Abstract 562: Altered Hippo Pathway Activity And Impaired Smooth Muscle Contractile Phenotype In Wnt16-Deficient Mice Worsens Ascending Aortic Aneurysm

Abstract

Wnt16 is expressed in mouse & human vascular smooth muscle (VSM). We assessed the impact of Wnt16 deficiency on angiotensin-II (AngII) -induced aneurysmal remodeling. AngII infusion for 4 weeks increased aortic Wnt16 2-fold & ascending thoracic aorta diameter by 60% (p = 1.5E-4, ANOVA p = 3E-6) in male LDLR-/- mice. Wnt16-/-;LDLR-/- cohorts exhibited worsened aorta dilatation (by 20%; Holm-Sidak p =0.03), indicating a protective role for Wnt16. Wnt16 deficiency reduced both pulse wave velocity & systolic blood pressure (Wnt16-null = 132+/- 3 vs. 146 +/- 6 mmHg, p < 0.05, n =11/genotype), suggesting altered contractile function. Since hypomorphic alleles encoding contractile proteins predispose to ascending aortic aneurysms, we studied the impact of Wnt16 on the contractile phenotype. VSM from Wnt16-null thoracic aorta exhibited reductions in Acta2 & SM22 (protein, mRNA; down 50%, p < 0.05), Myh11 (down 60%; p < 0.05), & Myocardin ( Myocd ; by 65%; p =0.0002). Inhibitory H3K27me3 histone methylation was selectively increased 3-fold in chromatin of genes encoding contractile proteins in Wnt16-/- VSM (p = 3E-5). DNA fragmentation was increased 22% in Wnt16-null VSM (p < 0.01). Label-free quantitative proteomic analyses of aorta revealed (a) upregulation of 14 mechanosensitive LIM proteins in Wnt16-null mice, with (b) > 90% reduction in Ankrd1 - a wound contraction protein & a prototypic target of Yap1-Taz/Wwtr1 activity in the Hippo pathway. Wnt16 deficiency significantly increased VSM cytoplasmic Yap1-Taz protein accumulation while reducing nuclear Yap1-Taz. siRNA targeting Taz, but not Yap1, reduced contractile gene expression, phenocopying the actions of Wnt16 siRNA or genetic deficiency. Antagonism of Lats1/2, Hippo pathway kinases that inhibit Yap1-Taz cytoplasmic-nuclear shuttling, upregulated VSM Myocd (4.4x), Acta2 (1.6x), Ankrd1 (2.5x; p < 0.01), mimicking actions of 8 nM Wnt16 treatment. Dosing mice with Wnt16 (0.25 mg/kg/day, 2 days) upregulated aortic contractile gene expression 1.6- to 1.8-fold (all p < 0.05). Wnt16 limits thoracic aortic remodeling & preserves the VSM contractile phenotype in part via the Hippo - Taz/Wwtr1 signal relay. Strategies that augment Wnt16 activity may mitigate enlargement of ascending aortic aneurysms.