Pigment Epithelium-derived Factor Inhibits Angiogenesis via Regulated Intracellular Proteolysis of Vascular Endothelial Growth Factor Receptor 1

Abstract

Pigment epithelium-derived factor (PEDF) has been identified as one of the most potent of endogenous negative regulators of blood vessel growth in the body. Here we report that PEDF is able to inhibit growth factor-induced angiogenesis in microvascular endothelial cells through a novel pathway requiring cleavage and intracellular translocation of the transmembrane domain of the VEGFR-1. Analysis of the subcellular distribution of VEGFR-1 revealed the appearance of an 80-kDa C-terminal domain in the cytosol of cells treated with VEGF and PEDF that correlated with a decrease of the full-length receptor in the nuclear and cytoskeletal fractions. This regulated intramembrane proteolysis is dependent on γ-secretase because inhibition of γ-secretase abolished the inhibitory effect of PEDF on VEGF-induced angiogenesis as well as VEGFR-1 cleavage. The addition of PEDF to microvascular endothelial cells significantly increases γ-secretase activity even in the absence of VEGF, showing that VEGF binding to VEGF-R1 is essential for substrate availability. This increase in activity was associated with translocation of presenilin 1 from the perinuclear region to the cell membrane. PEDF was also able to inhibit VEGF-induced phosphorylation of VEGFR-1. Taken together we have identified two novel pathways by which PEDF inhibits VEGF-induced angiogenesis: regulated intramembrane proteolysis and inhibition of phosphorylation. This confirms the importance of PEDF and VEGFR-1 in the negative regulation of angiogenesis. Pigment epithelium-derived factor (PEDF) has been identified as one of the most potent of endogenous negative regulators of blood vessel growth in the body. Here we report that PEDF is able to inhibit growth factor-induced angiogenesis in microvascular endothelial cells through a novel pathway requiring cleavage and intracellular translocation of the transmembrane domain of the VEGFR-1. Analysis of the subcellular distribution of VEGFR-1 revealed the appearance of an 80-kDa C-terminal domain in the cytosol of cells treated with VEGF and PEDF that correlated with a decrease of the full-length receptor in the nuclear and cytoskeletal fractions. This regulated intramembrane proteolysis is dependent on γ-secretase because inhibition of γ-secretase abolished the inhibitory effect of PEDF on VEGF-induced angiogenesis as well as VEGFR-1 cleavage. The addition of PEDF to microvascular endothelial cells significantly increases γ-secretase activity even in the absence of VEGF, showing that VEGF binding to VEGF-R1 is essential for substrate availability. This increase in activity was associated with translocation of presenilin 1 from the perinuclear region to the cell membrane. PEDF was also able to inhibit VEGF-induced phosphorylation of VEGFR-1. Taken together we have identified two novel pathways by which PEDF inhibits VEGF-induced angiogenesis: regulated intramembrane proteolysis and inhibition of phosphorylation. This confirms the importance of PEDF and VEGFR-1 in the negative regulation of angiogenesis. Pigment epithelium-derived factor (PEDF) 2The abbreviations used are: PEDF, pigment epithelium-derived factor; BRMEC, bovine retinal microvascular endothelial cell; MECBM, microvascular endothelial cell basal medium; VEGF, vascular endothelial growth factor; VEGFR-1, vascular endothelial growth factor receptor; PS, presenilin; TX-sol, Triton X-1000-soluble; TX-insol, Triton X-1000-insoluble; CTF, C-terminal fragment; EDANS, 5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid; 4-(4-dimethylaminophenylazo)benzoic acid. 2The abbreviations used are: PEDF, pigment epithelium-derived factor; BRMEC, bovine retinal microvascular endothelial cell; MECBM, microvascular endothelial cell basal medium; VEGF, vascular endothelial growth factor; VEGFR-1, vascular endothelial growth factor receptor; PS, presenilin; TX-sol, Triton X-1000-soluble; TX-insol, Triton X-1000-insoluble; CTF, C-terminal fragment; EDANS, 5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid; 4-(4-dimethylaminophenylazo)benzoic acid. was identified in the late 1980s by Tombran-Tink and Johnson (1Tombran-Tink J. Johnson L.V. Investig. Opthalmol. Vis. Sci. 1989; 30: 1700-1707PubMed Google Scholar) as a novel neurotrophic activity in conditioned medium from fetal pigmented epithelial cells. PEDF, a 50-kDa noninhibitory member of the serine protease inhibitor (Serpin) gene family (2Tombran-Tink J. Mazuruk K. Rodriguez I.R. Chung D. Linker T. Englander E. Chader G.J. Mol. Vis. 1996; 2: 11PubMed Google Scholar, 3Steele F.R. Chader G.J. Johnson L.V. Tombran-Tink J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1526-1530Crossref PubMed Scopus (419) Google Scholar) has now been identified in a wide variety of tissues throughout the body. The finding that PEDF is essential for maintaining avascularity (4Dawson D.W. Volpert O.V. Gillis P. Crawford S.E. Xu H. Benedict W. Bouck N.P. Science. 1999; 285: 245-248Crossref PubMed Scopus (1383) Google Scholar), coupled with recent observations that PEDF plays a critical role in preventing aberrant neovascularization (5Stellmach V. Crawford S.E. Zhou W. Bouck N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2593-2597Crossref PubMed Scopus (360) Google Scholar), has suggested that PEDF is a potent regulator of angiogenesis. PEDF has been shown to act by inhibiting the angiogenic response to factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (4Dawson D.W. Volpert O.V. Gillis P. Crawford S.E. Xu H. Benedict W. Bouck N.P. Science. 1999; 285: 245-248Crossref PubMed Scopus (1383) Google Scholar). This inhibitory effect was more profound than any of the other anti-angiogenic factors tested (i.e. angiostatin, thrombospondin-1, and endostatin). PEDF selectively inhibits the formation of new vessels from endothelial cells but does not appear to harm the existing vascular structure (6Bouck N. Trends. Mol. Med. 2002; 8: 330-334Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The inhibitory effect of PEDF on vessel formation appears to be reversible when a transient and regulated angiogenesis occurs in situations including tissue repair after injury (7Tonnesen M.G. Feng X. Clark R.A. J. Investig. Dermatol. Symp. Proc. 2000; 5: 40-46Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar). A recent study by Ogata et al. (8Ogata N. Nishikawa M. Nishimura T. Mitsuma Y. Matsumura M. Am. J. Ophthalmol. 2002; 133: 851-852Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) has provided clinical evidence to show that expression of VEGF and PEDF are inversely correlated during the development of retinal neovascularization, further supporting an anti-angiogenic role for PEDF in vivo. Although numerous studies have shown that VEGF mediates endothelial cell behavior via VEGFR-2 (9Ferrara N. J. Mol. Med. 1999; 77: 527-543Crossref PubMed Scopus (1067) Google Scholar, 10Takahashi T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (585) Google Scholar, 11Shalaby F. Ho J. Stanford W.L. Fischer K.D. Schuh A.C. Schwartz L. Bernstein A. Rossant J. Cell. 1997; 89: 981-990Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar), there is increasing evidence that VEGFR-1 plays a critical role in regulating VEGFR-2-mediated signaling (12Bussolati B. Dunk C. Grohman M. Kontos C.D. Mason J. Ahmed A. Am. J. Pathol. 2001; 159: 993-1008Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 13Rahimi N. Dayanir V. Lashkari K. J. Biol. Chem. 2000; 275: 16986-16992Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 14Roberts D.M. Kearney J.B. Johnson J.H. Rosenberg M.P. Kumar R. Bautch V.L. Am. J. Pathol. 2004; 164: 1531-1535Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 15Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Based on these observations, we explored the possibility that PEDF regulates angiogenesis through VEGFR-1 using an in vitro angiogenesis model exposing microvascular endothelial cells to PEDF in the presence or absence of VEGF. We are the first to demonstrate that the inhibitory effect of PEDF on VEGF-induced angiogenesis results from enhancing γ-secretase-dependent cleavage of the C terminus of VEGFR-1, which in turn inhibits VEGFR-2-induced angiogenesis. In addition, PEDF was also able to regulate the phosphorylation of VEGFR-1, which itself can regulate VEGFR-2 signaling. Reagents—Microvascular endothelial cell basal medium (MECBM) and growth supplement were from TCS Works (Buckingham, UK). Recombinant VEGF and γ-secretase activity kits were obtained from R & D Systems (Abingdon, UK). Recombinant PEDF was purchased from BioProducts MD (BioProducts MD LLC, MD). ProteoExtract™ subcellular proteome extraction kit was obtained from Merck. Neutralizing antibodies were initially a gift from Professor Asif Ahmed (Birmingham University, Birmingham, UK) and then obtained from Santa Cruz Biotechnology. All other materials were from Sigma retinal microvascular endothelial cells were and as J. S. J. Kontos C.D. M. Ahmed A. PubMed Scopus Google Scholar). bovine were by a on an the were medium and for The vessel were on a and in microvascular endothelial cell basal medium (MECBM) with growth supplement for The cells were used 1 and of cells were for in with VEGF in the presence or absence of PEDF for The cells to medium as an of PEDF were treated with VEGF for 1 by the addition of PEDF for 1 and were then to and In angiogenic activity of endothelial cells was using in vitro and formation as J. S. J. Kontos C.D. M. Ahmed A. PubMed Scopus Google Scholar, J. 1999; PubMed Scopus Google Scholar). were in in with growth supplement for The cells were to for which the medium was with basal medium fetal and the growth factors and the cells were for The cell was by were with growth factors for The cells were and on with and The medium was then and of was on the cells for to of by of medium for the of microvascular endothelial with fetal for The the were a and in The and of were in with growth supplement in of a were to cell the the cell were with for by with growth factors the presence of the cell were by a one was and fetal was the for the was and and a The were using and as the the and were using the that has shown evidence of in endothelial cells S. M. J. 2001; PubMed Scopus Google Scholar). of the were when the were to the for an and of were treated with of the and by with antibodies the VEGFR-1 C-terminal domain or the VEGFR-2 C-terminal domain by using antibodies a of Cruz The were and with Cruz Santa The was by and Based on results the were treated with of the and a The were then to growth factors for by of Analysis for VEGFR-1 of the have been J. S. J. Kontos C.D. M. Ahmed A. PubMed Scopus Google Scholar). cell of with VEGF PEDF for were to A the VEGFR-1 C-terminal domain Cruz was used a of The were and with Cruz The was by and were to two to Triton and as J. 1999; PubMed Scopus Google Scholar). The cells were first with of Triton 1 of and 1 of and of and Triton for The cell were for and the was as the The was further with of which was the as the Triton that of Triton The was for and then the was as the The from the and were by by Neutralizing and were by using antibodies as J. S. J. Kontos C.D. M. Ahmed A. PubMed Scopus Google Scholar). In were to antibodies to VEGFR-1 or VEGFR-2 for 1 by the addition of VEGF, PEDF, or a for The cell were then by to VEGFR-1 using antibodies the VEGFR-1 C-terminal domain or domain was in a using a γ-secretase kit to the The of the is that the cell is tested for activity by the addition of a to the and 4-(4-dimethylaminophenylazo)benzoic In the the from are by the of the which the of the by the and for the of a The of activity in the cell is to the in were with and for The cells were treated for with of fetal and γ-secretase inhibitor were with growth factors PEDF for by of or of to VEGFR-1 Analysis of 1 was from treated with growth factors by using and then using presenilin 1 were using as and as and for the of we bovine with and A of of were on a with and with and using of subcellular from treated with growth including and were obtained using ProteoExtract™ subcellular proteome extraction This kit the of the subcellular and during extraction to any of the subcellular and Analysis of 1 obtained as from treated with growth factors the were with of the domain Cruz for by the addition of of The was to and by the C-terminal domain Cruz to on were treated with growth factors the were with supplement fetal and then with for The cells were with Triton for by bovine the cells were for 1 with antibodies the terminus or Cruz by a or antibodies was to the The cells were with and the were The were on a using a TCS of the phosphorylation of VEGFR-1 in cell with VEGF PEDF the were from by of phosphorylation for by the addition of of Cruz The was to and by antibodies VEGFR-1 as in subcellular was with an phosphorylation to and by antibodies VEGFR-1. of the were The results are as the S.E. and formation was using a on The was used to of the VEGF microvascular endothelial cell and formation by to as with the cells PEDF in a of VEGF-induced endothelial cell and abolished and formation PEDF effect on or formation when with the negative and VEGFR-1 was shown to be a for PEDF inhibition of VEGF-induced angiogenesis using an VEGFR-1. that VEGFR-1 VEGFR-1 expression in in a with an of VEGFR-1 for VEGFR-2 VEGFR-1 effect on VEGFR-1 expression VEGFR-1 abolished the inhibitory effect of PEDF on VEGF-induced formation but effect on VEGF-induced of VEGFR-1 expression the VEGF-induced increase in even in the absence of PEDF VEGFR-1 effect on the inhibitory effect of VEGFR-1 also the inhibitory effect of PEDF in the and not results that PEDF inhibition of VEGF activity is dependent on VEGFR-1 in endothelial cells and VEGFR-1 is able to regulate vascular effect of on in vitro microvascular endothelial cells were treated with VEGFR-1 or The cells were to VEGF PEDF for two of for of the of formation was then a of showing formation in the presence of growth of in vitro formation of and using the of are shown in for VEGFR-1 and in for VEGFR-2 was a of The show the VEGFR-2 is in the inhibitory effect of PEDF on VEGF-induced VEGFR-2 was used to VEGFR-2 expression in In VEGFR-2 a inhibitory effect on VEGFR-2 expression with a of VEGFR-2 of VEGFR-1 VEGFR-2 not VEGFR-2 expression VEGFR-2 of VEGF-induced formation and of in the presence or the absence of growth factors the of VEGFR-1 in PEDF inhibitory activity is associated with a in VEGFR-1 receptor cell from treated with VEGF PEDF were to and then by an VEGFR-1. VEGFR-1 was as a the of of VEGFR-1 to tested the subcellular distribution of VEGFR-1 PEDF of endothelial we cells in Triton and the from the fractions. that full-length VEGFR-1 was in the in negative VEGF or PEDF PEDF in the presence of VEGF the appearance of a of a 80-kDa C-terminal domain of VEGFR-1 in the PEDF not appear to the domain of VEGFR-1 in the presence or absence of observations to that PEDF cleavage of VEGFR-1 the transmembrane or the intracellular in the intracellular domain of VEGFR-1 from the membrane. A VEGFR-1 to abolished translocation of the C-terminal with VEGFR-2 but not cleavage with the the finding that PEDF in retinal Zhou J. C. Investig. PubMed Scopus Google Scholar) to that cleavage of VEGFR-1 an the from treated with PEDF in the presence of VEGF were to of VEGFR-1 expression using the VEGFR-1 C-terminal The that by 1 after PEDF, a 80-kDa C-terminal domain of VEGFR-1 was in the with a by by the that a of the intracellular domain of VEGFR-1 to the we on to the cleavage of VEGFR-1 be by We first to γ-secretase activity was by of endothelial cells for in the presence of PEDF in a increase of γ-secretase activity in the cell to for PEDF with VEGF which not γ-secretase activity PEDF and VEGF not increase γ-secretase activity that with PEDF the addition of a γ-secretase the inhibitory effect of PEDF on VEGF-induced formation was abolished of cells with γ-secretase inhibitor also the translocation of the intracellular domain of VEGFR-1 to the by This that PEDF appears to γ-secretase activity to inhibition of by of VEGFR-1. is a of and the to which by This is to the from the in a J. PubMed Scopus Google Scholar). evidence has to an essential role for in the activity of γ-secretase J. PubMed Scopus Google Scholar). We an increase in γ-secretase activity by PEDF from an in was on from treated with VEGF PEDF using for bovine The of were on the showing that of to was not significantly tested and the of In the of we show that after PEDF results in an increase in the of full-length and C-terminal of with the and with of VEGF in the subcellular of PS, of a and a nuclear by were 1 a perinuclear VEGF PEDF translocation of to the cell We were then able to demonstrate the PEDF regulates the subcellular distribution of VEGFR-1 endothelial cells. subcellular for the domain of VEGFR-1 in cells and cells treated with VEGF, PEDF, and revealed the presence of an VEGFR-1 in and nuclear but not in the for the C-terminal domain of VEGFR-1 an VEGFR-1 in the and the nuclear fractions. 80-kDa C-terminal domain of VEGFR-1 was in the cytosol of cells treated with VEGF and PEDF the phosphorylation of the VEGFR-1 receptor and we with antibodies with a study by et al. T. H. Y. S. Y. Y. J. Shibuya M. M. M. Mol. 2002; Google Scholar), VEGF an increase in of VEGFR-1 with with and in cell PEDF VEGFR-1 phosphorylation in the and and was when PEDF was used in with VEGF PEDF VEGFR-1 phosphorylation by VEGF, were with VEGF for 1 and then with PEDF for A of and occurs after 1 of PEDF addition Analysis of the subcellular that VEGF an increase in of VEGFR-1 in and nuclear fractions. The the intracellular domain the and that the domain of VEGFR-1 the PEDF the VEGFR-1 phosphorylation in of the of the endothelial cells were in the presence or absence of VEGF of the 80-kDa VEGFR-1 was not in the This study a novel the by which PEDF via VEGFR-1 is able to inhibit VEGF-induced angiogenesis in microvascular endothelial cells. signaling pathways are regulated translocation of the intracellular domain of VEGFR-1 and VEGFR-1 phosphorylation. observations further a critical role for VEGFR-1 in the regulation of The of VEGFR-1 as a regulator of VEGFR-2-induced angiogenesis (12Bussolati B. Dunk C. Grohman M. 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A. 2004; Scopus Google Scholar), be to a to the In we have identified that VEGFR-1 plays a critical role in the regulation of angiogenesis and that PEDF as a potent inhibitor of VEGF-induced angiogenesis by the intracellular domain of VEGFR-1 and regulating the phosphorylation of VEGFR-1. This study has provided a novel the pathway by which PEDF regulates endothelial cell behavior and for to regulate angiogenesis.