Figure 3. Microvascular endothelial cells sprout to form capillary – like structures.

(a) – (i) Time – lapse images demonstrating cellular mechanism during early sprouting process (movie in supplementary material). Microvascular endothelial cells were cultured to form monolayer on collagen gel according to Methods and Materials. Directional migration and sprouting occurs in the microscopic view plane. Cells were cultured in VEGF enriched media (10 ng ml⁻¹) with a gradient in S1P (250 nM). Time indicated in hours and minutes refer to time after stimulation with VEGF and S1P. Scale bar shown in (a) - (d) represents 20 μm, those in 50 μm (e) - (i) (a) Initial root – like structure forms from membrane protrusion just below monolayer. (b) Filopodia lengthens to form highly branched structure. (c) Onset of nucleus translocation into thickens filopodia. (d) Filopodia further thickens, nucleus completely translocated into filopodia and forms cone – like structure. (e) Formation of lumen – like structure behind polarized cell. (e) Cell division on monolayer (f) – (i) single sprout elongation, lumen like structure lengths behind highly polarized cell. Particle trapped in lumen demonstrate it is a open structure. (j) EC monolayer cultured for several days (here 6 days) with soluble angiogenic stimuli (VEGF 10 ng ml⁻¹ enriched media, with a gradient in S1P (250 nM)) form complex multi-cellular capillary – like structure. End point sample tagged with fluorescent marker shows actin cytoskeleton (orange) and nuclei (green). (k) - (l) Another small microvascular sprout, with green fluorescent microspheres collected in lumen space.