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. Author manuscript; available in PMC: 2015 Oct 30.
Published in final edited form as: Cell Rep. 2015 Oct 17;13(4):746–759. doi: 10.1016/j.celrep.2015.09.028

Impaired LRP6-TCF7L2 activity enhances smooth muscle cell plasticity and causes coronary artery disease

Roshni Srivastava a, Gwang-woong Go a,1, Anand Narayanan a, Jiasheng Zhang a, Arya Mani a,b,2
PMCID: PMC4626307  NIHMSID: NIHMS723923  PMID: 26489464

SUMMARY

Mutations in Wnt signaling coreceptor LRP6 have been linked to coronary artery disease (CAD) by unknown mechanisms. Here we show that reduced LRP6 activity in LRP6R611C mice promotes loss of vascular smooth muscle cell (VSMC) differentiation, leading to aortic medial hyperplasia. Carotid injury augmented these effects and led to partial to total vascular obstruction. LRP6R611C mice on high fat diet displayed dramatic obstructive CAD, and exhibited an accelerated atherosclerotic burden on LDLR knockout background. Mechanistically, impaired LRP6 activity leads to enhanced non-canonical Wnt signaling, culminating in diminished TCF7L2 and increased Sp1-dependent activation of PDGF signaling. Wnt3a administration to LRP6R611C mice improved LRP6 activity, led to TCF7L2-dependent VSMC differentiation and rescued post carotid injury neointima formation. These findings demonstrate the critical role of intact Wnt signaling in the vessel wall, establish a causal link between impaired LRP6/TCF7L2 activities and arterial disease and identify Wnt signaling as a therapeutic target against CAD.

Graphical abstract

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INTRODUCTION

Aberrant Wnt signaling is implicated in pathogenesis of coronary artery disease and its metabolic risk factors. Rare, highly penetrant mutations with large effects in the Wnt signaling coreceptor LRP6 (low density lipoprotein receptor related protein 6) gene have been associated with autosomal dominant early onset CAD (OMIM: ADCADII) (Go et al., 2014; Mani et al., 2007; Singh et al., 2013b; Wang et al., 2012; Xu et al., 2014). The canonical Wnt signaling pathway consists of a cascade of events that initiate after binding of a Wnt-protein ligand to a Frizzled family receptor and phosphorylation of its coreceptors LRP5/6. This leads to stabilization of β Catenin and its translocation to the nucleus, where it interacts with TCF/LEF family transcriptional activators to promote gene expression that regulates cell cycle, cell growth and proliferation. Wnt proteins also activate different β-catenin independent signaling pathways that are collectively referred to as non-canonical Wnt signaling. This pathway involves activation of CAMKII, JNK, Rho, Rac, and ROCK. Recent studies suggest that canonical and non-canonical pathways reciprocally inhibit each other and exert opposing effects on common targets such as TCF7L2.

CAD is an extremely heterogeneous disorder with various etiologies. While arterial occlusive disease is generally attributed to lipid- and macrophage-rich atherosclerotic plaques, several lines of evidence implicate VSMC proliferation as a key event in CAD development (Ross and Glomset, 1973). Coronary and carotid artery occlusions in patients with autosomal dominant mutations in the smooth muscle alpha actin gene (SM α-actin, a.k.a., ACTA2) have been linked to excessive proliferation of VSM (Milewicz et al., 2010). Pathological studies in young subjects with death from myocardial infarction without a plaque rupture have revealed excessive VSMC proliferation and endothelial erosion in absence of overt inflammation (Virmani et al., 2000). In addition, recent data has implicated smooth muscle cell transdifferentiation in atherogenesis. New studies in human atherosclerotic lesions have shown that about 50% of foam cells and 40% of CD-68 positive cells are of VSMC origin (Allahverdian et al., 2014). Fate mapping in apolipoprotein E deficient mice has shown that VSMCs deficient for SMC markers undergo transformation into macrophage-like cells and account for major part of advanced atherosclerotic lesions (Feil et al., 2014). Finally, lineage tracing of SMC in Apoe−/− mice has shown that large number of macrophages and mesenchymal stem cells (MSCs) in advanced atherosclerotic lesions are SMC-derived (Shankman et al., 2015). These findings provide strong evidence for the critical role of VSMCs in CAD development.

Various indirect evidence has implicated Wnt signaling in regulation of VSMC plasticity (Mill and George, 2012). However, absence of an animal model has prohibited in-depth investigation into the role of Wnt signaling in regulation of VSMC plasticity in the context of CAD development. By introducing the human LRP6R611C mutation into the endogenous mouse LRP6 gene we have generated one of the few existing mouse models of CAD. Here we describe the mechanisms by which an impaired Wnt/LRP6/TCF axis alters VSMC phenotype, causes CAD, and promotes atherosclerosis.

RESULTS

LRP6R611C mice on chow diet develop aortic medial hyperplasia

The rare LRP6R611C mutation found in humans causes severe early onset coronary artery disease. To understand the role LRP6 in cardiovascular disease, we generated a knock-in mouse expressing this mutant in the endogenous LRP6 locus. VSMCs cultured from mice homozygote for LRP6R611C mutation (from now on referred to as LRP6R611C mice) exhibited reduced LRP6 activity measured by LRP6 phosphorylation levels and resulted in impaired canonical Wnt signaling activity, manifested by reduced expression of its downstream target cyclin D1 mRNA (Fig. 1A and B). The effect of LRP6 mutation on the aortic wall was assessed in 3–6 month old LRP6R611C mice on chow diet and compared with age- and gender-matched wildtype mice. LRP6R611C aortas revealed increased medial thickening (Fig. 1C–E) associated with disrupted elastic fibers (Fig. 1D), an unusual findings often observed after vascular injury. The medial thickening was associated with VSMC hyperplasia (Fig. 1F), but no significant changes in aortic lumen size (Fig. 1G). LRP6R611C VSMCs exhibited considerably lower expression of the contractile proteins - SM α-actin and SM-MHC and increased expression of undifferentiated VSMC marker vimentin compared to WT, assayed by immunostaining (Fig. 1H) and Western blot analysis (Fig. 1I). LRP6R611C aortic VSMCs showed decreased expression of myocardin (Fig. S1 A), and increased phosphorylation/activation of ELK1 (Fig. S1 B), which act as transcriptional activator and suppressor of SMC genes, respectively. SM α-actin mRNA levels were lower in isolated LRP6R611C VSMCs, which further reduced upon PDGF-BB stimulation, as compared to WT (Fig. 1J). Further, LRP6R611C VSMCs, showed increased proliferation upon PDGF-BB stimulation, as compared to WT (Fig. 1K).

Figure 1. Impaired Wnt signaling in LRP6R611C mice causes vascular smooth muscle cell proliferation.

Figure 1

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(A) Western blot of p-LRP6 levels in R611C and WT VSMC. (B) Cyclin D1 mRNA in R611C and WT VSMCs. (C) Elastin autofluorescence (green) and nuclei (blue) staining of aorta, (D) H&E and Trichrome staining showing medial thickening, increased cellularity and disrupted elastic laminae (green arrows in R611C aorta vs. WT, respectively, (n=10). (E, F, G) Quantification of aortic media cross sectional area, cell count and lumen size (n=10). (H) Immunofluorescence staining of aorta for SM α-actin (green), SM-MHC (red) and vimentin (green) (n=5). (I) Western blot of SM α-actin and vimentin in VSMC. (J) SM α-actin mRNA expression in of WT and R611C VSMCs, baseline and upon PDGF-BB stimulation. (K) BrdU positive cells WT and R611C VSMCs, baseline expression and upon PDGF-BB stimulation. M= media, L= lumen, p- LRP6= phosphorylated LRP6, R611C = LRP6R611C, WT= wildtype. Quantification of western blots and q-PCR were performed on data from three independent experiments. Data represent means ± SD. Scale bar: 25μm. **** p <0.0001, ** p< 0.005, * p<0.05. See also Fig. S1.

Since PDGF signaling is a master regulator of VSMC differentiation, we next examined the activity of this pathway. LRP6R611C mice exhibited increased expression of PDGF receptors β and α as well as their ligands PDGF-BB and –AA compared to WT mice both in aortic media (Fig. 2A–C) and isolated primary VSMC (Fig. 2D). In addition there was increased expression of IGF1 in the aortic media of LRP6R611C mice compared to WT (Fig. 2E). Study of the crystal structure of the LRP6 has shown that R611C substitution results in relaxation of the EGF2 domain (Cheng et al., 2011), determine which can explain its reduced affinity for ligands. We have previously shown that the LRP6R611C mutation causes reduced, but not a complete ablation of LRP6 signaling, and signaling can be rescued by high levels of ligand (Mani et al., 2007). To determine whether activation of LRP6 would reduce the growth factor levels in LRP6R611C mice aorta, recombinant mouse (rm) Wnt3a was administrated to LRP6R611C and WT mice on alternate days for 3 weeks. This resulted in dramatic reduction of PDGFRβ, PDGF–BB, -AA and IGF-1 expression in the aortic media of LRP6R611C mice (Fig. 2 A to C and E). Most strikingly, this treatment caused significant reduction of the aortic medial thickening (Fig. 2F). Taken together, these findings indicated that altered LRP6 function promotes VSMC phenotypic transformation by enhancing growth factor levels.

Figure 2. Impaired Wnt regulation of growth factor expression in LRP6R611C mice.

Figure 2

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Immunostaining for (A) PDGFRβ, (B) PDGF-BB, (C) PDGF-AA in aortic cross-sections of R611C and WT mice (n=7 each). (D) Western blot analysis of PDGFRβ, PDGFRα, PDGF-BB, PDGF-AA in R611C and WT primary VSMC, and its quantification from three independent experiments. (E) IGF-1 expression in R611C and WT mice aorta (n=7 each). (F) Aortic media area quantification in WT and R611C mice, (n=7 each). A= adventitia, M= media, L= lumen, RC and R611C = LRP6R611C, WT= wildtype. Date represents mean ± SD. Scale bar: 25μm. **p<0.02, ****p<0.0001.

LRP6 transcriptional regulation of Sp1 and its effect on VSMCs differentiation

The upregulation of vimentin and growth factor ligands and receptors in LRP6R611C VSMCs indicated a change in the function of a master regulator of VSMC differentiation. A common feature among these proteins is that they are all regulated by Sp1, a ubiquitously expressed transcription factor and an established regulator of VSMC plasticity (Lin et al., 1992; Park et al., 1998; Zhang et al., 2003). Strikingly, there was a dramatic increase in expression of Sp1 protein (Fig. 3A, B) and mRNA (Fig. 3C) in LRP6R611C VSMCs compared to WT. These findings strongly suggested transcriptional suppression of Sp1 by LRP6. To test this hypothesis we stimulated primary VSMCs with rmWnt3a (50 ng/ml), which resulted in significant reduction of Sp1 (Fig. 3C). These changes were associated with an increase in SM α-actin mRNA levels (Fig. 3D) in WT and LRP6R611C VSMCs. Most remarkably, administration of rmWnt3a in LRP6R611C mice also resulted in decreased expression of Sp1 (Fig. 3A) and Sp1 target genes such as PDGF ligands, PDGFRβ, and IGF-1 (Fig. 2 A to C and E) and increased expression of the contractile proteins SM α-actin and SM-MHC (MYH11) (Fig. 3E) in the aorta.

Figure 3. Impaired TCF7L2-dependent transcriptional regulation of Sp1 in LRP6R611C mice underlies loss of VSMC differentiation.

Figure 3

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(A) Sp1 expression (arrows) by immunostaining in aorta of R611C and WT mice (n=7 each). (B) Sp1 protein levels in aorta lysates from R611C and WT mice by Western blot. (C) SP1 mRNA levels (**p=0.001, ****p<0.0001) in primary VSMC of R611C and WT. (D) SM α-actin mRNA levels in primary VSMC of R611C and WT (*p= 0.02, **p=0.003). (E) SM α-actin and SM-MHC protein levels in aortic lysates from R611C and WT mice. (F) Western blot showing changes in LRP6 phosphorylation, TCF7L2 and β Catenin levels in Wnt3a stimulated VSMC. (G) TCF7L2 expression (arrows) in R611C and WT mice aorta by immunostaining, (H) mRNA expression of Sp1 and SM α-actin in R611C primary VSMCs, with or without TCF7L2 overexpression (**** p<0.0001, **p=0.005). (I) Western blot of Sp1 target genes PDGF-AA, PDGFRα and vimentin in control and TCF7L2 overexpressing R611C and WT VSMCs. (J) ChIP assay demonstrating TCF7L2 binding of T-C-A-A-A-G motif in Sp1 gene upon Wnt3a stimulation (***p<0.001). IF data are representative of n = 7 mice per group, quantification of western blot and q-PCR data are from three independent experiments. Error bars show mean ± SD. Scale bar: 25μm. M= media, L=lumen, RC, R611C = LRP6R611C, WT= wildtype, NS = non-specific target. See also Fig. S2.

LRP6 regulation of Sp1 is mediated by TCF7L2

Polymorphisms in the Wnt effector TCF7L2 gene have been associated with the prevalence and severity of CAD(Sousa et al., 2011). DNA array-based genome-wide analysis combined with reporter assays has identified multiple TCF7L2 binding sites have been identified in Sp1 promoter (Hatzis et al., 2008). There was decreased expression of TCF7L2 (Fig. 3 F and G) and both total and nuclear and cytosolic β Catenin (Fig. 3F, and S2 A, B) in primary VSMCs and the aortic media of LRP6R611C mice compared to WT. While rmWnt3a treatment (2h) had only modest effects on β Catenin expression in LRP6R611C mice (Fig. 3F), it significantly increased TCF7L2 expression in LRP6R611C VSMCs both in vitro and in vivo (Fig. 3F and G). The treatment time course with Wnt3a showed that TCF7L2 expression steadily increases and peaks at 8h and Sp1 expression reduces and reaches its lowest level at 8 h (Fig. S2 E). We then examined the potential role of TCF7L2 in inhibiting Sp1 expression in LRP6R611C VSMCs. TCF7L2 was overexpressed in LRP6R611C VSMCs and its effect on Sp1 mRNA expression was examined. TCF7L2 overexpression resulted in downregulation of Sp1 transcription (Fig. 3H), reduced expression of Sp1 downstream targets PDGF-AA, PDGFRβ and vimentin (Fig. 3I) and upregulation of SM α-actin (Fig. 3H). Taken together, these findings indicate that TCF7L2 acts as a transcriptional suppressor of Sp1.

To explore if TCF7L2 directly suppresses Sp1 through DNA binding a ChIP assay was carried out. Sp1 gene contains several conserved TCF binding motif T-C-A-A-A-G (Gustavson et al., 2004; Hatzis et al., 2008). The assay revealed that one motif downstream from transcription initiation site exhibits enhanced binding to TCF7L2 upon Wnt3a activation (Fig. 3J). An earlier study had shown that the position of T-cell factors binding motifs downstream or downstream of transcription start sites may determine whether T-cell factors act as activator or suppressor (Gustavson et al., 2004). Taken together, our data suggests that TCF7L2 binding of T-C-A-A-A-G motif downstream from transcription initiation site in Sp1 gene is activated by Wnt3a and possibly plays a role in inhibition of its transcription.

Loss of TCF7L2 in LRP6R611C is caused by increased non-canonical Wnt signaling activity

LRP6R611C aortic media exhibited reduced canonical Wnt signaling as shown by reduced total and phosphorylated LRP6 and cyclinD1 expression levels (Fig. 4A to C). Recent studies have shown that impaired activation of LRP6 can result in increased activation of non-canonical Wnt signaling pathways. A major difference between canonical and non-canonical Wnt signaling pathways is that the former increases β Catenin nuclear localization and promotes TCF7L2 activity and expression(Singh et al., 2013a; Wang et al., 2015), whereas the latter wields opposite effects by Nemo like kinase (NLK)-mediated phosphorylation and ubiquitination of TCF7L2 (Ishitani et al., 1999). Reduced TCF7L2 expression in LRP6R611C VSMCs suggested a shift toward increased activity of the non-canonical Wnt pathway at the expense of canonical Wnt. Extensive analysis of non-canonical Wnt signaling pathway revealed increased activation of the non-canonical RhoA, JNK, and NLK in aortic media (Fig. 4D to F) and aorta lysates (Fig. 4G) of LRP6R611C vs. WT mice. Administration of rmWnt3a to LRP6R611C mice resulted in increased LRP6 phosphorylation (Fig. 4B), enhanced TCF7L2 (Fig. 3F) and cyclinD1 (Fig. 4C) expression and reduced activities of non-canonical Wnt pathways RhoA, JNK, and NLK (Fig. 4D to F). NLK binds to and phosphorylates TCF7L2 DNA at threonine residues T178 and T189 (Ishitani T, Mol Cell Biol 2003). In absence of a reliable antibody, we co-immunostained aortic cross sections with phosphorylated threonine and TCF7L2-specific antibodies. The immunostaining images showed significant co-localization of TCF7L2 and phosphothreonines, suggestive of increased TCF7L2 threonine phosphorylation in LRP6R611C as compared to wildtype mice (Fig. S2 C). This finding was also confirmed by TCF7L2 immunoprecipitation and Western blot analysis using anti-phosphothreonine antibody (Fig. S2 D). In addition, there was an overall increase in phosphorylated threonine staining in LRP6R611C aorta as compared to wildtype mice, which is consistent with increased growth factor signaling activity. These results establish a link between LRP6, non-canonical Wnt and TCF7L2 in the vasculature and underlie their importance in vascular integrity.

Figure 4. Increased activation of non-canonical Wnt in R611C mice and its rescue by Wnt3a.

Figure 4

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(A–C) Immunofluorescence of aortic sections demonstrating LRP6 (A), phosphorylated LRP6 (B), and cyclinD1 in aorta (arrows) (C), p-RhoA (D), p-JNK (E, arrows), p- NLK (F) and its quantification in R611C and WT mice, (n=7 each). (G) Western blot analysis of aorta lysate from mice treated with or without i.p. Wnt3a analyzed for LRP6 mediated non-canonical Wnt regulation and its effect on TCF7L2 and its quantification (n=6). Scale bar: 25μm. M= media, N= neointima, L=lumen, RC, R611C = LRP6R611C, WT= wildtype, p= phosphorylated.

Increased neointima formation post carotid artery injury in LRP6R611C mice is rescued by Wnt3a

VSMC proliferation is a typical response in following carotid artery injury and a major cause of neointima formation in diverse disease states. We next used guide wire carotid injury in LRP6R611C mice to determine whether LRP6R611C augments neointima formation and if Wnt3a treatment would rescue it. Three weeks post injury, LRP6R611C carotid arteries showed significant neointima formation compared to WT mice, which had minimal neointima (Fig. 5A, B). Although LRP6 plays a critical role in regulation of endochondral metaplasia, staining of the injured carotid artery with Alizarin Red did not reveal any evidence for the same (data not shown). The hyperplastic response of the injured LRP6R611C carotid was accompanied by reduced TCF7L2 (Fig. 5C) and increased Sp1 expression (Fig. 5D) and consequently low expression levels of SM α-actin (Fig. 5E) in LRP6R611C mice carotid arteries. Consistent with our earlier results, a dministration of i.p. rmWnt3a resulted in significant protection against neointima formation in injured LRP6R611C mice as compared to untreated mice (Fig. 5 A to D). This finding correlated with the rise in TCF7L2 (Fig. 5C) and fall of Sp1 expression levels (Fig. 5D) compared to untreated LRP6R611C mice.

Figure 5. Wnt3a rescues post carotid injury neointima formation in LRP6R611C mice.

Figure 5

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(A) Elastin staining, (B) Quantification of neointima formation (n=7 each). IF staining of carotid for (C) TCF7L2 (arrows), (D) Sp1 (arrows) and (E) SM α-actin and CD31 (endothelium), dotted lines mark the area of carotid artery, in R611C and WT mice post guide wire injury with or without i.p. Wnt3a and its quantification (n=7). N= neointima, M=media, L= lumen. Dotted lines separate neointima from media. Error bars show mean ± SD. **** p <0.0001, ** p< 0.005, * p<0.05. Scale bar: 25μm. R611C = LRP6R611C, WT= wildtype.

LRP6R611C mice on high cholesterol diet exhibit arterial neointima formation and coronary artery disease

The most striking finding of our study was the development of a dramatic form of CAD in LRP6R611C mice fed with high cholesterol/high fat diet for 10 months, despite only modest elevation of VLDL/LDL in these mice (Go et al., 2014). The aortic root and coronary arteries of LRP6R611C mice showed extensive neointima formation (Fig. 6A, B), which stained intensely positive for SM α-actin (Fig. 6C). This observation was in contrast to our earlier findings in LRP6R611C mice on chow diet and suggested maturation of VSMC in later stages of the disease. Surprisingly, the neointima exhibited paucity of F4/80 positive cells (Fig. 6D) and no significant changes in plasma cytokine levels between LRP6R611C vs. WT mice (Fig S3 C). Furthermore, there were increased vimentin positive cells in all aortic layers (Fig. S3 A).

Figure 6. LRP6R611C mutation induces formation of arterial neointima on high cholesterol diet.

Figure 6

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(A) Cross sections of WT and R611C mice hearts on HCD showing hyperplasia of CA (delineated by dotted lines) and AR in R611C vs. WT mice (arrows). (B) H&E staining of AR and CA showing neointima formation (arrows). Immunofluorescence staining of the neointima of AR and CA for (C) SM α-actin (green) (AR and CA), (D) F4/80 (CA). Data are representative of n = 9 mice per group. Scale bar: 25μm. AR= aortic root, CA=coronary artery, L=lumen, A= adventitia, M= media, N= neointima, R611C = LRP6R611C, WT= wildtype. See also Fig. S3 and S4.

The lamina media in LRP6R611C mice was remarkably small, which could be in part explained by migration of VSMCs into intima. We speculate that the undifferentiated VSMCs proliferate and migrate to intima, and ultimately undergo differentiation, a hypothesis that can only be verified by fate mapping studies. In addition, there was increased apoptosis by TUNEL staining in the tunica media (and adventitia) of the coronary artery and aortic root of LRP6R611C vs. WT mice, which could be also accountable for the thinning of the tunica media (Fig, S3 B top row). The increase in apoptosis perfectly correlated with the overexpression of Sp1, which is a strong driver of apoptosis (Deniaud et al., 2009).

To further examine the role of VSMC phenotype switching on atherosclerotic lesion development, LRP6R611C mice were crossbred onto low-density lipoprotein receptor knockout (LDLR−/−) background and were fed HCD for 4 months. En face aorta preparations of LRP6R611C; LDLR−/− mice showed greater than twofold increase in atherosclerosis burden compared to LDLR−/− mice (48% vs. 20%) (Fig. 7A). LRP6R611C mice en face aorta preparations did not show any positive staining with Sudan IV (data not shown). Accordingly, there was a significant increase in atherosclerotic lesion size in the aortic roots of LRP6R611C; LDLR−/− vs. LDLR−/− mice (Fig. 7B). Examination of atherosclerotic lesions in the aortic root cross-sections of LRP6R611C; LDLR−/− mice, however, showed once again increased presence of SM α-actin and vimentin positive cells (Fig. 7C) and increased apoptosis (Fig. S3 B, bottom row) compared to LDLR−/− mice. In addition, the coronary arteries of the LRP6R611C; LDLR−/− mice were significantly enlarged and showed either luminal narrowing or occlusion, primarily accounted for by SM α-actin positive cells, (Fig. 7D). There was enhanced elastin staining in the atherosclerotic lesions of LRP6R611C; LDLR−/− vs. LDLR−/− mice, providing further evidence for the presence VSMCs in the lesion (Fig. 7G). Remarkably, the atherosclerotic lesions in LRP6R611C; LDLR−/− mice exhibited significantly fewer F4/80 or CD36 positive cells compared to LDLR−/− mice (Fig. 7 H and I). Accordingly, plasma cytokine profiling of LRP6R611C; LDLR−/− mice showed no significant difference in the plasma levels of inflammatory cytokines IL1α/β, IL6, IL10, IL17, IFNγ, TNFα but significantly lower plasma levels of MCP-1, a critical chemokine for monocyte recruitment and activation as compared to LDLR−/− mice (Fig. 8K). In comparison, there was an increase in the number of CD3+ T cells (Fig. 7J) and IL6 levels (Fig. S3 D) in the atherosclerotic lesions of LRP6R611C; LDLR−/− vs. LDLR−/− mice. No significant changes in MMP9, another inflammatory marker was observed (Fig. S3 C). We further examined the causes of increased atherosclerotic burden of LDLR−/− mouse by the mutant allele in absence of increased inflammation. Earlier studies by our group had shown increased cholesterol synthesis in diverse human and mice cell types expressing LRP6R611C. Thus, we compared the free cholesterol content of the aortic wall between LRP6R611C and WT mice as well as LRP6R611C; LDLR−/− vs. LDLR−/− mice. The result was striking, as the entire aortic wall in LRP6R611C and LRP6R611C; LDLR−/− mice was positive for filipin as compared to modest staining in WT and LDLR−/− mice (Fig. S4 A, B). Strikingly, LRP6R611C VSMC expressed significantly higher HMGCR protein, despite greater cholesterol content compared to WT mice. Altogether these findings suggested that VSMCs synthesize and accumulate free cholesterol and increase the atherosclerotic burden. While it is widely accepted that VSMCs form protective fibrous caps and stabilize atherosclerotic lesions, we demonstrate here that they can also augment atherosclerotic burden by proliferation and expansion of neointima and generation of occlusive disease.

Figure 7. LRP6R611C mutation increases the atherosclerosis burden in LDLR−/− mice.

Figure 7

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(A) Atherosclerotic lesions (red) in en face aorta preparation of LRP6R611C; LDLR−/− and LDLR−/− mice (n=7, 8). (B) Atherosclerotic burden in AR. (C) SM α-actin (top row, arrows) and vimentin (bottom row, arrows) staining of atherosclerotic lesions. (D) SM α-actin staining showing enlarged and occluded arteries in LRP6R611C; LDLR−/− vs. LDLR−/− mice. (E) Quantification of SM α-actin. (F–J) Staining of atherosclerotic lesions in AR for (F) elastin (black); (G) CD68 (brown); (H) F4/80 (red); (I) CD36 (green) and CD3 (red, arrows). (K) Plasma cytokine profiling of LRP6R611C; LDLR−/− vs. LDLR−/− mice. Data represent mean ± SD. Scale bar: 25μm. AR= aortic root, M= media, L= lumen, R611C = LRP6R611C, WT= wildtype. *p=0.02, **p< 0.01, ***p<0.001. See also Fig. S4.

DISCUSSION

Despite intensive investigations over the past decades, progress in identification of novel CAD risk factors has been incremental. Aberrant Wnt signaling has recently emerged as a risk factor for CAD and diabetes (Go et al., 2014; Mani et al., 2007; Singh et al., 2013b; Wang et al., 2012; Xu et al., 2014). In this study, we report that the CAD-associated LRP6R611C mutation causes increased non-canonical Wnt activation, alters VSMC phenotype, and leads to development of obstructive CAD in mice. This finding establishes the association between loss of function LRP6 mutations and CAD in humans and implies a critical role of non-canonical Wnt in development of arterial disease.

There have been several lines of evidence in support of VSMCs lack of terminal differentiation (Gomez and Owens, 2012). Most recent studies have provided strong evidence for VSMC transdifferentiation into macrophages in atherosclerotic mouse models (Allahverdian et al., 2014; Feil et al., 2014). Lineage tracing of SMC in Apoe−/− mice has shown that large number of macrophages and mesenchymal stem cells (MSCs) in advanced atherosclerotic lesions are SMC-derived (Shankman et al., 2015). The contribution of VSMCs to neointima formation in humans has been depicted by microscopic evidence of their migration through disrupted internal elastic lamina into the neointima (Schwartz et al., 1995). Most strikingly, the autopsy examination of coronary artery plaques in young men and women who had died from myocardial infarction have revealed massive proliferation of VSMCs in absence of inflammatory cells in nearly half of all cases (Farb et al., 1996; Virmani et al., 2000). This important clinical feature is rarely seen in mouse models of atherosclerosis. Our study in this novel mouse model of human mutation reveals medial and neointimal thickening, including occlusive lesions in the absence of excessive lipids or inflammation. This establishes the key role of VSMCs in CAD development and identifies LRP6 as critical regulator of their plasticity. These findings suggest cell autonomous effect of LRP6. Nonetheless, LRP6 is ubiquitously expressed and the effect of the mutant allele on the function of other cell types such as vascular endothelial cells and cells of myeloid lineage may have contributed to the disease. Several lines of evidence, however, supports the important role of LRP6 in VSMCs. Of note, mice with VSMC specific LRP6 knockout recently were shown to develop arterial calcification (Cheng et al., 2015). Interestingly, human mutation carriers have coronary artery calcification, but this trait was not observed in our mouse model

Earlier in vitro studies had implicated both canonical and non-canonical Wnt in VSMC proliferation but the underlying mechanisms were unclear. In the current study we show that non-canonical Wnt regulation of VSMC plasticity is TCF7L2-dependent and is exercised through modification of Sp1 expression levels. A ChIP assay demonstrated TCF7L2 binding to Sp1 gene downstream from transcription initiation site upon Wnt3a stimulation differentiation. Sp1 is a ubiquitously expressed transcription factor that regulates a diverse array of cellular processes, including VSMC differentiation. JNK has been also shown to increase Sp1 transcriptional activity by promoting its phosphorylation (Tan and Khachigian, 2009). Thus, enhanced non-canonical Wnt activity in LRP6R611C VSMCs may contribute to excess activity and availability of Sp1.

Common genetic variants in TCF7L2 have been associated with the risk for diabetes and hyperlipidemia and coronary artery disease (Muendlein et al., 2011), indicating the broader role of this transcription factor in diverse cardiovascular disorders of the general population. TCF7L2 activation and expression is triggered by the canonical Wnt (Singh et al., 2013a) and is inhibited by non-canonical Wnt activation of NLK (Ishitani et al., 1999). NLK phosphorylates TCF7L2, which results in inhibition of its DNA binding and its subsequent targeting to ubiquitination and degradation. The rescue of the vascular phenotype by Wnt3a was associated with reduced phosphorylation and increased expression of TCF7L2, further highlighting the critical role of non-canonical Wnt in this process. Interestingly, VSMC calcification has been recently shown to be triggered by loss of LRP6 and increased activation of non-canonical Wnt (Cheng et al., 2015).

Certain Wnt ligands are specific for canonical vs. non-canonical Wnt pathway, which are known to reciprocally inhibit each other (Nusse, 2012). Wnt3a has been surprisingly shown to activate both pathways, although, the specific circumstances and the mechanisms had not been explored (Nalesso et al., 2011). Our rescue studies in LRP6R611C mice shows the critical role of Wnt3a in regulation of non-canonical Wnt in the vessel wall. This rescue as we have previously shown is possible by larger Wnt3a dose to overcome reduced affinity of the mutant receptor for ligands. Our investigation of CAD development in humans and mice with LRP6R611C mutation indicates the pathological role of excess non-canonical Wnt activity in the VMSC.

One unexpected finding of our study was reduced expression of macrophage markers F4/80, CD68 and MCP-1 in LRP6R611C; LDLR−/− despite increased atherosclerotic burden compared to LDLR−/− mice. These findings are, however, logical and consistent with the established role of Wnt signaling in monocyte/macrophage maturation and promotion of inflammatory response (Pereira et al., 2009). In comparison, there was increased CD3+ T-cells in atherosclerotic lesion of LRP6R611C LDLR−/− vs. LDLR−/− mice. Earlier studies have shown that Wnt signaling is required for T cell proliferation arrest and negative regulation of regulatory T Cells (Shen et al., 2013) (van Loosdregt et al., 2013) and hence loss of Wnt signaling in LRP6R611C mice could explain increased T cell proliferation. There was also increased eotaxin levels in LRP6R611C; LDLR−/− vs., LDLR−/− mice, suggesting increased eosinophil activation. Increased eosinophil activation in patients with hypereosinophilia has been attributed to impaired Wnt signaling, measured by lower cyclin D1 and β-catenin levels. Interestingly, eotaxin has been shown to activate T cell infiltration, which may be another explanation for increased T cells in LRP6R611C; LDLR−/− vs., LDLR−/− lesions (Giannetti et al., 2014). Finally, increased T cell in LRP6R611C; LDLR−/− vs., LDLR−/− may have been contributed by increased IL-6, as IL-6 is known to promote T cell proliferation (Dienz and Rincon, 2009). The role of IL-6 in atherosclerosis, however, has been controversial and may be context dependent (Ait-Oufella et al., 2011). Nevertheless, contribution of CD3 and IL6 in LRP6R611C; LDLR−/− needs further investigations. Another limitation of our study is that it does not answer the disparities in VSMC phenotype i.e. undifferentiated VSMC in the aortic wall and highly differentiated VSMC in the coronary artery neointima and in atherosclerotic lesions. Based on recent studies, we believe these are VSMC that show significant plasticity at different stages of the development. A definitive answer, however, can be provided by fate mapping in these mice.

In summary, the LRP6R611C knock-in mouse constitutes one of the very few known rodent models of CAD. This model animal recapitulates features of human lesions and demonstrates the critical role of VSMCs in pathogenesis of CAD. In this model, we were able to show that altered function of Wnt/LRP6/TCF7L2 axis can induce VSMCs plasticity and initiate vascular wall remodeling. These findings identify LRP6 and TCF7L2 as regulators of vascular wall integrity and as potential targets for the pharmacotherapy of coronary artery disease.

EXPERIMENTAL PROCEDURES

Animals

Animal procedures were as per approved protocol of Yale University Institutional Animal Care and Use Committee (IACUC). Generation of homozygous LRP6R611C and LRP6R611C; LDLR−/− mice were previously described (Go et al., 2014). All mice used for the studies are homozygous and are referred to as either as LRP6R611C in the text or R611C or RC mice in the figures. All mice were fed ad libitum and housed at constant ambient temperature in 12hour light, 12hour dark cycle. For high cholesterol diet studies, at 6–8 weeks of age the mice were fed high cholesterol diet (40% fat, 1.25% cholesterol, 0.5% cholic acid) ad libitum (Research Diets Inc.) for4 or 10 months.

Chemicals and antibodies

Protease inhibitor cocktail (P8340), phosphatase inhibitor cocktail (P2850), Sudan IV and Oil red O were purchased from Sigma-Aldrich. Cell lysis buffer (9803) and antibodies for LRP6, p-LRP6(S1490), β actin, PDGFRβ, p- PDGFRβ (y751/y771), cKit were all purchased from Cell Signaling Technology. PromoFectin (PK-CT-2000-100) was purchased from Promokine. Wnt3a from R&D Systems; Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin streptomycin cocktail, Trypsin-EDTA solution, and TRIzol were purchased from GIBCO/Invitrogen; polyvinylidinefluoride membranes from Bio-Rad Laboratories, Filipin stain from Cayman Chemical. Antibodies for PDGFRα, β Catenin, TCF7L2, SP1, CD3 and protein A/G agarose gel were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies for PDGF-AA/-BB, F4/80, αSMA, SM-MHC were purchased from Abcam (Cambridge, MA), CD31 antibody from BD Pharmingen, secondary fluorescence tagged antibodies were purchased from Invitrogen.

Immunohistochemistry (IHC) and Immunofluorescence (IF)

IF staining was performed on 5μm frozen sections and fluorescence was measured using Nikon Eclipse80i using same laser output, gain and offset for each set of antibody tested. For atherosclerosis studies, whole aorta was trimmed off extraneous tissues and placed in formaldehyde sucrose solution and then pinned on wax pan and stained with Sudan IV and imaged. Aortic root sections were stained with Oil red O and. Images were and quantified using Image J.

VSMC isolation and culture

Isolation of aortic VSMCs was carried out as previously described (Ray et al., 2001). VSMCs were maintained in DMEM (4.5g/L glucose, glutamine, 100mg/L sodium pyruvate) supplemented with 20% FCS, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. For phosphorylation studies, cells were starved for 3 h in 0.2% FBS containing DMEM and were treated as follows: PDGF-BB at 10ng/mL for 15mins, Wnt3a (50ng/mL) for 1h prior to PDGF-BB stimulation. For Wnt3a dependent LRP6 phosphorylation and downstream targets cells were starved for 3 h in 0.2% FBS containing DMEM and were treated with Wnt3a for 2h. For mRNA studies, the cells were starved overnight in 0.2% FBS containing DMEM and treated with PDGF-BB (10ng/mL) or Wnt3a (50ng/mL) for 8h. For time course Wnt3a studies, cells were starved overnight in 0.2% FBS containing DMEM and treated with Wnt3a upto 8h.

In vitro TCF7L2 overexpression

Primary VSMCs were transfected with TCF7L2 plasmid (11031, Addgene) using PromoFectin transfection reagent according to the manufacturer’s instructions. Briefly, 1 μg of TCF7L2 plasmid DNA or empty vector control plasmid DNA were diluted in 50 μl of Opti-MEM, were mixed with 2μl of PromoFectin solution diluted in 50 μl of Opti-MEM, and incubated for 30 minutes at room temperature. The plasmid DNA solution was then added drop-wise into VSMC culture in antibiotic free medium. After 5h the medium was replaced with fresh antibiotic free medium. After 48 hours of transfection cells were starved overnight and harvested for analysis.

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed according to the manufacturer’s instructions (Pierce Agarose ChIP kit, 26156). Briefly, the chromatin/DNA protein complexes were prepared from mouse aortic smooth muscle cells treated with vehicle (PBS with 0.1% BSA) or Wnt3a (50 ng/ml) for 8hr. Chemical crosslinking of DNA-proteins was carried out using 1% formaldehyde for 10 min at room temperature and followed by addition of glycine solution. Cells were scraped into cold PBS containing Halt cocktail proteinase inhibitor. The cell suspension was centrifuged and the pellet was lysed and nuclei was digested using micrococcal nuclease to digest DNA to a length of approximately 200–1000 bp. Supernatant containing the digested chromatin was incubated with appropriate ChIP-grade TCF-4 (TCF7L2) antibody (sc-8631, Santa Cruz Biotechnology, Inc.) for immunoprecipitation overnight at 4°C with rotation, followed by ChIP-grade protein A/G agarose beads and incubation for 1 hr at 4°C with rotation. Anti –H3 antibody and β Actin primers were used as a positive control for assay technique and reagent integrity. The agarose resin was washed using buffers supplied with the kit. The eluted DNA was purified and analyzed by PCR to determine the binding of TCF7L2 to Sp1. The positions of TCF7L2 binding site in mouse Sp1 gene were determined (consensus sequence: TCAAAG) (Hatzis et al., 2008). The following primers were used to amplify the binding region: forward 5′-TGCAGCAGAATTGAGTCACC-3′, and Reverse 5′-CAGCCACAACATACTGCCCAC-3′. The primer sequences for β Actin promoter were: Forward 5′-GAGGGGAGAGGGGGTAAA -3′ and reverse 5′-GAAGCTGTGCTCGCG G -3′. Real-time PCR amplification was performed using iQ SYBR Green Supermix (Bio-Rad) and Eppendorf Mastercycler RealPlex2.

Carotid artery guide wire injury and intraperitoneal rmWnt3a administration

Mice were injected with 25 mg/kg i.p. rmWnt3a every other day for 3 weeks beginning one day prior to carotid wire injury. Similarly, control mice group received equal volumes of carrier buffer in which rmWnt3a was dissolved. The carotid artery guide wire injury was performed as previously described35. Three weeks post injury mice were euthanized and injured carotid arteries were excised from the arteriotomy site of external left carotid artery, including the internal left carotid artery and approximately 1cm of left common carotid artery. Similarly, right common carotids were harvested and used as uninjured controls. The arteries were embedded in OCT and serial tissue sections (5 μm) were obtained from left and right common carotid arteries, starting at the bifurcation (to external and internal carotids) and IF, IHC and morphometric analyses were performed. Neointima formation was measured in 10 sections (50μm apart) using images obtained by a bright-field microscope and quantified using ImageJ software (NIH, Bethesda, MD). Aorta and aortic roots were also harvested for studying Wnt3a effects on LRP6R611C as compared to the controls by immunoblotting and immunofluorescence studies.

Immunoblotting

Whole-cell lysates of primary VSMCs were separated by electrophoresis, transferred to PVDF membrane and probed using target primary antibodies followed by appropriate HRP-conjugated secondary antibodies. Blots were visualized using chemiluminescence reagents, imaged with BioRad gel doc system and quantified with Image J software.

Real-Time PCR

Total RNA was isolated from primary VSMC culture using TRIzol, and complementary DNA was generated using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Real- time PCR amplification was performed using specific primers and iQ SYBR Green Supermix in Eppendorf Mastercycler RealPlex2. Reactions were performed in quadruple with a β-actin internal control. Relative quantification of mRNA levels was expressed as fold increase relative to the control. The following mouse primer sequences were used for qRT-PCR:

  • SM α-actin Forward: 5′-CAGCTATGTGTGAAGAGGAAGACA-3′

  • SM α-actin Reverse: 5′-CCGTGTTCTATCGGATACTTCAG-3′

  • Sp1 Forward: 5′-CTGGTGGGCAGTATGTTGTG

  • Sp1 Reverse: 5′-TTGGTTTGCACCTGGTATGA-3′

  • CyclinD1 Forward: 5′-GCCTCTAAGATGAAGGAGACCA-3′ CyclinD1

  • Reverse: 5′-AGGAAGTGTTCGATGAAATCGT-3′

Apoptosis detection

To detect apoptosis, aortic root cross sections were fixed and stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) by using an ApopTag In Situ Apoptosis detection kit (S7111, Chemicon) and counterstained with DAPI, as per manufacturer’s protocol. Fluorescent apoptotic cells were visualized using fluorescein excitation and emission filters.

Cytokine analysis

Plasma cytokine detection and quantification was done at the Yale CytoPlex- multiplex core facility using the Multiplex System that analyses 23 mouse cytokines.

Statistical analyses

All in vivo studies included at least seven mice per genotype. For rescue studies using i.p. Wnt3a n=7 mice per group were used. All in vitro studies were carried out in three independent experiments in triplicate. Fluorescence and area measurements were done using Image J software (NIH, Bethesda, MD). Preparation of graphs and all statistical analyses were carried out using GraphPad Prism 6 Project software (GraphPad, La Jolla, CA). p <0.05 was considered significant. Data are presented as mean ±SD.

Highlights.

  • LRP6R611C mice exhibit aortic medial hyperplasia and coronary artery disease.

  • LRP6R611C mice VSMC have reduced TCF7L2 expression and are undifferentiated.

  • Activation of noncanonical Wnt is increased in LRP6R611C mice VSMC.

  • Wnt3a normalizes the noncanonial Wnt and TCF7L2 activities and rescues the phenotype.

Acknowledgments

The authors gratefully acknowledge Dr. Kathleen Martin for reading the manuscript and providing valuable comments.

Funding Sources: The study was supported by National Institutes of Health grant 1R01HL122830 and 1R01HL122822 (to A.M.).

Footnotes

Conflict of Interest Disclosures: None.

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