Placenta Growth Factor and Vascular Endothelial Growth Factor B and C Expression in Microvascular Endothelial Cells and Pericytes
1999; Elsevier BV; Volume: 274; Issue: 49 Linguagem: Inglês
10.1074/jbc.274.49.35172
ISSN1083-351X
AutoresHideto Yonekura, Shigeru Sakurai, Xiaoxu Liu, Hideyuki Migita, Hua Wang, Sho‐ichi Yamagishi, Motohiro Nomura, Md. Joynal Abedin, Hiroyuki Unoki‐Kubota, Yasuhiko Yamamoto, Hiroshi Yamamoto,
Tópico(s)Lymphatic System and Diseases
ResumoWe have shown previously that vascular endothelial growth factor (VEGF) synthesized by the cellular constituents of small vessels per se, viz.endothelial cells and pericytes, participates in the hypoxia-driven proliferation of both cell types (Nomura, M., Yamagishi, S., Harada, S., Hayashi, Y., Yamashima, T., Yamashita, J., Yamamoto, H. (1995)J. Biol. Chem. 270, 28316–28324; Yamagishi, S., Yonekura, H., Yamamoto, Y., Fujimori, H., Sakurai, S., Tanaka, N., and Yamamoto, H. (1999) Lab. Invest. 79, 501–509). In this study, we examined the expression of the recently isolated VEGF gene family members (placenta growth factor (PlGF), VEGF-B, and VEGF-C) in human dermal microvascular endothelial cells and bovine retinal pericytes cultured under various oxygen tensions. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the two cell types possess not only VEGF (VEGF-A) mRNA, but also VEGF-B, VEGF-C, and PlGF mRNAs. Among them, only VEGF-A mRNA was induced under hypoxia. Competitive reverse transcription-polymerase chain reaction showed that, under normoxic conditions, the rank order of mRNA content in endothelial cells was PlGF > VEGF-B > VEGF-C > VEGF-A and that mRNA coding for PlGF was expressed at >100-fold higher levels than VEGF-A mRNA. In pericytes, the rank order was VEGF-C > VEGF-A > VEGF-B > PlGF, and ∼7-fold higher levels of VEGF-C mRNA compared with VEGF-A mRNA were noted in this cell type. Furthermore, antisense inhibition of PlGF protein production lowered the endothelial cell synthesis of DNA under hypoxic conditions. The results suggest that these VEGF family members may also take active parts in angiogenesis. We have shown previously that vascular endothelial growth factor (VEGF) synthesized by the cellular constituents of small vessels per se, viz.endothelial cells and pericytes, participates in the hypoxia-driven proliferation of both cell types (Nomura, M., Yamagishi, S., Harada, S., Hayashi, Y., Yamashima, T., Yamashita, J., Yamamoto, H. (1995)J. Biol. Chem. 270, 28316–28324; Yamagishi, S., Yonekura, H., Yamamoto, Y., Fujimori, H., Sakurai, S., Tanaka, N., and Yamamoto, H. (1999) Lab. Invest. 79, 501–509). In this study, we examined the expression of the recently isolated VEGF gene family members (placenta growth factor (PlGF), VEGF-B, and VEGF-C) in human dermal microvascular endothelial cells and bovine retinal pericytes cultured under various oxygen tensions. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the two cell types possess not only VEGF (VEGF-A) mRNA, but also VEGF-B, VEGF-C, and PlGF mRNAs. Among them, only VEGF-A mRNA was induced under hypoxia. Competitive reverse transcription-polymerase chain reaction showed that, under normoxic conditions, the rank order of mRNA content in endothelial cells was PlGF > VEGF-B > VEGF-C > VEGF-A and that mRNA coding for PlGF was expressed at >100-fold higher levels than VEGF-A mRNA. In pericytes, the rank order was VEGF-C > VEGF-A > VEGF-B > PlGF, and ∼7-fold higher levels of VEGF-C mRNA compared with VEGF-A mRNA were noted in this cell type. Furthermore, antisense inhibition of PlGF protein production lowered the endothelial cell synthesis of DNA under hypoxic conditions. The results suggest that these VEGF family members may also take active parts in angiogenesis. vascular endothelial growth factor VEGF receptor placenta growth factor reverse transcription-polymerase chain reaction base pair(s) Angiogenesis, the process by which new vascular networks are formed from preexisting capillaries, is physiologically essential for embryogenesis, development, corpus luteum formation, ovulation, and wound repair (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4867) Google Scholar). It is also related to the progression of various pathological conditions such as cancer growth and metastasis, diabetic retinopathy, rheumatoid arthritis, and collateral path formation in occlusive vascular diseases (2Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar). Among the known angiogenic factors, vascular endothelial growth factor (VEGF)1 (3Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar) has emerged as a central regulator of the angiogenic process under both physiological and pathological conditions (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4867) Google Scholar, 4Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3164) Google Scholar, 5Nicosia R.F. Am. J. Pathol. 1998; 153: 11-16Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 6Korpelainen E.I. Alitalo K. Curr. Opin. Cell Biol. 1998; 10: 159-164Crossref PubMed Scopus (174) Google Scholar). We have previously shown that the VEGF gene is expressed in vascular endothelial cells and pericytes, the constituents of microvessels wherein angiogenesis takes place, and that the hypoxia-induced proliferation of both cell types is mediated by autocrine VEGF (7Nomura 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 (303) Google Scholar, 8Yamagishi S. Kawakami T. Fujimori H. Yonekura H. Tanaka N. Yamamoto Y. Urayama H. Watanabe Y. Yamamoto H. Microvasc. Res. 1999; 57: 329-339Crossref PubMed Scopus (63) Google Scholar). We have also reported that autocrine VEGF-A takes an active part in advanced glycation end product-driven angiogenesis (9Yamagishi S. Yonekura H. Yamamoto Y. Katsuno K. Sato F. Mita I. Ooka H. Satozawa N. Kawakami T. Nomura M. Yamamoto H. J. Biol. Chem. 1997; 272: 8723-8730Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) and that VEGF-A can also act on pericytes to stimulate their proliferation and migration (10Yamagishi S. Yonekura H. Yamamoto Y. Fujimori H. Sakurai S. Tanaka N. Yamamoto H. Lab. Invest. 1999; 79: 501-509PubMed Google Scholar). Recently, several VEGF-related genes, including placenta growth factor (PlGF) (11Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar), VEGF-B (12Olofsson B. Pajusola K. Kaipainen A. von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar), and VEGF-C (13Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar), have been isolated. The VEGF family members have been shown to share common receptors for their actions and have been implicated in the process of angiogenesis (4Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3164) Google Scholar, 5Nicosia R.F. Am. J. Pathol. 1998; 153: 11-16Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 6Korpelainen E.I. Alitalo K. Curr. Opin. Cell Biol. 1998; 10: 159-164Crossref PubMed Scopus (174) Google Scholar). Coexpression of plural members of the VEGF family has been reported in a variety of normal and tumoral tissues (14Enholm B. Paavonen K. Ristimäki 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 (387) Google Scholar, 15Salven P. Lymboussaki A. Heikkilä P. Jääskela-Saari H. Enholm B. Aase K. von Elder G. Eriksson U. Alitalo K. Joensuu H. Am. J. Pathol. 1998; 153: 103-108Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 16Sowter H.M. Corps A.N. Evans A.L. Clark D.E Charnock-Jones D.S. Smith S.K. Lab. Invest. 1997; 77: 607-614PubMed Google Scholar, 17Clark D.E. Smith S.K. Licence D. Evans A.L. Charnock-Jones D.S. J. Endocrinol. 1998; 159: 459-467Crossref PubMed Scopus (165) Google Scholar, 18Achen M.G. Gad J.M. Stacker S.A. Wilks A.F. Growth Factors. 1997; 15: 69-80Crossref PubMed Scopus (67) Google Scholar, 19Lagercrantz J. Farnebo F. Larsson C. Tvrdik T. Weber G. Piehl F. Biochim. Biophys. Acta. 1998; 1398: 157-163Crossref PubMed Scopus (69) Google Scholar). However, the expression and significance of these factors in vascular cells per se have not yet been determined. Elucidation of the expression of VEGF family members and their regulation in microvascular cells may provide important insights into the molecular mechanisms underlying blood vessel formation. In the study, we examined the expression of genes coding for PlGF, VEGF-B, and VEGF-C in human dermal microvascular endothelial cells and bovine retinal pericytes. Because the nucleotide sequences of bovine VEGF-B, VEGF-C, and PlGF mRNAs were not known, we first isolated the bovine cDNA equivalents and determined their primary structures. We then analyzed the expression of these factors in endothelial cells and pericytes cultured under various oxygen tensions and quantified their mRNA content in both cell types by competitive RT-PCR. Furthermore, since PlGF was found to be the most abundantly expressed VEGF family member in endothelial cells, we pursued its role in the hypoxia-driven proliferation of microvascular cells using an antisense approach. [3H]Thymidine (90 Ci/mmol), [γ-32P]ATP (6000 Ci/mmol), [α-32P]dCTP (6000 Ci/mmol), and Expre35S35S [35S]-protein labeling mixture (1175 Ci/mmol) were purchased from NEN Life Science Products. A cDNA library derived from bovine heart was purchased from CLONTECH (Palo Alto, CA). Restriction enzymes and Megalabel, a kit for 5′-end labeling of DNA, were from Takara (Kyoto, Japan). O2/CO2/N2 gas mixtures were from Nippon Sanso Corp. (Tokyo, Japan). Prime-It II, a kit for random DNA labeling, was from Stratagene (La Jolla, CA). Hybond-N+nylon membrane and protein G-Sepharose 4FF were from Amersham Pharmacia Biotech(Buckinghamshire, United Kingdom). Antigen affinity-purified goat anti-PlGF antibodies raised against recombinant human PlGF protein and the C-terminal 20-amino acid peptide of PlGF-1 were from Genzyme/Techne (Cambridge, MA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA), respectively. The bovine heart cDNA library was screened as described previously (20Yonekura H. Nata K. Watanabe T. Kurashina Y. Yamamoto H. Okamoto H. J. Biol. Chem. 1988; 263: 2990-2997Abstract Full Text PDF PubMed Google Scholar) using bovine VEGF-B, VEGF-C, or PlGF cDNA fragments that had been amplified by RT-PCR with bovine heart poly(A)+ RNA as a template. Primers used for the amplification corresponded to nucleotides 443–462 and 631–652 of human PlGF cDNA (11Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar), nucleotides 93–117 and 409–426 of human VEGF-B cDNA (12Olofsson B. Pajusola K. Kaipainen A. von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar), and nucleotides 968–987 and 1588–1609 of human VEGF-C cDNA (13Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar). Nucleotide sequences of cloned cDNAs were determined with an Applied Biosystems Model 373 DNA sequencer (Perkin-Elmer). Human microvascular endothelial cells isolated from neonatal dermis (Cascade Biologics, Inc., Portland, OR) were maintained in HuMedia-EB2 medium supplemented with 5% fetal bovine serum, 5 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, 10 ng/ml epidermal growth factor, 1 μg/ml hydrocortisone, and 39.3 μg/ml dibutyryl cyclic AMP according to the supplier's instructions (Kurabo Corp., Osaka, Japan). Cells at 5–10 passages were used for the experiments. Pericytes were isolated from bovine retina and maintained as described (21Yamagishi S. Kobayashi K. Yamamoto H. Biochem. Biophys. Res. Commun. 1993; 190: 418-425Crossref PubMed Scopus (90) Google Scholar). Cultures under low oxygen tensions were performed as described (7Nomura 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 (303) Google Scholar). Briefly, cells in the medium supplemented with 5% fetal bovine serum, 5 ng/ml basic fibroblast growth factor, and 10 μg/ml heparin in a cluster dish or flask were placed in a controlled atmosphere culture chamber (Bellco, Vineland, NJ), a humidified airtight incubation apparatus with inflow and outflow valves, into which a gas mixture containing 5% CO2 and 0, 2.5, 5, 10, or 20% O2 balanced with N2 was flushed for 5 min at a flow rate of 10 liter/min. The chamber was sealed to maintain a constant gas composition and kept at 37 °C. The gas phase was renewed every 24 h, and the O2 and CO2 in the culture chambers were immediately equilibrated to the set values by the gas flushing and kept constant for at least 24 h to an accuracy of ±5% (7Nomura 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 (303) Google Scholar). Poly(A)+ RNAs were isolated from cells that had been cultured under various oxygen tensions, using the QuickPrep micro mRNA purification kit (Amersham Pharmacia Biotech), and then analyzed by RT-PCR using a GeneAmp RNA PCR kit (Perkin-Elmer) as described previously (7Nomura 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 (303) Google Scholar, 8Yamagishi S. Kawakami T. Fujimori H. Yonekura H. Tanaka N. Yamamoto Y. Urayama H. Watanabe Y. Yamamoto H. Microvasc. Res. 1999; 57: 329-339Crossref PubMed Scopus (63) Google Scholar, 9Yamagishi S. Yonekura H. Yamamoto Y. Katsuno K. Sato F. Mita I. Ooka H. Satozawa N. Kawakami T. Nomura M. Yamamoto H. J. Biol. Chem. 1997; 272: 8723-8730Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). It is known that alternatively spliced products are generated from VEGF-A, VEGF-B, and PlGF genes (4Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3164) Google Scholar, 5Nicosia R.F. Am. J. Pathol. 1998; 153: 11-16Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The PCR primers were thus designed to discriminate between all the mRNA variants. The oligodeoxyribonucleotide primers and probes employed were as follows. The 5′- and 3′-primers and probe for human PlGF mRNA detection corresponded to nucleotides 628–647, 927–948, and 667–693 (11Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar), respectively; those for human VEGF-B mRNA corresponded to nucleotides 196–215, 495–514, and 350–378 (12Olofsson B. Pajusola K. Kaipainen A. von Euler G. Joukov V. Saksela O. Orpana A. Pettersson R.F. Alitalo K. Eriksson U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2576-2581Crossref PubMed Scopus (629) Google Scholar); for human VEGF-C mRNA, nucleotides 340–360, 724–748, and 606–634 (13Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar); for bovine PlGF mRNA, nucleotides 429–448, 755–775, and 639–664; for bovine VEGF-B mRNA, nucleotides 450–471, 825–844, and 654–685; and for bovine VEGF-C mRNA, nucleotides 261–283, 587–608 and 488–520. The primers for human and bovine VEGF-A mRNA amplification were the same as described previously (7Nomura 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 (303) Google Scholar) and corresponded to nucleotides 44–47 and 689–717 of the human cDNA (3Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar). The probes used for human and bovine VEGF-A corresponded to nucleotides 176–205 and 797–827 of the respective cDNAs (3Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar). The amounts of poly(A)+ RNA templates and cycle numbers for amplification were chosen in quantitative ranges where the reactions proceeded linearly, which had been determined by plotting the signal intensities as functions of the template amounts and cycle numbers; 30 ng of template and 35 cycles were chosen for amplifying human VEGF-A mRNA, 20 ng and 25 cycles for human PlGF mRNA, 30 ng and 25 cycles for human VEGF-B mRNA, 30 ng and 35 cycles for human VEGF-C mRNA, 30 ng and 25 cycles for bovine VEGF-A mRNA, 20 ng and 30 cycles for bovine PlGF mRNA, 20 ng and 30 cycles for bovine VEGF-B mRNA, and 10 ng and 25 cycles for bovine VEGF-C mRNA. Temperatures and time periods employed for melting and annealing/extension were 95 °C for 30 s and 60 °C for 30 s, respectively. An aliquot of each RT-PCR mixture was electrophoresed on a 2.5% agarose gel, transferred to a Hybond-N+ nylon membrane, and hybridized with specific oligodeoxyribonucleotide probes that had been32P-end-labeled with [γ-32P]ATP and polynucleotide kinase. The radioactivities of the hybridized bands were measured with a BAS1000 BioImage analyzer (Fuji PhotoFilm Co. Ltd., Hamamatsu, Japan). The RT-PCR products were sequence-verified. For competitive RT-PCR (22Wang A.M. Doyle M.V. Mark D.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9717-9721Crossref PubMed Scopus (1618) Google Scholar), each reaction should contain a known amount of a competitor RNA that undergoes amplification with the same primers as does the endogenous mRNA and should yield products distinguishable from those derived from endogenous templates on gel electrophoresis. To construct vectors from which such competitor RNAs are generated, composite primers were synthesized and used to amplify cloned bovine cDNA or human endothelial cell poly(A)+ RNA so that the resultant cDNAs would have ∼50–200-bp internal deletions at their 5′-sides. The composite primer for human and bovine VEGF-A mRNAs was made complementary to nucleotides 44–70 and 244–269 of the human cDNA, but devoid of the internal region 71–243; the primer for human PlGF mRNA corresponded to nucleotides 628–647 and 741–760; that for human VEGF-B mRNA corresponded to nucleotides 196–215 and 301–322; for human VEGF-C mRNA, to nucleotides 340–360 and 513–534; for bovine PlGF mRNA, to nucleotides 429–448 and 561–580; for bovine VEGF-B mRNA, to nucleotides 450–471 and 587–607; and for bovine VEGF-C mRNA to nucleotides 261–283 and 378–398. The resultant PCR products were ligated into a pCR2.1 vector to transform INVαF′ cells (Invitrogen, Carlsbad, CA). Competitor RNAs were then synthesized from purified plasmids by in vitrotranscription using a riboprobe combination system (Promega, Madison, WI). The RNA concentration was determined by measuring the absorption at 260 nm after denaturation at 65 °C. Two-fold serial dilutions of competitor RNA (ranging between 10 and 10−5 amol) plus 10–60 ng of poly(A)+ RNA underwent a series of RT-PCRs using the same primer set described under “Quantitative RT-PCR” and a GeneAmp RNA PCR kit or a GeneAmp EZ rTth RNA PCR kit (Perkin-Elmer). After amplification, aliquots of the reaction mixtures were electrophoresed on a 2.5% agarose gel and stained with SYBR Green (FMC Corp. BioProducts, Rockland, ME). Signal intensities of the bands were measured with an Epilight EP-250 (Aishin Cosmos Co. Ltd., Toyota, Japan). Subconfluent cultures of endothelial cells were incubated in the presence or absence of PlGF antisense or sense oligodeoxyribonucleotides for 24 h at 37 °C under 5% O2 and further incubated in methionine/cysteine-free Eagle's minimal essential medium (Sigma) supplemented with 0.5% fetal bovine serum and 200 μCi/ml [35S]methionine/cysteine mixture with or without the antisense or sense oligodeoxyribonucleotides for 24 or 48 h at 37 °C under 5% O2. After the incubation, the conditioned media were removed, and phenylmethylsulfonyl fluoride was added to a final concentration of 0.5 mm. After centrifugation at 10,000 × g at 4 °C for 10 min, the radioactivities of the supernatants were measured by the trichloroacetic acid precipitation method (23Yonekura H. Migita H. Sakurai S. Wang H. Harada S. Abedin Md J. Yamagishi S. Yamamoto H. Nucleic Acids Res. 1999; 27: 2591-2600Crossref PubMed Scopus (20) Google Scholar). Aliquots of the saved conditioned media containing the same radioactivities (∼1400 μl on average) were precleared with a 30-μl packed volume of protein G-Sepharose precoupled with normal goat IgG (Santa Cruz Biotechnology Inc.) at 4 °C for 2 h with gentle rotation. The precleared conditioned media were then immunoprecipitated with a 30-μl packed volume of protein G-Sepharose precoupled with 1 μg of goat anti-PlGF antibodies or normal goat IgG at 4 °C for 2 h with gentle rotation. We used two types of goat anti-PlGF antibodies for immunoprecipitation; one was a polyclonal antibody raised against recombinant human PlGF protein, and the other was raised against the C-terminal 20-amino acid peptide of human PlGF-1. The former antibody was expected to react with both PlGF-1 and PlGF-2 because the sequence of the bacterially expressed immunogen mostly overlapped between the two isoforms. On the other hand, since the sequence of the C-terminal 20 amino acids employed to raise the latter antibody was perfectly matched with PlGF-1, but only partly identical to PlGF-2 (12 out of 20) due to heterogeneity in that very region, this antibody was expected to scarcely recognize PlGF-2 and thereby to specify PlGF-1. Coupling of antibodies or normal IgG with protein G-Sepharose was performed in buffer A (10 mm Tris-HCl (pH 7.4), 0.15 m NaCl, 0.1% Triton X-100, and 1 mg/ml bovine serum albumin) at 4 °C for 2 h with gentle rotation. After immunoprecipitation, the gels were washed twice with buffer A and four times with 50 mmTris-HCl (pH 7.4), 0.15 m NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS. Immunoprecipitated materials were extracted from the Sepharose beads with SDS-polyacrylamide gel electrophoresis sample buffer (62.5 mm Tris-HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue) at 95 °C for 5 min and then electrophoresed on a 12.5% SDS-polyacrylamide gel under reducing conditions. For fluorography, gels were treated with Amplify (Amersham Pharmacia Biotech), dried and exposed to x-ray film at −80 °C. Phosphorothioate antisense oligodeoxyribonucleotides and their sense counterparts were synthesized and purified by two cycles of reverse-phase high pressure liquid chromatography at TOAGOSEI Co. (Tokyo). PlGF antisense and sense oligonucleotides corresponded to nucleotides 322–339 of human PlGF mRNA (11Maglione D. Guerriero V. Viglietto G. Delli-Bovi P. Persico M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9267-9271Crossref PubMed Scopus (843) Google Scholar), with their sequences then being 5′-CAGCCTCATGACCGGCAT-3′ and 5′-ATGCCGGTCATGAGGCTG-3′, respectively. VEGF-A antisense and sense oligonucleotides corresponded to nucleotides 57–78 of human VEGF-A mRNA (3Leung D.W. Cachianes G. Kuang W.-J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4466) Google Scholar), with their respective sequences being 5′-CCCAAGACAGCAGAAAGTTCAT-3′ and 5′-ATGAACTTTCTGCTGTCTTGGG-3′ (7Nomura 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 (303) Google Scholar). Antisense effects on endothelial cell production of PlGF proteins were assessed by immunoprecipitation of 35S-labeled proteins released into culture medium using the Genzyme/Techne anti-recombinant human PlGF protein antibodies as described above under “Metabolic Labeling and Immunoprecipitation.” Antisense effects on endothelial cell DNA synthesis were assessed as described previously (7Nomura 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 (303) Google Scholar). Briefly, human microvascular endothelial cells were seeded at a density of 2 × 104 cells/well of a Falcon 24-well plate and placed at 37 °C for 12 h. After cell attachment, media were changed to the HuMedia-EB2 medium supplemented with 5% fetal bovine serum containing antisense or sense oligodeoxyribonucleotides, and cells were incubated for 24 h. Then, [3H]thymidine was added to a final concentration of 5 μCi/ml, and cells were further incubated for 24 h. After incubation, cells were fixed with ice-cold 10% (w/v) trichloroacetic acid for 20 min. The resultant precipitates were washed twice with ice-cold 5% trichloroacetic acid and solubilized by mixing with 200 μl of 1 n NaOH at room temperature for 20 min, followed by neutralization with the same volume of 1 n HCl.3H radioactivity was measured by liquid scintillation counting. The bovine PlGF cDNA encodes a 149-amino acid protein with a 20-amino acid signal sequence (Fig.1 A). The nucleotide and deduced amino acid sequences of bovine and human cDNAs shared 74.9 and 75.2% identities, respectively. It has been reported that alternative splicing events give rise to three isoforms of human PlGF, namely PlGF-1, -2, and -3 (5Nicosia R.F. Am. J. Pathol. 1998; 153: 11-16Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 24Maglione D. Guerriero V. Viglietto G. Ferraro M.G. Aprelikova O. Alitalo K. Vecchio S.D. Lei K.-J. Chou J.Y. Persico M.G. Oncogene. 1993; 8: 925-931PubMed Google Scholar). In this study, all the bovine cDNAs isolated corresponded to PlGF-1, which lacks a heparin-binding domain that endows the ligand with the ability to adsorb the extracellular matrix (24Maglione D. Guerriero V. Viglietto G. Ferraro M.G. Aprelikova O. Alitalo K. Vecchio S.D. Lei K.-J. Chou J.Y. Persico M.G. Oncogene. 1993; 8: 925-931PubMed Google Scholar). The bovine VEGF-B cDNA encodes a 188-amino acid protein with a 21-amino acid signal sequence (Fig. 1 B). The mature VEGF-B protein of 167 amino acids had three basic amino acid clusters in its carboxyl-terminal region that may function as heparin-binding sites (Fig. 1 B). Another isoform (VEGF-B186) has been reported to occur in human also by alternative splicing (25Olofsson B. Pajusola K. von Euler G. Chilov D. Alitalo K. Eriksson U. J. Biol. Chem. 1996; 271: 19310-19317Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Four out of the five cDNA clones isolated from the heart library corresponded to VEGF-B167, and the nucleotide and deduced amino acid sequences of the bovine and human VEGF-B167 cDNAs shared 93.7 and 93.6% identities, respectively. The remaining one clone encoded VEGF-B186, which lacks putative heparin-binding sites. The bovine VEGF-C cDNA encodes a 420-amino acid protein with a 20-amino acid signal sequence (Fig. 1 C). The nucleotide and deduced amino acid sequences of bovine and human cDNAs shared 86.9 and 88.1% identities, respectively. As shown in Fig.2, quantitative RT-PCR analysis demonstrated that microvascular endothelial cells possess not only VEGF-A mRNA, but also VEGF-B, VEGF-C and PlGF mRNAs. RT-PCR with VEGF-A primers gave signals at 319 and 420 bp, which corresponded to VEGF-A121 and VEGF-A165 mRNAs, respectively. The levels of both VEGF-A121 and VEGF-A165 mRNAs significantly increased as the O2 concentration dropped to 2.5% (∼3–4-fold) and further to 0% (∼10-fold) (Fig. 2 A). PlGF primers yielded 321- and 384-bp products, which corresponded to PlGF-1 and PlGF-2 mRNAs, respectively (Fig. 2 B). This indicated that microvascular endothelial cells could generate both the soluble and heparin-binding forms of PlGF. VEGF-B primers gave signals at 395 and 496 bp, which corresponded to VEGF-B167 and VEGF-B186 mRNAs, respectively; the signal intensity of the latter was consistently stronger than that of the former regardless of the O2 tensions (Fig. 2 B), indicating that the soluble form of VEGF-B is predominant in microvascular endothelial cells. With VEGF-C primers, only a single band was noted at 409 bp (Fig. 2 C). The levels of mRNAs for VEGF-B, VEGF-C, and PlGF were not significantly changed in endothelial cells by exposure to hypoxia or anoxia (Fig. 2, B–D), in contrast with that of VEGF-A mRNA (Fig. 2 A). As shown in Fig. 3, quantitative RT-PCR analysis demonstrated the presence in bovine retinal pericytes of not only VEGF-A mRNA, but also VEGF-B, VEGF-C, and PlGF mRNAs, as in endothelial cells. RT-PCR with VEGF-A primers gave signals at 470 and 602 bp, which corresponded to VEGF-A121 and VEGF-A165 mRNAs, respectively (Fig. 3 A). The levels of both mRNA species increased significantly as the atmospheric O2 concentration decreased to 0% (∼8-fold) (Fig. 3 A). PlGF primers yielded 347- and 410-bp products, which corresponded to PlGF-1 and PlGF-2 mRNAs, respectively (Fig. 3 B). VEGF-B primers gave VEGF-B167- and VEGF-B186-corresponding signals at 395 and 496 bp, respectively (Fig. 3 C). In contrast with the case in endothelial cells, the former species was more prominent than the latter in pericytes (Fig. 3 C). This indicated that the heparin-binding form of VEGF-B predominated in pericytes. With VEGF-C primers, a single band was noted at 348 bp (Fig. 3 D), which exactly corresponded to the bovine mRNA. Under hypoxia, the PlGF, VEGF-B, and VEGF-C mRNA levels were essentially unchanged (Fig. 3, B–D). Since the RT-PCR analyses revealed that both endothelial cells and per
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