title: P2Y2 nucleotide receptor mediates arteriogenesis in a murine model of hind limb ischemia.
abstract: OBJECTIVE
  Arteriogenesis represents the maturation of preformed vascular connections in response to flow changes and shear stress. These collateral vessels can restore up to 60% of the native blood flow. Shear stress and vascular injury can induce the release of nucleotides from vascular smooth muscle cells and platelets that can serve as signaling ligands, suggesting they may be involved in mediating arteriogenesis. The P2Y2 nucleotide receptor (P2Y2R) has also been shown to mediate smooth muscle migration and arterial remodeling. Thus, we hypothesize that P2Y2R mediates arteriogenesis in response to ischemia.
  
  
  METHODS
  Hind limb ischemia was induced by femoral artery ligation (FAL) in C57Bl/6NJ or P2Y2R negative mice (P2Y2(-/-)). Hind limb perfusion was measured with laser Doppler perfusion imaging and compared with the sham-operated contralateral limb immediately and at 3, 7, 14, 21, and 28 days after ligation. Collateral vessel size was measured by Microfil casting. Muscle specimens were harvested and analyzed with immunohistochemistry for Ki67, vascular cell adhesion molecule, macrophages, and muscle viability by hematoxylin and essoin stain.
  
  
  RESULTS
  Hind limb ischemia induced by FAL in C57Bl/6NJ mice resulted in significant ischemia as measured by laser Doppler perfusion imaging. There was rapid recovery to nearly normal levels of perfusion by 2 weeks. FAL in P2Y2(-/-) mice resulted in severe ischemia with greater tissue loss. Recovery of perfusion was impaired, achieving only 40% compared with wild-type mice by 28 days. Collateral vessels in the P2Y2(-/-) mice were underdeveloped, with reduced vascular cell proliferation and smaller vessel size. The collaterals were ∼65% the size of wild-type collateral vessels (P = .011). Angiogenesis at 28 days in the ischemic muscle, however, was greater in the P2Y2(-/-) mice (P < .001), possibly related to persistent ischemia leading and angiogenic drive. Early macrophage recruitment was reduced by nearly 70% in P2Y2(-/-) despite significantly more myocyte necrosis. However, inflammation was greater at 28 days in the P2Y2(-/-) mice.
  
  
  CONCLUSIONS
  P2Y2R deficiency does not alter baseline collateral vessel formation but does significantly impair collateral maturation, with resultant persistent limb ischemia despite enhanced angiogenesis. These findings reinforce the importance of arteriogenesis in the recovery of perfusion in ischemic tissues compared with angiogenesis. They also support the role of P2Y2R in mediating this process. The mechanism by which P2Y2R mediates arteriogenesis may involve the recruitment of inflammatory cells to the ischemic tissues, which is essential to arteriogenesis. Approaches to target P2Y2R may yield new therapeutic strategies for the treatment of arterial occlusive disease.
authors.affiliation: Surgery Services, Department of Veterans Affairs Medical Center, Pittsburgh, Pa; Division of Vascular Surgery, Department of Surgery, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: McEnaney
authors.affiliation: Surgery Services, Department of Veterans Affairs Medical Center, Pittsburgh, Pa; Division of Vascular Surgery, Department of Surgery, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Ankur
authors.initials: A
authors.lastname: Shukla
authors.affiliation: Surgery Services, Department of Veterans Affairs Medical Center, Pittsburgh, Pa; Division of Vascular Surgery, Department of Surgery, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Madigan
authors.affiliation: Division of Vascular Surgery, Department of Surgery, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Ulka
authors.initials: U
authors.lastname: Sachdev
authors.affiliation: Surgery Services, Department of Veterans Affairs Medical Center, Pittsburgh, Pa; Division of Vascular Surgery, Department of Surgery, University of Pittsburgh, Pittsburgh, Pa. Electronic address: tzenge@upmc.edu.
authors.forename: Edith
authors.initials: E
authors.lastname: Tzeng
mesh_terms: D000818:Animals; D001158:Arteries / Q000378:metabolism* / Q000503:physiopathology; D001783:Blood Flow Velocity; D003097:Collateral Circulation; D004195:Disease Models, Animal; D006614:Hindlimb; D007511:Ischemia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D017078:Laser-Doppler Flowmetry; D008264:Macrophages / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000473:pathology; D009336:Necrosis; D018919:Neovascularization, Physiologic; D055420:Perfusion Imaging / Q000379:methods; D058473:Receptors, Purinergic P2Y2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012039:Regional Blood Flow; D015398:Signal Transduction; D013997:Time Factors
chemical_list: C546386:P2ry2 protein, mouse; D058473:Receptors, Purinergic P2Y2
references.citation: J Cell Biol. 2001 Apr 30;153(3):491-501
references.pmid: 11331301
references.citation: Arterioscler Thromb Vasc Biol. 2003 Jul 1;23(7):1143-51
references.pmid: 12676799
references.citation: Arterioscler Thromb Vasc Biol. 2007 Nov;27(11):2310-8
references.pmid: 17717295
references.citation: Physiol Genomics. 2007 Jul 18;30(2):179-91
references.pmid: 17426116
references.citation: J Leukoc Biol. 2006 Jul;80(1):59-65
references.pmid: 16684892
references.citation: Arterioscler Thromb Vasc Biol. 2013 Aug;33(8):1902-10
references.pmid: 23788761
references.citation: J Clin Invest. 2006 Sep;116(9):2344-55
references.pmid: 16932810
references.citation: Mol Cell Biochem. 2009 Feb;322(1-2):161-9
references.pmid: 18998200
references.citation: Am J Physiol. 1997 Sep;273(3 Pt 2):H1255-65
references.pmid: 9321814
references.citation: Circ Res. 2008 Mar 14;102(5):581-8
references.pmid: 18202316
references.citation: Circulation. 2002 Nov 19;106(21):2720-6
references.pmid: 12438299
references.citation: Biochem Biophys Res Commun. 1990 Jul 31;170(2):649-56
references.pmid: 2200403
references.citation: Am J Physiol. 1998 Oct;275(4):H1209-15
references.pmid: 9746468
references.citation: Br J Pharmacol. 1991 May;103(1):1203-5
references.pmid: 1652343
references.citation: Biochem Biophys Res Commun. 1994 Jan 14;198(1):213-9
references.pmid: 7507319
references.citation: Circulation. 2012 Jan 3;125(1):e2-e220
references.pmid: 22179539
references.citation: Circulation. 1996 Sep 1;94(5):1074-82
references.pmid: 8790049
references.citation: J Pathol. 2005 Jul;206(3):260-8
references.pmid: 15887283
references.citation: Circ Res. 2004 Sep 3;95(5):449-58
references.pmid: 15345667
references.citation: Pharmacol Rev. 2006 Sep;58(3):281-341
references.pmid: 16968944
references.citation: J Am Soc Nephrol. 2009 Aug;20(8):1724-32
references.pmid: 19478095
references.citation: Am J Physiol Heart Circ Physiol. 2003 Sep;285(3):H1123-31
references.pmid: 12915391
references.citation: J Mol Cell Cardiol. 2002 Jul;34(7):775-87
references.pmid: 12099717
references.citation: Physiol Genomics. 2010 Aug;42(3):469-79
references.pmid: 20551146
references.citation: Thromb Haemost. 2005 Apr;93(4):735-42
references.pmid: 15841322
references.citation: J Cardiovasc Pharmacol. 2001 Dec;38(6):900-8
references.pmid: 11707694
references.citation: J Biol Chem. 2012 Mar 23;287(13):10535-10543
references.pmid: 22298782
references.citation: FASEB J. 2003 Aug;17(11):1520-2
references.pmid: 12709411
references.citation: Circulation. 2008 Mar 4;117(9):1207-15
references.pmid: 18285563
references.citation: Arterioscler Thromb Vasc Biol. 2003 Aug 1;23(8):1370-6
references.pmid: 12791671
references.citation: PLoS One. 2012;7(11):e50654
references.pmid: 23209800
references.citation: J Biol Chem. 2003 Jul 4;278(27):24960-5
references.pmid: 12714597
references.citation: Arterioscler Thromb Vasc Biol. 2006 Mar;26(3):520-6
references.pmid: 16397137