Akt1/protein kinase Balpha 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.

Figure 1

Characterization of tissue and vascular expression of Akt1 and Akt2. (A) RT-PCR analysis of RNA isolated from mouse lung fibroblasts using Akt1-, Akt2-, and Hsp90-specific primers. (B) Expression of Akt1 and Akt2 protein in homogenates from heart and gastrocnemius muscle by Western blot with antibodies against Akt1, Akt2, or Hsp90 as a loading control. (C) Akt1 and Akt2 expression in various blood vessels by Western blot using antibodies against Akt1, Akt2, and β-actin as a loading control. (D) p-Akt levels in various blood vessels from WT, Akt1^–/–, and Akt2^–/– mice. Lysates from 3 animals per group were pooled and analyzed by Western blot using p-AktS473– and p-AktT308–specific Akt antibodies. (E) Relative expression of Akt isoforms in blood vessels. Lysates from WT mice were analyzed by SDS-PAGE and quantitative Western blot. Relative protein amounts of Akt1 and Akt2 in the vessels were quantified using standard curves obtained by running recombinant mouse Akt1 and Akt2 proteins. Data represent the mean of 2 pooled samples; n = 3.

Figure 2

Akt1^–/– mice have impaired ischemia-initiated blood flow recovery. (A) Blood flow in the gastrocnemius muscle was measured before, immediately after, and at 2 and 4 weeks after left femoral artery resection. Data are expressed as a ratio of the left (ischemic) to right (control) limb perfusion. WT, n = 14; Akt1^–/–, n = 12; Akt2^–/–, n = 6. (B) Akt1^–/– mice developed necrotic toes at 1 week to 2 weeks after left femoral artery resection while Akt2^–/– and WT littermate mice did not. (C) Clinical score at 4 weeks after femoral arteriectomy as an index of severity of limb ischemia: 0, normal; 1, pale foot or gait abnormalities; 2, less than half of foot necrotic; 3, more than half of foot necrotic without lower limb necrosis; 4, more than half of foot necrotic with some lower limb necrosis; 5, necrosis or autoamputation of entire lower limb. WT, n = 14; Akt1^–/–, n = 12; Akt2^–/–, n = 6. (D) Representative lectin staining of capillaries from sections of the gastrocnemius/soleus muscles 4 weeks after femoral ligation in WT, Akt1^–/–, and Akt2^–/– mice. Magnification, ×200. (E) Quantification of capillary density, calculated as the number of capillaries per muscle fiber. For each animal, 6–8 randomly selected fields (×200) from 3–4 sections were counted; n = 5. **P < 0.01; ***P < 0.001.

Figure 3

Ischemia-stimulated EPC mobilization is impaired in Akt1^–/– mice; administration of Akt1^–/– EPCs does not improve perfusion of ischemic limbs. (A) Representative photomicrographs showing Ac-Dil-LDL–positive EPCs isolated from peripheral blood mononuclear cells of WT and Akt1^–/– mice after femoral ligation. Magnification, ×200. (B) For each animal, Ac-Dil-LDL–positive cells in 10 low-power (×100) fields were counted. EPC counts were obtained at baseline and 6 days after induction of limb ischemia; n = 3–6. (C) Representative laser Doppler blood flow images of the limbs of WT mice at postoperative day 14 after femoral ligation and intravenous administration of medium, WT EPCs, or Akt1^–/– EPCs (see Methods). (D) Quantitative analysis of laser Doppler blood flow images. Data are expressed as a ratio of the left (ischemic) to right (control) limb perfusion; n = 4. **P < 0.01.

Figure 4

Akt1^–/– mice have impaired VEGF-induced vascular permeability and angiogenesis. (A) Vascular permeability assessed by Evans blue extravasation 30 minutes after subcutaneous injection of VEGF and saline (Basal) in the right and left ears, respectively. Data are expressed as ng Evans blue per mg dry weight of ear; n = 7. (B) Representative PECAM-1 staining of frozen ear sections showing impaired VEGF-induced angiogenesis in Akt1^–/– mice. Ad-VEGF or Ad–β-gal was applied by intradermal injection into the left and right ears, respectively, for 5 days. Frozen sections (7 μm thick) were stained for vessels with anti–PECAM-1 antibody and Alexa 488–conjugated secondary antibody (green). Tissues were counterstained with propidium iodide (red). Magnification, ×100. (C) Quantification of PECAM-1 staining. For each animal, 10 low-power (×100) fields from 5 sections were scored. n = 5. Angiogenic scores were calculated as percentage increases in PECAM-1 staining in Ad-VEGF versus Ad–β-gal–treated ears. *P < 0.05; **P < 0.01.

Figure 5

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.

Figure 6

Impaired cell migration and NO release in Akt1^–/– cells. (A) Migration of MLECs was examined in modified Boyden chambers using sphingosine-1-phosphate (S-1-P) as a chemoattractant. Migration of Akt1^–/– cells was reduced at all doses examined. Data are mean ± SEM; n = 4 from 3 independent experiments. (B) Migration of lung fibroblasts was examined in the modified Boyden chamber using serum-free DMEM (Basal) or 10% FBS as agonist. (C) Basal production of NO in MLECs (assayed as NO₂^– in the media) over a 24-hour period was determined by chemiluminescence. Levels of NO₂^– in media alone were subtracted out. (D) Cells were stimulated with VEGF (50 ng/ml) for 30 minutes, and NO release was quantified by chemiluminescence. For stimulated NO₂^– release, values were calculated by subtracting out levels obtained with nonstimulated cells. Data are mean ± SEM; n = 9–12 from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.