Akt1/protein kinase Bα is critical for ischemic and VEGF-mediated angiogenesis
Eric Ackah, Jun Yu, Stefan Zoellner, Yasuko Iwakiri, Carsten Skurk, Rei Shibata, Noriyuki Ouchi, Rachael M. Easton, Gennaro Galasso, Morris J. Birnbaum, Kenneth Walsh, William C. Sessa
Eric Ackah, Jun Yu, Stefan Zoellner, Yasuko Iwakiri, Carsten Skurk, Rei Shibata, Noriyuki Ouchi, Rachael M. Easton, Gennaro Galasso, Morris J. Birnbaum, Kenneth Walsh, William C. Sessa
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Research Article Vascular biology

Akt1/protein kinase Bα is critical for ischemic and VEGF-mediated angiogenesis

Abstract

Akt, or protein kinase B, is a multifunctional serine-threonine protein kinase implicated in a diverse range of cellular functions including cell metabolism, survival, migration, and gene expression. However, the in vivo roles and effectors of individual Akt isoforms in signaling are not explicitly clear. Here we show that the genetic loss of Akt1, but not Akt2, in mice results in defective ischemia and VEGF-induced angiogenesis as well as severe peripheral vascular disease. Akt1 knockout (Akt1–/–) mice also have reduced endothelial progenitor cell (EPC) mobilization in response to ischemia, and reintroduction of WT EPCs, but not EPCs isolated from Akt1–/– mice, into WT mice improves limb blood flow after ischemia. Mechanistically, the loss of Akt1 reduces the basal phosphorylation of several Akt substrates, the migration of fibroblasts and ECs, and NO release. Reconstitution of Akt1–/– ECs with Akt1 rescues the defects in substrate phosphorylation, cell migration, and NO release. Thus, the Akt1 isoform exerts an essential role in blood flow control, cellular migration, and NO synthesis during postnatal angiogenesis.

Authors

Eric Ackah, Jun Yu, Stefan Zoellner, Yasuko Iwakiri, Carsten Skurk, Rei Shibata, Noriyuki Ouchi, Rachael M. Easton, Gennaro Galasso, Morris J. Birnbaum, Kenneth Walsh, William C. Sessa

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Characterization of Akt-deficient ECs and fibroblasts. (A) Characterizat...
Characterization of Akt-deficient ECs and fibroblasts. (A) Characterization of MLECs. Lung ECs were isolated from Akt1–/–, Akt2–/–, and WT littermates. Some Akt1–/– ECs were reconstituted with a retrovirus expressing HA-tagged Akt1 (Akt1–/–rcAkt1) or with GFP (Akt1–/–rcGFP). WB, Western blot. (B) Characterization of mouse lung fibroblasts. (C) Relative expression of Akt isoforms in mouse cells. Lysates from MLECs, MLFs, and MASMCs were analyzed by SDS-PAGE and quantitative Western blot. Relative protein amounts of Akt1, Akt2, and Akt3 in the cells were quantified using standard curves obtained by running recombinant mouse Akt1, Akt2, and Akt3 proteins. (D) Basal phosphorylation of Akt and Akt substrates in MLECs. Cells were serum-starved for 48 hours and cell lysates subjected to SDS-PAGE and Western blot using the indicated antibodies. (E) Densitometric quantitation of p-protein to total protein for eNOS, GSK3β, and MDM2. For p-FKHR, data is expressed relative to β-actin loading control.