Ultrafine Particle Exposure Reveals the Importance of FOXO1/Notch Activation Complex for Vascular Regeneration.

AIMS: Redox active ultrafine particles (UFP, d < 0.2 µm) promote vascular oxidative stress and atherosclerosis. Notch signaling is intimately involved in vascular homeostasis, in which forkhead box O1 (FOXO1) acts as a co-activator of the Notch activation complex. We elucidated the importance of FOXO1/Notch transcriptional activation complex to restore vascular regeneration after UFP exposure. RESULTS: In a zebrafish model of tail injury and repair, transgenic Tg(fli1:GFP) embryos developed vascular regeneration at 3 days post amputation (dpa), whereas UFP exposure impaired regeneration (p < 0.05, n = 20 for control, n = 28 for UFP). UFP dose dependently reduced Notch reporter activity and Notch signaling-related genes (Dll4, JAG1, JAG2, Notch1b, Hey2, Hes1; p < 0.05, n = 3). In the transgenic Tg(tp1:GFP; flk1:mCherry) embryos, UFP attenuated endothelial Notch activity at the amputation site (p < 0.05 vs. wild type [WT], n = 20). A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) inhibitor or dominant negative (DN)-Notch1b messenger RNA (mRNA) disrupted the vascular network, whereas notch intracellular cytoplasmic domain (NICD) mRNA restored the vascular network (p < 0.05 vs. WT, n = 20). UFP reduced FOXO1 expression, but not Master-mind like 1 (MAML1) or NICD (p < 0.05, n = 3). Immunoprecipitation and immunofluorescence demonstrated that UFP attenuated FOXO1-mediated NICD pull-down and FOXO1/NICD co-localization, respectively (p < 0.05, n = 3). Although FOXO1 morpholino oligonucleotides (MOs) attenuated Notch activity, FOXO1 mRNA reversed UFP-mediated reduction in Notch activity to restore vascular regeneration and blood flow (p < 0.05 vs. WT, n = 5). Innovation and Conclusion: Our findings indicate the importance of the FOXO1/Notch activation complex to restore vascular regeneration after exposure to the redox active UFP. Antioxid. Redox Signal. 28, 1209-1223.

4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation.

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.