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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:68.)
© 2008 American Heart Association, Inc.

Integrative Physiology/Experimental Medicine

Phosphoinositide 3-Kinase Gene Knockout Impairs Postischemic Neovascularization and Endothelial Progenitor Cell Functions

Paolo Madeddu; Nicolle Kraenkel; Luciola S. Barcelos; Mauro Siragusa; Paola Campagnolo; Atsuhiko Oikawa; Andrea Caporali; Andrew Herman; Ornella Azzolino; Laura Barberis; Alessia Perino; Federico Damilano; Costanza Emanueli; Emilio Hirsch

From the Bristol Heart Institute (P.M., N.K., L.S.B., M.S., P.C., A.O., A.C., C.E.) and Cellular and Molecular Medicine (A.H.), University of Bristol, UK; the Molecular Biotechnology Center (O.A., L.B., A.P., F.D., E.H.), University of Turin, Italy; and INBB (N.K., A.O.), Osilo/Rome, Italy.

Correspondence to Paolo Madeddu, MD, CS, FAHA Bristol Heart Institute, University of Bristol, Upper Maudlin Street, Bristol, BS2 8HW, UK. E-mail madeddu{at}yahoo.com

Abstract

Objective— We evaluated whether phosphatidylinositol 3-kinase (PI3K) plays a role in reparative neovascularization and endothelial progenitor cell (EPC) function.

Methods and Results— Unilateral limb ischemia was induced in mice lacking the PI3K gene (PI3K^–/–) or expressing a catalytically inactive mutant (PI3K^KD/KD) and wild-type controls (WT). Capillarization and arteriogenesis were reduced in PI3K^–/– ischemic muscles resulting in delayed reperfusion compared with WT, whereas reparative neovascularization was preserved in PI3K^KD/KD. In PI3K^–/– muscles, endothelial cell proliferation was reduced, apoptosis was increased, and interstitial space was infiltrated with leukocytes but lacked cKit⁺ progenitor cells that in WT muscles typically surrounded arterioles. PI3K is constitutively expressed by WT EPCs, with expression levels being upregulated by hypoxia. PI3K^–/– EPCs showed a defect in proliferation, survival, integration into endothelial networks, and migration toward SDF-1. The dysfunctional phenotype was associated with nuclear constraining of FOXO1, reduced Akt and eNOS phosphorylation, and decreased nitric oxide (NO) production. Pretreatment with an NO donor corrected the migratory defect of PI3K^–/– EPCs. PI3K^KD/KD EPCs showed reduced Akt phosphorylation, but constitutive activation of eNOS and preserved proliferation, survival, and migration.

Conclusions— We newly demonstrated that PI3K modulates angiogenesis, arteriogenesis, and vasculogenesis by mechanisms independent from its kinase activity.

We demonstrated that PI3K is fundamental for reparative neovascularization and EPC function. PI3K is upregulated in ischemic limb muscles and hypoxic EPCs. After limb ischemia, PI3K-deficient mice show impaired revascularization and blood flow recovery, attributable to reduced EC proliferation and enhanced apoptosis. PI3K deficiency impairs EPC growth, survival, migration, and adhesion.

Key Words: limb ischemia • angiogenesis • vasculogenesis • endothelial progenitor cells • migration

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