FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2029452873> ?p ?o ?g. }

• W2029452873 endingPage "4405" @default.
• W2029452873 startingPage "4400" @default.
• W2029452873 abstract "Although several cytokines and growth factors have been shown to regulate vascular endothelial growth factor (VEGF) production, little is known about how VEGF may regulate growth factors that have known mitogenic and chemotactic actions on mesenchymal cells (which are involved in the maturation of the angiogenic process). We investigated the effect of VEGF on heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression in human umbilical vein endothelial cells. HB-EGF mRNA was induced by 8-fold after 2 h of VEGF stimulation, and it returned to base line within 6 h. VEGF did not alter the half-life of HB-EGF mRNA (55 min). Nuclear run-on experiments showed a 4.9-fold increase in HB-EGF gene transcription within 2 h of VEGF stimulation, and Western analysis demonstrated an associated increase in cellular HB-EGF protein. We found that platelet-derived growth factor-BB (PDGF-BB) mRNA was also induced 3-fold after 5 h of VEGF stimulation, whereas neither endothelin 1 nor transforming growth factor-β1 was regulated by VEGF. Finally, conditioned medium from VEGF-stimulated endothelial cells produced an increase in DNA synthesis in vascular smooth muscle cells, and this effect was blocked by a neutralizing antibody to PDGF. The induction of HB-EGF and PDGF-BB expression in endothelial cells may represent the mechanism by which VEGF recruits mesenchymal cells to form the medial and adventitial layers of arterioles and venules during the course of angiogenesis. Although several cytokines and growth factors have been shown to regulate vascular endothelial growth factor (VEGF) production, little is known about how VEGF may regulate growth factors that have known mitogenic and chemotactic actions on mesenchymal cells (which are involved in the maturation of the angiogenic process). We investigated the effect of VEGF on heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression in human umbilical vein endothelial cells. HB-EGF mRNA was induced by 8-fold after 2 h of VEGF stimulation, and it returned to base line within 6 h. VEGF did not alter the half-life of HB-EGF mRNA (55 min). Nuclear run-on experiments showed a 4.9-fold increase in HB-EGF gene transcription within 2 h of VEGF stimulation, and Western analysis demonstrated an associated increase in cellular HB-EGF protein. We found that platelet-derived growth factor-BB (PDGF-BB) mRNA was also induced 3-fold after 5 h of VEGF stimulation, whereas neither endothelin 1 nor transforming growth factor-β1 was regulated by VEGF. Finally, conditioned medium from VEGF-stimulated endothelial cells produced an increase in DNA synthesis in vascular smooth muscle cells, and this effect was blocked by a neutralizing antibody to PDGF. The induction of HB-EGF and PDGF-BB expression in endothelial cells may represent the mechanism by which VEGF recruits mesenchymal cells to form the medial and adventitial layers of arterioles and venules during the course of angiogenesis. Vascular endothelial growth factor induces heparin-binding epidermal growth factor-like growth factor in vascular endothelial cells.Journal of Biological ChemistryVol. 273Issue 15PreviewPages 4402 and 4403:Figs. 3 and 4 were not reproduced adequately. Full-Text PDF Open Access Angiogenesis is a crucial component of tumor growth and atherosclerosis (1Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6087) Google Scholar, 2O'Brien E.R. Garvin M.R. Dev R. Stewart D.K. Hinohora T. Simpson J.B. Schwartz S.M. Am. J. Pathol. 1994; 145: 883-894PubMed Google Scholar). During the formation of new blood vessels, endothelial cells are stimulated to migrate, proliferate, and invade surrounding tissue to form capillary tubules capable of carrying blood. An angiogenic stimulus is also necessary for the maturation of these vessels, which involves the migration and proliferation of pericytes and smooth muscle cells to form the basement membrane of capillaries, or smooth muscle cells and fibroblasts to form the tunica media and adventitia of arterioles and venules. Several growth factors and cytokines, such as vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α; PDGF, platelet-derived growth factor; PDGF-BB, platelet-derived growth factor-BB; ET1, endothelin 1; HB-EGF, heparin-binding epidermal growth factor-like growth factor; HUVEC, human umbilical vein endothelial cells; HCAEC, human coronary artery endothelial cells; HASMC, human aortic smooth muscle cells; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; EGF, epidermal growth factor; PBS, phosphate-buffered saline; RASMC, rat aortic smooth muscle cells. (1Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6087) Google Scholar), basic fibroblast growth factor, platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), and endothelin 1 (ET1) have been suggested to be important in the process of angiogenesis (3Connolly D.T. Huevelman D.M. Nelson R. Olander J.V. Eppley B.L. Delfino J.J. Siegel N.R. Leimgruber R.M. Feder J. J. Clin. Invest. 1989; 84: 1470-1478Crossref PubMed Scopus (1181) Google Scholar, 4Giaid A. Saleh D. Yanagisawa M. Clarke Forbes R.D. Transplantation. 1995; 59 (D.R. D. C.): 1308-1313RCrossref PubMed Scopus (32) Google Scholar). Among these, only VEGF is considered to be endothelial cell-specific, as its receptors, KDR/flk-1 and flt1, are expressed only by vascular endothelial cells in vivo (5Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4447) Google Scholar, 6Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Crossref PubMed Scopus (4161) Google Scholar, 7Berkman R.A. Merrill M.J. Reinhold W.C. Monacci W.T. Saxena A. Clark W.C. Robertson J.T. Ali I.U. Oldfield E.H. J. Clin. Invest. 1993; 91: 153-159Crossref PubMed Scopus (434) Google Scholar, 8Breier G. Albrecht U. Sterrer S. Risau W. Development. 1992; 114: 521-532Crossref PubMed Google Scholar, 9Patterson C. Perrella M.A. Hsieh C.-M. Yoshizumi M. Lee M.-E. Haber E. J. Biol. Chem. 1995; 270: 23111-23118Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Disruption of the KDR/flk-1 and flt1 genes in mice interferes with vasculogenesis, resulting in embryo death at days 8.5–9.5 and 9, respectively (10Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3360) Google Scholar, 11Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2216) Google Scholar). Similarly, mice deficient in VEGF die after 8.5–9 days of gestation and show impaired vasculogenesis and angiogenesis (12Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3458) Google Scholar). Despite these developmental studies and numerous reports that growth factors, cytokines, and physiologic stimuli (such as hypoxia and shear stress) up-regulate VEGF (13Klagsbrun M. Soker S. Curr. Biol. 1993; 3: 699-702Abstract Full Text PDF PubMed Scopus (142) Google Scholar, 14D'Amore P.A. Smith S.R. Growth Factors. 1993; 8: 61-75Crossref PubMed Scopus (146) Google Scholar, 15Neufeld G. Tessler S. Gitay-Goren H. Cohen T. Levi B.-Z. Prog. Growth Factor Res. 1994; 5: 89-97Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 16Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar, 17Brogi E. Schatteman G. Wu T. Kim E.A. Varticovski L. Keyt B. Isner J.M. J. Clin. Invest. 1996; 97: 469-476Crossref PubMed Scopus (341) Google Scholar, 18Koolwijk P. van Erck M.G.M. de Vree W.J.A. Vermeer M.A. Weich H.A. Hanemaaijer R. van Hinsbergh V.W.M. J. Cell Biol. 1996; 132: 1177-1188Crossref PubMed Scopus (267) Google Scholar, 19Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 2746-2753Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 20Brogi E. Wu T. Namiki A. Isner J.M. Circulation. 1994; 90: 649-652Crossref PubMed Scopus (536) Google Scholar, 21Cohen T. Nahari D. Cerem L.W. Neufeld G. Levi B.-Z. J. Biol. Chem. 1996; 271: 736-741Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar), there is a lack of information about the downstream effects of VEGF on growth factors that play a role in the maturation of angiogenesis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a 22-kDa protein that was originally purified from the conditioned medium of human macrophage-like U-937 cells (22Higashiyama S. Abraham J.A. Miller J. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1044) Google Scholar). The COOH terminus of HB-EGF contains six cysteine residues with spacing characteristic of members of the EGF family, and HB-EGF shares 40% sequence identity with EGF. HB-EGF binds to the EGF receptor and triggers its downstream signal transduction pathways (23Higashiyama S. Lau K. Besner G.E. Abraham J.A. Klagsbrun M. J. Biol. Chem. 1992; 267: 6205-6212Abstract Full Text PDF PubMed Google Scholar). HB-EGF is mitogenic and chemotactic for several primary cell types, such as vascular smooth muscle cells, keratinocytes, fibroblasts, and cardiomyocytes, but not for vascular endothelial cells. Vascular endothelial cells do express HB-EGF, however, and this expression can be induced by TNF-α and shear stress (24Temizer D.H. Yoshizumi M. Perrella M.A. Susanni E.E Quertermous T. Lee M.-E. J. Biol. Chem. 1992; 267: 24892-24896Abstract Full Text PDF PubMed Google Scholar, 25Perrella M.A. Yoshizumi M. Fen Z. Tsai J.-C. Hsieh C.-M. Kourembanas S. Lee M.-E. J. Biol. Chem. 1994; 269: 14595-14600Abstract Full Text PDF PubMed Google Scholar, 26Raab G. Higashiyama S. Hetelekidis S. Abraham J.A. Damm D. Ono M. Klagsbrun M. Biochem. Biophys. Res. Commun. 1994; 204: 592-597Crossref PubMed Scopus (71) Google Scholar, 27Morita T. Yoshizumi M. Kurihara H. Maemura K. Nagai R. Yazaki Y. Biochem. Biophys. Res. Commun. 1993; 197: 256-262Crossref PubMed Scopus (79) Google Scholar). HB-EGF is induced at the site of blastocyst implantation in the mouse uterus (28Das S.K. Wang X.-N. Paria B.C. Damm D. Abraham J.A. Klagsbrun M. Andrews G.K. Dey S.K. Development. 1994; 120: 1071-1083Crossref PubMed Google Scholar), a site of intensive angiogenesis. Up-regulation of HB-EGF expression also occurs in other pathological states that require angiogenesis, such as wound healing, and in human atherosclerotic plaques (29Miyagawa J. Higashiyama S. Kawata S. Inui Y. Tamura S. Yamamoto K. Nishida M. Nakamura T. Yamashita S. Matsuzawa Y. Taniguchi N. J. Clin. Invest. 1995; 95: 404-411Crossref PubMed Google Scholar, 30Marikovsky M. Breuing K. Liu P.Y. Eriksson E. Higashiyama S. Farber P. Abraham J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3889-3893Crossref PubMed Scopus (296) Google Scholar). Because VEGF is a critical factor in the development of new blood vessels, we speculated that VEGF may induce growth factors in vascular endothelial cells, which then act in a paracrine fashion to promote angiogenesis. We studied the regulation by VEGF of HB-EGF, PDGF-BB, ET1, and TGF-β1 in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC). HB-EGF and PDGF-BB mRNA were up-regulated by VEGF in distinct temporal patterns. Moreover, the increase in HB-EGF mRNA was regulated transcriptionally and translated to an increase in accumulation of cellular HB-EGF protein. Primary culture HUVEC, HCAEC, and HASMC were obtained from Clonetics (San Diego, CA). HUVEC and HCAEC were grown in M199 (JRH Biosciences, Lenexa, KS) supplemented with 20% fetal calf serum (HyClone Laboratories, Logan, UT), 100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml heparin sulfate (Sigma), and 50 μg/ml endothelial cell growth supplement (Collaborative Biomedical Products, Bedford, MA). HASMC were grown in M199 supplemented with 20% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were passaged every 3–5 days, and cells from passages 6–8 were used in the experiments described here. After the cells had grown to 80–90% confluence, they were placed in M199 medium supplemented with 5% fetal calf serum for 1 h before the experiments. Recombinant human VEGF (Collaborative Biomedical Products) was dissolved in PBS (JRH Biosciences) and stored at −80 °C until use. Total RNA from cells in culture was prepared by guanidinium isothiocyanate extraction and centrifugation through cesium chloride. An HB-EGF cDNA fragment from HUVEC RNA was amplified by the reverse transcription-polymerase chain reaction (31Yoshizumi M. Kourembanas S. Temizer D.H. Cambria R.P. Quertermous T. Lee M.-E. J. Biol. Chem. 1992; 267: 9467-9469Abstract Full Text PDF PubMed Google Scholar). The human PDGF-BB probe was obtained similarly by reverse transcription-polymerase chain reaction using forward (5′-CGTCTGGTCAGCGCCGAGGGG-3′) and reverse (5′-CGTCTTGTCATGCGTGTGCTT-3′) primers. The ET1 probe was obtained as described by Bloch et al. (32Bloch K.D. Friedrich S.P. Lee M.-E. Eddy R.L. Shows T.B. Quertermous T. J. Biol. Chem. 1989; 264: 10851-10857Abstract Full Text PDF PubMed Google Scholar). The TGF-β1 probe was a gift from G. S. Hotamisligil (Harvard School of Public Health, Boston, MA). Total RNA (10 μg) from cells in culture was fractionated on 1.3% formaldehyde-agarose gels and transferred to nitrocellulose filters. The filters were hybridized with a random-primed 32P-labeled HB-EGF cDNA probe as described (31Yoshizumi M. Kourembanas S. Temizer D.H. Cambria R.P. Quertermous T. Lee M.-E. J. Biol. Chem. 1992; 267: 9467-9469Abstract Full Text PDF PubMed Google Scholar). In some experiments, filters were also hybridized with PDGF-BB, ET1, and TGF-β1 probes. The hybridized filters were then washed in 30 mm sodium chloride, 3 mm sodium citrate, and 0.1% sodium dodecyl sulfate at 55 °C and autoradiographed with Kodak XAR film at −80 °C for 12–24 h or stored on phosphor screens for 6–8 h. To correct for differences in RNA loading, the filters were rehybridized with a radiolabeled 18 S oligonucleotide (5′-ACGGTATCTGATCGTCTTCGAACC-3′) probe. The filters were scanned and radioactivity was measured on a PhosphorImager running ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Subconfluent HUVEC were not stimulated (control) or stimulated with VEGF for 2 h. The cells were subsequently lysed, and nuclei were isolated as described by Perrella et al. (25Perrella M.A. Yoshizumi M. Fen Z. Tsai J.-C. Hsieh C.-M. Kourembanas S. Lee M.-E. J. Biol. Chem. 1994; 269: 14595-14600Abstract Full Text PDF PubMed Google Scholar). Nuclear suspension (200 μl) was incubated with 0.5 mm each of CTP, ATP, and GTP and with 125 μCi of 32P-labeled UTP (3,000 Ci/mmol, NEN Life Science Products). The samples were extracted with phenol/chloroform, precipitated, and resuspended at equal counts/min/ml in hybridization buffer (1.4 × 106 cpm/ml). Radiolabeled run-on transcripts were hybridized (40 °C) to denatured probes (HB-EGF and 18 S, 1 μg each) dot-blotted on nitrocellulose filters. The filters were then washed and scanned on a PhosphorImager running ImageQuant software (25Perrella M.A. Yoshizumi M. Fen Z. Tsai J.-C. Hsieh C.-M. Kourembanas S. Lee M.-E. J. Biol. Chem. 1994; 269: 14595-14600Abstract Full Text PDF PubMed Google Scholar). Subconfluent HUVEC were treated with VEGF or PBS for 2 h as described under “Cell Culture and mRNA Isolation.” Cells were lysed in radioimmune precipitation buffer (50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm phenylmethyl sulfonyl fluoride, 1 mm NaF, 1 mmNa3VO4, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) and disrupted by passage through a 22-gauge needle five times. Cell debris was removed by centrifugation at 14,000 ×g, and the supernatant was used as cell lysate for Western blotting. A Bradford-based assay kit (Bio-Rad) was used to measure protein concentrations. Equal amounts of protein from each time point were fractionated by 15% Tricine/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an Immobilon-P membrane. Western blotting was carried out using an ECL kit as described by the manufacturer (Amersham Life Science). HB-EGF was detected by chemiluminescence with a polyclonal primary antibody to HB-EGF (0.1 μg/ml, R&D Systems) and a horseradish peroxidase-conjugated secondary antibody to goat Ig. The film was scanned into Adobe Photoshop 3.0, and the signal density was measured with the NIH Image software. Cell migration was measured in a 48-well micro-Boyden chamber apparatus (Neuroprobe, Cabin John, MD) as described (33Bornfeldt K.E. Raines E.W. Nakano T. Graves L.M. Krebs E.G. Ross R. J. Clin. Invest. 1994; 93: 1266-1274Crossref PubMed Scopus (381) Google Scholar). Confluent HASMC were made quiescent by incubation in Dulbecco's modified Eagle's medium with 0.4% fetal calf serum for 72 h before the assay. HB-EGF and PBS (control) were diluted in Dulbecco's modified Eagle's medium with 0.25% bovine serum albumin and loaded into the lower wells of the Boyden chamber in triplicate. The wells were subsequently covered with a polyvinylpyrrolidone-free filter with 8-μm pores (Nucleopore, Palo Alto, CA) coated with type I collagen (Vitrogen, Collagen Corp., Palo Alto, CA). Cells were washed four times in PBS and trypsinized (0.01% trypsin, 0.11 mmEDTA) for the minimum period of time required to obtain a mononuclear suspension. The cells were washed twice in Dulbecco's modified Eagle's medium, 0.25% bovine serum albumin and resuspended at a density of 1 million cells/ml. Cells (50,000 cells in 50 μl) were loaded into the upper wells of the Boyden chamber. The chambers were incubated for 4 h at 37 °C in an atmosphere of 95% O2, 5% CO2. At the end of the incubation, cells that had attached to the filter were fixed and stained in Dif Quick (American Hospital Supply, McGaw Park, IL). Cells that had migrated to the lower side of the filter were counted under a microscope at 20× magnification. Chemotaxis was calculated as the difference between the number of cells that had migrated in the presence of the chemoattractant and the number that had migrated in its absence. Primary cultured rat aortic smooth muscle cells (RASMC) (25Perrella M.A. Yoshizumi M. Fen Z. Tsai J.-C. Hsieh C.-M. Kourembanas S. Lee M.-E. J. Biol. Chem. 1994; 269: 14595-14600Abstract Full Text PDF PubMed Google Scholar) were plated on 24-well plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and 25 mm Hepes (pH 7.4) and incubated for 24 h. Cells were made quiescent by incubation in Dulbecco's modified Eagle's medium plus 0.4% calf serum for 72 h before the experiments were performed. HUVEC were grown as described, and placed in M199 supplemented with 2% fetal calf serum for 1 h before stimulation with 20 ng/ml VEGF. After 2 h of VEGF treatment, the conditioned medium was harvested from HUVEC and used to stimulate quiescent RASMC (final proportion of 33% conditioned medium and 67% RASMC medium). Prior to its addition to RASMC, HUVEC conditioned medium was treated with either no antibody, anti-human PDGF polyclonal antibody (8 μg/ml, R&D Systems) or normal goat IgG (8 μg/ml, R&D Systems) for 1 h at 25 °C. Conditioned medium was applied to the RASMC for 24 h. During the last 2 h of this incubation period, the cells were labeled with [3H]thymidine (NEN Life Science Products) at 1 μCi/ml. Labeled cells were washed with phosphate-buffered saline, fixed in cold 10% trichloroacetic acid, and washed with 95% ethanol. Incorporated [3H]thymidine was extracted in 0.2 n NaOH and measured in a liquid scintillation counter. Northern blot analysis was performed with total RNA from HUVEC, and blots were hybridized with a human HB-EGF cDNA probe. In response to stimulation with 10 ng/ml VEGF (Fig. 1 A), HB-EGF mRNA levels increased rapidly to a maximum at 2 h. After 6 h, the HB-EGF mRNA level returned to base line. VEGF also induced HB-EGF mRNA in HUVEC in a dose-dependent manner (Fig. 1 B). To confirm that this response was not limited to HUVEC, we performed the same experiment in endothelial cells from another source, HCAEC. As in HUVEC, HB-EGF mRNA was induced by 10 ng/ml VEGF in HCAEC (data not shown). To determine whether induction of HB-EGF mRNA required protein synthesis de novo, we treated HUVEC with the protein synthesis inhibitor anisomycin (100 μm) for 1 h before adding VEGF. This dose of anisomycin completely inhibited leucine uptake in HUVEC (data not shown). Anisomycin did not prevent the induction of HB-EGF mRNA by VEGF (Fig. 2), suggesting that this process does not require new protein synthesis. We measured mRNA stability and rate of transcription to elucidate the mechanism by which VEGF up-regulates HB-EGF mRNA. The half-life of HB-EGF mRNA was determined in the presence of the transcription inhibitor actinomycin D. HUVEC were stimulated with VEGF (10 ng/ml) or vehicle (PBS) for 2 h. HB-EGF mRNA was then measured 0, 1, and 2 h after administration of actinomycin D (5 μg/ml). The half-life of HB-EGF mRNA at base line was approximately 55 min (Fig. 3 A); it was not altered by exposure to VEGF. Nuclear run-on experiments were then performed to assess the rate of transcription. VEGF increased transcription of the HB-EGF gene in HUVEC by 4.9-fold (Fig. 3 B). These experiments demonstrate that the increase in HB-EGF mRNA levels after VEGF stimulation was the result of an increase in HB-EGF gene transcription. To determine whether the increase in HB-EGF mRNA in HUVEC was associated with an increase in HB-EGF protein, we performed Western blot analysis with a polyclonal antibody specific for recombinant human HB-EGF. The level of HB-EGF protein increased in HUVEC stimulated with VEGF for 2 h (Fig. 4 A). This observation demonstrates that the effect of VEGF on HB-EGF mRNA in HUVEC is associated with an increase in HB-EGF protein. Because the maturation of new blood vessels requires cell migration and investment of capillary tubules with pericytes and smooth muscle cells, we measured the chemotactic effect of HB-EGF on vascular smooth muscle cells. HB-EGF induced a significant increase in HASMC migration (Fig. 4 B). To see if VEGF increased the expression of other factors known to affect angiogenesis (34Pepper M.S. Vassalli J.-D. Orci L. Montesano R. Exp. Cell Res. 1993; 204: 356-363Crossref PubMed Scopus (311) Google Scholar, 35Alberts G.F. Peifley K.A. Johns A. Kleha J.F. Winkles J.A. J. Biol. Chem. 1994; 269: 10112-10118Abstract Full Text PDF PubMed Google Scholar), we studied its effect on the mRNA levels of PDGF-BB, ET1, and TGF-β1 in HUVEC. Northern blot analysis showed that PDGF-BB mRNA increased as early as 2 h, and its induction reached 3-fold after 5 h of VEGF stimulation. TGF-β1 and ET1 mRNA levels were not altered by VEGF (Fig. 5). To further delineate the role of endothelium-derived growth factors on vascular smooth muscle cells, studies were performed using conditioned medium from HUVEC stimulated with VEGF. Compared with medium from vehicle-treated cells, medium from VEGF-stimulated HUVEC produced a significant increase in RASMC DNA synthesis (Fig. 6). Incubating the conditioned medium with a neutralizing antibody to PDGF prevented this increase in DNA synthesis. When control antiserum, normal goat IgG, was used in the same concentration as the PDGF antibody, the induction of DNA synthesis was not abrogated. A series of sequential events promote angiogenesis. These include (a) degradation of the vascular basement membrane and the interstitial matrix by endothelial cells, (b) endothelial cell migration and proliferation, (c) formation of new capillary tubes, and (d) formation of new basement membrane with investment of the tubule by pericytes and smooth muscle cells (1Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6087) Google Scholar,36Folkman J. N. Engl. J. Med. 1995; 333: 1757-1763Crossref PubMed Scopus (2249) Google Scholar). It has been established that VEGF is an endothelial cell-specific, multifunctional growth factor that plays a major role in the initiation of angiogenesis by acting directly as a mitogenic and a chemotactic factor, and indirectly by promoting remodeling of the extracellular matrix (16Dvorak H.F. Brown L.F. Detmar M. Dvorak A.M. Am. J. Pathol. 1995; 146: 1029-1039PubMed Google Scholar, 37Freeman M.R. Schneck F.X. Gagnon M.L. Corless C. Soker S. Niknejad K. Peoples G.E. Klagsbrun M. Cancer Res. 1995; 55: 4140-4145PubMed Google Scholar, 38D'Amore P.A. Semin. Cancer Biol. 1992; 3: 49-56PubMed Google Scholar, 39Nehls V. Schuchardt E. Drenckhahn D. Microvasc. Res. 1994; 48: 349-363Crossref PubMed Scopus (95) Google Scholar, 40Mandriota S.J. Seghezzi G. Vassalli J.-D. Ferrara N. Wasi S. Mazzieri R. Mignatti P. Pepper M.S. J. Biol. Chem. 1995; 270: 9709-9716Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Although pericytes and smooth muscle cells are essential in the late phase of angiogenesis, there is no clear explanation of how these cell types are recruited into the process by angiogenic factors such as VEGF (39Nehls V. Schuchardt E. Drenckhahn D. Microvasc. Res. 1994; 48: 349-363Crossref PubMed Scopus (95) Google Scholar, 41Nomura M. Yamagishi S. Harada S. Hayashi Y. Yamashima T. Yamashita J. Yamamoto H. J. Biol. Chem. 1995; 270: 28316-28324Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 42Nicosia R.F. Villaschi S. Lab. Invest. 1995; 73: 658-666PubMed Google Scholar, 43Orlidge A. D'Amore P.A. J. Cell Biol. 1987; 105: 1455-1462Crossref PubMed Scopus (526) Google Scholar). Suri et al. (44Suri C. Jones P.F. Patan S. Bartunkova S. Maisonpierre P.C. Davis S. Sato T.N. Yancopoulos G.D. Cell. 1996; 87: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (2394) Google Scholar) have reported recently that mice lacking the TIE2 tyrosine kinase ligand angiopoietin-1 exhibit abnormal vascular architecture, where the principal defect is a failure to recruit smooth muscle and pericyte precursors. Additionally, Lindahlet al. (45Lindahl P. Johansson B.R. Levéen P. Betsholtz C. Science. 1997; 277: 242-245Crossref PubMed Scopus (1746) Google Scholar) have shown that in PDGF-BB-deficient mouse embryos, endothelial cells of sprouting capillaries fail to attract pericyte progenitor cells. In a recent review article, Folkman and D'Amore (46Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar) have proposed a model of new vessel formation in which HB-EGF and PDGF-BB act as recruiting signals for mesenchymal cells during the late phase of angiogenesis. We demonstrate here in endothelial cells that VEGF induces HB-EGF and PDGF-BB, which have mitogenic and chemotactic effects on pericytes and smooth muscle cells. This demonstration links VEGF to early (endothelial cell recruitment) and late (smooth muscle cell migration and investment) stages of angiogenesis. Since the transcriptional mechanisms downstream of VEGF have not been described, this represents a first step toward defining those mechanisms. The induction of VEGF and its receptors (flk-1 and flt) in human atherosclerotic lesions and in animal models of arterial injury has also been described (37Freeman M.R. Schneck F.X. Gagnon M.L. Corless C. Soker S. Niknejad K. Peoples G.E. Klagsbrun M. Cancer Res. 1995; 55: 4140-4145PubMed Google Scholar, 47Kuzuya M. Satake S. Esaki T. Yamada K. Hayashi T. Naito M. Asai K. Iguchi A. J. Cell. Physiol. 1995; 164: 658-667Crossref PubMed Scopus (72) Google Scholar, 48Ruef J. Hu Z.Y. Yin L.-Y. Wu Y. Hanson S.R. Kelly A.B. Harker L.A. Rao G.N. Runge M.S. Patterson C. Circ. Res. 1997; 81: 24-33Crossref PubMed Scopus (155) Google Scholar). Lazarous et al. (49Lazarous D.F. Shou M. Scheinowitz M. Hodge E. Thirumurti V. Kitsiou A.N. Stiber J.A. Lobo A.D. Hunsberger S. Guetta E. Epstein S.E. Unger E.F. Circulation. 1996; 94: 1074-1082Crossref PubMed Scopus (355) Google Scholar) reported recently that VEGF given postoperatively in a canine model of left circumflex coronary artery occlusion and iliofemoral artery denudation induced further accumulation of neointima without inducing collateral blood vessel formation in the coronary circulation. These findings imply that VEGF may play a paracrine role in exacerbating neointima formation and vessel occlusion, both by inducing neovascularization in lesions and by stimulating the release of smooth muscle cell mitogens and chemoattractants, as suggested by Li et al. (50Li J. Perrella M.A. Tsai J.-C. Yet S.-F. Hsieh C.-M. Yoshizumi M. Patterson C. Endege W.O. Zhou F. Lee M.-E. J. Biol. Chem. 1995; 270: 308-312Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Our demonstration that VEGF induces the vascular smooth muscle cell mitogenic and chemotactic growth factors HB-EGF and PDGF-BB elucidates a mechanism by which this atherogenic process occurs. We have demonstrated that VEGF induces expression of angiogenic growth factors in vascular endothelial cells. VEGF up-regulates HB-EGF and PDGF-BB but not TGF-β1 or ET1. Induction occurs both in HUVEC and HCAEC (data not shown), suggesting that VEGF has this effect in vascular endothelial cells of various origins. HB-EGF and PDGF-BB are both chemotactic agents for vascular smooth muscle cells, and conditioned medium from endothelial cells treated with VEGF produces an increase in DNA synthesis in vascular smooth muscle cells. This growth-promoting effect of conditioned medium can be prevented by a neutralizing antibody to PDGF. Taken together, our data suggest that HB-EGF and PDGF-BB provide a critical endothelial cell-derived signal for the recruitment of pericytes and smooth muscle cells during angiogenesis, which is consistent with the model proposed by Folkman and D'Amore (46Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar). These findings may provide important insight into the process of new blood vessel formation and maturation. We thank Bonna Ith for technical assistance and Thomas McVarish for editorial assistance." @default.
• W2029452873 created "2016-06-24" @default.
• W2029452873 creator A5021501916 @default.
• W2029452873 creator A5057818292 @default.
• W2029452873 creator A5061886627 @default.
• W2029452873 creator A5070336079 @default.
• W2029452873 creator A5077119492 @default.
• W2029452873 creator A5077459772 @default.
• W2029452873 creator A5080657847 @default.
• W2029452873 creator A5081279505 @default.
• W2029452873 creator A5089346351 @default.
• W2029452873 date "1998-02-01" @default.
• W2029452873 modified "2023-09-28" @default.
• W2029452873 title "Vascular Endothelial Growth Factor Induces Heparin-binding Epidermal Growth Factor-like Growth Factor in Vascular Endothelial Cells" @default.
• W2029452873 cites W1514034386 @default.
• W2029452873 cites W1522794224 @default.
• W2029452873 cites W1524164104 @default.
• W2029452873 cites W1559595253 @default.
• W2029452873 cites W1563073913 @default.
• W2029452873 cites W1565444604 @default.
• W2029452873 cites W1957871175 @default.
• W2029452873 cites W1964068703 @default.
• W2029452873 cites W1967365012 @default.
• W2029452873 cites W1970083280 @default.
• W2029452873 cites W1973418567 @default.
• W2029452873 cites W1978638754 @default.
• W2029452873 cites W1983763042 @default.
• W2029452873 cites W1985804036 @default.
• W2029452873 cites W1986559037 @default.
• W2029452873 cites W1988655450 @default.
• W2029452873 cites W1993593600 @default.
• W2029452873 cites W1999933929 @default.
• W2029452873 cites W2012390831 @default.
• W2029452873 cites W2015344488 @default.
• W2029452873 cites W2016904444 @default.
• W2029452873 cites W2024681203 @default.
• W2029452873 cites W2026325139 @default.
• W2029452873 cites W2029320522 @default.
• W2029452873 cites W2037143824 @default.
• W2029452873 cites W2038086562 @default.
• W2029452873 cites W2038750685 @default.
• W2029452873 cites W2045247659 @default.
• W2029452873 cites W2046516822 @default.
• W2029452873 cites W2047561204 @default.
• W2029452873 cites W2056242981 @default.
• W2029452873 cites W2057918209 @default.
• W2029452873 cites W2066888615 @default.
• W2029452873 cites W2081399863 @default.
• W2029452873 cites W2082157250 @default.
• W2029452873 cites W2104231986 @default.
• W2029452873 cites W2105255713 @default.
• W2029452873 cites W2108145145 @default.
• W2029452873 cites W2108337314 @default.
• W2029452873 cites W2118678876 @default.
• W2029452873 cites W2122226633 @default.
• W2029452873 cites W2127150853 @default.
• W2029452873 cites W2132475459 @default.
• W2029452873 cites W2141610498 @default.
• W2029452873 cites W2324677651 @default.
• W2029452873 cites W4376595434 @default.
• W2029452873 doi "https://doi.org/10.1074/jbc.273.8.4400" @default.
• W2029452873 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9468491" @default.
• W2029452873 hasPublicationYear "1998" @default.
• W2029452873 type Work @default.
• W2029452873 sameAs 2029452873 @default.
• W2029452873 citedByCount "75" @default.
• W2029452873 countsByYear W20294528732012 @default.
• W2029452873 countsByYear W20294528732013 @default.
• W2029452873 countsByYear W20294528732014 @default.
• W2029452873 countsByYear W20294528732015 @default.
• W2029452873 countsByYear W20294528732016 @default.
• W2029452873 countsByYear W20294528732017 @default.
• W2029452873 countsByYear W20294528732018 @default.
• W2029452873 countsByYear W20294528732019 @default.
• W2029452873 countsByYear W20294528732022 @default.
• W2029452873 crossrefType "journal-article" @default.
• W2029452873 hasAuthorship W2029452873A5021501916 @default.
• W2029452873 hasAuthorship W2029452873A5057818292 @default.
• W2029452873 hasAuthorship W2029452873A5061886627 @default.
• W2029452873 hasAuthorship W2029452873A5070336079 @default.
• W2029452873 hasAuthorship W2029452873A5077119492 @default.
• W2029452873 hasAuthorship W2029452873A5077459772 @default.
• W2029452873 hasAuthorship W2029452873A5080657847 @default.
• W2029452873 hasAuthorship W2029452873A5081279505 @default.
• W2029452873 hasAuthorship W2029452873A5089346351 @default.
• W2029452873 hasBestOaLocation W20294528731 @default.
• W2029452873 hasConcept C103041312 @default.
• W2029452873 hasConcept C130287650 @default.
• W2029452873 hasConcept C146285616 @default.
• W2029452873 hasConcept C15215337 @default.
• W2029452873 hasConcept C167734588 @default.
• W2029452873 hasConcept C170493617 @default.
• W2029452873 hasConcept C185592680 @default.
• W2029452873 hasConcept C2775960820 @default.
• W2029452873 hasConcept C2776362946 @default.
• W2029452873 hasConcept C2777025900 @default.
• W2029452873 hasConcept C502942594 @default.
• W2029452873 hasConcept C55493867 @default.

• next