (causing an attenuation in fibrosis) (Madan et al., 2016b). Novel PORCN inhibitors, such as compound 53 have shown promising preliminary results, but their physicochemical and pharmacokinetic properties restrict their applicability. Thus, more research is required for their improvement, before they are further studied in vitro and in vivo (Xu et al., 2016). Lastly, one very important point that should be taken into consideration when using factors acting at the level of PORCN is their relative nonselectivity. Targeting PORCN usually leads to a global WNT secretion suppression, both the ones forming the β-catenin-dependent pathway (WNT1, -2, -3, -8A/B, and -10A/B), as well as the β-catenin-independent WNTs-4, -5A/5B, -6, -7A/B, and -11), thus there is always the danger for off-target effects.

WNT16 expression can be directly affected by a compound called 4,4-di-O-methylellagic acid (4,4-DiOMEA). The expression of WNT16 mRNA levels is correlated with concentrations of 4,4-DiOMEA, and this molecule exhibits robust antiproliferative effects on colon cancer cell lines, even ones that are resistant to well-established chemotherapy drugs such as 5-FU (Ramirez de Molina et al., 2015).

B. Extracellular Pharmacological Targeting of WNT Signaling: WNT-FZD Interaction

The interaction of the WNT ligands with their corresponding FZD receptors is the hallmark event initiating the signal transduction. A long list of compounds have been tested and have been found to influence the WNT-FZD interaction, whereas a probably even longer list has been identified via high-throughput methods (Koval and Katanaev, 2012) and awaits further investigation. As discussed in section III.E, the interaction of WNT and FZD components is complex. The scientific community has been assuming a rather simplified view of the kinetics, the selectivity of some WNTs for some FZD, the nature and varied types of complexes formed, the cofactors playing secondary roles in the complex formation, the role of G proteins, etc. Nevertheless, it appears that our knowledge is far from complete and more research is needed in our understanding of receptor complex formation. Thus, one should make sure to take into account all factors playing a major role in the formation of the WNT-FZD complex, before devising a plan to prevent or promote the complex formation (Schulte, 2015).

Blockade of FZD receptors with antibodies is an attractive therapeutic strategy, because antibodies usually bind their target with high affinity and selectivity. Various groups have used antibodies directed against several FZD isoforms. In a seminal study, Gurney et al. (2012) showed that the antibody OMP-18R5 (vantictumab) could be a novel tool in blocking multiple FZD receptor targets. Initially, OMP-18R5 was thought to bind to FZD₇ only; however, it was later shown to act antagonistically against FZD₁, _-2, _-5, and _-8 as well. OMP-18R5 is effective against a wide range of human cancers (lung, pancreas, breast, teratocarcinoma), and it was shown to act synergistically with first-line chemotherapy (Gurney et al., 2012). A chimeric humanized monoclonal antibody directed against FZD₁₀ was developed, code-named OTSA101. The plain (nonradiolabeled) antibody is a weak antagonist of FZD₁₀; however, when radiolabeled with Yttrium 90 (OTSA101-DTPA-90Y), it exhibits a robust antitumor effect. Even a single intravenous injection of the labeled monoclonal antibody suppressed the growth of sarcoma tumor cells in a mouse xenograft model, without leading to substantial toxicity (Fukukawa et al., 2008). Currently, OTSA101 is in Phase I clinical trial stage (https://clinicaltrials.gov/ct2/show/NCT01469975; “SYNFRIZZ”) with patients suffering from synovial sarcoma. Furthermore, Nagayama et al. (2005) described a polyclonal antibody (TT641 pAb) against FZD₁₀ that acts on its extracellular domain. An attenuated growth of synovial sarcoma tumor cells in vitro, as well as of xenografts after injection in mice was observed using this antibody. In addition, Paes et al. (2011) generated an FZD₄ monoclonal antibody (1.99.25). The antibody binds the FZD₄ CRD and thus neutralizes the WNT3A-mediated β-catenin signaling in a retinopathy mouse model. A polyclonal antibody directed against FZD₅ has been developed, with evidence showing that it attenuates the proinflammatory cytokines IL-6, IL-15, and RANKL in rheumatoid arthritis (Sen et al., 2001), while a blocking antibody against FZD₇ has been used both in vitro and in vivo in Wilm’s tumor, with beneficial effects (Pode-Shakked et al., 2011).

OMP-54F28 (ipafricept) is a novel fusion protein that targets FZD₈-interacting WNT ligands. It has been constructed by fusing the CRD of FZD₈ with the Fc domain of immunoglobulin. The latter competes with the native FZD₈ receptor for its ligands and thereby antagonizes WNT signaling (thus acting more as a scavenger of WNT ligands, rather than an antagonist of FZD). The first preclinical studies showed beneficial effects of OMP-54F28 against a variety of solid tumors (Le et al., 2015). Currently there is one clinical trial that has been completed, one is in progress, and two are recruiting patients, in the context of the use of OMP-54F28 in various human cancers (https://www.clinicaltrials.gov/ct2/results?term=omp54f28&Search=Search). The first data show beneficial therapeutic effects against ovarian cancer when the antibody is used along with paclitaxel and carboplatin (Fischer et al., 2015), whereas the antibody did not exhibit pronounced toxicity in patients with advanced solid tumors, although bone effects were observed (Jimeno et al., 2014).

Our laboratory has been active for more than a decade in developing interventions aiming at the WNT-FZD interaction. We focused on identifying molecules that block FZD proteins, thereby preventing the WNT and FZD complex formation, eventually leading to the attenuation of signal transduction. We previously showed that UM206, a 13-amino acid-long peptide fragment of WNT3A/WNT5A, can have profound effects on FZD-receptor blockade, leading to a suppression of WNT signaling pathway (Laeremans et al., 2011). UM206 possesses two Cys residues and it was shown that substituting both of them with another amino acid can have detrimental effects for the activity of the molecule, which is indicative for a possible covalent binding between the two Cys of UM206 and the Cys-rich domain of FZD. Furthermore, UM206 may be rather selective, as shown by the fact that it blocked FZD₁ and FZD₂ receptors in the presence of WNT3A, but not FZD₃ or FZD₄. Due to these pronounced effects of UM206 on the WNT signaling pathway, we decided to investigate its effects following MI in mice. UM206 treatment demonstrated beneficial effects on cardiac remodeling and prevented HF development 35 days post-MI, an outcome attributed to increased myofibroblast numbers in the infarct area and better scar healing. Reduced dilatation of the left ventricle and improved infarct healing were also observed in a pig MI model where UM206 was infused for 5 weeks post-MI, albeit that the myofibroblasts numbers were actually reduced in this study (Uitterdijk et al., 2016). It has to be noted, however, that direct binding of UM206 to the CRD of FZD has not been observed, and, given that the peptide is derived from a region of WNT5A that appears to be not in direct contact with FZD (Janda et al., 2012), it is more likely that peptide inhibits the formation of the complex of ligand and (co)-receptors in a different but yet unidentified way.

Foxy5 is a formylated hexapeptide that was shown to act via FZD₅ receptors, leading to impaired migration of MDA-MB-468 breast cancer tumor cells. The authors pretreated cells with a FZD₅-specific blocking antibody and observed an attenuation of the effects of Foxy5, a phenomenon implying that the compound is most probably mediating its effects via the FZD₅ receptor but not via FZD₂ (Safholm et al., 2006, 2008). The same group investigated later another compound called Box5, which shares the same amino acid sequence with Foxy5 but possesses a butyloxycarbonyl group (instead of a formyl group) at the N terminal of its hexapeptide. Box5 directly antagonizes the WNT5A-mediated cellular signaling by attenuating WNT5A-mediated Ca²⁺ and PKC signaling but, although it was not proven, there is the assumption that is also acts via a blockade of FZD₅ receptor similar to Foxy5 (Jenei et al., 2009).

Recently, a nonpeptidergic compound was described that can interact with FZD₄. The compound, named FzM1, was shown to bind to the intracellular loop three of FZD₄ and blocking the Norrin-stimulated activation of TCF/LEF with micromolar affinity. Interestingly, the compound is also able to act as a chaperone for the misfolded FZD₄ protein observed in familiar exudative vitreoretinopathy, restoring the impaired transport of this mutated receptor to the plasma membrane (Generoso et al., 2015).

Lee et al. (2015) identified five different novel and very potent molecules (1094-0205, 2124-0331, 3235-0367 and the two most potent ones, NSC36784 and NSC654259) that bind to the CRD of FZD₈ and can regulate WNT signaling. All five compounds could inhibit the WNT3A-mediated signaling in low micromolar concentrations. Interestingly, all five compounds showed LRP6-inhibitory activity, although the authors could not exclude that the FZD CRD inhibitors might be able to target other FZD receptor’s CRDs (other than FZD₈).

Niclosamide is an anthelmintic drug, used against tapeworm infestations; nevertheless, its antitumor properties have been demonstrated in a variety of cancers. It has been shown to act via WNT signaling (Li et al., 2014c), albeit not exclusively, and the targeting appears to be dependent on the context: it caused internalization of FZD₁ in an osteosarcoma cell line (U2OS) (Chen et al., 2009b) and attenuation of DVL2 in a colorectal cell line (Osada et al., 2011). Conversely, studies on prostate and breast cancer cell lines suggested that LRP6 suppression is a direct effect of treatment with niclosamide (Lu et al., 2011b). These findings show it is important to make the choice of niclosamide treatment heavily dependent on the type of cancer to be treated (Osada et al., 2011). Interestingly, curcumin (which will be discussed in more depth later) was found to suppress the expression of FZD₁ eightfold in MDA-1986 squamous cell carcinoma cell line, a finding that was paradoxical, because curcumin was always regarded to work more downstream (Yan et al., 2005). In this context, Zheng et al. (2017) very recently showed that curcumin might have additional inhibitory effects on WNT3A and LRP6. Previously, it was shown that WNT10B, FZD₂, and LRP5 might be activated, leading to amplification of the cascade (Ahn et al., 2010). These findings strongly suggest that curcumin could also exhibit its effects more upstream in the pathway, affecting the binding of the WNT ligand to its receptor (FZD) and to its coreceptor (LRP). In addition, there is evidence stressing potential deleterious effects of curcumin (even promoting cancer in some cases), thus more research is urgently needed to ascertain the toxicologic profile of the compound and its benefit-to-risk ratio (Burgos-Moron et al., 2010).

Sclerostin is an endogenous antagonist of the WNT cascade and is expressed practically exclusively by osteocytes. Sclerostin binds LRP5/6 (Li et al., 2005) and disrupts the LRP5/6-FZD complex formation. In fact, it binds more strongly to LRP6, whereas antisclerostin antibodies can reverse the sclerostin-mediated LRP6 inhibition (van Dinther et al., 2013) and prevent noxious effects on the bones (Tian et al., 2011).

Because phosphorylation of LRP6 is an essential element of WNT-mediated activation, interaction with the LRP5/6 coreceptor is another major point where pharmacological interventions could act on to influence the signaling cascade. Salinomycin is a potassium ionophore compound that was shown to possess WNT antagonistic activity. Salinomycin was found to inhibit the WNT1/FZD₅/LRP6 and WNT3/FZD₅/LRP6 complexes, to robustly reduce LRP6 protein levels, and to induce degradation of LRP6 in chronic lymphocytic leukemia cells (Lu et al., 2011a). Similar results were demonstrated by Lu and Li (2014), which also implicated mTORC1 and a activation of GSK3β in an LRP6-mediated cross-talking mechanism, leading to antitumor effects in prostate and breast cancer.

Leptin has been shown to possess WNT signaling-mediating capabilities. This weight-regulating hormone could induce the phosphorylation of LRP6 in Leptin KO mice (which show an attenuated LRP6 expression), whereas DKK1 had the opposite effect. Furthermore, leptin was shown to affect GSK3β levels as well as axin2 expression, implying that it possesses an activator role in the signaling (Benzler et al., 2013).

Silibinin, is a naturally occurring compound that exhibits in vitro and in vivo antitumor effects. Silibinin attenuated the endogenous LRP6 expression at the transcription level and blocks its phosphorylation in a variety of prostate and breast cancer cell lines (Lu et al., 2012). Some research groups have provided evidence for the potential of anti-LRP6 antibodies as blockers (Ettenberg et al., 2010; Gong et al., 2010) or inducers (Gong et al., 2010) of WNT binding via LRP6-mediated mechanisms. Lastly, a 28-amino acid peptide mimicking the LRP5/6 domain was described to interfere with the LRP5/6 and N-cadherin complex in osteoblasts. The peptide led to an activation of WNT signaling and its effects were dependent on N-cadherin (Hay et al., 2012).

The utilization of endogenous mediators of the WNT signaling pathway is also another means of interfering with the cascade. DKK1 is a natural inhibitor of the cascade (Mao et al., 2001), whereas sFRP isoforms demonstrate variable activities (see section IV.2) (Shi et al., 2007). DKK1 and DKK2 bind LRP6 at domains different to the ones required for the WNT/FZD complex formation (Mao et al., 2001). Neutralizing the effects of DKK1 by a polyclonal antibody was shown to inhibit tumor cell growth and cell invasion in vitro; the treatment was found to be relative safe (Sato et al., 2010b). A humanized monoclonal antibody against DKK1 called BHQ880 had beneficial effects in a mouse myeloma model, where it exhibited not only antitumor effects but also promoted bone formation (Fulciniti et al., 2009). Thus far, BHQ880 has been investigated in three clinical trials in myeloma patients (https://clinicaltrials.gov./ct2/results?term=BHQ880+&Search=Search). The trials have been completed; however, the drug is not yet available. It was reported that patients have been tolerating the treatment and that the antibody has caused meaningful improvements in the quality of their lives (Iyer et al., 2014).

Furthermore, active immunization with a DKK1 vaccine showed impressive results in boosting of T-cell production and protecting mice from myeloma or treating mice that were suffering from a myeloma tumor. The beneficial effects of the active vaccine could be further improved by the addition of B7H1-blocking or OX40-agonist mAbs.

sFRP manipulations can also regulate WNT signaling. Iminooxothiazolidines (compound 1–34) are reported to be potential inhibitors of SFRP1 and to promote osteogenesis (Shi et al., 2009). Following a detailed screening of approximately 440,000 compounds, Bodine et al. (2009) also described an inhibitor of sFRP1 called WAY-316606 that led to increased bone formation; the same group proposed the use of several other similar inhibitory compounds (Moore et al., 2010).

C. Intracellular Compartment

D. β-Catenin and Gene Transcription

Once β-catenin translocates into the nucleus, it forms complexes with several TCF/LEF transcription factors, as well as coactivators (namely p300, CBP, BCL9, Pygo, and others) (Cadigan and Waterman, 2012). Following this crucial step, it drives the transcription of a long list of β-catenin target genes, including AXIN2, CD44, CCND1 (cyclin-D1), MMP2/9, MYC, VEGF, and others (Herbst et al., 2014). The fact that the β-catenin transcriptional complex plays a key role in a wide range of pathologies (especially in cancer), explains the strong pursuit of new compound intervening with its action.

E. Conclusions and Future Directions

In the last decade, there has been a remarkable growth in the number of compounds targeting the Wnt signaling pathway. Based on the mutations identified in different components of the WNT/β-catenin pathway in multiple cancers, the majority of these compounds is designed to aim at this branch of the WNT signaling cascade. In cardiovascular conditions, however, alterations in the regulation of the pathway rather than mutations in its components are likely to contribute to the disease process. Therefore, for cardiovascular conditions the preferential sites of intervention in the pathway would be more upstream, at the level of WNT proteins or their interaction with the receptor complex. Fortunately, the number of compounds allowing upstream intervention is increasing and several of them have been tested, particularly in models of MI. As discussed in detail in section VIII, interventions in PORCN (WNT-974 and GNF-6231) or at the level of the receptor complex (UM206) showed beneficial effects on infarct healing. Moreover, also interventions aiming at the β-catenin signaling branch (BIO, ICG-001, and XAV939) improved the wound healing following MI, suggesting an involvement of β-catenin-mediated signaling; however, this does not exclude the involvement of non-β-catenin-mediated signaling in infarct healing. Unfortunately, pharmacological interventions have been only sparsely tested in other cardiovascular conditions, so it is recommended to perform drug interventions at different levels of the pathway in these models in the near future, preferably in a single experiment.

A major concern for the global inhibition of WNT signaling is the occurrence of side effects. Several rapidly dividing cells, e.g., in intestinal epithelium and skin, rely on WNT signaling, and an intervention therefore could disturb their normal proliferation, causing, e.g., diarrhea. Surprisingly, such side effects have rarely been observed where these drugs were administered. A potential explanation for this could be that the amount of WNT signaling needed by these epithelial cells is limited, allowing a partial blockade to be without harmful side effects on these cells. Obviously, further research will be needed on this topic before these compounds can be successfully applied in patients. Moreover, these studies should go hand in hand with the development and validation of biomarkers (such as circulating DKK) for WNT signaling activity, to obtain a quick surrogate end point for the effectiveness of the therapy in individual patients.

Recently, several registered drugs and food supplements have been shown to act as inhibitors of the WNT pathway. An obvious advantage of these compounds is that they can be readily applied in man, allowing a rapid translation of preclinical findings to patients. However, a downside is that the dose needed to block WNT signaling usually is quite high, which may give rise to side effects. This problem could be addressed by administering several different drugs at a lower dose. These drugs can be chosen to target the pathway at different levels, which can have an additive (or even synergistic) effect and result in sufficient inhibition of WNT signaling, without the occurrence of severe side effects. On the other hand, the newly developed drugs that target Wnt signaling in a more selective way should be tested and optimized for selectivity, toxicity, and pharmacokinetic profile to develop them toward use in the clinic.