Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner - PubMed (original) (raw)

Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner

Y Fujio et al. J Biol Chem. 1999.

Abstract

Regulation of endothelial cell apoptosis is a critical modulator of normal and pathological angiogenesis. In this study, we examined the role of the protein kinase Akt/PKB in endothelial cell survival in response to growth factor and matrix attachment signals. Vascular endothelial growth factor(VEGF)-induced cytoprotection of endothelial cell monolayers correlated with the wortmannin-sensitive induction of Akt activity. Transfection of an adenovirus expressing a dominant-negative Akt mutant decreased endothelial cell viability in the presence of VEGF. Conversely, adenoviral transduction of wild-type Akt facilitated the cell survival effects of VEGF, whereas transduction of constitutively active Akt conferred endothelial cell survival in the absence of VEGF. Constitutively active Akt also conferred survival to endothelial cells in suspension culture, whereas stimulation with VEGF did not. In suspension cultures, VEGF stimulation was unable to activate Akt, and Akt protein levels were repressed in cells undergoing anoikis. These data suggest that cross-talk between growth factor- and anchorage-dependent signaling pathways are essential for Akt activation and endothelial cell survival.

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Figures

Fig. 1. VEGF protects HUVEC cultures against cell death induced by the serum depletion

A, dose dependence of VEGF cytoprotection. Cells were plated in 24-well dishes overnight and then cultured in DMEM containing 20% fetal bovine serum (open bar) or the indicated concentrations of VEGF (closed bars). After 21 h in culture, cell viability was determined by the trypan blue exclusion assay (31, 32) performed in quadruplicate. B, VEGF reduces the frequencies of pyknotic nuclei in HUVECs cultured under serum depletion conditions. HU-VECs were incubated in the presence (+) or absence (−) of VEGF (100 ng/ml) for 21 h. Cells were stained with Hoechst 33342 as described under “Materials and Methods.”

Fig. 2. Wortmannin abrogates the cytoprotection of VEGF

Cells were plated overnight and then cultured in DMEM containing the indicated concentrations of wortmannin in the presence (closed bars) or absence (open bar) of VEGF (100 ng/ml). A, after a 21-h culture, viability was determined by the trypan blue exclusion assay performed in quadruplicate. Data are shown as the mean ± S.E. (n = 4). B, effects of wortmannin on VEGF-mediated reduction in the frequency of HUVECs with pyknotic nuclei. Data are shown as the mean ± S.E. (n = 4). Cells were cultured in the presence or absence of VEGF with or without wortmannin for 21 h. Cells were stained with Hoechst 33342, and cells with pyknotic nuclei were counted.

Fig. 3. VEGF activates Akt in HUVEC cultures in a wortmannin-dependent manner

HUVEC cultures were preincubated in serum-free media in the presence (lane 3) or absence (lanes 1, 2, and 4) of wortmannin for 30 min. Cells were then stimulated with VEGF (100 ng/ml) (lanes 2, 3, and 4) for 15 min. Cell lysates were prepared and immunoprecipitated with anti-Akt antibody with (lane 4) or without (lanes 1, 2, and 3) competitor peptide. Kinase activity was measured with histone H2B as a substrate.

Fig. 4. Adenovirus constructs expressing wild-type Akt and its mutants

A, structures of adenovirus vectors expressing wild-type, dominant-negative, and constitutively active Akt are indicated. B, HUVECs were infected with adenovirus vector expressing Akt or _β_-galactosidase at multiplicity of infection 50 as described under “Materials and Methods.” After 30 min of serum starvation, cells were stimulated with VEGF (100 ng/ml) for 15 min. Cell lysates were immunoprecipitated with anti-HA antibody. Kinase activity was measured with histone H2B as a substrate (top panel). Anti-HA-immunoprecipitated protein was Western blotted with anti-Akt antibody (bottom panel).

Fig. 5. Dominant-negative Akt abrogates VEGF-mediated cell survival

HU-VEC cultures were infected with adenovirus expressing _β_-galactosidase (_β_-gal) or dominant-negative Akt (dnAkt). Cells were incubated with (+) or without (−) VEGF (100 ng/ml) for 21 h (A). Viable cells were counted using a trypan blue exclusion assay. Data are shown as the mean ± S.E. (n = 4) (B). Floating and attached cells were collected, and DNA fragmentation was analyzed as described under “Materials and Methods.”

Fig. 6. Akt promotes endothelial cell survival

A, HUVEC cultures were transfected with adenovirus expressing _β_-galactosidase (_β_-gal) or wild-type Akt (wtAkt) at a multiplicity of infection of 50. Cells were cultured in the indicated concentrations of VEGF for 21 h. Viable cells were determined by trypan blue exclusion assay. Data are shown as the mean ± S.E. (n = 4). B, HUVEC cultures were transfected with adenovirus expressing _β_-galactosidase (_β_-gal) or constitutively active Akt (myrAkt). Cells were cultured in the serum-depleted condition for 21 h. Cells were fixed and stained with Hoechst 33342. The percentage of cells with pyknotic nuclei is demonstrated. Data are shown as the mean ± S.E. (n = 4).

Fig. 7. Akt activation is essential but not sufficient for VEGF-mediated induction of Bcl-2

HUVECs were transfected with adenovirus expressing _β_-galactosidase (_β_-gal), dominant-negative Akt (dnAkt) or constitutively active Akt (myrAkt). Cells were cultured in media containing 2% fetal calf serum in the presence or absence of VEGF (100 ng/ml) for 24 h. Cells were harvested, and cell lysates were analyzed by Western blot with anti-Bcl-2 or anti-_α_-tubulin antibody.

Fig. 8. Constitutively active Akt confers resistance to apoptosis induced by cell detachment

Cells were transfected with adenovirus expressing _β_-galactosidase (_β_-gal) or constitutively active Akt (myrAkt). Cells were collected with EDTA treatment and cultured in a suspension system with (+) or without (−) VEGF (100 ng/ml) as described under “Materials and Methods.” A, cells were cultured for 12 h in suspension system and examined with a phase contrast microscope. B, cells were cultured in suspension system for 21 h and then MTS assays were performed. Data are shown as the mean ± S.E. (n = 5).

Fig. 9. VEGF cannot activate Akt in suspension culture

Cells were cultured in either monolayer (M) or suspension (S) conditions for 15 min (A) or 21 h (B). After 45 min of serum deprivation, cells were cultured with (+) or without (−) VEGF (100 ng/ml) for 15 min (A) or 21 h (B). Cell lysates were prepared and immunoblotted with anti-phosphospecific Akt (p-Akt), anti-Akt (Akt), or anti-tubulin antibody.

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Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner - PubMed (original) (raw)