Hemodynamic shear stress is intimately linked with endothelial metabolic effects, regulating key mechanisms in endothelial function, homeostasis, and repair. In-vivo modulation of shear stress signaling pathways in the zebrafish model allows for identification of mechanisms with potential therapeutic implications. The following studies combine in-vivo zebrafish models with in-vitro shear stress studies to characterize the mechanisms whereby shear stress regulates vascular development and repair.
We investigated shear stress-modulated genes via microarray, and identified Angiopoietin-2 (Ang-2), a well known regulator of angiogenesis and vascular development, as a potential target for further mechanistic study. Oscillatory shear stress (OSS) induced Ang-2 mRNA expression in a Wnt-signaling dependent manner. Inhibition of Wnt signaling or Ang-2 expression supressed endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2 treatment. These results were recapitulated in the embryonic zebrafish model using heat-shock inducible transgenic Tg(hsp70l:dkk1-GFP) and Tg(kdrl:GFP) embryos injected with Ang-2 morpholino. Inhibition of Wnt signaling with IWR-1 also impaired vascular repair after tail amputation, which was rescued by injection of zAng-2 mRNA. Taken together, this data demonstrated shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and recapitulated shear stress-Wnt-Ang-2 signaling vascular repair in the zebrafish model.
Shear stress-modulated gene- and protein-based mechanisms result in significant changes at the metabolomic level. In the second part of my thesis, we examined the role of emerging mechano-sensitive metabolic pathways in vascular repair. Metabolomic analysis revealed both pulsatile shear stress (PSS) and OSS significantly increased endothelial glycolytic metabolites, but decreased gluconeogenic metabolites. Additionally, both OSS and PSS up-regulated the expression of PKCɛ. We therefore tested whether shear stress modulates endothelial metabolomics to promote vascular repair via PKCɛ-mediated glycolytic metabolites. Treatment with pro-glyocolytic metabolites in-vitro rescued tube formation following treatment with siPKCɛ. To recapitulate vascular repair in transgenic Tg(flk1:GFP) zebrafish embryos, we decreased viscosity and fluid shear stress by micro-injection of GATA-1a morpholino oligonucleotide (MO). In the zebrafish tail amputation model, GATA-1a MO impaired and delayed vascular repair. Co-injection of PKCɛ mRNA with GATA-1a MO rescued this phenomenon. Injection of epo mRNA to increase viscosity resulted in enhanced tail repair. Overall, our studies revealed that shear responsive VEGFR- PKCɛ-metabolomic signaling modulates glycolytic metabolites to influence vascular repair.
Mitochondria are the metabolic center of the cell, and mitochondrial state is intimately linked to endothelial function. To further investigate the effect of shear stress on endothelial metabolic function, we examined the effect of physiological pulsatile shear stress (PSS) on mitochondrial membrane potential (ΔΨm) and the role of Mn-SOD expression on ΔΨm. PSS induced a dynamic increase in ΔΨm, while silencing Mn-SOD attenuated PSS-mediated ΔΨm increase. Mn-SOD mimetic MnTMPyP increased ΔΨm to the similar extent as induced by PSS. Our findings suggest that PSS modulates mitochondrial function by increasing mitochondrial ΔΨm, in part, via Mn-SOD up-regulation.
In addition to using the embryonic zebrafish model to investigate vascular repair, we worked to develop technologies enabling the use of the adult zebrafish as a model for genetic and chemically-induced cardiomyopathies. To address the confounding effects from sedation of fish and removal from the aquatic habitat for micro-electrocardiogram (?ECG) measurements, we developed waterproof and wearable flexible electronic sensors to uncover the circadian variation in heart rate (HR) and heart rate variability (HRV). Innovations including an ultra-soft silicone integrated jacket, matching Young’s modulus to the fish surface, and embedded micro-glass spheres to reduce the effective density enabled physiological ECG telemetry in the fish’s natural habitat without the need for sedation. The novel features of the flexible silicon jacket for ?ECG telemetry unraveled the biological clock and normalization of QT intervals at following ventricular resection at 26 days post ventricular amputation, providing the first evidence of new physiological phenomena during cardiac injury and repair. Additionally, we revealed Amiodarone-mediated QTc prolongation, HR reduction and HRV increase otherwise masked by sedation. This light weight and waterproof design holds promise to advance the next generation of mobile health and drug discovery.