Simvastatin stimulates Akt protein kinase activity. a, Representative autoradiogram of phosphorylated histone H2B (2 μg) by immunoprecipitated Akt. Akt was immunoprecipitated from lysate prepared from HUVEC treated with simvastatin (SIM) or 100 ng/ml VEGF for 30 min. Under some conditions, cells were infected with adenovirus construct carrying a dominant-negative form of Akt1 (Ad-dnAkt) 24 h before drug treatment. Immunoprecipitated Akt was incubated with histone H2B (2 μg) for 30 min at 30°C and reaction was terminated by adding SDS-sample buffer. Proteins were separated by SDS–PAGE and the extent of histone H2B phosphorylation was visualized by autoradiography. b, Analysis of Akt protein kinase activity towards eNOS peptides. Akt was immunoprecipitated and incubated with 25 μg eNOS peptide (wild: eNOS peptide corresponding to positions including its functional site, serine 1179 residue. S1179A: the mutant peptide where the serine residue phosphorylated by Akt changed to alanine). Treatment with wortmannin (WM, 500 nM) was performed 1 h before addition of simvastatin. Results are presented as mean ± S.E.M. (n = 5–7, * P < 0.05).

Simvastatin promotes endothelial cell survival through an Akt-dependent pathway. a, Representative images showing the cell-survival effects by simvastatin as detected by double-staining with annexin-V (green) and propidium iodide (red). HUVEC cultures were plated on chamber slides at a density of 4 × 10⁴ cells/well. HUVEC were incubated in serum-depleted media for 3 h and subjected to stimulation with simvastatin (1 μM) for an additional 5 h. Parallel cultures were infected with Ad-dnAkt 24 h before the change to serum-free media. Some cultures were treated with 500 nM wortmannin (WM) for 1 h before each stimulation with simvastatin. b, Quantitative analysis of simvastatin-promoted endothelial survival by counting pyknotic nuclei stained by Hoechst 33342. HUVEC were examined in serum-free media as described in (a) to assess the effects of 1 μM simvastatin (SIM) or VEGF (100μg/ml) on survival. Data are shown as the mean ± S.E.M. (n = 4–6, *P < 0.05).

Statin administration or enhanced Akt signaling in endothelial cells promotes blood vessel formation in the rabbit hindlimb in response to unilateral femoral artery resection. a, The femoral artery and its main branches were dissected. Adenovirus-mediated gene transfer to the endothelium was achieved by infusing saline containing Ad-Bgal or Ad-myrAkt through the distal end of the femoral artery and incubating for 15 min in the limb by temporarily clamping the femoral vein. b, Gastrocnemius muscle was excised 3 days after surgery and perfusion with Ad-βgal, and stained with X-gal to determine transgene distribution in hematoxylin and eosin-stained tissue. c, Internal iliac angiography was performed on the different treatment groups to assess collateral vessel formation. Angiograms at 40 days after femoral artery resection showing enhanced collateral vessel formation in animals that received 0.1 mg/kg/d of simvastatin by intraperitoneal injection (i.p.) relative to control animals that underwent surgery but received saline. Quantitative measurements of collateral vessels were performed on the control group, the simvastatin-treated group and a group that received an intramuscular injection (i.m.) of Ad-VEGF. Angiographic score was also assessed in the experimental groups receiving infusions of saline, Ad-βgal or Ad-myrAkt at 31 days after surgery. d, Alkaline phosphatase staining of the adductor muscle from ischemic limbs showing greater capillary density in the simvastatin-treated animals than in control animals at 40 days post-surgery. Average capillary density for all experimental groups is reported. Data in each experiment are presented as mean ± S.E.M. (n = 6 rabbits for each treatment group, *P < 0.05 relative to the saline-infused or saline-injected groups compared by one-way analysis of variance).

Statins promote Akt phosphorylation in endothelial cells. Representative western immunoblots of the effects of simvastatin on Akt phosphorylation concentration are shown under various conditions as follows: a, Dose-dependent phosphorylation of Akt by simvastatin. b, Time-dependent changes in Akt phosphorylation following stimulation by simvastatin (1 μM). c, Reversal of simvastatin-induced Akt phosphorylation by mevalonate (200 μM). d, Sensitivity of simvastatin-induced Akt phosphorylation to wortmannin. e, Sensitivity of simvastatin-, pravastatin- and VEGF-induced Akt phosphorylation to LY294002. The extent of Akt phosphorylation was detected by anti-phosphorylated serine 473 residue of Akt1 specific antibody (phospho-Akt (Ser473)). HUVEC were treated with simvastatin for 30 min except for time course experiment shown in (a). (c, d, and e), HUVEC were pretreated with mevalonate (200 μM), wortmannin (500 nM) or LY294002 (10 μM) for 1 h, before 30 min stimulation with 1 μM simvastatin, 1 μM pravastatin or 100 ng/ml VEGF.

Simvastatin induces Akt-mediated phosphorylation of eNOS in intact cells. a, Simvastatin (SIM) increases phosphorylation of endogenous eNOS, which is abrogated by overexpression of dominant negative Akt1. HUVEC were radiolabeled with ³²P-orthophosphate and stimulated with simvastatin (1 μM) or VEGF (100 ng/ml) for 30 min. Alternatively, HUVEC were infected with adenovirus expressing constitutively-active Akt (myrAkt) 24 h before radiolabeling and assayed for endogenous eNOS phosphorylation in the absence of Akt agonist. Parallel cultures were mock infected or infected with Ad-dnAkt 24 h before simvastatin or VEGF activation. b, Simvastatin-induced eNOS phosphorylation is sensitive to wortmannin. After 1 h pre-treatment with 500 nM wortmannin, HUVEC were stimulated with simvastatin or VEGF in the presence or absence of 500 nM wortmannin. Wortmannin was added to cultures 1 h before stimulation with simvastatin or VEGF. Endogenous eNOS protein was immunoprecipitated, separated by SDS–PAGE (7.5%), and its phosphorylation was visualized by autoradiography. c, COS-7 cells co-transfected with eNOS (wt: wild-type eNOS, S1179A: serine 1179 mutant) and Akt (HA-Akt) expression plasmids. Following transfection, cells were incubated in serum-depleted media for 48 h and then subjected to radiolabeling with ³²P-orthophosphate and treatment with wortmannin and simvastatin. Immunoprecipitates of wild-type or mutant eNOS were examined for ³²P-orthophosphate incorporation by autoradiography following SDS–PAGE (7.5%).

Statins promote vascular structure formation in an in vitro Matrigel assay. HUVEC were seeded on growth factor-reduced Matrigel in the presence of simvastatin (SIM, 0.1 μM), pravastatin (PRV, 0.1 μM), VEGF (100 ng/ml) or no addition (control) in the absence of serum. Parallel HUVEC cultures were infected with adenoviral vectors (50 MOI) carrying either (dnAkt) or (myrAkt) or treated with LY294002 (10μM) at the time of seeding. a, Cultures were photographed after 8 h. b, Quantitation assessment of the extent of tube formation. *P < 0.01 relative to control.