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.
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.
Woodward
Heather N
HN
Department of Pediatrics, 12700 E. 19th Ave., University of Colorado Denver, Research Complex 2, Box B131, Aurora, CO 80045, USA.
Anwar
Adil
A
Riddle
Suzette
S
Taraseviciene-Stewart
Laimute
L
Fragoso
Miguel
M
Stenmark
Kurt R
KR
Gerasimovskaya
Evgenia V
EV
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*
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
J Biol Chem. 2002 Nov 22;277(47):44638-50
12244041
J Physiol. 1999 Apr 1;516 ( Pt 1):67-74
10066923
J Physiol. 2004 Jul 15;558(Pt 2):479-88
15155793
Biochem Biophys Res Commun. 2005 Jun 17;331(4):1429-38
15883034
J Cardiovasc Pharmacol. 2001 Dec;38(6):900-8
11707694
J Biol Chem. 2001 Aug 31;276(35):32925-32
11387334
Cardiovasc Res. 2007 Sep 1;75(4):649-58
17631284
Nat Rev Mol Cell Biol. 2003 Apr;4(4):285-94
12671651
PLoS One. 2008 Jul 30;3(7):e2801
18665255
Front Biosci. 2008 Jan 01;13:759-76
17981586
Nat Med. 2007 Aug;13(8):913-9
17632526
Am J Physiol Cell Physiol. 2002 Feb;282(2):C289-301
11788340
Circ Res. 2005 Jul 22;97(2):185-91
15961717
Am J Physiol Heart Circ Physiol. 2003 Aug;285(2):H793-803
12714321
Am J Pathol. 2006 Jun;168(6):1793-807
16723696
Exp Mol Med. 2004 Dec 31;36(6):524-33
15665585
Trends Cardiovasc Med. 2005 Nov;15(8):302-8
16297768
J Biol Chem. 1999 Apr 2;274(14):9129-32
10092582
Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1874-9
14769914
Am J Physiol Cell Physiol. 2008 Jul;295(1):C231-41
18495810
J Neurochem. 1998 Jun;70(6):2347-56
9603199
Nihon Yakurigaku Zasshi. 2004 Jun;123(6):403-11
15170080
J Biol Chem. 2006 May 19;281(20):13957-63
16543245
Pharmacol Rev. 2006 Sep;58(3):281-341
16968944
J Clin Invest. 2005 Jul;115(7):1785-96
15965500
Mol Pharmacol. 2003 Oct;64(4):785-95
14500734
J Mammary Gland Biol Neoplasia. 2005 Oct;10(4):291-8
16900393
Cell Mol Life Sci. 2007 Aug;64(16):2053-65
17530172
J Physiol. 2001 May 1;532(Pt 3):759-69
11313444
J Biol Chem. 2007 Sep 28;282(39):28749-28758
17627942
Annu Rev Biochem. 2002;71:537-92
12045106
Am J Physiol Heart Circ Physiol. 2007 Sep;293(3):H1646-53
17545472
Exp Cell Res. 1999 Nov 25;253(1):239-54
10579926
Trends Cell Biol. 2001 Dec;11(12):471-7
11719051
Circ Res. 1994 Mar;74(3):401-7
8118948
Biochem Biophys Res Commun. 2003 Sep 5;308(4):764-9
12927784
Nature. 1998 Jul 16;394(6690):295-9
9685162
Circ Res. 1999 May 28;84(10):1186-93
10347093
J Cell Biochem. 2005 Jul 1;95(4):827-39
15838884
Am J Physiol Gastrointest Liver Physiol. 2004 Apr;286(4):G538-46
14604861
Circ Res. 2006 Sep 29;99(7):675-91
17008597
Biochem Biophys Res Commun. 2000 Mar 16;269(2):633-40
10708606
J Gen Physiol. 2002 Jun;119(6):511-20
12034759
Angiogenesis. 2008;11(2):169-82
18071915
Science. 2006 Dec 15;314(5806):1792-5
17170310
Pharmacol Ther. 2006 Nov;112(2):358-404
16784779
Nature. 2004 Oct 21;431(7011):1007-11
15496927
Arterioscler Thromb Vasc Biol. 2002 Mar 1;22(3):364-73
11884276
J Biol Chem. 2003 Jan 10;278(2):1354-62
12414798
J Biol Chem. 1996 Sep 6;271(36):21691-4
8702960
Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1208-18
15790881
Nat Rev Mol Cell Biol. 2008 Jun;9(6):455-63
18500256
Biochim Biophys Acta. 2008 May;1783(5):673-94
18302942
Arterioscler Thromb Vasc Biol. 2003 Feb 1;23(2):211-7
12588761
Endocrinology. 1997 Jun;138(6):2615-20
9165056
J Biol Chem. 1997 Dec 26;272(52):32966-71
9407076
Cancer Res. 2004 Dec 15;64(24):8994-9001
15604264
Oncol Rep. 2006 May;15(5):1147-52
16596177
Biophys J. 1997 May;72(5):1954-62
9129800
Invest Ophthalmol Vis Sci. 1994 May;35(6):2783-90
8188473
Cell Physiol Biochem. 2000;10(5-6):313-20
11125211
J Am Coll Cardiol. 2004 Dec 21;44(12):2293-300
15607389