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Hypoxia and Vascular Endothelial Growth Factor Selectively Up-regulate Angiopoietin-2 in Bovine Microvascular Endothelial Cells

1999; Elsevier BV; Volume: 274; Issue: 22 Linguagem: Inglês

10.1074/jbc.274.22.15732

ISSN

1083-351X

Autores

Hideyasu Oh, Hitoshi Takagi, Kiyoshi Suzuma, Atsushi Otani, Miyo Matsumura, Yoshihito Honda,

Tópico(s)

Retinal Diseases and Treatments

Resumo

Recent studies have shown that the angiopoietin-Tie2 system is a predominant regulator of vascular integrity. In this study, we investigated the effect of two known angiogenic stimuli, hypoxia and vascular endothelial growth factor (VEGF), on these molecules. VEGF induced both a time- and concentration-dependent increase in angiopoietin-2 (Ang2) mRNA expression in bovine microvascular endothelial cells. This up-regulation was derived primarily from an increased transcription rate as evidenced by nuclear run-on assay and mRNA decay study. The increased Ang2 expression upon VEGF treatment was almost totally abolished by inhibition of tyrosine kinase or mitogen-activated protein kinase and partially by suppression of protein kinase C. Hypoxia also directly increased Ang2 mRNA expression. In contrast, Ang1 and Tie2 responded to neither of these stimuli. The enhanced Ang2 expression following VEGF stimulation and hypoxia was accompanied by de novo protein synthesis as detected by immunoprecipitation. In a mouse model of ischemia-induced retinal neovascularization, Ang2 mRNA was up-regulated in the ischemic inner retinal layer, and remarkable expression was observed in neovascular vessels. These data suggest that both hypoxia- and VEGF-induced neovascularization might be facilitated by selective induction of Ang2, which deteriorates the integrity of preexisting vasculature. Recent studies have shown that the angiopoietin-Tie2 system is a predominant regulator of vascular integrity. In this study, we investigated the effect of two known angiogenic stimuli, hypoxia and vascular endothelial growth factor (VEGF), on these molecules. VEGF induced both a time- and concentration-dependent increase in angiopoietin-2 (Ang2) mRNA expression in bovine microvascular endothelial cells. This up-regulation was derived primarily from an increased transcription rate as evidenced by nuclear run-on assay and mRNA decay study. The increased Ang2 expression upon VEGF treatment was almost totally abolished by inhibition of tyrosine kinase or mitogen-activated protein kinase and partially by suppression of protein kinase C. Hypoxia also directly increased Ang2 mRNA expression. In contrast, Ang1 and Tie2 responded to neither of these stimuli. The enhanced Ang2 expression following VEGF stimulation and hypoxia was accompanied by de novo protein synthesis as detected by immunoprecipitation. In a mouse model of ischemia-induced retinal neovascularization, Ang2 mRNA was up-regulated in the ischemic inner retinal layer, and remarkable expression was observed in neovascular vessels. These data suggest that both hypoxia- and VEGF-induced neovascularization might be facilitated by selective induction of Ang2, which deteriorates the integrity of preexisting vasculature. The formation of blood vessels requires a series of events, including differentiation of endothelial cells, tube formation, and vascular maturation (1Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (5991) Google Scholar). Two processes termed vasculogenesis and angiogenesis take place during the formation of a mature vascular network (2Risau W. Flamme I. Annu. Rev. Cell Dev. Biol. 1995; 11: 73-91Crossref PubMed Scopus (1320) Google Scholar, 3Folkman J. D'Amore P.A. Cell. 1996; 87: 1153-1155Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar, 4Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4755) Google Scholar). Previous studies have revealed some of the molecular mechanisms involved, and two families of largely endothelial cell-specific receptor tyrosine kinases are known to play crucial roles in these processes. The vascular endothelial growth factor receptor (VEGFR) 1The abbreviations used are: VEGFR, vascular endothelial growth factor receptor; VEGF, vascular endothelial growth factor; Ang1, angiopoietin-1; Ang2, angiopoietin-2; BRECs, bovine retinal endothelial cells; BAECs, bovine aortic endothelial cells; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PCR, polymerase chain reaction1The abbreviations used are: VEGFR, vascular endothelial growth factor receptor; VEGF, vascular endothelial growth factor; Ang1, angiopoietin-1; Ang2, angiopoietin-2; BRECs, bovine retinal endothelial cells; BAECs, bovine aortic endothelial cells; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PCR, polymerase chain reaction family is composed of Flt-1 (f ms-liketyrosine kinase-1; VEGFR-1) (5Shibuya M. Yamaguchi S. Yamane A. Ikeda T. Tojo A. Matsushime H. Sato M. Oncogene. 1990; 5: 519-524PubMed Google Scholar), Flk-1/KDR (fetal liverkinase/kinase domain-containingreceptor; VEGFR-2) (6Terman B.I. Carrion M.E. Kovacs E. Rasmussen B.A. Eddy R.L. Shows T.B. Oncogene. 1991; 6: 1677-1683PubMed Google Scholar), and Flt-4 (f ms-liketyrosine kinase-4; VEGFR-3) (7Pajusola K. Aprelikova O. Korhonen J. Kaipainen A. Pertovaara L. Alitalo R. Alitalo K. Cancer Res. 1992; 52: 5738-5743PubMed Google Scholar). Targeted gene disruption for Flk-1 in embryonic mice leads to loss of endothelial cells and embryonic death at embryonic day 8.5 and thus indicates the requisite role of this receptor in differentiation of hemangioblasts into endothelial cells (8Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3308) Google Scholar). Mice lacking Flt-1, despite the presence of normal hematopoietic precursors and endothelial cells, also die at embryonic day 8.5, and the absence of tube formation strongly suggests a major role for this receptor in endothelial cell-cell or cell-matrix interactions (9Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2185) Google Scholar). VEGF, a potent angiogenic factor and the common ligand for these two receptors, is distinctive in that its mitogenic effect is highly specific for endothelial cells (10Gospodarowicz D. Abraham J.A. Schilling J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7311-7315Crossref PubMed Scopus (552) Google Scholar) and its expression is up-regulated by hypoglycemia (11Stein I. Neeman M. Shweiki D. Itin A. Keshet E. Mol. Cell. Biol. 1995; 15: 5363-5368Crossref PubMed Scopus (415) Google Scholar) and by hypoxia (12Shweiki D. Itin A. Soffer D. Keshet E. 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This is further substantiated by experiments in which inhibition of either VEGF or Flk-1/KDR resulted in suppression of pathologic angiogenesis (17Millauer B. Shawver L.K. Plate K.H. Risau W. Ullrich A. Nature. 1994; 367: 576-579Crossref PubMed Scopus (1158) Google Scholar, 18Kim K.J. Li B. Winer J. Armanini M. Gillett N. Phillips H.S. Ferrara N. Nature. 1993; 362: 841-844Crossref PubMed Scopus (3311) Google Scholar), which validates the hypothesis that the VEGF signal transduction system is a viable target for antiangiogenic therapeutic intervention. Another family of receptor tyrosine kinases, designated the Tie (tyrosine kinase that containsimmunoglobulin-like loops and epidermal growth factor-similar domains) family, has also been studied and found to be expressed primarily on cells of endothelial lineage (19Dumont D.J. Yamaguchi T.P. Conlon R.A. Rossant J. Breitman M.L. Oncogene. 1992; 7: 1471-1480PubMed Google Scholar, 20Partanen J. Armstrong E. Makela T.P. Korhonen J. Sandberg M. Renkonen R. Knuutila S. Huebner K. Alitalo K. Mol. Cell. Biol. 1992; 12: 1698-1707Crossref PubMed Scopus (292) Google Scholar). Recent studies revealed that mice lacking either of these receptors, Tie1 or Tie2, die later than do those lacking VEGF or VEGFRs, indicating that this family exerts its effect in the later stages of embryonic blood vessel formation (21Dumont D.J. Gradwohl G. Fong G.H. Puri M.C. Gertsenstein M. Auerbach A. Breitman M.L. Genes Dev. 1994; 8: 1897-1909Crossref PubMed Scopus (807) Google Scholar, 22Sato T.N. Tozawa Y. Deutsch U. Wolburg-Buchholz K. Fujiwara Y. Gendron-Maguire M. Gridley T. Wolburg H. Risau W. Qin Y. Nature. 1995; 376: 70-74Crossref PubMed Scopus (1488) Google Scholar). The Tie1 signal has been implicated in the control of fluid exchange and hemodynamic stress resistance (22Sato T.N. Tozawa Y. Deutsch U. Wolburg-Buchholz K. Fujiwara Y. Gendron-Maguire M. Gridley T. Wolburg H. Risau W. Qin Y. Nature. 1995; 376: 70-74Crossref PubMed Scopus (1488) Google Scholar, 23Puri M.C. Rossant J. Alitalo K. Bernstein A. Partanen J. EMBO J. 1995; 14: 5884-5891Crossref PubMed Scopus (411) Google Scholar), even though its ligand remains unidentified. In contrast, Tie2 appears to regulate the capability of endothelial cells to recruit stromal cells around the endothelial tubes and stabilizes vascular integrity (24Vikkula M. Boon L.M. Carraway K.R. Calvert J.T. Diamonti A.J. Goumnerov B. Pasyk K.A. Marchuk D.A. Warman M.L. Cantley L.C. Mulliken J.B. Olsen B.R. Cell. 1996; 87: 1181-1190Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). The phenotype of Tie2 knockout mice is distinct from that of mice lacking VEGFRs. Endothelial cells are detected in normal numbers, and tube formation occurs; but the distinction between large and small vessels is obscure, and encapsulation by periendothelial cells is absent (22Sato T.N. Tozawa Y. Deutsch U. Wolburg-Buchholz K. Fujiwara Y. Gendron-Maguire M. Gridley T. Wolburg H. Risau W. Qin Y. Nature. 1995; 376: 70-74Crossref PubMed Scopus (1488) Google Scholar). Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are newly identified ligands for the Tie2 receptor, and both bind to Tie2 receptors with similar affinity (25Suri 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 (2352) Google Scholar, 26Maisonpierre P.C. Suri C. Jones P.F. Bartunkova S. Wiegand S.J. Radziejewski C. Compton D. McClain J. Aldrich T.H. Papadopoulos N. Daly T.J. Davis S. Sato T.N. Yancopoulos G.D. Science. 1997; 277: 55-60Crossref PubMed Scopus (2899) Google Scholar, 27Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1660) Google Scholar). Ang1 induces autophosphorylation of Tie2 and has a remarkable chemotactic effect on endothelial cells, whereas Ang2 competitively inhibits this effect (26Maisonpierre P.C. Suri C. Jones P.F. Bartunkova S. Wiegand S.J. Radziejewski C. Compton D. McClain J. Aldrich T.H. Papadopoulos N. Daly T.J. Davis S. Sato T.N. Yancopoulos G.D. Science. 1997; 277: 55-60Crossref PubMed Scopus (2899) Google Scholar,28Witzenbichler B. Maisonpierre P.C. Jones P. Yancopoulos G.D. Isner J.M. J. Biol. Chem. 1998; 273: 18514-18521Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Moreover, Ang2-overexpressing transgenic mice mimic the phenotype of knockout mice of Ang1 and Tie2, suggesting that Ang2 is a natural antagonist for Tie2 (26Maisonpierre P.C. Suri C. Jones P.F. Bartunkova S. Wiegand S.J. Radziejewski C. Compton D. McClain J. Aldrich T.H. Papadopoulos N. Daly T.J. Davis S. Sato T.N. Yancopoulos G.D. Science. 1997; 277: 55-60Crossref PubMed Scopus (2899) Google Scholar). Ang1 has been reported to be down-regulated by treatment with serum or several cytokines in human lung fibroblasts (29Ristimaki A. Narko K. Enholm B. Joukov V. Alitalo K. J. Biol. Chem. 1998; 273: 8413-8418Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 30Enholm B. Paavonen K. Ristimaki A. Kumar V. Gunji Y. Klefstrom J. Kivinen L. Laiho M. Olofsson B. Joukov V. Eriksson U. Alitalo K. Oncogene. 1997; 14: 2475-2483Crossref PubMed Scopus (384) Google Scholar) and also by hypoxia in rat glioma cells (30Enholm B. Paavonen K. Ristimaki A. Kumar V. Gunji Y. Klefstrom J. Kivinen L. Laiho M. Olofsson B. Joukov V. Eriksson U. Alitalo K. Oncogene. 1997; 14: 2475-2483Crossref PubMed Scopus (384) Google Scholar). Regulation of Ang2 and Tie2 expression, however, has not yet been well characterized. This study addresses that both hypoxia and VEGF selectively up-regulate Ang2 expression in bovine endothelial cells despite the stable expression of Ang1 and Tie2 and that Ang2 expression is up-regulatedin vivo in a mouse model of ischemia-induced retinal angiogenesis. Primary cultures of bovine retinal endothelial cells (BRECs) were isolated by homogenization and a series of filtration steps as described previously (31King G.L. Goodman A.D. Buzney S. Moses A. Kahn C.R. J. Clin. Invest. 1985; 75: 1028-1036Crossref PubMed Scopus (398) Google Scholar). Cells were grown on fibronectin (Sigma)-coated dishes (Iwaki Glass, Tokyo, Japan) containing Dulbecco's modified Eagle's medium with 5.5 mmglucose, 10% platelet-derived horse serum (Wheaton, Pipersville, PA), 50 mg/ml heparin, and 50 units/ml endothelial cell growth factor (Roche Molecular Biochemicals). Bovine aortic endothelial cells (BAECs) were also isolated from bovine aorta and cultured in Dulbecco's modified Eagle's medium containing 5% calf serum and 10% platelet-derived horse serum. Cells were cultured in 5% CO2 at 37 °C, and media were changed every 3 days. Cells were characterized for their endothelial homogeneity by immunoreactivity for factor VIII antigen and remained morphologically unchanged under these conditions, as confirmed by light microscopy. Only cells from passages 4 to 7 were used for the experiments. For the kinetic studies of VEGF treatment, cells were incubated with VEGF (0–125 ng/ml; Genzyme, Cambridge, MA) for the indicated time points. To determine the roles of tyrosine kinase, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) in VEGF-induced Ang2 mRNA expression, BRECs were pretreated with genistein (40 μm; LC Laboratories, Boston, MA), GF 109203X (5 μm; LC Laboratories), and PD 098059 (25 μm; Upstate Biotechnology, Inc., Lake Placid, NY), respectively, followed by stimulation with 25 ng/ml VEGF. These drug levels of the inhibitors have been shown to block each target selectively and effectively in endothelial cells (32Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (522) Google Scholar, 33Rousseau S. Houle F. Landry J. Huot J. Oncogene. 1997; 15: 2169-2177Crossref PubMed Scopus (712) Google Scholar). cDNA templates for PCR were synthesized by reverse transcriptase (first strand kit, Invitrogen, Carlsbad, CA) from human umbilical vein endothelial cells (Kurabo, Osaka, Japan) according to the method recommended by the manufacturer. For Ang1, Ang2, and Tie2 cDNAs, a standard PCR was performed (PCR optimizer kit, Invitrogen) using 5′-AGA ACC ACA CGG CTA CCA TGC T-3′ (Ang1 sense primer corresponding to nucleotides +671 to +692), 5′-TGT GTC CAT CAG CTC CAG TTG C-3′ (Ang1 antisense primer), 5′-AGC TGT GAT CTT GTC TTG GC-3′ (Ang2 sense primer corresponding to nucleotides +377 to +396), 5′-GTT CAA GTC TCG TGG TCT GA-3′ (Ang2 antisense primer corresponding to nucleotides +802 to +821), 5′-GCC TTA ATG AAC CAG CAC CAG G-3′ (Tie2 sense primer corresponding to nucleotides +335 to +356), and 5′-ACT TCT GGG CTT CAC ATC TCC G-3′ (Tie2 antisense primer corresponding to nucleotides +773 to +794). These cDNAs were cloned by the reverse transcription-PCR method recommended by the manufacturer. The PCR products were then subcloned into a vector (pCRII, Invitrogen) and sequenced in their entirety, and comparison with the published human sequences revealed complete sequence identity. These cDNA probes were used for hybridization. Total RNA was isolated using acid-guanidium thiocyanate, and Northern blot analysis was performed as described previously (34Otani A. Takagi H. Suzuma K. Honda Y. Circ. Res. 1998; 82: 619-628Crossref PubMed Scopus (258) Google Scholar). Total RNA (20 μg) was electrophoresed through 1% formaldehyde-agarose gels and then transferred to a nylon membrane (BNRG3R, Pall BioSupport Division, East Hills, NY). Radioactive cDNA probes were generated by use of labeling kits (Megaprime DNA labeling systems, Amersham International, Buckinghamshire, United Kingdom) and [32P]dCTP (NEG-513, Bio-Rad). After ultraviolet cross-linking using a UV cross-linker (FS-1500, Funakoshi, Tokyo), blots were prehybridized; hybridized with the indicated cDNA probe; and washed in 0.5% SSC and 5% SDS at 65 °C, with changing of the solution four times over 1 h in a rotating hybridization oven (Taitec, Saitama, Japan). All signals were scanned and analyzed utilizing a densitometer (BAS-2000II, Fuji Film, Tokyo), and lane loading differences were normalized by means of 36B4 control cDNA (35Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4675) Google Scholar) (generously provided by Dr. Lloyd P. Aiello). BRECs were treated with vehicle or VEGF (25 ng/ml) for 2 h. The cells were lysed in a solubilization buffer (10 mm Tris-HCl, 10 mmNaCl, 3 mm MgCl2, and 0.5% Nonidet P-40), and the nuclei were isolated. ATP, CTP, and GTP (50 mm each) and 3.7 MBq of [32P]UTP (Amersham International) were added to the nuclear suspension (100 μl) and incubated for 30 min. The samples were extracted with phenol/chloroform and precipitated. cDNA probes (Ang2 and 36B4, 10 μg each) were then slot-blotted onto nitrocellulose filters (Schleicher & Schüll, Dassel, Germany) and hybridized with the precipitated samples of equal counts/min/ml in hybridization buffer at 45 °C for 48 h. The filters were washed, and the radioactivity was measured using the BAS-2000II densitometer. The level of Ang2 mRNA was normalized to that of 36B4 mRNA. BRECs were treated with 25 ng/ml VEGF for 2 h prior to mRNA stability experiments. Thereafter, half of the plates were returned to Dulbecco's modified Eagle's medium without VEGF, and actinomycin D (10 μg/ml; Wako, Osaka) was added to all plates. Total RNA was isolated at 0, 3, and 6 h after the addition of actinomycin D, and Northern blot analysis was performed. BRECs or BAECs were exposed to hypoxic conditions of 1% oxygen using an advanced computer-controlled infrared water-jacketed multigas incubator (Model BL-M10, Jujikagaku, Tokyo). All cells were maintained at 37 °C in a constant 5% CO2atmosphere with oxygen deficit induced by nitrogen replacement. Cells maintained under these conditions for periods exceeding 24 h showed no morphologic changes by light microscopy and could subsequently be passaged normally. Cells incubated under standard normoxic conditions (95% air and 5% CO2) from the same batch and passage were used as controls. To study the effect of hypoxia-induced VEGF on Ang2 expression, BRECs were incubated under hypoxic or normoxic conditions for 2 h with or without anti-VEGF neutralizing antibody (10 μg/ml; R&D Systems, Minneapolis, MN). BRECs were treated with 25 ng/ml VEGF for the VEGF study or subjected to hypoxic conditions for the hypoxic study for 12 h in serum-free, methionine-free medium with 35S (100 μCi/ml; Amersham International). Cells were washed three times with cold phosphate-buffered saline and lysed in a solubilization buffer (50 mm Hepes, pH 7.4, 10 mm EDTA, 100 mm NaF, 10 mm sodium pyrophosphate, 1% Triton X-100, 10 mm NaVO4, 20 μmleupeptin, 1.5 μm aprotinin, and 2 mmphenylmethylsulfonyl fluoride) at 4 °C for 1 h. To clear the protein extract, protein A-Sepharose (20 μl of a 50% suspension; Pharmacia Biotech, Uppsala, Sweden) was added to the cell lysates, after which they were incubated for 1 h, followed by centrifugation and collection of the supernatant. Protein concentrations were measured by a protein assay (BCA protein assay, Pierce). A specific goat anti-human Ang2 antibody (5 μg; Santa Cruz Biotechnology, Santa Cruz, CA) was added and rocked with the protein sample (500 μg) at 4 °C for 1.5 h; 10 μg of protein A-Sepharose was then added, and the sample was rocked for another 1.5 h at 4 °C. For denaturation, protein A-Sepharose antigen-antibody conjugates were separated by centrifugation, washed five times, and boiled for 3 min in Laemmli sample buffer. The samples were separated on 7.5% SDS-polyacrylamide gel (Bio-Rad), and the gel was vacuum-dried. Results were visualized and analyzed by densitometric scanning (BAS-2000II). The well established mouse model of ischemia-induced retinal neovascularization was created as described previously (36Smith L.E. Wesolowski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Invest. Ophthalmol. Visual Sci. 1994; 35: 101-111PubMed Google Scholar). Briefly, litters of 7-day-old (postnatal day 7) C57BL/6J mice were exposed to 75 ± 2% oxygen for 5 days and then returned to room air at postnatal day 12 to produce retinal neovascularization. Mice of the same age maintained in room air served as controls. For in situ hybridization studies, mice at different time points during the induction of neovascularization were deeply and intraperitoneally anesthetized with sodium pentobarbital and killed by perfusion through the left ventricle with 4% paraformaldehyde in phosphate-buffered saline. Eyes were enucleated, fixed in 4% paraformaldehyde at 4 °C overnight, and embedded in paraffin. Serial 5-μm sections of the whole eyes were placed on microscope slides. Slides were treated with 0.2 n HCl for 20 min, followed by washing in phosphate-buffered saline containing 0.01% diethyl pyrocarbonate, digestion with 20 μg/ml proteinase K at 37 °C for 10 min, and fixation in 4% paraformaldehyde for 5 min. Blocking was performed in phosphate-buffered saline containing 50% formamide and 2× SSC at room temperature for 1 h. Sense and antisense Ang2 cRNA probes were generated from the same plasmid used for Northern hybridization and labeled with digoxigenin-dUTP (DIG RNA labeling kit, Roche Molecular Biochemicals) as recommended by the manufacturer. The efficiency of labeling was confirmed by agarose gel electrophoresis. The probe was used at a concentration of 50 ng/section. Hybridization was performed at 45 °C for 16 h. After extensive sequential washings in 2×, 1×, and 0.5× SSC, the unhybridized probe was digested with ribonuclease (Promega, Madison, WI) in 0.5× SSC. The hybridization product was detected after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody (1:500 dilution; Roche Molecular Biochemicals) overnight at 4 °C, followed by development in 4-tetrazolium chloride (1:50 dilution; Roche Molecular Biochemicals) overnight at room temperature. All determinations were performed in triplicate, and results are expressed as means ± S.D. One-way analysis of variance, followed by Fisher's t test, was used to evaluate significant differences, and p < 0.05 was selected as the statistically significant value. To investigate the effect of VEGF treatment on Ang1, Ang2, and Tie2 expression, BRECs were exposed to VEGF (25 ng/ml), and Northern blot analysis was performed. Increased Ang2 mRNA expression was observed after 1 h of stimulation and was time-dependent, with a maximal response of a 4.6 ± 0.7-fold (p = 0.0031) increase after 2 h of stimulation (Fig. 1 A). In contrast, both Ang1 and Tie2 mRNA expression remained stable. To analyze dose dependence, cells were treated with various concentrations of VEGF for 2 h. A dose-dependent increase in Ang2 mRNA was observed with an EC50 of ≈12.5 ng/ml and peaked at 25 ng/ml (p < 0.0001) (Fig. 1 B). These data suggest that VEGF increases Ang2 mRNA expression in both a time- and dose-dependent manner. Since expression of angiopoietins has been reported to be cell-type dependent (28Witzenbichler B. Maisonpierre P.C. Jones P. Yancopoulos G.D. Isner J.M. J. Biol. Chem. 1998; 273: 18514-18521Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar) and to confirm a similar effect of VEGF on macrovascular endothelial cells, we also tested the effect of VEGF on BAECs. The resultant response revealed a 2.6 ± 0.3-fold (p = 0.0026) increase in Ang2 mRNA expression after 2 h of VEGF stimulation (25 ng/ml) (Fig. 1 C). As in BRECs, VEGF had no significant effects on Ang1 or Tie2 mRNA expression in BAECs. We investigated whether the VEGF-induced increase in Ang2 mRNA is derived from up-regulation of transcription or from increased mRNA stability. Nuclear run-on transcription analysis was employed to determine whether VEGF leads to an increase in the transcription initiation rate. Nuclei prepared from cells treated with VEGF (25 ng/ml) or vehicle were evaluated. VEGF treatment increased the rate of Ang2 gene transcription by 3.8-fold compared with that of controls (Fig. 2). To determine whether VEGF affects the stability of Ang2 mRNA, we evaluated the half-life of Ang2 mRNA with the aid of actinomycin D to inhibit de novo gene transcription. The half-life of Ang2 mRNA was 4.2 h when treated with VEGF and 3.8 h in unstimulated controls (Fig. 3). No significant difference was observed. These findings, together with the data from nuclear run-on transcription analysis, clearly demonstrate that the VEGF-induced increase in Ang2 mRNA was derived mainly from an increase in the transcription rate. Since previous reports have shown that tyrosine kinase and PKC (32Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (522) Google Scholar) and MAPK (37Waltenberger J. Mayr U. Pentz S. Hombach V. Circulation. 1996; 94: 1647-1654Crossref PubMed Scopus (232) Google Scholar) have significant roles in VEGF-induced intracellular signaling pathways, we determined whether these molecules could have effects on VEGF-induced Ang2 mRNA expression. BRECs were treated with 25 ng/ml VEGF for 2 h after pretreatment with a protein kinase inhibitor: genistein, a tyrosine kinase inhibitor (40 μm); GF 109203X, a PKC-specific inhibitor (5 μm); or PD 098059, a MAPK kinase inhibitor (25 μm) (Fig.4). The addition of these agents abolished the VEGF-induced increase in Ang2 mRNA expression by 84.6 ± 13.1% (p = 0.0009), 65.4 ± 16.8% (p = 0.0029), and 92.4 ± 8.5% (p= 0.0005), respectively. The 0.1% Me2SO carrier used to solubilize these inhibitors did not significantly alter Ang2 mRNA expression (data not shown). These data indicate that MAPK and tyrosine phosphorylation have a predominant role in VEGF-induced Ang2 mRNA expression and that the PKC-dependent pathway makes a more minor contribution. To investigate the effects of hypoxia on the angiopoietin-Tie2 system, BRECs were exposed to hypoxic conditions. Ang2 mRNA expression revealed a time-dependent increase that peaked at 2 h (3.6 ± 0.09-fold, p < 0.0001) and returned to the basal level after 4 h of stimulation (Fig.5 A). In contrast, Ang1 and Tie2 mRNA expression remained stable despite hypoxia. As in the VEGF stimulation study, we found a similar response in BAECs. Two hours of hypoxia induced a 2.1 ± 0.1-fold (p = 0.0327) increase in Ang2 mRNA expression, whereas no significant effect on Ang1 or Tie2 mRNA expression was observed (Fig. 5 B). Since hypoxia is the major stimulus for VEGF induction and our result showed that VEGF increases Ang2 expression, we investigated whether hypoxia-induced VEGF is involved in the observed hypoxic regulation of Ang2 in BRECs. The anti-VEGF neutralizing antibody (10 μg/ml) exhibited no significant effect on Ang2 mRNA under not only normoxic, but also hypoxic conditions (Fig.6). These results suggest that the increase in Ang2 expression under hypoxic conditions is the direct effect of hypoxia and is not mediated by VEGF induction.Figure 6Effect of anti-VEGF neutralizing antibody on hypoxia-induced Ang2 mRNA expression in BRECs . Total RNA was isolated at 2 h after stimulation by hypoxia or normoxia with or without 10 μg/ml anti-VEGF antibody, and Northern blot analysis was performed. Three triplicate experiments were performed, and the representative blots are shown (upper panel). Results were quantified by densitometric analysis of the autoradiogram derived from the upper panel after normalization to the 36B4 control cDNA signals. Values are presented as a percentage of the control and are expressed as means ± S.D. (lower panel).kb, kilobases.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether the increase in Ang2 mRNA was accompanied by an increase in new protein synthesis, we precipitated the35S-labeled cell lysates with a specific goat anti-human Ang2 antibody. The molecular mass of Ang2 protein has been reported to range from 55 to 70 kDa, due to glycosylation (27Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1660) Google Scholar). The detected size of Ang2 protein was ∼55 kDa, and the expressio

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