Oxidized Low-Density Lipoprotein Induces WNT5A Signaling Activation in THP-1 Derived Macrophages and a Human Aortic Vascular Smooth Muscle Cell Line

The pathogenesis of atherosclerosis is complex, evolves, and involves many cell types. Macrophages and vascular smooth muscle cells (VSMCs) are critically involved in atherosclerosis development and progression. Several studies have shown that WNT5A protein is abundantly expressed in human atherosclerotic lesions; however, the mechanism and role of WNT signaling pathway activation is not clearly known. Using THP-1 derived macrophages, and human aortic VSMC cells, we evaluated in vitro how oxidized low-density lipoprotein (oxLDL) and WNT5A signaling interact in these two cell lines. We used western blot, scratch assay, metabolic proliferation assay, as well as immunostaining to analyze the effect of Wnt signaling activation. The results demonstrated that oxLDL, as well as WNT5A (control), induced Disheveled-2 (DVL2) activation and Kif26b degradation, indicating activation of non-canonical Wnt signaling. We found that oxLDL and WNT5A induced FZD5-ROR2 co-localization at the cellular membrane in vitro in THP-1 derived macrophages. Box5 (FZD5 receptor antagonist) inhibited oxLDL-induced DVL2/JNK activation secondary to newly secreted WNT protein from THP-1 derived macrophages. We found that WNT3A (canonical Wnt) and WNT5A showed different roles in this VSMC cell line. These findings indicate that WNT5A is upregulated by oxLDL, promotes foam cell formation, and affects VSMC phenotype and migration in these two cell lines. Also, in these cell lines FZD5 signaling seems to be necessary for lipid accumulation and, through this mechanism, WNT5A could modulate foam cell formation. Thus, our results suggest that WNT5A may contribute to the pathogenesis of vascular disease through modulating macrophage and VSMC behavior.


INTRODUCTION
Wnt signaling pathway activation has been reported in many cardiovascular diseases and as a consequence, the modulation of Wnt signaling has aroused scientific interest (1)(2)(3)(4)(5). Both non-canonical WNT signaling, as well as canonical signaling have been described to be activated in atherosclerosis (6)(7)(8). However, the exact mechanism of non-canonical WNT5A signaling in the atherosclerotic plaque remains elusive. The complex pathogenesis of atherosclerosis involves a cascade of biological events including endothelial dysfunction, monocyte transmigration, vascular smooth muscle cell (VSMC) proliferation and migration, as well as foam cell formation, among others (9). Macrophages and VSMC perform crucial roles at different stages in the pathogenesis of atherosclerosis (9,10). At the beginning, macrophages and VSMC are associated with the development of foam cells, whereas in advanced stages of the disease, macrophages and VSMC are involved in plaque stability and vulnerability (10,11).
WNTs primarily act as short-range ligands in autocrine and paracrine signaling, leading to the activation of pathways that regulate diverse cellular biological processes including cell proliferation, cell polarity, cell differentiation, and apoptosis (12). WNT signaling is classified into two general pathways: a canonical pathway involving β-catenin and the non-canonical pathway which is β-catenin independent. The latter consists of two sub-groups: the WNT/Ca 2+ and the Planar Cell Polarity (PCP) pathways (12,13). Specific WNT/receptor/co-receptor combinations are particularly important in dictating the resulting downstream signaling effects of a given WNT (12). For example, the co-receptor receptor tyrosine kinase-like orphan receptor 2 (ROR2) is critical for activation of the PCP signaling pathway by WNT5A (14,15). In this pathway, WNT5A binds to the receptor complex consisting of a Frizzled (FZD) receptor (i.e., FZD5) and co-receptor ROR (i.e., ROR2) (16,17). Unique combinations of the many receptor/co-receptor/ligand binding often correlate to specific downstream effects by a given WNT ligand (12,18). Downstream of these receptors, reliable readouts for non-canonical signaling activation have been lacking. Three Disheveled (DVL) proteins are integral to the downstream signaling activation of WNT proteins (14,15). DVL 1, 2, 3 are not specific to a given pathway; however, DVL2 and c-Jun N-terminal kinase (JNK) are important indicators of non-canonical WNT5A/PCP signaling activation (15,19). Recently, Susman et al. described that Kinesin Family Member 26B (Kif26b) is rapidly degraded in response to non-canonical WNT signaling activation and is a highly specific marker of non-canonical signaling activation (20,21).
The interaction between oxidized low-density lipoprotein (oxLDL) and macrophages and VSMC is a critical step in the pathogenesis of atherosclerosis, specifically in the formation of foam cells (22,23). Foam cells are a critical cell type in atherosclerosis development and VSMC's contribute to a large portion (∼50%) of the total foam cell population within the atherosclerotic plaque (23). Activation of macrophages triggers WNT secretion by macrophages and subsequent WNT signaling activation in neighboring cells (24). In this context, our group described increased transcription of WNT5A mRNA in mouse and human macrophages upon exposure to lipopolysaccharide (LPS) and oxLDL (2,6). Subsequently, we demonstrated that oxLDL increases the expression of CD36 in a FZD5 dependent mechanism (25). CD36 is a key scavenger receptor in macrophages, resulting in lipid uptake and foam cell formation (26). VSMCs also play other important roles in the pathogenesis of atherosclerosis, such as fibrous cap formation. A biological characteristic of VSMCs is their capacity to switch from contractile toward a synthetic phenotype, also referred to as myofibroblast phenotype, involving increased production of extracellular matrix (ECM) and increased proliferation. WNT proteins are implicated in migration and differentiation of VSMCs from the media to the intima which is crucial for fibrous cap formation, an important feature of stable plaques (27)(28)(29)(30)(31)(32). These findings demonstrate the importance of macrophages and VSMCs in the atherosclerotic process.
The present study seeks to dissect the effect of oxLDL and WNT5A in THP-1 derived macrophages and a human aortic VSMCs line., Our central hypothesis is that atherogenic stimuli (oxLDL) induce WNT signaling dependent cellular events associated with pathophysiological events in vascular disease.

MATERIALS AND METHODS
In-vitro THP-1 and Derived Macrophages Cell Culture THP-1 cells (Human Monocytic Leukemia cell line) were purchased (American Type Culture Collection, TIB-202 TM , Manassas, VA), and were cultured in RPMI-1640 supplemented with 10% FBS (complete RPMI) at 37 • C, 5% CO 2 as described previously (6,33). To induce monocyte-to-macrophage differentiation, THP-1 cells were stimulated with 50 ng/ml Phorbol-12-Myristate-13-Acetate (PMA) for 24 h. After activation and once adherent, the culture medium was removed and replaced with treatment medium (described below). Controls were treated with medium alone plus vehicle where applicable and maintained for the duration of all experiments. At the end of experiments, cell culture medium was collected and centrifuged (900xg) at 4 • C for 10 min to remove cell debris. The supernatant was then concentrated 40-fold using Amicon ultra centrifugal filter units (UFC801024, Millipore-Sigma, Burlington, MA). Briefly, supernatant (2 mL) was centrifuged at 7,500xg at 4 • C for 10 min (∼50 µL final volume). The supernatant was then stored at −80 • C before western blotting.

In-vitro Human Aortic Vascular Smooth Muscle Cell Line Culture
Human aortic vascular smooth muscle cells (VSMCs) were purchased from American Type Culture Collection (T/G HA-VSMC, CRL-1999 TM , Manassas, VA), and were cultured according to the supplier's instructions at 37 • C, 5% CO 2 . To make the complete growth medium, the following components (final concentrations) were added to the F-12K base medium

Migration and Wound Healing Assays
VSMCs were grown in complete growth medium to confluence in 6-well plates and then serum starved (to induce quiescence) for 24 h in serum-free medium (SFM) before treatment. After quiescence/starvation, the cell monolayer was subjected to two parallel scratches on a pre-drawn cross under the plate as a reference point. After the scratch, VSMCs were washed twice with PBS and incubated in 100 ng/mL rWNT5A FIGURE 1 | Box5 Blocks rWNT5A and oxLDL-Mediated Non-canonical Wnt Signaling in THP-1-Derived Macrophages. Representative western blots of THP-1-derived macrophages. THP-1-derived macrophages were pre-treated, or not, with Box5 (100 mM) for 1 h or not followed by treatment with rWNT5A (100 ng/mL), nLDL, or oxLDL (both 100 µg/mL) for an additional 24 h. Box5 significantly inhibited rWNT5A-induced and oxLDL-induced DVL2 activation (black arrow). Box5 also inhibited rWNT5A-induced JNK activation but not oxLDL-induced JNK activation, although overall activation was reduced. Experiments were performed in triplicate and western blot band intensities were quantified and are presented as bar graphs (mean ± SEM) of the ratios of DVL2-p/GAPDH, WNT5A/GAPDH, JNK-P/JNK-T expression relative to respective controls (UT or UT-Box5). ns = not significant compared to untreated control (UT or UT-Box5) (p > 0.05); * = compared to UT control (p < 0.05); *** = compared to UT control (p < 0.001); % = compared to Box5 UT control (p < 0.05); & = compared to nLDL control (p < 0.05); &&& = compared to nLDL control (p < 0.001). $ = compared to rWNT5A (p < 0.05). or rWNT3A supplemented treatment medium. To quantify migration, wells were photographed, including reference points, immediately following wounding and every 6 h for 24 h after wounding to measure the wound area migrated by the VSMCs. Phase-contrast images were acquired on a Nikon Eclipse TS100 microscope. Images were processed using analyzed with ImagePro Plus 7 software (Rockville, MD, USA) as follows: the Lopass large spectral filter (width 5, height 4, passes 3) was applied followed by automatic contrast enhancement (Brightness= 76, Contrast= 67, Gamma= 1.0) and finally threshold into binary output for wound area as a percent of total area. Colored vertical lines (red, blue, black) are estimated wound borders for clarity and do not indicate analyzed border data. Five independent experiments were performed.

Proliferation Assay
VSMCs were grown in complete growth medium to ∼60% confluence and then incubated for 24 h in SFM to induce quiescence. After 24 h, cells were then incubated with treatment medium supplemented with 100 ng/mL rWNT5A, 100 ng/mL rWNT3A or 10 ng/mL PDGF-BB and replenished every 24 h. At the end of the experiments, cell proliferation was assayed using CellTiter 96 R AQueous One Solution Cell

Lipid Accumulation
Neutral lipid droplets were analyzed using Invitrogen's HCS LipidTOX TM Phospholipidosis and Steatosis Detection Kit for image-based high-content screening assays (Invitrogen, H34476, ThermoFisher Scientific, Waltham, Massachusetts). Staining was prepared following the protocol of the manufacturer. Briefly, VSMCs were fixed in formalin and incubated with HCS LipidTOX TM Red Neutral Lipid Stain. Plates were then sealed without washing and imaged. Images were captured on a Nikon Microphot-SA fluorescence microscope or Nikon Eclipse Confocal Microscope A1 Ti-E (images were recorded at 512 × 512 or 2,048 × 2,048 pixels with a pixel dwell of 3.1) using Nikon NIS-Elements software (Nikon Melville, NY). At least ten high-power fields per biological replicate were collected. Three independent experiments were performed.  Representative western blots evaluating the expression of WNT signaling components in response to oxLDL. In response to oxLDL, there was a significant increase in WNT5A and DVL2 (black arrow) expression (p < 0.05, p < 0.01; respectively). There was also a significant decrease in Kif26b expression (p < 0.01), indicative of activation of non-canonical WNT signaling in oxLDL-treated VSMCs. There was a significant decrease in WNT3A expression (p < 0.01), in the oxLDL treated group. An overall increasing trend in WNT3A and active β-catenin expression in the Box5 pre-treated groups was found. All protein experiments were performed in triplicate and western blot band intensities were quantified and are presented as bar graphs (mean ± SEM) of the ratio of control normalized (protein of interest)/tubulin protein. ns = not significant to untreated control (UT or UT-Box5); * = compared between groups (p < 0.05); **= compared between groups (p < 0.01). against FZD5 and ROR2 (described in the Immunochemistry section) diluted in 1% BSA-PBST. On separate coverslips, species-matched IgG at the same concentration was used as an isotype control for the primary antibodies. After overnight incubation, the primary antibodies were carefully aspirated, and the cells were washed three times with PBS for 5 min each wash. The cells were then incubated with species-matched secondary antibody (1/1,500, A11008, A11005, Invitrogen) diluted in 1% BSA-PBS at room temperature for 1.5 h. The secondary antibody solution was then decanted and cells washed three times with PBS, 5 min each, in the dark. Coverslips were counterstained with DAPI and mounted on glass slides. Images were captured on a Nikon Eclipse Confocal Microscope A1 Ti-E (images were recorded at 512 × 512 and 2,048 × 2,048 pixels with a pixel dwell of 3.1) using Nikon NIS-Elements software. Experiments were performed in triplicate with at least 10 images per biological replicated captured at 20x, 60x, and 100x. For each experiment and biological replicate, over 100 cells were evaluated.

Statistical Analysis
GraphPad Prism5 software was used to generate graphs and for statistical analysis. For MTS proliferation data, one-way analysis of variance (ANOVA) was used to determine differences between groups followed by Tukey post-hoc analyses. Lipid accumulation analysis is described above. Migration analysis was performed using a two-way analysis of variance (ANOVA) to determine differences between groups at each time-point followed by Tukey's post-test to compare means. For densitometric analysis of western blots, one-way ANOVA was used to compare means of groups normalized by loading controls. For all analyses, p < As an approach to investigate the role of oxLDL on WNT signaling in macrophages, THP-1 derived macrophages were used. Results suggest that oxLDL induces WNT5A expression and DVL2 activation in THP-1 derived macrophages (Figure 1). To investigate the mechanism, we inhibited at the receptor level (FZD5) and inhibited endogenous WNT secretion.
First, FZD5 signaling was inhibited using the FZD5specific inhibitor Box5. Box5 has been described to be a competitive inhibitor of WNT5A through binding FZD5 receptor (34,35). The results in Figure 1 show that Box5 pre-treatment inhibited rWNT5A induced DVL2 activation andoxLDL-induced DVL2 activation in THP-1 derived macrophages (Figure 1). Further downstream in the WNT signaling pathway, rWNT5A and oxLDL increased activation of JNK, suggesting non-canonical WNT/PCP pathway activation. In the VSMCs line studied, oxLDL also significantly increased WNT5A expression and DVL2 activation (Figure 2, both p < 0.05), bul also Box5 pretreatment inhibited WNT5A signaling activation. Interestingly, oxLDL significantly decreased WNT3A expression in VSMCs and a reduction in active β-catenin relative to UT control (Figure 2, p < 0.01). Kif26b was significantly decreased in response to oxLDL compared to UT and nLDL (both, p < 0.01), confirming an upregulation of non-canonical WNT signaling.
Second, endogenous WNT secretion was inhibited to investigate if oxLDL-induced DVL2 activation was specific to the WNT signaling pathway, or an independent effect of oxLDL. To accomplish this, we used the inhibitor of Wnt Production 2 (IWP-2). In THP-1 derived macrophages there was reduced WNT5A detection in the cell culture supernatant after treatment with increasing concentration of IWP-2, with no changes in WNT5A expression in the whole-cell lysate ( Figure 3A). We then evaluated the intracellular activation of DVL2 in cells pretreated for 24 h with IWP-2 (5 µM DMSO) and then treated with nLDL or oxLDL. Compared to control cells, there was a clear decrease in DVL2 activation in response to oxLDL in In response to rWNT5A (100 ng/mL), expression of CD36 in VSMCs was significantly increased (**, p < 0.01), which was blocked by pre-treatment with Box5. *** = compared to UT control (p < 0.001); $ = compared to rWNT5A (p < 0.05).
the IWP-2 group (Figure 3B). Supplementary Figure 1 shows that WNT5A activated DVL2 over time, as evidenced by the electrophoretic mobility shift from ∼90 to 95 kDa. Together, these results demonstrate that oxLDL-induced DVL2 activation requires intact WNT secretion.

WNT5A Regulates THP-1 Derived Macrophages and VSMC-Derived Foam Cell Formation
To confirm that oxLDL results in lipid accumulation and foam cell development, THP-1 derived macrophages were treated with 100 µg/mL nLDL or oxLDL and stained with LipidTox Red and Oil Red O. Results demonstrated significant lipid accumulation in macrophages treated with oxLDL (Supplementary Figure 1). LipidTox staining of VSMCs treated with rWNT5A or oxLDL also resulted in a strong increase in lipid droplet accumulation within the cytoplasm (Figure 4A). When VSMCs were pretreated with Box5, the lipid accumulation in response to WNT5A and oxLDL was significantly blocked (Figure 4A, p < 0.001). Further, VSMCs increase the expression of CD36 increased in VSMCs in response to rWNT5A (Figure 4B, p < 0.01). Importantly, this increase was also blocked by Box5, suggesting a FZD5-specific mechanism for lipid accumulation.
The role of canonical signaling in foam cell formation is unknown. Our results demonstrated that WNT3A decreased  Representative phase-contrast microscopy images of migrating VSMCs in response to rWNT5A or rWNT3A. Cells were grown to confluence and subjected to wound-healing scratch assay. After the scratch, VSMC were washed 2x with PBS and incubated in rWNT5A or rWNT3A supplemented medium (both 100 ng/mL). VSMCs were then imaged every 6 h. Colored vertical lines are the estimated wound borders for clarity. Quantification was performed on phase-contrast images. rWNT5A promoted migration in comparison to untreated control (UT), as evidenced by a significant decrease in % wound area (*p < 0.01, n = 5 experiments). Additionally, rWNT3A inhibited migration compared to UT (*p < 0.01, n = 5 experiments). (B) Results of MTS proliferation assays in VSMCs. Cells were seeded at ∼60% confluence then incubated for 24 h in basal medium to induce quiescence. After 24 h, cells were then incubated with the indicated treatment medium. Date were normalized to a standard curve to determine cell number and represented as fold change. rWNT5A inhibited cell proliferation relative to control and complete medium, whereas rWNT3A induced VSMC proliferation almost to the same degree as the positive control (PDGF-BB). Data are representative of five independent experiments with each group performed in triplicate, $ p < 0.05 relative to complete medium; ***p < 0.001; ns = not significant relative to control for each time point.
lipid droplets in macrophages (Supplementary Figure 3). There was an increase in lipid accumulation in the WNT5A-treated group, confirming our previous report (Supplementary Figure 3). In the same experiment, macrophages were treated with WNT5A/WNT3A concurrently (both 100 ng/mL) to evaluate if there would be any opposing effects. WNT5A/WNT3A co-treatment demonstrated a similar level of lipid accumulation as untreated (UT) cells, which suggested opposing effects of WNT5A/WNT3A in regulating lipid droplet accumulation.
rWNT5A and oxLDL Promote FZD5 and ROR2 Co-localization in-vitro in THP-1 Derived Macrophages Receptor availability and localization are important for ligand-mediated signaling cascades. We next investigated the mechanism of WNT5A and oxLDL in the co-localization of FZD5 and ROR2 receptors in-vitro. Figure 5 demonstrates the expression of these receptors in THP-1derived macrophages in response to rWNT5A (100 ng/mL), nLDL (100 µg/mL), and oxLDL (100 µg/mL) for 24 h. Results in Figure 5 show a strong increase in expression of FZD5 and ROR2 in response to rWNT5A, nLDL, and oxLDL. Importantly, there was strong co-localization of these receptors at the cellular membrane in response to WNT5A and oxLDL (Figure 5, Pearson's Coefficients of r = 0.78 and r = 0.875, respectively). Isotype and negative controls are available in the data supplement (Supplementary Figure 2). These results provide evidence to suggest that in response to oxLDL non-canonical WNT5A signaling was activated.

WNT5A and WNT3A Differentially Regulate VSMC Migration and Proliferation
A wound healing/scratch assay was used to evaluate the migration in vitro of human VSMC cell line in response to rWNT5A or rWNT3A. Figure 6A showed representative phasecontrast microscopy images of migrating VSMCs in response to rWNT5A or rWNT3A with approximated migrating border (red, blue, black) for clarity. Quantification of migration demonstrated that, in response to rWNT5A (100 ng/mL), VSMCs migrated significantly more compared to control cells (18 and 24 h, p < 0.01) (Figure 6A). Further, in response to rWNT3A (100 ng/mL), VSMC migration was reduced relative to control (18 and 24 h, p < 0.01) (Figure 6A). These results suggested contrasting roles for WNT5A and WNT3A responses in VSMC migratory capacity.  Proliferation of VSMCs in response to rWNT5A and rWNT3A was evaluated using MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, salt] assay. rWNT5A inhibited cell proliferation relative to control and complete medium, whereas rWNT3A induced VSMC proliferation to a similar magnitude as a positive control (PDGF-BB) (Figure 6B). These results suggested distinct functions of WNT5A and WNT3A in VSMC proliferation.

WNT5A and WNT3A Perform Contrasting Functions in Determining Phenotypic Markers in the Human VSMC Line Studied
VSMCs were incubated in medium supplemented with 100 ng/mL rWNT5A or rWNT3A for 24 h. Protein lysates were then isolated and assayed by western blot. Results demonstrated that rWNT3A significantly increased the expression of plateletderived growth factor receptor beta (PDGF-R-β) and β-catenin ( Figure 7A, p < 0.001and p < 0.01, respectively). Figure 7A also demonstrated that rWNT3A decreased markers of contractile phenotypic differentiation (calponin, p = 0.01 and smoothelin, p = ns). Concomitantly, rWNT3A increased expression of the synthetic marker, collagen I (p < 0.05). Conversely, rWNT5A decreased (p < 0.05) collagen I expression (p < 0.05). Furthermore, rWNT5A decreased detection of β-catenin, which was significantly different from WNT3A (p < 0.01), and rWNT3A significantly increased β-catenin expression relative to control (p < 0.01), confirming significant signaling crosstalk of these two WNT proteins ( Figure 7A). We next wanted to evaluate if these WNT proteins, given their apparent opposing effects, directly competed to regulate VSMC phenotypic markers.
To determine if WNT signaling pathways interacted, we first chose to test if archetypal WNTs, WNT5A, and WNT3A, interact to determine VSMC phenotypic markers. VSMCs were incubated for 24 h with varying concentrations of recombinant WNT5A/3A proteins. Intriguingly, we found a clear WNT concentration-dependent gradient of the VSMC phenotypic markers investigated (Figure 7B). In summary, under higher relative concentrations of WNT5A, VSMC's may be directed toward a more contractile/quiescent phenotype, whereas with higher relative concentrations of WNT3A, VSMCs may be induced toward a synthetic, proliferative phenotype.

DISCUSSION
The interaction between oxLDL, macrophages, and VSMC are critical in the pathogenesis of atherosclerosis, specifically during atheroma formation. In a previous study, our group demonstrated that human peripheral blood macrophages increase transcription of WNT5A mRNA upon exposure to oxLDL (6). More recently, we have reported that WNT5A increases expression of CD36, resulting in oxLDL/lipid uptake within macrophages (25). The results presented in this study expand on this previous work. Our data suggest that WNT5A signaling activation through the FZD5 receptor is a potencial mechanism involved in foam cell formation in THP-1 derived macrophages and human aortic VSMC cell line used in this study. Our results cannot rule out the effects of blocking alternative FZD5 ligands; however, WNT5A, via FZD5 binding, remains an attractive target for therapeutic intervention.
WNT signaling is a complex system of multiple pathways, and the understanding of non-canonical WNT signaling is less well-described. In this study, we investigated the involvement of non-canonical WNT signaling pathways using Kif26b as a specific marker of non-canonical WNT signaling activation (20). Kif26b is specifically targeted by ubiquitination and degradation in a non-canonical WNT signaling-specific manner. Using this approach, we confirmed activation of non-canonical WNT5A signaling activation in VSMCs upon treatment with oxLDL. oxLDL also promotes WNT5A protein expression and secretion in THP-1 macrophages. Thus, non-canonical WNT signaling likely plays a role in arterial wall vascular biology.
WNT signaling activation is dependent on the receptor and cell context; here, we studied two receptors involved in noncanonical WNT signaling, FZD5 and ROR2 (36). Our results showed a high degree of FZD5 and ROR2 co-localization in foam cells in vitro. FZD5/ROR2 co-localization was inducible in response to oxLDL and WNT5A; interestingly, this effect was more pronounced with oxLDL treatment. Additionally, our results demonstrated for the first time that oxLDLinduced activation of WNT5A/DVL2 signaling was dependent on WNT5A secretion, suggesting an autocrine, positive-feedback loop (oxLDL/WNT5A/DVL2) where WNT5A signaling was activated in response to oxLDL. Dissecting further down in the WNT signaling pathway, our results showed that oxLDLinduced JNK-p46 activation was only partially blocked by Box5, indicating that oxLDL was likely inducing JNK activation via a secondary pathway independent of FZD5. This result is in line with results reported by Kim et al. where they demonstrated WNT5A induces JNK and NF-κB activation in THP-1 cells (37).
Although evidence has implicated non-canonical WNT signaling as pro-atherogenic (4,6,25), numerous reports demonstrated a critical role of the canonical WNT signaling pathway in cardiovascular disease (38)(39)(40). Other studies support the concept of an intricate balance of canonical vs. non-canonical WNT signaling pathways and their importance in vascular biology (41,42). VSMC phenotypic switching was described as an important initiating factor and key pathophysiological change in proliferative vascular diseases (43,44). VSMCs have a high capacity to differentiate/de-differentiate from a contractile to a synthetic phenotype, as evidenced by changes in protein markers in response to physical or molecular stimuli from the microenvironment. VSMC phenotypic differentiation involves proliferation, migration, and extracellular matrix (ECM) deposition. The current study showed the effect of WNT signaling on VSMC phenotypic markers. Interestingly, our results showan increase in collagen I protein in response to rWNT3A; this result is in line with previous reports that demonstrated increases collagen I RNA in mouse VSMCs line CRL-2797 in response to WNT3A (5). Further, our data demonstrated that archetypal canonical and non-canonical WNT proteins (WNT3A and WNT5A, respectively) induced proliferation (WNT3A) or migration (WNT5A). Co-treatment experiments demonstrated that the effects of WNT5A and WNT3A can be reversed in a concentration-dependent manner. The phenotypic switching of VSMCs has been associated to fibrous cap thickness, which dictated plaque vulnerability (8,31,32). Previous reports in the literature also regard VSMCs as important contributors to the total foam cell population (23). In line with this, our results also show that WNT5A may promote differentiation of thehuman VSMCs line used in the study into foam cells. All of these results suggest that WNT signaling plays a role in vascular smooth muscle cell biology. A limitation of our study is that our in vitro models used immortalized cell lines, these studies need to be extended using different models to comprehensively evaluate these novel mechanisms in humans.
In summary, this study implicated WNT5A signaling in THP-1 derived macrophages, human aortic VSMCs, and foam cells with the following lines of evidence. First, oxLDL promoted FZD5 and ROR2 co-localization in THP-1 derived macrophages, providing crucial novel information regarding WNT/FZD5 receptor/co-receptor combinations in foam cells. Further, oxLDL-induced DVL2 activation was dependent on WNT secretion, demonstrating for the first time a mechanism whereby WNT5A signaling was activated in response to oxLDL. Results also suggested that a balance of canonical/noncanonical WNT signaling activation may regulate switching the VSMCs phenotype. Taken together, these results suggest that WNT signaling overall may be an important player in thepathophysiology of vascular disease, making modulation of WNT signaling a potential therapeutic tool to improve cardiovascular disease outcomes.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
IA, CS, and RM designed the study and prepared the manuscript, with input from all authors. IA and CS performed experiments and data analyses. MS and RM guided the interpretation of the results. All authors contributed to the article and approved the submitted version.

FUNDING
The authors would like to thank the National Institutes of Health and the National Heart, Lung, and Blood Institute (NHLBI-R15HL133885) awarded to RM for helping to fund portions of this work. Other portions of this work would not have been possible without the Kopchick MCB/TBS Research Fellowship awarded to IA. Stipend and fellowship support to IA generously provided by the Osteopathic Heritage Foundations' Dual Degree Program at Ohio University Heritage College of Osteopathic Medicine, Athens, OH.