title: PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells.
abstract: We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1-3% O2) resulted in time- and O2-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.
authors.affiliation: Department of Pediatrics, 12700 E. 19th Ave., University of Colorado Denver, Research Complex 2, Box B131, Aurora, CO 80045, USA.
authors.forename: Heather N
authors.initials: HN
authors.lastname: Woodward
authors.forename: Adil
authors.initials: A
authors.lastname: Anwar
authors.forename: Suzette
authors.initials: S
authors.lastname: Riddle
authors.forename: Laimute
authors.initials: L
authors.lastname: Taraseviciene-Stewart
authors.forename: Miguel
authors.initials: M
authors.lastname: Fragoso
authors.forename: Kurt R
authors.initials: KR
authors.lastname: Stenmark
authors.forename: Evgenia V
authors.initials: EV
authors.lastname: Gerasimovskaya
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D015687:Cell Hypoxia / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004338:Drug Combinations; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D008297:Male; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D014651:Vasa Vasorum / Q000166:cytology*; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D000255:Adenosine Triphosphate; D003094:Collagen; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
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