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• W2000709661 abstract "Angiogenesis, the growth of new blood vessels, is regulated by a number of factors, including hypoxia and vascular endothelial growth factor (VEGF). Although the effects of hypoxia have been studied intensely, less attention has been given to other extracellular parameters such as pH. Thus, the present study investigates the consequences of acidic pH on VEGF binding and activity in endothelial cell cultures. We found that the binding of VEGF165 and VEGF121 to endothelial cells increased as the extracellular pH was decreased from 7.5 to 5.5. Binding of VEGF165 and VEGF121 to endothelial extracellular matrix was also increased at acidic pH. These effects were, in part, a reflection of increased heparin binding, because VEGF165 and VEGF121 showed increased retention on heparin-Sepharose at pH 5.5 compared with pH 7.5. Consistent with these findings, soluble heparin competed for VEGF binding to endothelial cells under acidic conditions. However, at neutral pH (7.5) low concentrations of heparin (0.1–1.0 μg/ml) potentiated VEGF binding. Extracellular pH also regulated VEGF activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). VEGF165 and VEGF121 activation of Erk1/2 at pH 7.5 peaked after 5 min, whereas at pH 6.5 the peak was shifted to 10 min. At pH 5.5, neither VEGF isoform was able to activate Erk1/2, suggesting that the increased VEGF bound to the cells at low pH was sequestered in a stored state. Therefore, extracellular pH might play an important role in regulating VEGF interactions with cells and the extracellular matrix, which can modulate VEGF activity. Angiogenesis, the growth of new blood vessels, is regulated by a number of factors, including hypoxia and vascular endothelial growth factor (VEGF). Although the effects of hypoxia have been studied intensely, less attention has been given to other extracellular parameters such as pH. Thus, the present study investigates the consequences of acidic pH on VEGF binding and activity in endothelial cell cultures. We found that the binding of VEGF165 and VEGF121 to endothelial cells increased as the extracellular pH was decreased from 7.5 to 5.5. Binding of VEGF165 and VEGF121 to endothelial extracellular matrix was also increased at acidic pH. These effects were, in part, a reflection of increased heparin binding, because VEGF165 and VEGF121 showed increased retention on heparin-Sepharose at pH 5.5 compared with pH 7.5. Consistent with these findings, soluble heparin competed for VEGF binding to endothelial cells under acidic conditions. However, at neutral pH (7.5) low concentrations of heparin (0.1–1.0 μg/ml) potentiated VEGF binding. Extracellular pH also regulated VEGF activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). VEGF165 and VEGF121 activation of Erk1/2 at pH 7.5 peaked after 5 min, whereas at pH 6.5 the peak was shifted to 10 min. At pH 5.5, neither VEGF isoform was able to activate Erk1/2, suggesting that the increased VEGF bound to the cells at low pH was sequestered in a stored state. Therefore, extracellular pH might play an important role in regulating VEGF interactions with cells and the extracellular matrix, which can modulate VEGF activity. Angiogenesis is the process by which endothelial cells sprout and migrate from pre-existing blood vessels to form new capillaries (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4846) Google Scholar). Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR-2 cells, VEGFR-2 expressing NIH-3T3 cells; BAECs, bovine aortic endothelial cells; CHO-K1, Chinese hamster ovary K1 cells; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; Erk1/2, extracellular regulated kinases 1 and 2; FGF, fibroblast growth factor; HS, heparan sulfate; HSPGs, heparan sulfate proteoglycan; Parent cells, NIH-3T3 cells; PBS, phosphate buffered saline; BSA, bovine serum albumin; GF, growth factor; ANOVA, analysis of variance. is a potent mitogen for endothelial cells and has been shown to be an important growth factor in initiating angiogenesis (2Veikkola T. Karkkainen M. Claesson-Welsh L. Alitalo K. Cancer Res. 2000; 60: 203-212PubMed Google Scholar). Several isoforms of VEGF exist that are formed by alternative splicing. At least six VEGF proteins have been discovered: 121, 145, 165, 183, 189, and 206 (3Robinson C.J. Stringer S.E. J. Cell Sci. 2001; 114: 853-865Crossref PubMed Google Scholar). VEGF165, VEGF121, and VEGF189 are the most abundantly expressed isoforms. These isoforms vary in their ability to interact with heparan sulfate proteoglycans (HSPGs). VEGF121 is the only isoform that is not able to bind to HSPGs directly. It lacks exon 7, which encodes for the heparin-binding domain (3Robinson C.J. Stringer S.E. J. Cell Sci. 2001; 114: 853-865Crossref PubMed Google Scholar). The remaining isoforms vary in their affinity for HSPGs. VEGF189 and VEGF206 have been found sequestered in the extracellular matrix (ECM) and have a higher affinity for HSPGs than VEGF165, which is able to interact with HSPGs but has not been found to be sequestered in the ECM (4Park J.E. Keller G.A. Ferrara N. Mol. Biol. Cell. 1993; 4: 1317-1326Crossref PubMed Scopus (962) Google Scholar). HSPGs are expressed in most tissues and are major components of cell surfaces and extracellular matrices (5Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2323) Google Scholar). HSPGs can participate in physiological processes such as cell adhesion, migration, proliferation, differentiation, and lipoprotein uptake (5Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2323) Google Scholar, 6Wight T.N. Kinsella M.G. Qwarnstrom E.E. Curr. Opin. Cell Biol. 1992; 4: 793-801Crossref PubMed Scopus (339) Google Scholar, 7Woods A. Oh E.S. Couchman J.R. Matrix Biol. 1998; 17: 477-483Crossref PubMed Scopus (78) Google Scholar). HSPGs consist of a core protein that has generally 1–4 covalently linked heparan sulfate chains consisting of repeating disaccharide units containing N-acetylglucosamine and uronic acid, which can vary in sulfation and epimerization (8Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). The heparan sulfate (HS) chains can bind various types of ligands such as growth factors and ECM molecules (9Sasisekharan R. Ernst S. Venkataraman G. Angiogenesis. 1997; 1: 45-54Crossref PubMed Scopus (76) Google Scholar). The negatively charged sulfate groups of HSPGs participate in interactions with basic regions of growth factors. HSPGs can act as suppressors or activators of growth factor activity. Cell surface HSPGs may localize growth factors near their receptors or act as coreceptors for growth factors, whereas ECM HSPGs may act as sites for growth factor storage, sequestering them from cell surface receptors (10Turnbull J. Powell A. Guimond S. Trends Cell Biol. 2001; 11: 75-82Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 11Esko J.D. Lindahl U. J. Clin. Invest. 2001; 108: 169-173Crossref PubMed Scopus (788) Google Scholar, 12Nugent M.A. Iozzo R.V. Int. J. Biochem. Cell Biol. 2000; 32: 115-120Crossref PubMed Scopus (400) Google Scholar). For example, basic fibroblast growth factor (FGF-2) has been shown to bind to HSPGs with high affinity, and these interactions potentiate FGF-2 binding to FGF receptors (13Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2086) Google Scholar, 14Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1291) Google Scholar, 15Fannon M. Forsten K.E. Nugent M.A. Biochemistry. 2000; 39: 1434-1445Crossref PubMed Scopus (140) Google Scholar). In addition, it has been shown that VEGF binding to VEGF receptors is dependent on HSPGs (16Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar, 17Tessler S. Rockwell P. Hicklin D. Cohen T. Levi B.Z. Witte L. Lemischka I.R. Neufeld G. J. Biol. Chem. 1994; 269: 12456-12461Abstract Full Text PDF PubMed Google Scholar). Moreover, VEGF binding to HSPGs restores VEGF activity after oxidative damage (18Gengrinovitch S. Berman B. David G. Witte L. Neufeld G. Ron D. J. Biol. Chem. 1999; 274: 10816-10822Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). It has also been found that low concentrations of heparin potentiate VEGF binding to endothelial cells, whereas high concentrations of heparin inhibit VEGF binding (16Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar). HSPGs have been identified to be up-regulated at active sites of angiogenesis (19Iozzo R.V. San Antonio J.D. J. Clin. Invest. 2001; 108: 349-355Crossref PubMed Scopus (402) Google Scholar, 20Sharma B. Handler M. Eichstetter I. Whitelock J.M. Nugent M.A. Iozzo R.V. J. Clin. Invest. 1998; 102: 1599-1608Crossref PubMed Scopus (175) Google Scholar). In addition, the HSPG glypican-1 has been shown to be expressed in pancreatic cancer cells and the surrounding fibroblasts where it is believed to play a role in tumor growth and progression (21Kleeff J. Ishiwata T. Kumbasar A. Friess H. Buchler M.W. Lander A.D. Korc M. J. Clin. Invest. 1998; 102: 1662-1673Crossref PubMed Scopus (313) Google Scholar). Thus, HSPGs likely play important roles in regulating VEGF activity during angiogenesis. Active angiogenesis has been found to play important roles in many pathological and physiological processes, such as tumor progression, diabetic retinopathy, rheumatoid arthritis, and wound healing (22Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7218) Google Scholar). Environments surrounding tumors and wounds have been shown to be hypoxic in nature (23Tannock I.F. Br. J. Radiol. 1972; 45: 515-524Crossref PubMed Scopus (222) Google Scholar). It has been well established that VEGF and its receptors are up-regulated in response to hypoxia (24Shweiki D. Neeman M. Itin A. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 768-772Crossref PubMed Scopus (539) Google Scholar, 25Brogi 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, 26Gerber H.P. Condorelli F. Park J. Ferrara N. J. Biol. Chem. 1997; 272: 23659-23667Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 27Akimoto T. Liapis H. Hammerman M.R. Am. J. Physiol. 2002; 283: R487-R495Google Scholar). Due to the hypoxia, tumor cells undergo high rates of anaerobic glycolysis leading to the production of lactate (28Yamagata M. Hasuda K. Stamato T. Tannock I.F. Br. J. Cancer. 1998; 77: 1726-1731Crossref PubMed Scopus (184) Google Scholar). These conditions contribute to the acidity of the extracellular environment surrounding tumor cells. In fact, the extracellular space within malignant tissues has been measured to be as low as pH 5.8 (29Wike-Hooley J.L. Haveman J. Reinhold H.S. Radiother. Oncol. 1984; 2: 343-366Abstract Full Text PDF PubMed Scopus (822) Google Scholar, 30Wike-Hooley J.L. Van der Zee J. van Rhoon G.C. Van den Berg A.P. Reinhold H.S. Eur. J. Cancer Clin. Oncol. 1984; 20: 619-623Abstract Full Text PDF PubMed Scopus (36) Google Scholar). It is possible that the acidic extracellular pH would impact the activity of angiogenic factors within tumors. Indeed, it was found that the rate of VEGF-stimulated microvessel growth is increased at acidic pH (6.9) in an endothelial cell culture model system (31Burbridge M.F. West D.C. Atassi G. Tucker G.C. Angiogenesis. 1999; 3: 281-288Crossref PubMed Scopus (36) Google Scholar). Reduced extracellular pH also inhibits apoptotic death in endothelial cells (32D'Arcangelo D. Facchiano F. Barlucchi L.M. Melillo G. Illi B. Testolin L. Gaetano C. Capogrossi M.C. Circ. Res. 2000; 86: 312-318Crossref PubMed Scopus (140) Google Scholar). Thus, local changes in the extracellular environment, such as acidification, might participate in directing angiogenesis to poorly vascularized sites under both normal and pathological conditions. While changes in extracellular pH would certainly impact cell function, there has been little attention focused on the role of pH in regulating angiogenesis or more specifically VEGF activity. Thus, it is possible that decreased local pH could contribute to recruiting new blood vessels to tumors and other poorly vascularized regions. This type of process might involve direct alterations in VEGF interactions and activity in endothelial cells. To begin to elucidate how local pH changes might affect VEGF, we investigated the impact of pH on VEGF binding and activity in vascular endothelial cells. We found that, as pH is decreased, VEGF165 and VEGF121 binding to cell surface and extracellular matrix sites increased. We also found that VEGF165 and VEGF121 binding to heparin increased as pH was decreased. We further found that VEGF stimulation of Erk1/2 was modulated by extracellular pH. Thus, changes in extracellular pH might dramatically impact VEGF-mediated angiogenesis. Materials—Human recombinant VEGF165 was obtained from R&D Systems (Minneapolis, MN). Human recombinant VEGF121 was from Reliatech (Braunschweig, Germany). Heparinase III from Flavobacterium heparinum was a generous gift from Biomarin Pharmaceuticals (Montreal, Canada). Heparin, phenylmethylsulfonyl fluoride, sodium orthovanadate, and secondary antibody raised against rabbits and conjugated with horseradish peroxidase were obtained from Sigma (St. Louis, MO).125I-Bolton-Hunter reagent was obtained from PerkinElmer Life Sciences (Boston, MA). Phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), F-12 Ham's medium, penicillin/streptomycin, l-glutamine, and 1 m HEPES buffer were from Invitrogen (Rockville, MD). Fetal bovine serum and calf serum were from HyClone (Logan, UT). Primary antibody for phospho-Erk1/2 was purchased from New England BioLabs (Beverly, MA). Primary antibody for total Erk1/2 was obtained from Upstate Biotechnology (Lake Placid, NY). ECL detection kit, heparin-Sepharose CL-6B, and Sepharose CL-6B was purchased from Amersham Biosciences (Upp-sala, Sweden). Cell Culture—Bovine aortic endothelial cells (BAECs) were a gift from Dr. Elazer Edelman at Massachusetts Institute of Technology (Cambridge, MA). Chinese hamster ovary (CHO-K1) cells were from Dr. Jeffrey Esko at University of Alabama Birmingham (Birmingham, AL). NIH-3T3 cells (Parent cells) and NIH-3T3 cells expressing VEGFR-2 (VEGFR-2 cells) generated by retroviral infection were obtained from Dr. Nader Rahimi at Boston University (Boston, MA). BAECs were maintained in low glucose DMEM supplemented with 10% calf serum, 5 mm glutamine, 0.1 unit/ml penicillin G, and 0.1 μg/ml streptomycin sulfate. CHO-K1 cells were maintained in F-12 Ham's supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin. NIH-3T3 cells were maintained in low glucose DMEM supplemented with 10% fetal bovine serum, 5 mm glutamine, 100 unit/ml penicillin G, and 100 μg/ml streptomycin sulfate. For experiments, BAECs were used at confluence from passages 8 to 16. For experiments, NIH-3T3 cells were used at subconfluency, to prevent cell lifting from the substratum, from passages 4 to 15. CHO-K1 cells were used at confluence. Cell number was determined with a Coulter Counter (Miami, FL). Radiolabeling of VEGF—125I-VEGF165 and125I-VEGF121 were prepared by using a modified Bolton-Hunter procedure (33Nugent M.A. Edelman E.R. Biochemistry. 1992; 31: 8876-8883Crossref PubMed Scopus (205) Google Scholar). Lyophilized VEGF was dissolved in 100 mm sodium phosphate buffer, pH 8.5 (final concentration of 285 μg/ml). An aliquot (30 μl) was added to dry Bolton-Hunter reagent (1 mCi, 0.6 μmol) and incubated on ice for 2.5 h. The reaction was quenched by adding 200 μl of 0.2 m glycine and incubating on ice for 45 min. Twenty microliters of 10 mg/ml BSA in PBS and 250 μl of 1 mg/ml BSA in PBS were added. The sample was applied to a PD-10 column equilibrated and run in PBS containing 1 mg/ml BSA to separate unincorporated radiolabel from the radiolabeled VEGF. SDS-PAGE and autoradiography revealed125I-VEGF165 and125I-VEGF121 at ∼45 and ∼35 kDa, respectively. After radiolabeling,125I-VEGF165 retained its ability to bind to a heparin-Sepharose column and was eluted with high concentrations of salt. Also, both125I-VEGF165 and125I-VEGF121 were able to stimulate activation of Erk1/2 on BAECs, indicating that the radiolabeling procedure did not adversely disrupt VEGF structure. 125I-VEGF Binding—Equilibrium binding assays were carried out with confluent cell cultures as previously described (15Fannon M. Forsten K.E. Nugent M.A. Biochemistry. 2000; 39: 1434-1445Crossref PubMed Scopus (140) Google Scholar, 16Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar, 34Moscatelli D. J. Cell Biol. 1988; 107: 753-759Crossref PubMed Scopus (219) Google Scholar). BAECs were seeded at 75,000 cells/well in 24-well dishes (Corning Inc., Corning, NY). CHO-K1 cells were seeded at 100,000 cells/well in 24-well dishes. NIH-3T3 cells and VEGFR-2 cells were seeded at 50,000 cells/well in 24-well dishes. Cells were grown for 20–26 h. Binding assays conducted at various pHs were carried out in binding buffer consisting of 25 mm HEPES adjusted to the indicated pH (7.5–5.5) in DMEM (without bicarbonate) containing 0.1% BSA. Cells were washed once with ice-cold binding buffer. Binding buffer was added to cells and incubated at 4 °C for 10 min to inhibit endocytosis and binding site turnover.125I-VEGF165 (0.12 nm) or125I-VEGF121 (0.14 nm) was added to cells. Cells were incubated for 2.5 h at 4 °C. After the binding period, unbound125I-VEGF was removed by washing the cells three times with ice-cold binding buffer. To dissociate VEGF interactions involving HSPGs, cells were exposed to a high salt buffer (25 mm HEPES, pH 7.5, 2 m NaCl) for ∼5 s and then rinsed with PBS. Cells were then solubilized with 1 n NaOH to account for the remaining interactions that are presumably VEGF-bound to receptor. It has been shown that FGF-2 can be dissociated from HSPGs on cell surfaces by using a 2 m NaCl wash (34Moscatelli D. J. Cell Biol. 1988; 107: 753-759Crossref PubMed Scopus (219) Google Scholar). It has been established that VEGF elutes from a heparin-Sepharose column with 0.69 m NaCl (35Keyt B.A. Berleau L.T. Nguyen H.V. Chen H. Heinsohn H. Vandlen R. Ferrara N. J. Biol. Chem. 1996; 271: 7788-7795Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). Consistent with these findings, we have established that 0.75 mm NaCl is enough to sufficiently remove VEGF from the first binding fraction. Moreover, increasing the ionic strength up to 2 m NaCl did not remove additional VEGF. Therefore, we conclude that the first wash contains VEGF that is able to interact with HSPGs through ionic interactions and that the remaining VEGF-receptor interactions are observed by solubilizing the cells in 1 n NaOH. 125I-VEGF binding was quantified by counting in a Cobra Auto-Gamma 5005 γ-counter (Packard Instruments, Meridian, CT). Nonspecific binding was measured using a 500-fold excess of unlabeled VEGF, and the calculated value was subtracted from each sample. Replicate cells were maintained under the same conditions as sample cells in the absence of125I-VEGF and were used to measure final experimental pH. It was found that the pH of the various buffers did not change during the course of the binding experiments. Also, cell numbers were determined after the binding incubation and did not vary between different pHs. All binding data was normalized to cell number, however, it is important to note that we observed some experiment to experiment variability in the absolute amount of VEGF bound per cell. This variability was likely the result of differences in various preparations of 125I-VEGF, cell passage number, and the extent to which the cells were confluent in each individual experiment. In addition, toxicity experiments were conducted in cells maintained in the various pH buffers for up to 4 h at 37 °C. Cell number and viability (trypan blue exclusion) did not vary over the 4-h incubation across the pH range of 7.5–5.5. To determine the effects of removing heparan sulfate proteoglycans, cells were treated with 0.5 unit/ml heparinase III for 1 h at 37 °C prior to conducting the binding studies. Heparinase and digestion products were removed by washing two times with binding buffer. To determine the effects of heparin, various concentrations were added to cells prior to the addition of125I-VEGF. All conditions were conducted in triplicate and each experiment was repeated at least three separate times. Preparation of ECM-coated Dishes—ECM-coated dishes were prepared as previously described (33Nugent M.A. Edelman E.R. Biochemistry. 1992; 31: 8876-8883Crossref PubMed Scopus (205) Google Scholar, 36Vlodavsky I. Folkman J. Sullivan R. Fridman R. Ishai-Michaeli R. Sasse J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2292-2296Crossref PubMed Scopus (841) Google Scholar, 37Bashkin P. Doctrow S. Klagsbrun M. Svahn C.M. Folkman J. Vlodavsky I. Biochemistry. 1989; 28: 1737-1743Crossref PubMed Scopus (516) Google Scholar). BAECs were plated at 25,000 cells per well in 24-well dishes. Cells were grown for 3 days and reached confluence. The cells were lysed by incubating the cultures for 3 min at 23 °C in a solution containing 0.5% Triton X-100, 20 mm NH4OH in phosphate-buffered saline, leaving the ECM associated with the culture surface. Subsequently the ECM was washed four times with PBS. The ECM remained intact and was characterized to contain HSPGs by35S labeling of HS chains (data not shown). Heparin-Sepharose Columns—Heparin-Sepharose affinity chromatography was used to assess VEGF165 and VEGF121 binding to heparin directly (35Keyt B.A. Berleau L.T. Nguyen H.V. Chen H. Heinsohn H. Vandlen R. Ferrara N. J. Biol. Chem. 1996; 271: 7788-7795Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar).125I-VEGF165 (0.01 μm),125I-VEGF121 (0.011 μm), or125I-EGF (0.05 μm) was incubated in column buffers (150 mm NaCl, 25 mm HEPES, pH 7.5 or 5.5) for 15 min. Columns (1 ml packed with heparin-Sepharose CL-6B) were equilibrated with the column buffers corresponding to those that the growth factor (GF) was incubated by passing 3 ml of buffer through the columns. Individual columns were prepared for each sample condition. After the 15-min incubation, the125I-GF samples were applied to the columns, and the column was washed with the same buffer (1 ml) in which it was equilibrated. Flow-through was collected. The125I-GF was then eluted with either the same buffer in which it was incubated or the other pH buffer. This eluant was collected and labeled pH wash. Fractions were trichloroacetic acid-precipitated to remove any free125I from125I-GF. Samples were quantitated in a γ counter. The fraction of125I-GF bound to the column was quantified by counting the entire column in a γ counter. Activation of Erk1/2—BAECs were plated at 20,000 cells per well in 6-well dishes. After 24 h, the medium was replaced with DMEM containing 0.5% calf serum for 24 h, to make the cells quiescent. Prior to stimulation with VEGF, cells were washed with binding buffer at pH 7.5, 6.5, or 5.5 and remained in the binding buffer for 90 min. VEGF165 (0.6 nm) or VEGF121 (0.7 nm) was added to the cells at various time points. Binding buffer with VEGF was removed, and cells were extracted in 0.1% Triton X-100, 150 mm NaCl, 10 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet P-40 containing 1 mm phenylmethylsulfonyl fluoride, and 0.2 mm sodium orthovanadate. Cell lysates were spun at 13,000 × g for 10 min at 4 °C. Supernatants were collected. BCA protein assays were conducted to determine the total protein content. An equal amount of protein from each sample was subjected to SDS-PAGE (12% gel) and transferred to Immobilon membranes (Millipore Corp., Bedford, MA). Membranes were blocked with 5% BSA in Tris-buffered saline with 0.05% Tween 20. Subsequently, the membranes were incubated with anti-phospho-Erk1/2 or anti-Erk1/2. Immunoreactive bands were visualized with chemiluminescence using horseradish peroxidase-conjugated anti-rabbit IgG and ECL reagent. Autoradiograms were analyzed using Scion Image for Windows (Scion Corp., Frederick, MD) to determine relative band intensities. Membranes were stained with Ponceau S to evaluate the total protein loaded. pH did not vary over time at 37 °C. Experiments were repeated for NIH-3T3 cells plated at 50,000 cells per well in 6-well dishes. VEGF165and VEGF121Binding to Cell Surfaces Are Altered by pH—At sites of angiogenesis, such as tumors and wounds, the environment is rather hypoxic. Hypoxia increases expression of VEGF thereby promoting increased rates of migration and proliferation of endothelial cells. Hypoxic environments also lead to decreases in extracellular pH. Although the intracellular signaling events occurring under hypoxic conditions in response to VEGF have been of major focus, there has been little attention to how acidity affects VEGF outside of cells. Therefore, to investigate the role of extracellular pH on VEGF165 and VEGF121 interactions with cell surfaces, binding assays were conducted with confluent BAECs at various pHs ranging from pH 7.5 to 5.5 (Fig. 1). It was found that, as pH decreased, VEGF165 and VEGF121 binding to BAECs increased dramatically. HSPG-mediated VEGF165 binding at pH 5.5 was ∼2.5-fold greater than that at pH 7.5, whereas VEGF121 binding increased 20-fold. At pH 7.5, only ∼40% of the total bound VEGF165 bound through the HSPGs component, where at pH 5.5, greater than 50% of the total VEGF165 bound occurred through the HSPGs component. For VEGF121, there appeared to be a more dramatic specificity for binding to the HSPGs component at the lower pHs. At pH 7.5 ∼40% of total bound VEGF121 occurred through the HSPGs component, whereas at pH 5.5 ∼80% bound through the HSPGs component. Similar binding experiments were conducted with CHO-K1 cells and NIH-3T3 (Parent) cells, which are cell types that do not express endogenous VEGF receptors, and VEGFR-2 cells, which are NIH-3T3 cells engineered to express VEGFR-2. A similar increase in binding was observed at pH 5.5 compared with pH 7.5 for VEGF165 and VEGF121 with all three cell types (Fig. 2). These results indicate that the increased VEGF binding at acidic pH does not depend on the expression of VEGF receptors.Fig. 2125I-VEGF165 and125I-VEGF121 binding to CHO-K1 cells, NIH-3T3 cells, and VEGFR-2 expressing cells at pH 7.5 or 5.5. Binding assays were conducted on confluent CHO-K1 cells (A) or subconfluent Parent cells (gray bars) and VEGFR-2 cells (black bars) (B). Cells were incubated at pH 7.5 or 5.5 at 4 °C for 2.5 h. Binding assays were performed with125I-VEGF165 or125I-VEGF121. Samples were quantitated in a γ counter. Samples were normalized to cell number. Representative data are presented as the mean of triplicate determinations ± S.E. Two-tailed paired t tests were conducted to evaluate the binding differences observed at pH 5.5 and 7.5. The increased binding observed for VEGF121 and VEGF165 at pH 5.5 compared with pH 7.5 on CHO, NIH-3T3, and VEGFR-2 cells was found to be significant (p < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine if HSPGs play a role in the pH-induced changes in cell surface binding, endothelial cells were treated with heparinase III to degrade heparan sulfate chains prior to the addition of VEGF. We found that heparinase III treatment to BAECs reduced VEGF165 and VEGF121 binding under neutral (pH 7.5) and acidic (pH 5.5) conditions by ∼60 and ∼30%, respectively (Table I). Thus, HSPGs are involved in VEGF165 and VEGF121 interactions with endothelial cell surfaces at neutral and acidic pH.Table IHeparinase III pretreatment to BAEC- and endothelial cell-deposited ECM reduced 125I-VEGF165 and 125I-VEGF121 bindingVEGF boundDecreasedNativeHeparinase III-treatedfmole/105cells%BAECSpH 7.5 VEGF1650.267 ± 0.0070.103 ± 0.02961.4pH 5.5 VEGF1651.242 ± 0.0720.776 ± 0.04337.6pH 7.5 VEGF1210.037 ± 0.0050.013 ± 0.00764.9pH 5.5 VEGF1210.384 ± 0.0180.254 ± 0.01033.9Extracellular matrixpH 5.5 VEGF1650.534 ± 0.0120.332 ± 0.02437.8pH 5.5 VEGF1212.194 ± 0.1671.458 ± 0.55533.6 Open table in a new tab Changes in Extracellular pH Alter VEGF165and VEGF121Interactions with Extracellular Matrices—HSPGs are a major component of extracellular matrices. Because the previous results suggest that the acidic pH-mediated binding of VEGF may be independent of VEGF receptors, we wanted to determine if binding of VEGF to extracellular matrix would be affected by changes in pH. To investigate the role of pH on VEGF interactions within the extracellular matrix, VEGF binding to BAEC-deposited extracellular matrices was characterized. BAECs were grown for 3 days until confluent. Cell layers were extracted leaving BAEC-deposited extracellular matrix-coated dishes. Extracellular matrices were labeled with 35SO4 to determine if HSPGs are a component of these matrices. Matrices were treated with heparinase III after labeling, and it was found that35S radioactivity decreased by 66% after heparinase III treatment, confirming that HSPGs were a component of the matrices. Matrices were washed" @default.
• W2000709661 created "2016-06-24" @default.
• W2000709661 creator A5003340871 @default.
• W2000709661 creator A5074638151 @default.
• W2000709661 date "2003-05-01" @default.
• W2000709661 modified "2023-10-15" @default.
• W2000709661 title "Regulation of Vascular Endothelial Growth Factor Binding and Activity by Extracellular pH" @default.
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