Research ArticleCARDIOMETABOLIC DISEASE

Adipose tissue–derived WNT5A regulates vascular redox signaling in obesity via USP17/RAC1-mediated activation of NADPH oxidases

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Science Translational Medicine  18 Sep 2019:
Vol. 11, Issue 510, eaav5055
DOI: 10.1126/scitranslmed.aav5055
  • Fig. 1 Gene expression of Wnt ligands in AT and plasma concentration of WNT5A/SFRP5 in obesity.

    (A to C) Gene expression of the 19 Wnt ligands in human (A) perivascular AT (PVAT), (B) thoracic AT (ThAT), and (C) subcutaneous AT (ScAT) in n = 54 patients of study 1. (D to F) Circulating plasma concentrations of (D) WNT5A (range, 1 to 112 ng/ml), (E) decoy receptor SFRP5, and (F) WNT5A/SFRP5 ratio in individuals of study 1. (G to I) Gene expression of (G) WNT5A, (H) SFRP5, and (I) the ratio of WNT5A/SFRP5 in ThAT of study 1 participants. Study 1 participants per group: 25 (BMI ≤20 kg/m2), 217 (BMI = 20.1 to 24.9 kg/m2), 432 (BMI = 25 to 29.9 kg/m2), 233 (BMI = 30 to 34.9 kg/m2), and 57 (BMI ≥35 kg/m2). Data are presented as mean ± SEM (A to C) or median [25th to 75th percentile] (D to I). P values in (A) to (C) are calculated by Friedman tests; P values in (D) to (I) are calculated by Kruskal-Wallis tests. AT, adipose tissue.

  • Fig. 2 Interactions between obesity, WNT5A/SFRP5, and vascular disease.

    (A to C) Gene expression of (A) WNT5A, (B) SFRP5, and (C) the ratio of WNT5A/SFRP5 in internal mammary arteries (IMAs) according to BMI. (D to F) Gene expression of (D) WNT5A, (E) SFRP5, and (C) the ratio of WNT5A/SFRP5 in PVAT according to BMI. (G to I) Gene expression of Wnt receptors (G) FZD2, (H) FZD5, and (I) ROR1 in IMAs according to BMI. (J to L) Circulating plasma concentrations of (J) WNT5A, (K) SFRP5, and (L) WNT5A/SFRP5 ratio in patients with coronary artery disease (CAD) and healthy controls (n = 70). (M) Table of multivariable regression analysis of the association of circulating WNT5A, SFRP5, WNT5A/SFRP5, cardiovascular risk factors, and the presence of CAD in study 2 (n = 70). (N) Plasma WNT5A concentration in patients with or without coronary calcium score (CCS) progression (ΔCCS ≥ 1 = HU, 61.8 [22.6 to 234.6], n = 68, study 3). (O) Plasma WNT5A concentration in patients with or without new-onset calcification (follow-up CSS: 21.7 [2.5 to 27.1], n = 38). (N) and (O) are presented as median [25th to 75th percentile]. Data are presented as median [25th to 75th percentile]. P values in (A) to (N) are calculated by Kruskal-Wallis tests. Study 1 participants with IMA/PVAT samples available for (A) to (I) were as follows: 33 (BMI <25 kg/m2), 77 (BMI = 25 to 29.9 kg/m2), 42 (BMI = 30 to 34.9 kg/m2), and 13 (BMI >35 kg/m2).

  • Fig. 3 WNT5A regulates vascular redox state in humans.

    (A) Basal and (B) NADPH-stimulated superoxide (O2•−) production in IMA segments according to BMI (study 1). (C) Basal and (D) NADPH-stimulated O2•− production in IMAs according to circulating plasma WNT5A/SFRP5 ratio tertiles. (E) Basal and (F) NADPH-stimulated O2•− production in IMAs according to WNT5A/SFRP5 ratio tertiles in PVAT. (G) Basal and (H) NADPH-stimulated O2•− production in IMAs according to WNT5A/SFRP5 ratio tertiles in IMAs. (I) Basal, (J) NADPH-stimulated, and (K) Vas2870-inhibitable O2•− production in IMAs in the presence and absence of WNT5A and SFRP5 in ex vivo IMA segments (n = 5 to 10 pairs per intervention). (L) Dihydorethidium (DHE) staining in IMAs incubated with or without Vas2870, WNT5A, and SFRP5 (specific oxidized DHE fluorescence corresponds to the red signal; the green signal reflects tissue autofluorescence). (M) Basal or (N) NADPH-stimulated O2•− production in IMAs preincubated (preinc.) with Vas2870 and treated with WNT5A (n = 5 to 10 pairs per intervention). (O) Circulating plasma WNT5A (n = 11 to 14 mice per group) and (P) basal O2•−, (Q) NADPH-stimulated O2•−, and (R) Vas2870-inhibitable O2•− in TetOWNT5A mouse aortas [(O), n = 5 per group; (P) to (R), n = 7 per group]. Data are presented as median [25th to 75th percentile] (A to H) or as mean ± SEM (I to K and N to R). P values in (A) to (H) are calculated by Kruskal-Wallis tests; *P < 0.05 versus control in (I) and (J) and (M) and (N) by Wilcoxon signed rank tests; P values are calculated by Mann Whitney U tests in (O) to (R).

  • Fig. 4 WNT5A triggers RAC1 activation, resulting in NADPH oxidases in the human vascular wall.

    Fold change of phosphorylated c-jun N-terminal kinase (JNK) in (A) human IMA segments ex vivo (n = 5 of paired samples) in the presence and absence of WNT5A and SFRP5 and in (B) Wnt5a+/rtTA+ mouse aortas in vivo (n = 5 per group). (C) Activation of RAC1 and membrane translocation of (D) RAC1 and (E) P47phox subunits of NADPH oxidases in human IMAs (n = 5 of paired samples) ex vivo in the presence or absence of WNT5A and SFRP5. (F) Basal, (G) NADPH-stimulated, and (H) Vas2870-inhibitable superoxide (O2•−) anion production in IMA segments with or without WNT5A and NSC23766, a specific RAC1 inhibitor (n = 8 to 10 pairs per intervention). (I) Basal, (J) NADPH-stimulated, and (K) Vas2870-inhibitable superoxide (O2•−) anion production in Wnt5a+/rtTA+ mouse aortas incubated with or without WNT5A and NSC23766 (n = 5 to 7 per group). Data are presented as mean ± SEM. *P < 0.05 versus control in all panels by Wilcoxon signed rank tests.

  • Fig. 5 WNT5A is secreted by adipocytes and enhances NADPH oxidase activity in human VSMCs via RAC1 activation.

    Fold change of (A) phosphorylated JNK (n = 5), (B) activated RAC1 (n = 5), and (C) activated β-catenin (n = 6) in VSMCs in the presence or absence of WNT5A and SFRP5. (D) Basal, (E) NADPH-stimulated, and (F) Vas2870-inhibitable superoxide (O2•−) production in VSMCs (n = 6 to 11 pairs per intervention) in the presence or absence of WNT5A and SFRP5. (G) Knockdown of WNT5A in human immortalized preadipocytes (n = 3). (H) Basal and (I) gp91-dstat–inhibitable O2•− production in VSMCs cocultured with or without WNT5A-KO preadipocytes displayed lower (n = 5). Data are presented as mean ± SEM. *P < 0.05 versus control by Wilcoxon signed ranks tests (A to F, H, and I) or paired t test (G).

  • Fig. 6 The pro-oxidant effects of WNT5A in human VSMCs are mediated by Fzd2 and Fzd5 receptors.

    (A) Fluorescence lipofectamine RNAimax imaging (BLOCK-iT Alexa Fluor Red Fluorescence) against cell nuclei stained with DAPI (4′,6-diamidino-2-phenylindole) (blue signal). (B) Expression fold change, and (C) Western blotting of transfection efficiency of knockdown of FZD2 and FZD5 genes in VSMCs (~96% down-regulation of FZD2, ~65% down-regulation of FZD5; n = 3). (D) Basal, (E) NADPH-stimulated, and (F) Vas2870-inhibitable O2•− production in siRNA-FZD2–treated VSMCs (n = 5 to 6 pairs per intervention) in the presence or absence of WNT5A. (G) Basal, (H) NADPH-stimulated, and (I) Vas2870-inhibitable O2•− production in siRNA-FZD5–treated VSMCs (n = 6 pairs per intervention) in the presence or absence of WNT5A. Data are presented as mean ± SEM. *P < 0.05 versus control by Wilcoxon signed rank tests (D to I) or paired t test (B).

  • Fig. 7 WNT5A triggers redox-dependent migration of human vascular smooth muscle cells.

    (A) Hierarchical clustering of 136 WNT5A differentially expressed genes (DEGs; fold change >1 or <−1; P ≤ 0.05) in WNT5A-treated VSMCs from n = 5 patients. DEGs were annotated with cell motility function in the Gene Ontology database. (B) Enriched signaling pathways of WNT5A cell motility DEGs identified through ConsensusPathDB. (C) Microscopy and (D) quantitation of Boyden chamber assay for cell migration, using VSMCs treated with or without recombinant WNT5A and SFRP5 (n = 30). (E) Proliferation of VSMCs in the presence of WNT5A (n = 6 to 9 per time point per group, evaluated by two-way ANOVA for repeated measures). Data are presented as mean ± SEM in (D) and (E). *P < 0.05 versus control by Wilcoxon signed rank tests; +P < 0.05 versus WNT5A by Wilcoxon signed rank test. FC, fold change.

  • Fig. 8 USP17 as a link between WNT5A and vascular redox signaling.

    (A) Migration of VSMCs incubated with peg-SOD, an intracellular scavenger of superoxide (O2•−) production (n = 5 to 8 pairs per intervention), and treated with WNT5A. (B) A subset of the WNT5A cell motility DEGs of the microarray analysis were at least partially rescued by superoxide (O2•−) anion scavenging with superoxide dismutase (peg-SOD) resulting in P values >0.05 (n = 5 pairs, genes presented by descending mean fold change). (C) USP17 expression in VSMCs incubated with peg-SOD and treated with WNT5A (n = 10). (D) RAC1 activation in HeLa cells transfected with shUSP17 and treated with WNT5A (n = 8). Data are presented as mean ± SEM in (A), (C), and (D). *P < 0.05 versus control by Wilcoxon signed rank test in (A) and (C). +P < 0.05 versus untreated empty vector control by Wilcoxon signed rank test; NS by Wilcoxon signed rank test for WNT5A-treated versus untreated shUSP17 cells in (D).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/510/eaav5055/DC1

    Materials and methods

    Fig. S1. WNT5A and SFRP5 expression in ScAT, IMAs, and PVAT.

    Fig. S2. Ex vivo effect of WNT5A on NOX isoform gene expression in IMAs.

    Fig. S3. Association of plasma WNT5A/SFRP5 with arterial NOX expression.

    Fig. S4. Effects of WNT5A on endothelial function and eNOS coupling.

    Fig. S5. Phenotyping of the TetO-Wnt5a rtTA mouse model.

    Fig. S6. Phenotyping of isolated primary VSMCs.

    Fig. S7. WNT5A dysregulated genes and pathways and effects on VSMC migration.

    Fig. S8. WNT5A and VSMC phenotypic switch.

    Fig. S9. USP17 as the maximally up-regulated target in response to WNT5A and USP17 transfection in HeLa cells.

    Fig. S10. Schematic diagram with proposed mechanism.

    Table S1. Demographic characteristics of the study participants.

    Table S2. Demographic characteristics of study 1 participants per BMI group.

    Table S3. Multivariate linear regression models of calcified plaque progression and new-onset calcification.

    Table S4. Multivariate linear regression models of arterial NADPH-stimulated superoxide (O2•−) production.

    Data file S1. Individual subject-level data (Excel file).

    References (4247)

  • The PDF file includes:

    • Materials and methods
    • Fig. S1. WNT5A and SFRP5 expression in ScAT, IMAs, and PVAT.
    • Fig. S2. Ex vivo effect of WNT5A on NOX isoform gene expression in IMAs.
    • Fig. S3. Association of plasma WNT5A/SFRP5 with arterial NOX expression.
    • Fig. S4. Effects of WNT5A on endothelial function and eNOS coupling.
    • Fig. S5. Phenotyping of the TetO-Wnt5a rtTA mouse model.
    • Fig. S6. Phenotyping of isolated primary VSMCs.
    • Fig. S7. WNT5A dysregulated genes and pathways and effects on VSMC migration.
    • Fig. S8. WNT5A and VSMC phenotypic switch.
    • Fig. S9. USP17 as the maximally up-regulated target in response to WNT5A and USP17 transfection in HeLa cells.
    • Fig. S10. Schematic diagram with proposed mechanism.
    • Table S1. Demographic characteristics of the study participants.
    • Table S2. Demographic characteristics of study 1 participants per BMI group.
    • Table S3. Multivariate linear regression models of calcified plaque progression and new-onset calcification.
    • Table S4. Multivariate linear regression models of arterial NADPH-stimulated superoxide (O2•−) production.
    • References (4247)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual subject-level data.

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