Regulation of Vascular Endothelial Growth Factor Binding and Activity by Extracellular pH*
Adrienne L. Goerges and
Matthew A. Nugent
From the
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
Received for publication, November 3, 2002
, and in revised form, March 11, 2003.
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ABSTRACT
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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 (0.11.0 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.
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INTRODUCTION
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Angiogenesis is the process by which endothelial cells sprout and migrate from pre-existing blood vessels to form new capillaries (1). Vascular endothelial growth factor (VEGF)1 is a potent mitogen for endothelial cells and has been shown to be an important growth factor in initiating angiogenesis (2). 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 (3). 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 (3). 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 (4).
HSPGs are expressed in most tissues and are major components of cell surfaces and extracellular matrices (5). HSPGs can participate in physiological processes such as cell adhesion, migration, proliferation, differentiation, and lipoprotein uptake (5, 6, 7). HSPGs consist of a core protein that has generally 14 covalently linked heparan sulfate chains consisting of repeating disaccharide units containing N-acetylglucosamine and uronic acid, which can vary in sulfation and epimerization (8). The heparan sulfate (HS) chains can bind various types of ligands such as growth factors and ECM molecules (9). 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 (10, 11, 12). 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 (13, 14, 15). In addition, it has been shown that VEGF binding to VEGF receptors is dependent on HSPGs (16, 17). Moreover, VEGF binding to HSPGs restores VEGF activity after oxidative damage (18). It has also been found that low concentrations of heparin potentiate VEGF binding to endothelial cells, whereas high concentrations of heparin inhibit VEGF binding (16). HSPGs have been identified to be up-regulated at active sites of angiogenesis (19, 20). 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 (21). 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 (22). Environments surrounding tumors and wounds have been shown to be hypoxic in nature (23). It has been well established that VEGF and its receptors are up-regulated in response to hypoxia (24, 25, 26, 27). Due to the hypoxia, tumor cells undergo high rates of anaerobic glycolysis leading to the production of lactate (28). 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 (29, 30). 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 (31). Reduced extracellular pH also inhibits apoptotic death in endothelial cells (32). 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.
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EXPERIMENTAL PROCEDURES
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MaterialsHuman 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 CultureBovine 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 VEGF125I-VEGF165 and125I-VEGF121 were prepared by using a modified Bolton-Hunter procedure (33). 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 BindingEquilibrium binding assays were carried out with confluent cell cultures as previously described (15, 16, 34). 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 2026 h. Binding assays conducted at various pHs were carried out in binding buffer consisting of 25 mM HEPES adjusted to the indicated pH (7.55.5) 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 (34). It has been established that VEGF elutes from a heparin-Sepharose column with 0.69 M NaCl (35). 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
-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 7.55.5. 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 DishesECM-coated dishes were prepared as previously described (33, 36, 37). 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 ColumnsHeparin-Sepharose affinity chromatography was used to assess VEGF165 and VEGF121 binding to heparin directly (35).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
counter. The fraction of125I-GF bound to the column was quantified by counting the entire column in a
counter.
Activation of Erk1/2BAECs 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 x 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.
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RESULTS
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VEGF165 and VEGF121 Binding to Cell Surfaces Are Altered by pHAt 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.

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FIG. 1. 125I-VEGF165 and125I-VEGF121 binding to BAECs at various pHs. BAECs were incubated at the pH indicated at 4 °C for 2.5 h. Binding assays were performed with125I-VEGF165 (A and C) or125I-VEGF121 (B and D). VEGF interactions involving HSPGs were determined with a high salt, neutral pH wash (A and B). Following the high salt wash, the remainder of VEGF bound to cell surface receptors was accounted for by extracting cells in 1 N NaOH (C and D). Samples were quantitated in a counter. Cells maintained in exact conditions as experiments without the addition of radiolabeled VEGF were trypsinized and counted in a Coulter counter to normalize samples to cell number. Representative data are presented as the mean of triplicate determinations ± S.E. A one-way analysis of variance (ANOVA) was run for all data sets, and the differences across pH within each set were found to be highly significant with F = 142, 229, 1353, and 252 for sets A, B, C, and D, respectively, and p < 0.001 for all data sets. A linear regression analysis was also run to determine if there was a relationship between the pH and the level of VEGF binding. The regression was highly significant for data sets A, B, and C with R2 = 0.94, 0.95, and 0.85, respectively (p < 0.001), and the regression coefficient for pH was significantly different than zero with t = -14.7, -15.1, -8.4, respectively (p < 0.001).
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FIG. 2. 125I-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 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).
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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.
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TABLE I
Heparinase III pretreatment to BAEC- and endothelial cell-deposited ECM reduced 125I-VEGF165 and 125I-VEGF121 binding
BAEC and ECM were treated with 0.5 unit/ml heparinase III for 1 h at 37 °C prior to conducting the binding assays with 125I-VEGF165 and 125I-VEGF121 at pH 5.5 and 7.5. After 125I-VEGF165 and 125I-VEGF121 binding occurred, the cells and matrices were washed with a high salt, neutral pH buffer. Samples were quantitated in a counter. Representative data are presented as the mean of triplicate determinations ± S.E., and the percentage decreased was defined as [(VEGF bound in native - VEGF bound in heparinase III-treated)/VEGF bound in native] x 100. Two-tailed paired t tests were conducted, and the binding differences observed between the untreated and the heparinase III treated samples were determined to be significant (p < 0.05).
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Changes in Extracellular pH Alter VEGF165 and VEGF121 Interactions with Extracellular MatricesHSPGs 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 with cold binding buffer at various pHs (7.5, 7.0, 6.5, 6.0, and 5.5), and 125I-VEGF165 and125I-VEGF121 binding were measured (Fig. 3, A and B). It was observed that as pH decreased VEGF165 and VEGF121 binding increased by
5-fold. At pH 5.5, heparinase III treatment decreased VEGF165 and VEGF121 binding to the BAEC-deposited matrices by
30% indicating that HSPGs contribute to the acidic-pH mediated binding of VEGF to ECM (Table I). Together, these observations suggest that VEGF interactions with the ECM are regulated by extracellular pH.

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FIG. 3. 125I-VEGF165 and125I-VEGF121 binding to BAEC-deposited ECM at various pH levels. BAECs were grown to confluence for 3 days in culture. Cells were extracted leaving behind the deposited ECM. Binding assays were conducted on ECM. Matrices were incubated at various pHs as indicated at 4 °C for 2.5 h. Binding assays were performed with125I-VEGF165 (A) or125I-VEGF121 (B). VEGF interactions involving HSPGs were determined with a high salt, neutral pH wash. Samples were quantitated in a counter. Data are presented as the mean of triplicate determinations ± S.E. ANOVA was conducted and revealed that the differences in binding over the range of pH values tested were significant with F = 22 and 7 for data sets A and B, respectively, and p < 0.01.
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pH Alters the Affinity of VEGF165 and VEGF121 for HeparinThe increased binding of VEGF to endothelial cells and the extracellular matrix at low pH suggest that VEGF interactions with heparan sulfate are enhanced under acidic conditions. To assess how pH directly affects VEGF-HS interactions, VEGF165 and VEGF121 binding to heparin-Sepharose was analyzed at pH 7.5 and 5.5 (Fig. 4, A and B). VEGF165 and VEGF121 were incubated in pH 7.5 or 5.5 binding buffer for 15 min. Heparin-Sepharose columns were equilibrated in the pH solution in which the VEGF was incubated. VEGF was applied to the column and then eluted with either the pH 7.5 or 5.5 solutions. It was observed that 65% of VEGF165 bound to the column at pH 7.5 and 80% bound at pH 5.5. Also, when VEGF165 was incubated and applied to the column at pH 5.5 and subjected to a pH 7.5 wash, 30% of the bound VEGF165 was released. These data suggest that VEGF165 has a higher affinity for heparin at pH 5.5 and that these interactions are reversible when returned to neutral pH. Only 20% of VEGF121 was bound to the column at pH 7.5, consistent with earlier observations that this isoform does not show significant heparin affinity. However, at pH 5.5, 45% of the applied VEGF121 was bound. Furthermore, when VEGF121 was bound to the column at pH 5.5 and then washed with pH 7.5, 50% of the bound VEGF121 was released from the column. These data suggest that acidic conditions can reversibly convert VEGF121 to a heparin-binding protein. As a control, VEGF165 and VEGF121 were passed through Sepharose CL-6B columns to ensure that they were interacting with the heparin and not the Sepharose at low pH. It was found that both VEGF165 and VEGF121 passed freely through Sepharose CL-6B at pH 7.5 and 5.5 (data not shown). Moreover, to conclude that pH does not have some general nonspecific effects on protein adsorption to heparin-Sepharose, EGF, a non-heparin binding growth factor, was eluted through heparin-Sepharose columns at pH 7.5 and 5.5 (Fig. 4C). EGF did not show increased binding to the column at acidic pH. Therefore, increased binding of VEGF165 and VEGF121 to heparin at acidic pH is not a general phenomenon for all proteins.

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FIG. 4. VEGF165, VEGF121, and EGF elution through heparin-Sepharose column at pH 7.5 and 5.5. Heparin-Sepharose columns were equilibrated in pH 7.5 or 5.5 buffers.125I-VEGF165 (A),125I-VEGF121 (B), or125I-EGF (C) were incubated in either pH 7.5 or 5.5 buffers for 20 min and then applied to the columns. Columns were washed with the incubation buffer and collected as flow-through (black bars). Columns were then washed with either pH 7.5 or 5.5 buffer and collected (white bars). The remaining125I-VEGF bound to the columns was quantitated (gray bars). EGF was only washed with the incubation buffer (white bars). Data from one experiment are presented as being representative of similar results from three separate experiments. Comparisons (t test) of the flow-through values when VEGF was applied at pH 5.5 versus 7.5 and the bound fractions at pH 5.5 versus 7.5 revealed that the VEGF121 and VEGF165 profiles were statistically significantly different at the two pH levels tested (p < 0.05). EGF flow-through and bound fractions were not statistically significantly different at pH 5.5 versus 7.5 (p = NS).
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Exogenous Heparin Inhibits VEGF Binding at Acidic pH HSPGs have been shown to play a role in modulating VEGF activity. Here we show that VEGF-HS interactions were altered by pH. Thus, we wanted to determine how the addition of heparin would affect VEGF165 and VEGF121 binding to BAECs at various pHs (Fig. 5). Binding assays were conducted at pH 7.5, 6.5, and 5.5. Various concentrations of heparin (0.1500 µg/ml) were added to cells prior to the addition of125I-VEGF, and cell surface binding was determined. It was found that VEGF165 binding to BAECs at the three pHs was inhibited at high concentrations of heparin (50500 µg/ml) (Fig. 5A). At pH 7.5, low concentrations of heparin (0.125 µg/ml) potentiated VEGF165 binding significantly (
3-fold). Interestingly, 0.1 µg/ml heparin increased VEGF165 binding at pH 7.5 to levels that were equivalent to those at pH 5.5 in the absence of heparin. At pH 6.5, 0.1 µg/ml heparin maintained the same amount of VEGF165 bound as VEGF165 bound in the absence of heparin. At pH 5.5, 0.1 µg/ml heparin decreased VEGF165 binding. Interestingly, the trends for VEGF165 binding at pH 7.5, 6.5, and 5.5 in the presence of 1500 µg/ml heparin were almost superimposable. It appears that low concentrations of heparin had the same affect on VEGF165 binding as did reduced pH. However, the effects of heparin on VEGF121 binding at pH 7.5, 6.5, and 5.5 were quite different (Fig. 5, C and D). It was observed that all concentrations of heparin decreased VEGF121 binding at pH 6.5 and 5.5. At pH 7.5, low concentrations of heparin (0.150 µg/ml) were able to potentiate VEGF121 binding (Fig. 5D), however, the potentiation was unable to reach levels of VEGF121 binding at pH 6.5 or 5.5 in the absence of heparin. Interestingly, there appeared to be a shift in the amount of heparin required to potentiate VEGF121 binding in relation to VEGF165 binding under neutral conditions. Maximal VEGF165 binding occurred in the presence of 0.1 µg/ml heparin, whereas maximal VEGF121 binding occurred at 1 µg/ml heparin (Fig. 5, B and D). Therefore, heparin and pH may coordinate to regulate VEGF activity.
Erk1/2 Activation by VEGF Is Affected by Decreases in Extracellular pHVEGF stimulates a number of signaling molecules. One that has been well characterized in response to VEGF is activation of the extracellular signal-regulated kinases (Erk1/2) (38). Because extracellular pH modulates VEGF interactions with BAE cell surfaces, we wanted to determine how these differences might relate to activation of Erk1/2. Quiescent BAECs at various pHs were stimulated with VEGF, and the activation of Erk1/2 was analyzed by Western blot (Fig. 6). At pH 7.5, VEGF165 stimulated Erk1/2 activation with a peak activation time of 5 min. At pH 6.5, Erk1/2 activation peaked at 10 min. At pH 5.5, Erk1/2 did not appear to be phosphorylated in response to VEGF165 (Fig. 6A). Total Erk1/2 was not affected by pH. VEGF121 displayed a similar pattern of Erk1/2 activation as that of VEGF165 (Fig. 6B). Cell number was determined under each condition and was identical at the three different pH levels tested. Moreover, the Erk1/2 pathway is functional at pH 5.5, because the addition of heparin alone could activate Erk1/2 under these conditions, likely via heparin receptors (data not shown). These experiments were repeated on NIH-3T3 cells and NIH-3T3 cells transfected with VEGFR-2. It was found that VEGF165 stimulated Erk1/2 phosphorylation in the VEGFR-2-expressing cells at pH 7.5 but was unable to stimulate activation at pH 5.5. VEGF165 was unable to activate Erk1/2 at pH 7.5 or 5.5 in the parent NIH-3T3 cells (data not shown).

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FIG. 6. Erk1/2 phosphorylation in response to VEGF165 and VEGF121 at pH 7.5, 6.5, and 5.5. Effect of pH on VEGF-stimulated Erk1/2 activation. Subconfluent BAECs were treated with pH 7.5, 6.5, or 5.5 buffers for 90 min at 37 °C prior to the addition of 0.6 nM VEGF165 (A) or 0.7 nM VEGF121 (B) and incubated at 37 °C for the various times indicated. Cells were extracted, and SDS-PAGE (12%) was conducted and transferred to Immobilon membranes. Blots were hybridized with anti-phospho-Erk1/2 antibody and visualized with ECL reagent. Blots were stripped of antibodies and rehybridized with anti-Erk1/2 antibody and visualized with ECL reagent. Quantitation of band intensities by densitometry revealed that VEGF165 showed a 4-fold greater level of Erk1/2 activation (activation = density of activated Erk1/2 in VEGF treated/density of activated Erk1/2 in corresponding untreated well) after 2 min at pH 7.5 compared with pH 6.5. After 10-min treatment, the levels of activation were similar (activation = 8.4 versus 6.7 at pH 7.5 and 6.5, respectively). At pH 5.5 VEGF165 induced no Erk1/2 activation (activation = 1.05 and 1.03 at 2 and 10 min, respectively). For VEGF121, activation peaked after 5-min treatment at pH 7.5 (activation = 15.0) and after 10 min at pH 6.5 (activation = 17.1), whereas VEGF121 induced only minimal activation at the 5- and 10-min time points at pH 5.5 (activation = 1.2 and 1.6, respectively).
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To ensure that the acidic pH (5.5) did not damage VEGF in such a way as to eliminate activity, experiments were conducted where VEGF165 was incubated at pH 5.5 for 20 min prior to the addition of cells at pH 7.5 (Fig. 7A). Preincubating VEGF165 at acidic pH showed similar Erk1/2 activation as untreated VEGF165 with a peak activation time of 5 min. Therefore, the pH affects on VEGF165 activity were reversible. Also, to ensure that BAECs activity could be recovered after being exposed to acidic pH, experiments were conducted where BAECs were incubated at pH 5.5 for 30 min at 37 °C followed by pH 7.5 incubation for 60 min and then stimulated with VEGF165 for 2, 5, and 10 min (Fig. 7B). It was found that cells could be stimulated in response to VEGF165 to the same extent as cells that were not exposed to acidic pH. Thus, the low pH treatment did not damage VEGF165 or BAECs.

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FIG. 7. Recovery of Erk1/2 phosphorylation in response to VEGF165 incubated at pH 5.5. A, effect of pH on VEGF165 activation of Erk1/2. Subconfluent BAECs were treated with pH 7.5 or 5.5 buffers for 90 min at 37 °C. VEGF165 (23.8 nM) was incubated at pH 5.5 for 20 min prior to the addition to cells at a final concentration of 0.6 nM. Cells were incubated at 37 °C for the various times indicated. Acid-treated VEGF165 stimulated Erk1/2 activation at pH 7.5 but not at pH 5.5 (activation = 10.1 and 1.3 at the 5-min point for pH 7.5 and 5.5, respectively). B, effect of pH on BAECs activity. Subconfluent BAECs were incubated at pH 5.5 or 7.5 for 30 min followed by 60-min incubation at pH 5.5 or 7.5. VEGF165 (0.6 nM) was added to cells for 2, 5, or 10 min. Cells were extracted and subjected to SDS-PAGE (12%) and transferred to Immobilon membranes. Blots were hybridized with anti-phospho-Erk1/2 antibody and visualized with ECL reagent. Blots were stripped of antibodies and rehybridized with anti-Erk1/2 antibody and visualized with ECL reagent. Erk1/2 activation levels were similar for cells maintained at pH 7.5 (activation = 3.3) versus those preincubated at pH 5.5 and then switched to pH 7.5 (activation = 4.3).
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DISCUSSION
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Low extracellular pH is a common feature of solid tumors. Endothelial cells are exposed to this environment while undergoing angiogenesis under many pathological and physiological conditions. pH could have an impact on extracellular proteins, which would ultimately influence cell activity. Forsten et al. (39) discovered that low extracellular pH increases cell surface binding and nuclear localization of IGF-1. Recently, Wahl et al. (40) found that, under acidic conditions, angiostatin decreased endothelial cell migration and increased cell death. However, D'Arcengelo et al. (32) found that endothelial cells are protected from apoptosis in an acidic environment. These results provide evidence for the importance of pH in biological systems. Our results demonstrate that a consequence of decreased local pH is an increase in cell surface and ECM binding of VEGF165 and VEGF121. The increased VEGF binding at low pH could be disrupted by high salt and neutral pH. Thus, acidic conditions appear to alter VEGF and/or VEGF binding sites reversibly, suggesting that VEGF deposited within hypoxic and acidic regions of tissues would be rapidly released as the pH increases, where it could stimulate nearby endothelial cells.
The mechanism by which acidic pH increases VEGF binding to cells and ECM appears to involve increased binding to HSPGs and is not dependent on VEGF receptors. The acidic pH increase in binding was observed with VEGFR-deficient cells, CHO and NIH-3T3, as well as with acellular ECM. Moreover, the increased binding to cells and ECM was reduced by pretreatment with the heparan sulfate-degrading enzyme heparinase III, indicating that HSPGs are involved in this process. However, heparinase treatment of the ECM only caused VEGF binding to decrease by
30% indicating that there are other ECM components involved in the acidic pH binding component of VEGF. Recently it was found that VEGF165 binds to fibronectin (41). Therefore, VEGF interactions with fibronectin might also be modulated by pH. Moreover, the ability of fibronectin to bind to heparin and heparan sulfate suggests the possibility that VEGF, HSPGs, and fibronectin may participate in the formation of a high affinity ternary complex within the matrix that might be stabilized by low pH. The increased interaction of VEGF with HSPGs at low pH does not likely involve the traditional heparin-binding domain on VEGF165, because increased binding is observed with VEGF121 as well. Thus, reduced pH could stabilize VEGF structure for optimal interaction or may create new heparin-binding sites within VEGF. Indeed, within the shared regions of VEGF121 and VEGF165 sequence there are several histidine residues near basic amino acids that could comprise regions of positive charge at acidic pH.
Consistent with the model that acidic pH induces the formation of a new heparin-binding domain within amino acids 1121 in VEGF, we observed a greater relative effect of pH on VEGF121 binding to BAECs compared with that with VEGF165. Although VEGF165 binds HSPGs well under neutral conditions, a new heparin-binding site under acidic conditions may enhance this existing property. In contrast, VEGF121 does not contain the neutral pH heparin-binding domain; thus, the generation of the property to bind heparin at low pH results in
20-fold increased binding. This property might be conferred by the generation of a new binding site under acidic conditions, or, alternatively, it might result from a conformational change in the growth factor by positioning basic residues properly to bind to heparin.
The addition of exogenous heparin had different affects on VEGF165 and VEGF121 binding to endothelial cells. At neutral pH, low concentrations of heparin potentiated VEGF binding while high concentrations inhibited VEGF binding. This data is supported by Gitay-Goren et al. (16), who have found that exogenous heparin potentiates VEGF binding to bovine aortic endothelial cells. However, at acidic pH, all concentrations of heparin attenuated VEGF binding. For VEGF165, the addition of low concentrations of heparin enhanced VEGF165 binding at neutral pH to levels that were equivalent to those for VEGF165 binding at low pH. The addition of exogenous heparin had different affects on VEGF121 binding. High concentrations of heparin inhibited VEGF121 binding at neutral and acidic pH. Low concentrations of heparin enhanced VEGF121 binding. However, unlike VEGF165, heparin could not enhance VEGF121 binding to the high levels observed at acidic pH. The differences in heparin effects on VEGF165 and VEGF121 binding might reflect the generation of a new heparin-binding domain in VEGF at acidic pH.
The mitogen-activated protein kinase pathway has been shown to be activated in response to VEGF in endothelial cells (38). One component of this pathway is the activation of Erk1/2. In this study, we utilized the activation of Erk1/2 as a marker of biological response to VEGF under different extracellular pHs. It appeared that VEGF-mediated Erk1/2 activation at pH 5.5 was reduced to basal levels. These data correlate with other results showing that pro-angiogenic activity is reduced under acidic pH (31, 40). Moreover, exposing the cells to an acidic pH did not damage cells, because cells that were returned to neutral pH could once again be stimulated by VEGF165. Also, acidic pH did not appear to induce a permanent change in VEGF structure, in that, once VEGF returned to neutral pH, it was able to stimulate Erk1/2 activation.
Our data suggest the possibility that extracellular pH might participate in directing angiogenesis by establishing gradients of VEGF stored within the ECM. In environments where the extracellular pH is decreased, such as at sites of injury and tumors, VEGF may be sequestered in the ECM via its potential new heparin-binding site. As new vasculature is recruited to these sites, the pH would increase and the stored VEGF would be released from the ECM to further activate endothelial cells. In conclusion, we have found that extracellular pH can dramatically modulate VEGF binding to and activity in endothelial cells. Increased VEGF deposition within the ECM in hypoxic and locally acidic tissue environments might participate in guiding the growth of new blood vessels to these undervascularized regions.
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FOOTNOTES
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* This work was supported by National Institutes of Health Grant HL56200. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, Boston, MA 02118. Tel.: 617-638-4169; Fax: 617-638-5339; E-mail: nugent{at}biochem.bumc.bu.edu.
1 The 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. 
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ACKNOWLEDGMENTS
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We thank Dr. Elizabeth Denhom at Biomarin Pharmaceuticals for generously providing heparinase III. We are grateful to Dr. Nader Rahimi for providing valuable reagents and advice during these studies. We thank Dr. Kimberly Forsten-Williams for helpful discussions.
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