Exploring the Uncharted Waters of Endothelial Tip Cell Migration: A Story of Aquaporins, Hydrostatic Pressure, and Angiogenesis
Posted by Li-Kun Phng, on 2 April 2025
Written by Li-Kun Phng
Behind the Paper Story of “Combined forces of hydrostatic pressure and actin polymerisation drive endothelial tip cell migration and sprouting angiogenesis”.
A critical function of blood vessels is the transportation of plasma, blood cells, nutrients, and metabolites efficiently across the body. The formation and maintenance of blood vessels as hollow tubes is thus important for their function and is a research interest in our lab. One long-standing question in this subject is how apical membranes expand at luminal patches, which form between endothelial cells, at the initial phase of lumen formation. We questioned whether this could occur through physical forces generated from within the luminal patch that would drive the expansion of apical membranes to form a bigger lumen. This led to the hypothesis that endothelial cells may expel water at its apical surface into the luminal space to build up hydrostatic pressure that would consequently inflate the lumen. While we were exploring this idea, an scRNAseq screen in my lab identified two endothelial water channels, aqp1a.1 and aqp8a.1, to be differentially expressed within blood vascular networks of the developing zebrafish embryo. While aqp1a.1 mRNA is ubiquitously expressed in all blood vessels, aqp8a.1 mRNA expression is confined to the posterior trunk vessels and in the ventral regions of intersegmental vessels (ISVs). We therefore speculated that these two Aquaporins may have distinct roles in blood vessel formation and function.
Igor, the first author of the paper, subsequently generated zebrafish mutants using CRISPR/Cas9 to investigate the function of Aqp1a.1 and Aqp8a.1. Our initial analysis was focused on determining whether blood vessel lumens formed normally in aqp1a.1 and aqp8a.1 mutants. To address this, we performed microangiography experiments to examine lumen patency at 2 and 3 days post fertilisation (dpf), after trunk vessels have lumenised. This analysis showed a decrease in the number of ISVs that are fully perfused and an increase in the number of partially perfused ISVs (Fig. 1), which supported our hypothesis that Aquaporin-mediated water flow may have a role in lumen formation. However, we needed to exclude the possibility that the decrease in fully perfused ISVs may have arisen from a defect in an earlier phase of ISV formation. We therefore examined sprouting angiogenesis in aqp1a.1 and aqp8a.1 mutants. This took a long time as we needed to cross three mutant zebrafish lines (aqp1a.1-/-, aqp8a.1-/- and aqp1a.1-/-;aqp8a.1-/-) to a transgenic endothelial reporter line to visualise endothelial cell shape and behaviours. Eventually, through time-lapse live imaging, we discovered that endothelial tip cells lacking endothelial Aquaporins take longer to emerge from the dorsal aorta (or not at all), generate fewer membrane protrusions and migrate more slowly, thus impairing sprouting angiogenesis1. This was an unexpected finding since it is a commonly held perception that endothelial cell migration depends predominantly on the remodelling of actin cytoskeleton. Our findings instead implicate Aquaporin-mediated water flow as an alternative mechanism of endothelial cell migration in vivo, and the defects in lumen perfusion observed is secondary to incomplete ISV formation.

How do we demonstrate that Aquaporins mediate water flow in endothelial tip cells in vivo?
Aquaporins are widely known to increase water permeation by allowing water to flow across the plasma membrane up an osmotic gradient2. However, we did not know in which direction water flows as endothelial tip cells migrate. This was (and still is) a technical challenge since there is no available method to track water flow inside cells in a living organism. We thus attempted to address whether the loss of Aquaporin function would lead to changes in cytoplasmic viscosity by tracking (Video 1) and calculating the diffusion coefficient (Fig. 2) of genetically encoded multimeric nanoparticles, which self-assemble into spherical particles with a diameter of 50nm (50nm-GEMs)3. Unfortunately, we were unable to observe a difference in 50nm-GEM mobility between wildtype and aqp1a.1-/-;aqp8a.1-/- endothelial tip cells. Still, this did not necessarily mean that Aquaporins do not mediate water flow in endothelial cells since the diffusion coefficient of particles depends on their size4. In our experiment, the 50nm-GEMs may not be sensitive enough to detect small changes in cytoplasmic viscosity that is altered by water influx or efflux, or that the changes in viscosity may be confined to local regions of the cell. We next resorted to measuring tip cell volume, with the assumption that cytoplasmic volume would increase if Aquaporins mediate water influx or decrease if they mediate water efflux. Using this approach, we found that the cytoplasmic volume of tip cells decreased when both Aqp1a.1 and Aqp8a.1 were depleted; conversely, tip cell volume increased when Aqp1a.1 was overexpressed. Coupled with a reduction in tip cell elongation in aqp1a.1-/-;aqp8a.1-/- embryos, we concluded that Aquaporins mediate water influx into tip cells and in doing so, increase cytoplasmic hydrostatic pressure.

What controls the direction of water flow?
That there is directed water flow into tip cells points towards the generation of an osmotic gradient by endothelial tip cells. Upon one of the reviewers’ suggestions, we investigated whether the anion channel, SWELL1 (also known as LRRC8A), may generate an osmotic gradient across the cell membrane to control water flow. To our surprise, chemical inhibition of SWELL1 phenocopied many of the defects found in aqp1a.1-/-;aqp8a.1-/- zebrafish including decreased membrane protrusions, migration velocity and ISV length. All in all, our work on endothelial Aquaporin function uncovered a novel role of osmotic pressure-driven water inflow and increased hydrostatic pressure in driving endothelial tip cell migration and sprouting angiogenesis in vivo 1.
These new findings took me back to my post-doctoral work more than 10 years ago, when I discovered that endothelial tip cells continued to migrate, albeit more slowly, after the inhibition of actin polymerisation and in the absence of filopodia5. (This finding was another surprise as it challenged the previously held dogma that filopodia are necessary for polarised tip cell migration.) Curiously, tip cells were able to generate lamellipodia-type membrane protrusions under the low dose of Latrunculin B (Lat. B) treatment used to inhibit filopodia formation. Back then, I had assumed that the low dose of Lat. B used did not inhibit all actin polymerisation events so that some actin-based membrane protrusions could still be generated to drive the slower migration observed. However, our new results suggested that local increases in hydrostatic pressure in the cytoplasm could instead be the driving force for the observed residual migration. To support this, we inhibited both actin polymerisation and hydrostatic pressure by treating aqp1a.1-/-;aqp8a.1-/- embryos with Lat. B and discovered that tip cell migration and ISV formation were more severely impaired compared to their individual inhibition. This final experiment cemented the conclusion that endothelial tip cells employ two modes of migration – actin polymerisation and hydrostatic pressure – to ensure robust sprouting angiogenesis in physically confined tissues.
In sum, the journey behind our paper is one of twists and turns with a revisit of a past observation. Such discovery-based science filled with unexpected findings is what makes research exciting, and I look forward to uncovering more surprises in endothelial cell behaviours!
References:
1. Kondrychyn, I., He, L., Wint, H., Betsholtz, C. & Phng, L.-K. Combined forces of hydrostatic pressure and actin polymerization drive endothelial tip cell migration and sprouting angiogenesis. eLife 13, RP98612 (2025).
2. Agre, P. et al. Aquaporin water channels – from atomic structure to clinical medicine. J. Physiol. 542, 3–16 (2002).
3. Hernandez, C. M., Duran-Chaparro, D. C., Eeuwen, T. van, Rout, M. P. & Holt, L. J. Development and Characterization of 50 nanometer diameter Genetically Encoded Multimeric Nanoparticles. bioRxiv 2024.07.05.602291 (2024) doi:10.1101/2024.07.05.602291.
4. Sakai, K., Kondo, Y., Goto, Y. & Aoki, K. Cytoplasmic fluidization contributes to breaking spore dormancy in fission yeast. Proc. Natl. Acad. Sci. 121, e2405553121 (2024).
5. Phng, L. K., Stanchi, F. & Gerhardt, H. Filopodia are dispensable for endothelial tip cell guidance. Development 140, 4031–4040 (2013).