Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction

Abstract

Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7-10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. d-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes.

Fig. 1

Increasing d-glucose concentrations decrease endothelial cell (EC) proliferation independent of osmotic changes or cell death. A: human umbilical vein ECs (HUVEC) were seeded at 1,000 cells/well and grown in endothelial growth medium containing baseline control (5 mM) and high (20 and 40 mM) concentrations of d-glucose. Cell counts were performed through 15 days. Log phase of growth was between days 7 and 10. Number of proliferating HUVEC on day 8 were significantly lower when cultured in 20 (P < 0.01) and 40 mM (P < 0.01) d-glucose compared with control (5 mM d-glucose). B: mannitol at 5, 20, and 40 mM did not change HUVEC proliferation. C: percentage of cells undergoing necrosis was not significantly different among groups. Relative percentage of apoptotic cells was lower than necrotic cells in 5 mM d-glucose (*P < 0.05). There was an increase in percentage of apoptotic cells in 40 mM d-glucose vs. 5 mM d-glucose (#P < 0.01).

Fig. 2

Concentration of 40 mM d-glucose decreases phospho-phosphatidylinositol 3-kinase (PI3k) expression in ECs. Immunoblots were performed for tyrosine-phosphorylated proteins after immunoprecipitation of PI3k from lysates obtained from HUVEC cultured in 5, 20, and 40 mM d-glucose. Total PI3k was evaluated by reprobing membranes. Bar graph shows the mean relative band intensity ± SE of tyrosine-phosphorylated PI3k in lysates. *P < 0.05 compared with control (5 mM d-glucose).

Fig. 3

A: high d-glucose concentration does not alter total Akt expression in ECs. HUVEC were cultured in 5, 20, or 40 mM d-glucose and in 5, 20, and 40 mM mannitol. Bar graphs show the means relative band intensity ± SE of Akt in lysates. B: high d-glucose concentration does not alter expression of phosphorylated Akt at Serine 473 in ECs. C: high d-glucose concentration decreases expression of phosphorylated Akt at Threonine 308 in ECs. *P < 0.05, #P < 0.01.

Fig. 4

High d-glucose concentration decreases Akt activity in ECs. Akt was immunoprecipitated from HUVEC lysates using immobilized Akt antibody slurry. Immunoprecipitate was incubated with GSK-3 fusion protein in presence of ATP and kinase buffer. Phosphorylation of GSK-3 was used as a measure of Akt activity. Bar graphs show the means relative band intensity ± SE of phospho-GSK-3α/β (Ser 21/9) in lysates. *P < 0.05 and #P < 0.001 compared with control (5 mM d-glucose).

Fig. 5

Pharmacological inhibition of PI3k-Akt inhibits EC proliferation. A: EC proliferation was attenuated by wortmannin at all concentrations (P < 0.05 vs. 5 mM d-glucose). B: LY294002 produced dose-dependent reduction in EC proliferation (P < 0.05 for 1 μM and P < 0.001 for 10 μM vs. 5 mM d-glucose).

Fig. 6

Overexpression of active Akt increases proliferation and protects EC from deleterious effects of elevated d-glucose. A: successful transfection was confirmed by immunoblotting with anti-myc and anti-Akt antibodies. B: constitutively active Akt+/+ significantly increased proliferation of HUVEC grown in 5 mM d-glucose compared with nontransfected EC, and EC transfected with empty vector or Akt−/− mutant. C: Akt+/+ protected against proliferative impairment induced by 20 mM d-glucose and restored proliferation to control levels (5 mM d-glucose). D: Akt+/+ protected against proliferative impairment induced by 40 mM d-glucose but did not restore proliferation to control levels (5 mM d-glucose).