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The role of AMP-activated protein kinase in the functional effects of vascular endothelial growth factor-A and -B in human aortic endothelial cells.

Reihill JA, Ewart MA, Salt IP - (2011)

Bottom Line: Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner.Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation.VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cardiovascular & Medical Sciences, College of Medicine, Veterinary & Life Sciences, University of Glasgow, Glasgow UK. ian.salt@glasgow.ac.uk.

ABSTRACT

Background: Vascular endothelial growth factors (VEGFs) are key regulators of endothelial cell function and angiogenesis. We and others have previously demonstrated that VEGF-A stimulates AMP-activated protein kinase (AMPK) in cultured endothelial cells. Furthermore, AMPK has been reported to regulate VEGF-mediated angiogenesis. The role of AMPK in the function of VEGF-B remains undetermined, as does the role of AMPK in VEGF-stimulated endothelial cell proliferation, a critical process in angiogenesis.

Methods: Human aortic endothelial cells (HAECs) were incubated with VEGF-A and VEGF-B prior to examination of HAEC AMPK activity, proliferation, migration, fatty acid oxidation and fatty acid transport. The role of AMPK in the functional effects of VEGF-A and/or VEGF-B was assessed after downregulation of AMPK activity with chemical inhibitors or infection with adenoviruses expressing a dominant negative mutant AMPK.

Results: Incubation of HAECs with VEGF-B rapidly stimulated AMPK activity in a manner sensitive to an inhibitor of Ca2+/calmodulin-dependent kinase kinase (CaMKK), without increasing phosphorylation of endothelial NO synthase (eNOS) phosphorylation at Ser1177. Downregulation of AMPK abrogated HAEC proliferation in response to VEGF-A or VEGF-B. However, activation of AMPK by agents other than VEGF inhibited proliferation. Downregulation of AMPK abrogated VEGF-A-stimulated HAEC migration, whereas infection with adenoviruses expressing constitutively active mutant AMPK stimulated chemokinesis. Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner. Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation.

Conclusions: These data suggest that VEGF-B stimulates AMPK by a CaMKK-dependent mechanism and stimulation of AMPK activity is required for proliferation in response to either VEGF-A or VEGF-B and migration in response to VEGF-A. AMPK activation alone was not sufficient, however, to stimulate proliferation in the absence of VEGF. VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.

No MeSH data available.


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VEGF-B stimulates AMPK in HAECs. HAECs were incubated in the presence or absence of A-C) the indicated concentrations of VEGF-A or VEGF-B for 5 min or D) 100 ng/ml VEGF-B for the durations indicated and lysates prepared. Lysates were subsequently subjected to A) assay of AMPK activity or B,C,D) immunoblotting with the antibodies indicated. A) The results are expressed as the mean ± SEM% basal AMPK activity for four independent experiments. B,D) Representative immunoblots are shown, repeated with similar results on three independent sets of lysates. C) Quantification of immunoblots. *p < 0.05 relative to value in absence of VEGF, **p < 0.01 relative to value in absence of VEGF.
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Figure 1: VEGF-B stimulates AMPK in HAECs. HAECs were incubated in the presence or absence of A-C) the indicated concentrations of VEGF-A or VEGF-B for 5 min or D) 100 ng/ml VEGF-B for the durations indicated and lysates prepared. Lysates were subsequently subjected to A) assay of AMPK activity or B,C,D) immunoblotting with the antibodies indicated. A) The results are expressed as the mean ± SEM% basal AMPK activity for four independent experiments. B,D) Representative immunoblots are shown, repeated with similar results on three independent sets of lysates. C) Quantification of immunoblots. *p < 0.05 relative to value in absence of VEGF, **p < 0.01 relative to value in absence of VEGF.

Mentions: Incubation of HAECs for 5 minutes with 10 or 100 ng/ml VEGF-B significantly stimulated AMPK activity, yet the magnitude of this stimulation was less than that elicited by 10 ng/ml VEGF-A (Figure 1A). VEGF-B also significantly stimulated phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC) (Figure 1B &1C), which reached a maximum after 2-5 min before returning to basal levels of phosphorylation (Figure 1D). Unlike VEGF-A, VEGF-B had no significant effect on eNOS Ser1177 phosphorylation in HAECs (Figure 1B-D). We have previously reported that VEGF-A stimulates AMPK by a CaMKK-mediated mechanism [14]. We therefore determined whether the stimulation of AMPK by VEGF-B was sensitive to the CaMKK inhibitor, STO-609. Preincubation of HAECs with STO-609 completely inhibited VEGF-B and VEGF-A-stimulated AMPK phosphorylation at Thr172, yet had no effect on AMPK Thr172 phosphorylation stimulated by AICAR (Figure 2A), which stimulates AMPK phosphorylation by a CaMKK-independent mechanism [12-14,17]. Incubation of HAECs with VEGF-B had no additional effect on VEGF-A-stimulated AMPK Thr172 or ACC phosphorylation (Figure 2B).


The role of AMP-activated protein kinase in the functional effects of vascular endothelial growth factor-A and -B in human aortic endothelial cells.

Reihill JA, Ewart MA, Salt IP - (2011)

VEGF-B stimulates AMPK in HAECs. HAECs were incubated in the presence or absence of A-C) the indicated concentrations of VEGF-A or VEGF-B for 5 min or D) 100 ng/ml VEGF-B for the durations indicated and lysates prepared. Lysates were subsequently subjected to A) assay of AMPK activity or B,C,D) immunoblotting with the antibodies indicated. A) The results are expressed as the mean ± SEM% basal AMPK activity for four independent experiments. B,D) Representative immunoblots are shown, repeated with similar results on three independent sets of lysates. C) Quantification of immunoblots. *p < 0.05 relative to value in absence of VEGF, **p < 0.01 relative to value in absence of VEGF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094250&req=5

Figure 1: VEGF-B stimulates AMPK in HAECs. HAECs were incubated in the presence or absence of A-C) the indicated concentrations of VEGF-A or VEGF-B for 5 min or D) 100 ng/ml VEGF-B for the durations indicated and lysates prepared. Lysates were subsequently subjected to A) assay of AMPK activity or B,C,D) immunoblotting with the antibodies indicated. A) The results are expressed as the mean ± SEM% basal AMPK activity for four independent experiments. B,D) Representative immunoblots are shown, repeated with similar results on three independent sets of lysates. C) Quantification of immunoblots. *p < 0.05 relative to value in absence of VEGF, **p < 0.01 relative to value in absence of VEGF.
Mentions: Incubation of HAECs for 5 minutes with 10 or 100 ng/ml VEGF-B significantly stimulated AMPK activity, yet the magnitude of this stimulation was less than that elicited by 10 ng/ml VEGF-A (Figure 1A). VEGF-B also significantly stimulated phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC) (Figure 1B &1C), which reached a maximum after 2-5 min before returning to basal levels of phosphorylation (Figure 1D). Unlike VEGF-A, VEGF-B had no significant effect on eNOS Ser1177 phosphorylation in HAECs (Figure 1B-D). We have previously reported that VEGF-A stimulates AMPK by a CaMKK-mediated mechanism [14]. We therefore determined whether the stimulation of AMPK by VEGF-B was sensitive to the CaMKK inhibitor, STO-609. Preincubation of HAECs with STO-609 completely inhibited VEGF-B and VEGF-A-stimulated AMPK phosphorylation at Thr172, yet had no effect on AMPK Thr172 phosphorylation stimulated by AICAR (Figure 2A), which stimulates AMPK phosphorylation by a CaMKK-independent mechanism [12-14,17]. Incubation of HAECs with VEGF-B had no additional effect on VEGF-A-stimulated AMPK Thr172 or ACC phosphorylation (Figure 2B).

Bottom Line: Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner.Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation.VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cardiovascular & Medical Sciences, College of Medicine, Veterinary & Life Sciences, University of Glasgow, Glasgow UK. ian.salt@glasgow.ac.uk.

ABSTRACT

Background: Vascular endothelial growth factors (VEGFs) are key regulators of endothelial cell function and angiogenesis. We and others have previously demonstrated that VEGF-A stimulates AMP-activated protein kinase (AMPK) in cultured endothelial cells. Furthermore, AMPK has been reported to regulate VEGF-mediated angiogenesis. The role of AMPK in the function of VEGF-B remains undetermined, as does the role of AMPK in VEGF-stimulated endothelial cell proliferation, a critical process in angiogenesis.

Methods: Human aortic endothelial cells (HAECs) were incubated with VEGF-A and VEGF-B prior to examination of HAEC AMPK activity, proliferation, migration, fatty acid oxidation and fatty acid transport. The role of AMPK in the functional effects of VEGF-A and/or VEGF-B was assessed after downregulation of AMPK activity with chemical inhibitors or infection with adenoviruses expressing a dominant negative mutant AMPK.

Results: Incubation of HAECs with VEGF-B rapidly stimulated AMPK activity in a manner sensitive to an inhibitor of Ca2+/calmodulin-dependent kinase kinase (CaMKK), without increasing phosphorylation of endothelial NO synthase (eNOS) phosphorylation at Ser1177. Downregulation of AMPK abrogated HAEC proliferation in response to VEGF-A or VEGF-B. However, activation of AMPK by agents other than VEGF inhibited proliferation. Downregulation of AMPK abrogated VEGF-A-stimulated HAEC migration, whereas infection with adenoviruses expressing constitutively active mutant AMPK stimulated chemokinesis. Neither VEGF-A nor VEGF-B had any significant effect on HAEC fatty acid oxidation, yet prolonged incubation with VEGF-A stimulated fatty acid uptake in an AMPK-dependent manner. Inhibition of eNOS abrogated VEGF-mediated proliferation and migration, but was without effect on VEGF-stimulated fatty acid transport, ERK or Akt phosphorylation.

Conclusions: These data suggest that VEGF-B stimulates AMPK by a CaMKK-dependent mechanism and stimulation of AMPK activity is required for proliferation in response to either VEGF-A or VEGF-B and migration in response to VEGF-A. AMPK activation alone was not sufficient, however, to stimulate proliferation in the absence of VEGF. VEGF-stimulated NO synthesis is required for the stimulation of proliferation by VEGF-A or VEGF-B, yet this may be independent of eNOS Ser1177 phosphorylation.

No MeSH data available.


Related in: MedlinePlus