A Novel Function of Plasminogen Activator Inhibitor-1 in Modulation of the AKT Pathway in Wild-type and Plasminogen Activator Inhibitor-1-deficient Endothelial Cells

Cell proliferation, an event associated with angiogenesis, involves coordinated activities of a number of proteins. The role of plasminogen activator inhibitor-1 (PAI-1) in angiogenesis remains controversial. Utilizing proliferating PAI-1−/− endothelial cells (EC), the impact of a host PAI-1 deficiency on Akt activation was evaluated. Hyperactivation of Akt(Ser(P)473) was observed in PAI-1−/− EC, and this was probably due to enhanced inactivation of tumor suppressor PTEN, thus rendering the cells resistant to apoptotic signals. Higher levels of inactivated caspase-9 in PAI-1−/− EC led to lower levels of procaspase-3 and cleaved caspase-3, thereby promoting survival. These effects were reversed when recombinant PAI-1 was added to PAI-1−/− EC. Additional studies demonstrated that regulation of proliferation is dependent on its interaction with low density lipoprotein receptor-related protein. Thus, PAI-1 is a negative regulator of cell growth, exerting its effect on the phosphatidylinositol 3-kinase/Akt pathway and allowing controlled cell proliferation. Cell proliferation, an event associated with angiogenesis, involves coordinated activities of a number of proteins. The role of plasminogen activator inhibitor-1 (PAI-1) in angiogenesis remains controversial. Utilizing proliferating PAI-1−/− endothelial cells (EC), the impact of a host PAI-1 deficiency on Akt activation was evaluated. Hyperactivation of Akt(Ser(P)473) was observed in PAI-1−/− EC, and this was probably due to enhanced inactivation of tumor suppressor PTEN, thus rendering the cells resistant to apoptotic signals. Higher levels of inactivated caspase-9 in PAI-1−/− EC led to lower levels of procaspase-3 and cleaved caspase-3, thereby promoting survival. These effects were reversed when recombinant PAI-1 was added to PAI-1−/− EC. Additional studies demonstrated that regulation of proliferation is dependent on its interaction with low density lipoprotein receptor-related protein. Thus, PAI-1 is a negative regulator of cell growth, exerting its effect on the phosphatidylinositol 3-kinase/Akt pathway and allowing controlled cell proliferation. Angiogenesis is an important biological process involving the formation of new capillaries from preexisting blood vessels, and it occurs in normal as well as pathophysiological settings. It involves a highly orchestrated series of events, including the stimulation of endothelial cells (EC) 2The abbreviations used are: EC, endothelial cell(s); LRP, low density lipoprotein receptor-related protein; PAI-1, plasminogen activator inhibitor-1; rPAI-1, recombinant PAI-1; PAI-1−/−, plasminogen activator inhibitor-1 homozygous deficient; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial cell growth factor; WM, wortmannin; WT, wild-type; PI3K, phosphatidylinositol 3-kinase; CM, complete medium. by growth factors, degradation of the extracellular matrix, migration and proliferation of EC, and formation of new capillary tubes (1Carmeliet P. Nat. Med. 2000; 62: 389-395Crossref Scopus (3467) Google Scholar, 2Chavakis E. Dimmeler S. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 887-893Crossref PubMed Scopus (233) Google Scholar). Components of the fibrinolytic pathway have been implicated in the formation of angiogenic vessels, notably plasminogen activators, viz. urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. The activation product, plasmin, a trypsin-like serine protease, facilitates degradation of the extracellular matrix, either directly or indirectly through activation of matrix metalloproteases. This process allows migration of EC and subsequent events leading to neovessel formation. However, specific inhibitors control proteolysis of the extracellular matrix, and PAI-1 is an important inhibitor of the plasminogen activation system (3Loskutoff D.J. van Mourik J.A. Erickson L.A. Lawrence D.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2956-2960Crossref PubMed Scopus (264) Google Scholar, 4Fay W.P. Shapiro A.D. Shih J.L. Schleef R.R. Ginsburg D. N. Engl. J. Med. 1992; 327: 1729-1733Crossref PubMed Scopus (231) Google Scholar). Considering the important role of the plasminogen activator-PAI-1 system in angiogenesis and the availability of genetically engineered mice lacking various components of this system, several investigators have utilized transgenic approaches to establish the functional potential of components of the plasminogen activator-PAI-1 system in angiogenesis. The role of PAI-1 in angiogenesis and tumor formation has been extensively studied, but results were determined to be dependent on the experimental setting (5Bajou K. Noel A. Gerard R.D. Masson V. Brunner N. Holst-Hansen C. Skobe M. Fuseing N.E. Carmeliet P. Collen D. Foidart J.-M. Nat. Med. 1998; 4: 923-928Crossref PubMed Scopus (613) Google Scholar, 6Gutierrez L.S. Schulman A. Brito-Robinson T. Noria F. Ploplis V.A. Castellino F.J. Cancer Res. 2000; 60: 5839-5847PubMed Google Scholar, 7Devy L. Blacher S. Grignet-Debrus C. Bajou K. Masson V. Gerard R.D. Gils A. Carmeliet G. Carmeliet P. Declerk P.J. Noel A. Foidart J.-M. FASEB J. 2002; 16: 147-154Crossref PubMed Scopus (251) Google Scholar, 8Lambert V. Munaut C. Noel A. Frankenne F. Bajou K. Gerard R.D. Carmeliet P. Defresne M.P. Foidart J.-M. Rakic J.M. FASEB J. 2001; 15: 1021-1027Crossref PubMed Scopus (86) Google Scholar), the stage of cancer progression, and the origin of the cells (9Eitzman D.T. Krauss J.C. Shen T. Cui J. Ginsburg D. Blood. 1996; 87: 4718-4722PubMed Google Scholar, 10Almholt K. Nielsen B.S. Frandsen T.L. Brunner N. Dano K. Johnsen M. Oncogene. 2003; 22: 4389-4397Crossref PubMed Scopus (63) Google Scholar, 11Stefansson S. Petitclerc E. Wong M.K. McMahon G.A. Brooks P.C. Lawrence D.A. J. Biol. Chem. 2001; 276: 8135-8141Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 12McMahon G.A. Petitclerc E. Stefansson S. Smith E. Wong M.K. Westrick R.J. Ginsburg D. Brooks P.C. Lawrence D.A. J. Biol. Chem. 2001; 276: 33964-33968Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 13Bajou K. Maillard C. Jost M. Lijnen R.H. Gils A. Declerck P. Carmeliet P. Foidart J.-M. Noel A. Oncogene. 2004; 41: 6986-6990Crossref Scopus (140) Google Scholar). It has been demonstrated that implanted malignant keratinocytes were unable to induce vascularization of tumors in PAI-1-deficient mice (PAI-1−/−) (5Bajou K. Noel A. Gerard R.D. Masson V. Brunner N. Holst-Hansen C. Skobe M. Fuseing N.E. Carmeliet P. Collen D. Foidart J.-M. Nat. Med. 1998; 4: 923-928Crossref PubMed Scopus (613) Google Scholar). Similarly, T241 fibrosarcoma tumor growth was suppressed in PAI-1−/− mice relative to WT mice (6Gutierrez L.S. Schulman A. Brito-Robinson T. Noria F. Ploplis V.A. Castellino F.J. Cancer Res. 2000; 60: 5839-5847PubMed Google Scholar). In vitro studies demonstrated a lack of microvessel generation in an aortic ring assay derived from aortas of PAI-1−/− mice (7Devy L. Blacher S. Grignet-Debrus C. Bajou K. Masson V. Gerard R.D. Gils A. Carmeliet G. Carmeliet P. Declerk P.J. Noel A. Foidart J.-M. FASEB J. 2002; 16: 147-154Crossref PubMed Scopus (251) Google Scholar). Laser-induced choroidal neoangiogenesis was also reduced in PAI-1−/− mice (14Lambert V. Munaut C. Carmeliet P. Gerard R.D. Declerck P.J. Gils A. Claes C. Foidart J.-M. Noel A. Rakic J.M. Invest. Ophthalmol. Vis. Sci. 2003; 44: 2791-2797Crossref PubMed Scopus (42) Google Scholar). However, both of these observations were reversed when the experimental system was exposed to This is in with studies that levels of PAI-1 with and reduced from a of tumors N. U. U. A. C. F. M. J. Biol. 2000; 15: PubMed Scopus Google Scholar). from these studies that PAI-1 is However, have also been a PAI-1 deficiency impact on of cells (9Eitzman D.T. Krauss J.C. Shen T. Cui J. Ginsburg D. Blood. 1996; 87: 4718-4722PubMed Google or on tumor cancer K. Nielsen B.S. Frandsen T.L. Brunner N. Dano K. Johnsen M. Oncogene. 2003; 22: 4389-4397Crossref PubMed Scopus (63) Google Scholar). In an effect of PAI-1 was observed through the of growth angiogenesis in the assay S. Petitclerc E. Wong M.K. McMahon G.A. Brooks P.C. Lawrence D.A. J. Biol. Chem. 2001; 276: 8135-8141Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). studies have that the of PAI-1 tumor growth and angiogenesis in in mice in a G.A. Petitclerc E. Stefansson S. Smith E. Wong M.K. Westrick R.J. Ginsburg D. Brooks P.C. Lawrence D.A. J. Biol. Chem. 2001; 276: 33964-33968Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). a effect of PAI-1 has been in vitro (7Devy L. Blacher S. Grignet-Debrus C. Bajou K. Masson V. Gerard R.D. Gils A. Carmeliet G. Carmeliet P. Declerk P.J. Noel A. Foidart J.-M. FASEB J. 2002; 16: 147-154Crossref PubMed Scopus (251) Google and in (14Lambert V. Munaut C. Carmeliet P. Gerard R.D. Declerck P.J. Gils A. Claes C. Foidart J.-M. Noel A. Rakic J.M. Invest. Ophthalmol. Vis. Sci. 2003; 44: 2791-2797Crossref PubMed Scopus (42) Google of angiogenesis. the of PAI-1 in angiogenesis is controversial. vascular EC the of angiogenesis, highly cells in to the role of PAI-1 in cell associated with the angiogenic This has that a complete deficiency of PAI-1 is associated with proliferation of aortic EC in vitro V.A. R.D. J. N. D. D. J.A. Castellino F.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), an effect associated with levels of is to be by the interaction of growth with thereby promoting EC A. J. M. V. N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Res. 2000; PubMed Scopus Google Scholar). In it has been demonstrated that vascular EC growth the Akt and an in cell K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the role of has been in proliferation, and of EC activation of the and A. F. M. F. S. Blood. PubMed Scopus Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google Scholar). a number of studies have on the effect of PAI-1 or its deficiency on tumor and and is the to the effect of PAI-1 on EC the pathway V.A. R.D. J. N. D. D. J.A. Castellino F.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google that a in proliferating PAI-1−/− EC activation of the on an of the of this enhanced The results of these The was from and from PTEN, and were from Cell the used was and The was a from D. K. was used the complete was with of of of endothelial growth and EC was from Cell WT PAI-1 and the recombinant PAI-1 and recombinant PAI-1 were and as R.D. Lawrence D.A. Castellino F.J. Ploplis V.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). levels were the levels of the PAI-1 used were The of PAI-1−/− mice have been P. L. L. van A. G. M. Collen D. J. Invest. PubMed Scopus Google Scholar, P. J.M. L. van M. Collen D. J. Invest. PubMed Scopus Google Scholar). PAI-1−/− mice were the WT mice were from the and were used as mice and of were utilized this The mice were with a of of and of of were by the and of the of The of aortas and the and of of EC have been V.A. R.D. J. N. D. D. J.A. Castellino F.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). cell were in a have been used Cell proliferation were as V.A. R.D. J. N. D. D. J.A. Castellino F.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). EC in a density of were added to and to The was and EC growth was added The cells were to and the in medium. proliferation the of PAI-1 was the cells were as but was added to the The cells were cell were the cells were in and with The cells were with a and were the and the Cell were determined in proliferation involving of PI3K, EC were a density of in a and to The was and complete EC growth was added The cells were to and with this the was and cells were with and in with and were of and cell were The of apoptotic cells was determined the were a density of in a and to this the was and EC growth was were and by was added to WT and PAI-1−/− EC, were used to the WT and PAI-1−/− cells were in of endothelial cell growth a density of and with the inhibitor of and The cells were with by a with were on the system the This assay was also EC with this both and proliferation assay was as of EC the WT and PAI-1−/− cells the or of were caspase-9 the caspase-9 assay as the of cells were in and the of the was of of was in cell and with and were in a This assay was also EC with were as of WT were a density of in a and to this the was and EC growth was The cells were to and with were in EC and with or The cells were directly with of of the levels of Akt and of the cell was on a on a and in the Akt or of caspase-3, PTEN, and cells were added to and and The cell were and were on on and to the WT was added to proliferating cells and the cells were and caspase-9 were by the cell on of the cleaved caspase-9 the was the the of the and the was an of were the from on the from WT and PAI-1−/− EC was the levels were by and to a The was the The was on a with to the WT and PAI-1−/− cells a of were and was on a was and by The was in The was exposed to the of cleaved or of WT and PAI-1−/− cell were with or of in a of with this of of was added and the The were with cell and in of and were an was used to results the in were of were to be Akt and in WT and PAI-1−/− Akt is in the and the and growth Akt to the Akt is and both is activation of Akt K. Res. 2002; PubMed Scopus Google Scholar). the of was in PAI-1−/− EC with WT cells the levels of in the were as well as the levels of a that facilitates of The of has been M. C. V. L. J. S. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). Akt activation is by PI3K, proliferating WT and PAI-1−/− EC were with a inhibitor of and demonstrated that of or of Akt in both cell levels of Akt were These results as in WT Akt activation in PAI-1−/− cells is by It has been that is to growth as well as in of EC. it has been demonstrated that effects due to of in EC Res. 2000; PubMed Scopus Google Scholar). Similarly, is to the of growth V. R.J. J. PubMed Scopus Google and growth in cells PubMed Scopus Google Scholar), as well as in EC A. J. M. V. N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of in the proliferating WT and PAI-1−/− EC were with and cell the cell in both WT and PAI-1−/− EC. The in cell be due to of in In the of an in cell was These results the that the in WT and PAI-1−/− EC is the of enhanced Akt(Ser(P)473) in proliferating PAI-1−/− the negative PTEN, was the pathway by Akt to the a that is allowing activation of Akt T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). it has been that the of its thus in of and Akt F. S. N. Biol. 2000; PubMed Scopus Google Scholar). the of in proliferating WT and PAI-1−/− EC was with enhanced Akt levels of the inactivated of PTEN, were observed in PAI-1−/− cells with WT cells and These results that regulation of an important role in proliferation. Akt in PAI-1−/− EC with and observations that is an of PAI-1 and activation of cells in in PAI-1 demonstrated an in activation of and of S. M. T. Res. Res. 2001; PubMed Scopus Google Scholar). It has been that and were when PAI-1 was added to cells J. K. Declerck P.J. J. 2000; PubMed Google Scholar). 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PubMed Google Scholar), is by cells cell migration and proliferation. it has been demonstrated that vascular cell proliferation and and with the and in of the and of uPAR, studies were to the of aortic EC used the in both WT and PAI-1−/− cells also be in cell of both In to effects of PAI-1 on cell proliferation, WT and PAI-1−/− EC were to that a of was from complete to complete the EC growth and cells were to this cells were to in the or of In the of PAI-1, the cell of PAI-1−/− EC to the of WT cells However, the of PAI-1 to WT cells in in cell of the growth of WT and PAI-1−/− EC in the and of WT and PAI-1−/− cells were a density of in in in complete and to this the was and the cells were to in complete endothelial cell growth The cells were either exposed to PAI-1 or were with and PAI-1−/− EC exposed to PAI-1 PAI-1−/− EC exposed to of the growth of WT and PAI-1−/− EC in the and of of were as in and PAI-1 WT and of were added to the Cell were of with PAI-1−/− EC PAI-1 and PAI-1−/− EC exposed to WT PAI-1−/− EC exposed to WT and cells exposed to PAI-1 the by PAI-1 proliferation is due to its or to its to to LRP, EC were with PAI-1 were added to proliferating WT and PAI-1−/− EC. These were either in the of plasminogen activator or was R.D. Lawrence D.A. Castellino F.J. Ploplis V.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the of PAI-1 to WT cells the cell However, in PAI-1−/− the reduced the cell WT PAI-1, when added to the cells the the cells of PAI-1−/− EC to WT proliferating PAI-1−/− EC were with PAI-1 and the This was due to observations that enhanced Akt(Ser(P)473) was in PAI-1−/− EC, cells to PAI-1 the effects of from these studies that the PAI-1−/− EC with WT and the cell to WT levels the with PAI-1−/− EC proliferation of PAI-1−/− EC. These results that regulation of cell proliferation by PAI-1 is dependent on its interaction with and on its to the of PAI-1 to PAI-1−/− EC the components of the Akt levels of PTEN, and procaspase-3 and cleaved were cell from cells in the of PAI-1 the levels of Akt(Ser(P)473) as well as that of inactivated in PAI-1−/− of procaspase-3 and cleaved levels was observed in PAI-1−/− cells with relative to WT the the of PAI-1 to WT cells have an effect on the levels of and also WT and PAI-1−/− cells exposed to caspase-9 levels and in caspase-9 was observed in PAI-1−/− cells with The of also the of apoptotic of PAI-1−/− cells effect on either caspase-9 or in WT these results that PAI-1 be as a negative growth regulator of aortic EC. Thus, PAI-1−/− EC a WT and the of PAI-1 the growth through the in cell proliferation. from these studies also that PAI-1 the activation of Akt levels of caspase-3, of cell proliferation and involves a of growth factors, and the by the interaction of growth and the pathway has to be important cell in cell activation of this pathway with the and of Nat. 2002; PubMed Scopus Google Scholar). on observations that enhanced proliferation of PAI-1−/− EC is probably due to enhanced activation of the Akt pathway V.A. R.D. J. N. D. D. J.A. Castellino F.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), were to the effects of this on The activation of the pathway results in the of a number of Akt that important in the cell and P.J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). In both WT and PAI-1−/− EC, the activation of Akt is dependent on PI3K, levels of Akt(Ser(P)473) were low levels of The to the cell in both WT and PAI-1−/− EC in the of is extracellular and and of in cell proliferation. It is that is in PAI-1−/− EC, be by the that the cell of PAI-1−/− cells to the as WT of is a in the of the effects of growth A. S. Res. 1998; PubMed Scopus Google Scholar). It has been that and of Akt in a and effects A. J. M. V. N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Res. 2000; PubMed Scopus Google Scholar). of both and is activation of was in PAI-1−/− cell levels of were in both the activation of Akt in PAI-1−/− cells be the levels of inactivated in Akt This that regulation of in WT cells in the cells by the and It has been that is a Akt(Ser(P)473) and of cancer in Cancer 2004; PubMed Scopus (42) Google Scholar). PTEN, as a tumor suppressor was to be inactivated in several of tumors and in activation of the pathway Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that lower of procaspase-3 and cleaved in the PAI-1−/− cells with to be a effect of levels of Akt(Ser(P)473) and the inactivated of levels of in in both its is lower in PAI-1−/− EC The levels in caspase-9 were in PAI-1−/− EC in levels of cleaved The that and inactivation of caspase-9 be of several by Akt to cell survival. The activation of Akt an important role in EC and proliferation. that the of host or PAI-1 inactivation of PTEN, in activation of Akt serine a regulator of cell events, Akt a number of including is well of caspase-9 by Akt results in inactivation and to the PAI-1−/− EC were of to PAI-1, suppressed PAI-1 the levels of Akt(Ser(P)473) as well as the levels of procaspase-3 in PAI-1−/− It has been that of the D.J. 1992; Full Text PDF PubMed Scopus Google and R.R. P. J.A. D.A. J. PubMed Scopus Google of with have that PAI-1 is an inhibitor of and that the of PAI-1 is the interaction PAI-1 and R.J. D.J. J. 2004; PubMed Scopus Google Scholar). that the of host PAI-1 is associated with lower levels of procaspase-3 and cleaved caspase-3, allowing the EC to be resistant to This effect was reversed by the of Similarly, studies have demonstrated that PAI-1 the pathway in EC as well as in vascular cells through of cell by PAI-1 N. T. T. N. Biol. Chem. 2003; PubMed Scopus Google Scholar). studies have that PAI-1 and of and cells J. K. Declerck P.J. J. 2000; PubMed Google Scholar). of by PAI-1 was also observed in cell vascular cells R.J. D.J. J. 2004; PubMed Scopus Google and S. M. T. Res. Res. 2001; PubMed Scopus Google Scholar), and this was reversed by of In tumor levels of PAI-1 the of the these observations that PAI-1 in a it has been demonstrated that a in proliferation of and occurs in of PAI-1-deficient mice with WT this enhanced in PAI-1-deficient mice was to levels of Akt(Ser(P)473) PubMed Scopus Google Scholar). Thus, the that PAI-1 an important role in the process and PAI-1 be in growth a is a regulator of it is an inhibitor of cell growth, studies have that it also In vitro proliferation studies that from growth mice a with cells from WT growth cell proliferation by an and also has the to of components of the extracellular matrix and C. L. A. J. 1998; PubMed Scopus Google Scholar). PAI-1 is a protein. from its role as a serine it also to S. Lawrence D.A. 1996; PubMed Scopus Google Scholar). it has an that facilitates the of and its by PAI-1 J. J. 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These observations a potential by PAI-1 the activation of the Akt in cell proliferation and van and the used in this also and in cell