Abstract 10487: Shear Stress-Activated Wnt-Angiopoeitin-2 Signaling Recapitulates Vascular Development and Repair in Zebrafish Embryos

Abstract

Objective: Fluid shear stress and mechano-signal transduction intimately impact on vascular endothelial repair. Here, we seek to assess the mechanisms underlying shear stress-activated developmental genes such as Angiopoietin-2 (Ang-2) in human aortic endothelial cells (HAEC) via the Wnt/β-catenin signaling pathway with a translational implication in endothelial repair in the zebrafish system.

Approach and Results: HAEC were subjected to atherogenic oscillatory shear stress occurring at the arterial branches (OSS=0 ± 3 dyn[[Unable to Display Character: ]]cm-2 at 1 Hz for 4 hours). Both TOPflash Wnt reporter activities and Ang-2 mRNA expression were significantly up-regulated by 2.3 fold and 1.4 fold, respectively. (p < 0.05, n=3). OSS up-regulated Ang-2 mRNA expression by was attenuated in response to wnt inhibitor IWR-1. In parallel, Wnt signaling inhibitor DKK-1 down-regulated Ang-2 mRNA expression in a dose- and time-dependent manner (p < 0.05, n=3), similarly, Wnt3a agonist up-regulated Ang-2 also in a dose-dependent manner (p < 0.05, n=3). in HAEC. Both DKK-1 and Ang-2 siRNA knock-down inhibited endothelial repair in terms of HAEC migration and tube formation, whereas treatment with recombinant Ang-2 protein restored endothelial repair. To further elucidate Wnt-Ang-2 signaling , we used Tg(hsp70l:dkk1-GFP) transgenic zebrafish embryos. We demonstrated that Ang-2 mRNA was down-regulated in response to heat-shock inducible DKK-1 at 72 hours post fertilization (hpf). Ang-2 morpholino (MO) injections also impaired subintestinal vessel (SIV) and intersegmental vessel (ISV) development in the Tg(kdrl:gfp) transgenic embryos at 72 hpf. Next, we corroborated that inhibition of Wnt signaling with IWR-1 impaired vascular repair after tail amputation in the Tg(kdrl:gfp) transgenic embryos, whereas injection of Ang-2 mRNA plasmids at 1-cell stage rescued both SIV and ISV development as well as tail repair.

Conclusion: Shear stress-reactivated Wnt-Ang-2 signaling in the mature endothelial cells is recapitulated in the zebrafish embryos with a translational implication in vascular repair.