Significance of the Wnt signaling pathway in coronary artery atherosclerosis

Abstract

Introduction: The progression of coronary atherosclerosis is an active and regulated process. The Wnt signaling pathway is thought to play an active role in the pathogenesis of several cardiovascular diseases; however, a better understanding of this system in atherosclerosis is yet to be unraveled.

Methods: In this study, real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to quantify the expression of Wnt3a, Wnt5a, and Wnt5b in the human coronary plaque, and immunohistochemistry was used to identify sites of local expression. To determine the pathologic significance of increased Wnt, human vascular smooth muscle cells (vSMCs) were treated with Wnt3a, Wnt5a, and Wnt5b recombinant proteins and assessed for changes in cell differentiation and function.

Results: RT-PCR and Western blotting showed a significant increase in the expression of Wnt3a, Wnt5a, Wnt5b, and their receptors in diseased coronary arteries compared with that in non-diseased coronary arteries. Immunohistochemistry revealed an abundant expression of Wnt3a and Wnt5b in diseased coronary arteries, which contrasted with little or no signals in normal coronary arteries. Immunostaining of Wnt3a and Wnt5b was found largely in inflammatory cells and myointimal cells. The treatment of vSMCs with Wnt3a, Wnt5a, and Wnt5b resulted in increased vSMC differentiation, migration, calcification, oxidative stress, and impaired cholesterol handling.

Conclusions: This study demonstrates the upregulation of three important members of canonical and non-canonical Wnt signaling pathways and their receptors in coronary atherosclerosis and shows an important role for these molecules in plaque development through increased cellular remodeling and impaired cholesterol handling.

Keywords: RT-PCR; cholesterol efflux; human; immunohistochemistry; vascular smooth muscle cells.

Figure 1

Coronary plaques have greater mRNA and protein expression of Wnt signaling proteins compared with healthy tissues. (A) An increased mRNA expression of Wnt3a and Wnt5b in diseased coronary arteries. The data are represented as mean ± SEM. *P < 0.05 compared with healthy tissues; n = 11 for coronary plaques and n = 7 for healthy tissues. Student’s t-test was used for statistical analysis. (B) A representative Western blot of Wnt proteins in diseased and healthy coronary arteries. (C) A densitometry analysis of the Western blot of Wnt protein expression in diseased and healthy coronary arteries. The data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with normal tissues; n = 9 for coronary plaques and n = 5 for normal tissues. Student’s t-test was used for statistical analysis. (D) An increased mRNA expression of FZD2, FZD9, and FZD10 in diseased coronary arteries. The data are represented as mean ± SEM. *P < 0.05 compared with healthy tissues; n = 11 for coronary plaques and n = 7 for healthy tissues. Student’s t-test was used for statistical analysis. CAD, coronary artery disease.

Figure 2

Coronary plaques have greater immunostaining of Wnt proteins compared with histologically normal tissues. (A) A negative stain control of the coronary artery. Very little Wnt3a (B), Wnt5a (C), and Wnt5b (D) immunostaining is seen in histologically normal coronary arteries. A strong Wnt3a staining is seen in the shoulder of the lesion around the lipid core and in the fibrous cap (E), and colocalized with αSMA (H) Wnt3a is colocalized with CD68 immunostaining around the lesion (I,L, respectively). Some Wnt5a immunostaining around the lipid core and in the fibrous cap (F) colocalized with αSMA (H) no significant colocalization of Wnt5a and CD68 immunostaining (J,L, respectively). A strong Wnt5b staining is seen in and around the lesion and in the fibrous cap (G) and colocalized with αSMA (H) colocalization of Wnt5b and CD68 immunostaining around the lesion (K,L, respectively). Scale bar = 200 μm. *Lipid core, ℓ = lumen. The arrows refer to significant immunostaining in and around the lesion.

Figure 3

Wnt proteins induced an osteogenic differentiation of vascular smooth muscle cells and increased oxidative stress, cell migration, and calcification. (A) an mRNA expression of vSMC, osteogenic, and macrophage markers after treatment with 200 ng/mL Wnt proteins in vSMCs for 7 days. The data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 compared with controls; n = 3 for all experimental groups. One-way ANOVA, followed by Dunnett's post hoc multiple comparisons test, was used for statistical analysis. Relative fluorescence intensity for the detection of (B) cellular reactive oxygen species and (C) mitochondria superoxides after treatment with 200 ng/mL Wnt proteins for 2 h. The data are represented as mean ± SEM. *P < 0.05 and **P < 0.01 compared with controls; n = 3 for all experimental groups. One-way ANOVA, followed by Dunnett's post hoc multiple comparisons test, was used for statistical analysis. (D) The migrated cell count of SMCs after treatment with 200 ng/mL Wnt proteins in serum-free media for 24 h. The data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with controls; n = 3 for all experimental groups. One-way ANOVA, followed by Dunnett's post hoc multiple comparisons test, was used for statistical analysis. Representative images (E) and quantification of a calcification stain (F) of vSMCs after treatment with 200 ng/mL Wnt proteins in OSM for 7 days. The data are represented as mean ± SEM. **P < 0.01 and ***P < 0.001 compared with controls; n = 3 for all experimental groups. One-way ANOVA, followed by Dunnett's post hoc multiple comparisons test, was used for statistical analysis. OSM, osteogenic media; SFM, serum-free media; vSMC, vascular smooth muscle cells.

Figure 4

Wnt proteins impaired cholesterol handling in vascular smooth muscle cells. (A) Cholesterol ester to total cholesterol ratio in vSMCs treated with 200 µg/mL oxLDL and/or 200 ng/mL Wnt proteins for 7 days. The data are represented as mean ± SEM. *P < 0.01 and **P < 0.01 compared with controls; n = 3 for all experimental groups. Student's t-test was used for statistical analysis. (B) ApoA1-mediated cholesterol efflux in vSMCs treated with 200 ug/mL oxLDL and/or 200 ng/mL Wnt proteins for 48 h. The data are represented as mean ± SEM. *P < 0.01 and **P < 0.01 compared with controls; n = 3 for all experimental groups. Student's t-test was used for statistical analysis. (C) an mRNA expression of cholesterol handling genes after treatment with 200 µg/mL oxLDL and/or 200 ng/mL Wnt proteins for 7 days. The data are represented as mean ± SEM. *P < 0.01, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with controls; n = 3 for all experimental groups. One-way ANOVA, followed by Dunnett's post hoc multiple comparisons test, was used for statistical analysis. vSMC, vascular smooth muscle cells.