Role of ATP during the initiation of microvascularization: acceleration of an autocrine sensing mechanism facilitating chemotaxis by inorganic polyphosphate.

The in vitro tube formation assay with human umbilical vein endothelial cells (HUVEC) was applied to identify the extra- and intracellular sources of metabolic energy/ATP required for cell migration during the initial stage of microvascularization. Extracellularly, the physiological energy-rich polymer, inorganic polyphosphate (polyP), applied as biomimetic amorphous calcium polyP microparticles (Ca-polyP-MP), is functioning as a substrate for ATP generation most likely via the combined action of the alkaline phosphatase (ALP) and the adenylate kinase (AK). The linear Ca-polyP-MP with a size of 40 phosphate units, close to the polyP in the acidocalcisomes in the blood platelets, were found to increase endothelial cell tube formation, as well as the intracellular ATP levels. Depletion of extracellular ATP with apyrase suppressed tube formation during the initial incubation period. Inhibition experiments revealed that inhibitors (levamisole and Ap5A) of the enzymes involved in extracellular ATP generation strongly reduce the Ca-polyP-MP-induced tube formation. The stimulatory effect of Ca-polyP-MP was also diminished by the glycolysis inhibitor oxamate and trifluoperazine which blocks endocytosis, as well as by MRS2211, an antagonist of the P2Y13 receptor. Oligomycin, an inhibitor of the mitochondrial F0F1-ATP synthase, displayed no effect at lower concentrations on tube formation. Electron microscopic data revealed that after cellular uptake, the Ca-polyP-MP accumulate close to the cell membrane. We conclude that in HUVEC exposed to polyP, ATP is formed extracellularly via the coupled ALP-AK reaction, and intracellularly during glycolysis. The results suggest an autocrine signaling pathway of ATP with polyP as an extracellular store of metabolic energy for endothelial cell migration during the initial vascularization process.