Sprouting angiogenesis is an essential vascularization mechanism consisting of sprouting and remodelling. The remodelling phase is driven by rearrangements of endothelial cells (ECs) within the post-sprouting vascular plexus. Prior work has uncovered how ECs polarize and migrate in response to flow-induced wall shear stress (WSS). However, the question of how the presence of erythrocytes (widely known as red blood cells (RBCs)) and their impact on haemodynamics affect vascular remodelling remains unanswered. Here, we devise a computational framework to model cellular blood flow in developmental mouse retina. We demonstrate a previously unreported highly heterogeneous distribution of RBCs in primitive vasculature. Furthermore, we report a strong association between vessel regression and RBC hypoperfusion, and identify plasma skimming as the driving mechanism. Live imaging in a developmental zebrafish model confirms this association. Taken together, our results indicate that RBC dynamics are fundamental to establishing the regional WSS differences driving vascular remodelling via their ability to modulate effective viscosity.
Sprouting angiogenesis is an essential vascularisation mechanism and consists of two phases: sprouting and remodelling. The remodelling phase is driven by rearrangements of endothelial cells (ECs) within the primitive vascular plexus. Prior work has uncovered how ECs polarise and migrate in response to flow-induced wall shear stress (WSS). However, the question of how the presence of red blood cells (RBCs), and their profound impact on microvascular haemodynamics, affect vascular remodelling has not been addressed. Here, we extend our computational framework to model blood flow in developmental mouse retina as a suspension of RBCs. Our results demonstrate a previously unreported highly heterogeneous distribution of RBCs in the post-sprouting vascular network. Furthermore, we report a strong association between vessel regression and RBC depletion, and identify plasma skimming as the driving mechanism. Live imaging in a developmental zebrafish model confirms this association. Taken together, our results indicate that RBC dynamics are fundamental for establishing the regional WSS differences driving vascular remodelling via their ability to modulate effective viscosity.
Previous studies have shown that Vasohibin-1 (Vash1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo. Recently, Vash1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash1 knockdown. Vash1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing endothelial cell divisions and cell number in the sprouts. We show that altering secondary sprout numbers and structure upon Vash1 depletion leads to defective lymphatic vessel formation and ectopic lymphatic progenitor specification in the zebrafish trunk.
The generation and maintenance of genome edited zebrafish lines is typically labor intensive due to the lack of an easy visual read-out for the modification. To facilitate this process, we have developed a novel method that relies on the inclusion of an artificial intron with a transgenic marker (InTraM) within the knock-in sequence of interest, which upon splicing produces a transcript with a precise and seamless modification. We have demonstrated this technology by replacing the stop codon of the zebrafish fli1a gene with a transcriptional activator KALTA4, using an InTraM that enables red fluorescent protein expression in the heart.
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