AMP-activated Protein Kinase (AMPK) Signaling in Endothelial Cells Is Essential for Angiogenesis in Response to Hypoxic Stress

AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that is regulated by hypoxia and other cellular stresses that result in diminished cellular ATP levels. Here, we investigated whether AMPK signaling in endothelial cells has a role in regulating angiogenesis. Hypoxia induced the activating phosphorylation of AMPK in human umbilical vein endothelial cells (HUVECs), and AMPK activation was required for the maintenance of pro-angiogenic Akt signaling under these conditions. Suppression of AMPK signaling inhibited both HUVEC migration to VEGF and in vitro differentiation into tube-like structures in hypoxic, but not normoxic cultures. Dominant-negative AMPK also inhibited in vivo angiogenesis in Matrigel plugs that were implanted subcutaneously in mice. These data identify AMPK signaling as a new regulator of angiogenesis that is specifically required for endothelial cell migration and differentiation under conditions of hypoxia. As such, endothelial AMPK signaling may be a critical determinant of blood vessel recruitment to tissues that are subjected to ischemic stress. AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that is regulated by hypoxia and other cellular stresses that result in diminished cellular ATP levels. Here, we investigated whether AMPK signaling in endothelial cells has a role in regulating angiogenesis. Hypoxia induced the activating phosphorylation of AMPK in human umbilical vein endothelial cells (HUVECs), and AMPK activation was required for the maintenance of pro-angiogenic Akt signaling under these conditions. Suppression of AMPK signaling inhibited both HUVEC migration to VEGF and in vitro differentiation into tube-like structures in hypoxic, but not normoxic cultures. Dominant-negative AMPK also inhibited in vivo angiogenesis in Matrigel plugs that were implanted subcutaneously in mice. These data identify AMPK signaling as a new regulator of angiogenesis that is specifically required for endothelial cell migration and differentiation under conditions of hypoxia. As such, endothelial AMPK signaling may be a critical determinant of blood vessel recruitment to tissues that are subjected to ischemic stress. Angiogenesis is central feature of normal embryonic and post-natal development, and this process plays a critical role in the neovascularization that is associated with tumor growth and occlusive vascular diseases (1Breier G. Damert A. Plate K.H. Risau W. Thromb. Haemost. 1997; 78: 678-683Crossref PubMed Scopus (123) Google Scholar, 2Ferrara N. Alitalo K. Nat. Med. 1999; 5: 1359-1364Crossref PubMed Scopus (917) Google Scholar, 3Carmeliet P. Nat. Med. 2000; 6: 389-395Crossref PubMed Scopus (3494) Google Scholar). A large body of evidence has shown that vascular angiogenic growth factors promote angiogenesis through activation of MAP kinase (4Pages G. Milanini J. 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AMP-activated protein kinase (AMPK) 1The abbreviations used are: AMPK, AMP-activated kinase; AMPKK, AMP-activated kinase kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; ACC, acetyl-CoA; eNOS, endothelial nitric-oxide synthase; HUVECs, human umbilical vein endothelial cells; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; GFP, green fluorescence protein; EBM, endothelial cell basal medium; EGM, endothelial cell growth medium; SNP, sodium nitroprusside; l-NAME, Nω-nitro-l-arginine methyl ester; m.o.i., multiplicity of infection; VEGF, vascular endothelial growth factor. is a metabolite-sensing protein kinase that shares amino acid sequence homology with yeast SNF1 (6Carling D. Aguan K. Woods A. Verhoeven A.J. Beri R.K. Brennan C.H. Sidebottom C. Davison M.D. Scott J. J. Biol. Chem. 1994; 269: 11442-11448Abstract Full Text PDF PubMed Google Scholar, 7Mitchelhill K.I. Stapleton D. Gao G. House C. Michell B. Katsis F. Witters L.A. Kemp B.E. J. Biol. 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Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar, 28Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.Z. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar) and AMPK (29Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (720) Google Scholar). Phosphorylation of eNOS at this residue leads to enzyme activation and nitric oxide (NO) production. AMPK is reported to phosphorylate eNOS in cardiac myocytes under hypoxic conditions (29Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (720) Google Scholar), but it is not clear whether NO production by endothelial cells is regulated by this reaction. Recently, AMPK was detected in human umbilical vein endothelial cells (HUVECs) (30Dagher Z. Ruderman N. Tornheim K. Ido Y. Biochem. Biophys. Res. Commun. 1999; 265: 112-115Crossref PubMed Scopus (83) Google Scholar). The pharmacological stimulator of AMPK, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), protects HUVECs from apoptosis under hyperglycemic conditions (31Ido Y. Carling D. Ruderman N. Diabetes. 2002; 51: 159-167Crossref PubMed Scopus (297) Google Scholar). Interestingly, AICAR was found to reverse the inhibitory effect of hyperglycemia on mitogen-stimulated Akt In this we whether AMPK signaling plays a role in angiogenesis. we whether AMPK is for endothelial cell differentiation into and NO production under both normoxic and hypoxic conditions. also whether AMPK signaling is required for angiogenesis in Matrigel plugs that were subcutaneously implanted in mice. The of this that AMPK and Akt is for angiogenesis under conditions of hypoxic but for angiogenic cellular responses in normoxic endothelial and and were from (ACC) and was from protein was from Akt and eNOS were from Nω-nitro-l-arginine methyl sodium and human vascular endothelial growth were from AMPK was a from Birnbaum of the AMPK residue was to J. Brozinick Jr., J.T. Valladares O. Bucan M. Birnbaum M.J. Mol. Cell. 2001; 7: 1085-1094Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar), with the of and were Y. Walsh K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. K. T. Y. Walsh K. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). The are to the green fluorescence protein was from and umbilical vein cells (HUVECs), endothelial cell basal and endothelial cell growth were from HUVECs were in with and HUVECs at or HUVECs were with the at a multiplicity of of for The was to to the of by a in cells were in a hypoxia to the for the of conditions of of and of by D. Y. E. M. H. A. T. N. Y. K. H. M. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). HUVECs at or was as D. E. H. M. T. Walsh K. M. M. A. M. Y. Circ. Res. 2000; PubMed Scopus Google Scholar). In cell were with and sodium by The were with the at a by the with at a was used for were as a B. Y. Z. A. D. Walsh K. J. 1999; 104: PubMed Scopus Google Scholar). HUVECs with the were in cells were with with and in VEGF was into the of the with with were used to the and cells in the the of were to both The were for at under normoxic or hypoxic conditions. were and the cells to the were by through the on the of were with for with and were was in and were of structures by HUVECs on growth Matrigel was as Y. Z. I. A. D. Sessa W.C. Walsh K. Nat. Med. 2000; 6: PubMed Scopus Google Scholar). were with Matrigel to the The HUVECs were on at in VEGF and at for under normoxic or hypoxic conditions. In was also was were with a The of was by the of in from the was for was enzyme from to the cells were in to the effect of VEGF was to the in the or of NO synthase were under hypoxic or normoxic conditions for to the was in and of were Angiogenesis of new in vivo was by Matrigel as J. M.J. R. H. M. B. A. M.D. Circ. Res. 1995; PubMed Scopus Google Scholar, C. A. T. Y. Birnbaum M. Jr., Walsh K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). these of Matrigel and GFP, AMPK, or Akt was on and subcutaneously into the of mice. were after the The Matrigel plugs with tissues were by in with and in in and for were on was used at a by of at a The was used for were in was were as the were of with for AMPK by Hypoxia in Endothelial whether AMPK is activated in endothelial cells under conditions of HUVEC were in a hypoxia for of and were on that is for AMPK phosphorylation at of the A basal of AMPK phosphorylation was detected in cell under normoxic conditions, and hypoxia a in AMPK phosphorylation A of this is shown phosphorylation leads to AMPK activation, and the of AMPK signaling by hypoxia was by the phosphorylation of at which is a substrate for Hypoxia also to a in AMPK as by phosphorylation with AMPK not and phosphorylation of was cells were at with a of AMPK that a A and with the protein at a with AMPK also inhibited the phosphorylation of AMPK Hypoxia not the of in a it the of protein not Hypoxia to a in basal Akt phosphorylation at which is required for kinase M. B. P. N. P. EMBO J. 1996; PubMed Scopus Google Scholar, A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) with AMPK Akt Akt phosphorylation at The of the by AMPK as the of to hypoxia of eNOS Phosphorylation and NO by production of endothelial NO is for vascular and angiogenesis E. E. J. J. D. I. Am. J. Physiol. 1998; PubMed Google Scholar, T. T. M. C. H. C. M. D. J. 1998; PubMed Scopus Google Scholar, Y. S. R. Circ. Res. 1999; PubMed Scopus Google Scholar, D. T. A. Y. J. D.G. R.K. Acad. Sci. S. A. 2001; PubMed Scopus Google Scholar, Y. K. S. M. T. W. N. M. Thromb. Biol. 2002; PubMed Scopus Google Scholar). VEGF promotes Akt signaling in NO through the activating phosphorylation of eNOS at residue 1177 (27Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar). AMPK is also reported to phosphorylate eNOS at residue 1177 in cardiac myocytes (29Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (720) Google Scholar), the of AMPK to eNOS phosphorylation was in HUVEC under normoxic and hypoxic conditions In VEGF not the of AMPK or phosphorylation not However, with AMPK eNOS phosphorylation under conditions of it effect in normoxic HUVEC basal of AMPK in normoxic endothelial cells has effect on the of eNOS 1177 AMPK signaling was for phosphorylation under these conditions A Akt was used as a in these it is to eNOS phosphorylation in normoxic endothelial cell (27Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar). In to AMPK, with Akt inhibited eNOS phosphorylation at in normoxic cultures. with Akt also inhibited eNOS phosphorylation in hypoxic cultures. However, it effect on phosphorylation under Recently, it has shown that eNOS is regulated through phosphorylation of (29Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (720) Google Scholar, B.J. Z. T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, I. Fisslthaler B. S. Kemp B.E. Busse R. Circ. Res. 2001; PubMed Scopus Google Scholar). In normoxic VEGF to a in phosphorylation A and with a B.J. Z. T. Stapleton D. Katsis F. Power D.A. Sim A.T. Kemp B.E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The in phosphorylation by VEGF was by with AMPK in phosphorylation were also in hypoxic but the of the VEGF effect was that in normoxic cultures. AMPK effect on in under conditions of hypoxia. The production of NO by eNOS was in HUVEC by the of C. A. T. Y. Birnbaum M. Jr., Walsh K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Under the conditions of these was HUVEC under normoxic and hypoxic conditions Under both conditions, was inhibited by the eNOS with AMPK inhibited NO in hypoxic cultures. was a NO in normoxic with AMPK, but this was not In with Akt inhibited NO in both hypoxic and normoxic HUVEC cultures. with Akt or AMPK not basal NO production in the of VEGF in hypoxic or normoxic AMPK for Endothelial under the role of AMPK signaling on endothelial cell HUVEC were with and for to VEGF in a Under hypoxic conditions, with AMPK cell migration to cells with in cell migration were detected and the in cell migration not to be to cell Under these cell conditions cells not increases in the of or was a in not of sodium NO the inhibitory of AMPK on HUVEC migration to VEGF in the hypoxic were also at and not However, with a of Akt was the NO at the by AMPK in hypoxic to AMPK SNP, and not to with AMPK effect on HUVEC migration to VEGF under normoxic conditions. In with Akt migration under normoxic conditions, with M. G. G. Fujio Y. Walsh K. Sessa W.C. Circ. Res. 2000; PubMed Scopus Google Scholar), and also this in hypoxic cultures. AMPK Endothelial under the role of AMPK signaling on endothelial cell differentiation into vascular structures in HUVECs were on in the of VEGF after with GFP, AMPK or and was under normoxic and hypoxic conditions was after with AMPK to a as Akt under hypoxic conditions. The of by AMPK was by the of in the were at and not with Akt was the NO in the of by AMPK to AMPK SNP, and not to In with but not AMPK, on in normoxic AMPK in Matrigel in the role of AMPK signaling on angiogenesis in in vivo plugs were subcutaneously implanted on the of mice. In this the as a for the and endothelial cells that the and C. A. T. Y. Birnbaum M. Jr., Walsh K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). after plugs were and subjected to to identify structures and the endothelial cell Matrigel plugs with structures were of endothelial and plugs of a of cells in the plugs with plugs with structures and a of AMPK is as a regulator of cellular in to hypoxia or vigorous contraction in muscle (8Kudo N. Barr A.J. Barr R.L. Desai S. Lopaschuk G.D. J. Biol. Chem. 1995; 270: 17513-17520Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 9Kudo N. Gillespie J.G. Kung L. Witters L.A. Schulz R. Clanachan A.S. Lopaschuk G.D. Biochim. Biophys. Acta. 1996; 1301: 67-75Crossref PubMed Scopus (218) Google Scholar, 10Chen Z. Heierhorst J. Mann R.J. Mitchelhill K.I. Michell B.J. Witters L.A. Lynch G.S. Kemp B.E. Stapleton D. FEBS Lett. 1999; 460: 343-348Crossref PubMed Scopus (114) Google Scholar, W.W. Hardie D.G. Am. J. Physiol. 1996; 270: E299-E304Crossref PubMed Google Scholar, 12Hutber C.A. Hardie D.G. Winder W.W. Am. J. Physiol. 1997; 272: E262-E266PubMed Google Scholar, 13Hayashi T. Hirshman M.F. Kurth E.J. Winder W.W. Goodyear L.J. Diabetes. 1998; 47: 1369-1373Crossref PubMed Scopus (709) Google Scholar). Here, a new for AMPK in the vascular is AMPK signaling is induced by in endothelial and this is for angiogenic cellular responses under these conditions. of AMPK signaling through of a of the inhibited endothelial cell migration VEGF and in vitro endothelial cell differentiation into tube-like structures in hypoxic cultures. migration and differentiation of endothelial cells are critical of vessel a Matrigel angiogenesis was to the role of endothelial AMPK signaling in in In this Matrigel as a for that endothelial cells as the the of intracellular protein on C. A. T. Y. Birnbaum M. Jr., Walsh K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Dominant-negative AMPK inhibited the of Matrigel evidence that this signaling is for angiogenesis in A of this is that AMPK signaling has or effect on endothelial cell or NO production are to These the of the AMPK this angiogenic cellular responses under cell conditions associated with AMPK these data that the basal of AMPK detected in normoxic to the angiogenic these of AMPK in normoxic cells to fatty acid and in the normal Z. Ruderman N. Tornheim K. Ido Y. Circ. Res. 2001; PubMed Scopus Google Scholar). with this of normoxic endothelial cells with AMPK the phosphorylation of ACC, a regulator of fatty acid (16Carling D. Clarke P.R. Zammit V.A. Hardie D.G. Eur. J. Biochem. 1989; 186: 129-136Crossref PubMed Scopus (344) Google Scholar, 21Davies S.P. Sim A.T. Hardie D.G. Eur. J. Biochem. 1990; 187: 183-190Crossref PubMed Scopus (220) Google Scholar). A Akt was used as a in this Akt signaling is as regulator of NO production (27Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar, 28Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.Z. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar, Z. Fujio Y. Y. G. D. Sessa W.C. Walsh K. J. 2000; PubMed Scopus Google Scholar), Y. Walsh K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. M. N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), migration M. G. G. Fujio Y. Walsh K. Sessa W.C. Circ. Res. 2000; PubMed Scopus Google Scholar, S. E. Zeiher FEBS Lett. 2000; PubMed Scopus Google Scholar, Z. H. S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and differentiation Y. Z. I. A. D. Sessa W.C. Walsh K. Nat. Med. 2000; 6: PubMed Scopus Google Scholar, E. B. L. H. Walsh K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) in normoxic endothelial However, the role of Akt signaling in hypoxic endothelial cells has not a of this is that Akt signaling is for angiogenic cellular responses in hypoxic endothelial Hypoxia a in Akt phosphorylation at this in Akt activation, signaling through this signaling was for endothelial cell migration and NO under conditions of with Akt also inhibited in Matrigel plugs implanted in mice. a of shown that activation of Akt signaling is to promote angiogenesis in vivo Y. Z. I. A. D. Sessa W.C. Walsh K. Nat. Med. 2000; 6: PubMed Scopus Google Scholar, A. Y. J. Z. K. D. Y. D. Walsh K. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, M. Acad. Sci. S. A. 2000; PubMed Scopus Google Scholar), the data the evidence that Akt signaling is for neovascularization in of evidence that AMPK and Akt signaling is of the cellular to hypoxic stress was found that of AMPK signaling the activating phosphorylation of Akt at in hypoxic endothelial The that the of endothelial cell migration and differentiation by AMPK be by with Akt is also with may also in the of eNOS activation by with Akt or AMPK eNOS phosphorylation and NO from hypoxic endothelial these data that eNOS is in hypoxic endothelial cells and the phosphorylation of eNOS at and the of Akt and AMPK, and the of that cells are to which in the of both of these signaling Under the conditions of the phosphorylation of eNOS by AMPK to to eNOS phosphorylation or endothelial NO However, eNOS phosphorylation at may are under conditions of hypoxia or is by D. Walsh K. R.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). of AMPK and Akt and the may on the process that angiogenesis in ischemic The maintenance of NO under hypoxic conditions may to the pro-angiogenic of NO is a of angiogenesis E. E. J. J. D. I. Am. J. Physiol. 1998; PubMed Google Scholar, T. T. M. C. H. C. M. D. J. 1998; PubMed Scopus Google Scholar, Y. S. R. Circ. Res. 1999; PubMed Scopus Google Scholar, D. T. A. Y. J. D.G. R.K. Acad. Sci. S. A. 2001; PubMed Scopus Google Scholar, Y. K. S. M. T. W. N. M. Thromb. Biol. 2002; PubMed Scopus Google Scholar), the by which NO this effect is In the reported AMPK diminished eNOS phosphorylation on and NO under conditions of effect on these in normoxic cultures. the of NO to the the of AMPK on endothelial cell differentiation and migration to VEGF in hypoxic cultures. However, with Akt was the NO at the inhibitory of These data that NO has a effect on angiogenic cellular AMPK signaling angiogenesis of NO production from of the of angiogenesis by glycogen synthase C. A. T. Y. Birnbaum M. Jr., Walsh K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In we that the activation of a pro-angiogenic in endothelial cells to hypoxia is a new for AMPK The that AMPK the of implanted Matrigel plugs the of AMPK in angiogenesis in that may endothelial AMPK in vivo in that are associated with tumor growth or occlusive vessel diseases (29Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (720) Google Scholar, 2000; Google Scholar). In angiogenesis associated with normal growth and be to that of A. Y. J. Z. K. D. Y. D. Walsh K. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, B. L. E. H. D. K. J. P. M. S. G. M. P. H. T. L. N. H. R. F. D. C. P. F. Plate K.H. W. D. P. Nat. 2001; PubMed Scopus Google Scholar). these data endothelial AMPK signaling may be feature of the angiogenic to ischemic on angiogenic endothelial cell responses or normal endothelial AMPK may a pharmacological for the of angiogenesis associated with for in the Richard and Ruderman for and