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Originally published In Press as doi:10.1074/jbc.M308482200 on October 21, 2003

J. Biol. Chem., Vol. 279, Issue 3, 2307-2315, January 16, 2004
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pH Regulates Vascular Endothelial Growth Factor Binding to Fibronectin

A MECHANISM FOR CONTROL OF EXTRACELLULAR MATRIX STORAGE AND RELEASE*

Adrienne L. Goerges and Matthew A. Nugent{ddagger}

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, August 1, 2003 , and in revised form, September 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypoxia is one of the major signals that induces angiogenesis. Hypoxic conditions lead to reduced extracellular pH. Vascular endothelial growth factor (VEGF) binding to endothelial cells and the extracellular matrix (ECM) increases at acidic pH (7.0-5.5). These interactions are dependent on heparan sulfate proteoglycans, but do not depend on the presence of VEGF receptors. Here we report that VEGF165 and VEGF121 binding to fibronectin also increased at acidic pH, and that these interactions are further enhanced by the addition of heparin. These results reveal that the accepted non-heparin-binding isoform of VEGF (VEGF121) is converted into a heparin-binding growth factor under acidic conditions. Interestingly, we did not observe increased binding of VEGF to collagen type I at acidic pH in the presence or absence of heparin, indicating that this effect is not a general property of all heparin-binding ECM proteins. The high level of VEGF binding at acidic pH was also rapidly reversed as demonstrated by increased rates of VEGF dissociation from fibronectin and fibronectin-heparin matrices as the pH was raised. The VEGF released from fibronectin retained its ability to stimulate the activation of extracellular-regulated kinase 1/2 in endothelial cells. These results suggest that VEGF may be stored in the extracellular matrix via interactions with fibronectin and heparan sulfate in tissues that are in need of vascularization so that it can aid in directing the dynamic process of growth and migration of new blood vessels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis is the process by which new vessels develop from pre-existing vessels (1). Under normal conditions, the vascular endothelium is quiescent in adults, but it can be initiated to respond by a number of normal and pathological stimuli, such as, injury and cancer (2, 3). In the development of malignant cancers, it has been suggested that a switch occurs to trigger an angiogenic response (4, 5). Some of the signals that trigger the switch include metabolic stress, mechanical stress, and genetic mutations, such as, low oxygen, low pH, and the activation of oncogenes that control the production of angiogenic regulators (6, 7). An imbalance between the demand and supply of oxygen and nutrients in tissues promotes angiogenesis in pathological disorders, such as tumor metastasis and diabetic retinopathy. Vascular endothelial growth factor (VEGF)1 is a major regulator of endothelial cell activity and angiogenesis (8). Several isoforms exist for VEGF that differ in their ability to interact with heparan sulfate proteoglycans (HSPG) (9). The most predominant isoforms are VEGF121, VEGF165, and VEGF189. VEGF121 lacks the native heparin-binding domain and is unable to directly interact with heparan sulfate (HS). VEGF165 interacts directly with HS and is dependent on HS for maximal activity (10). VEGF189 is predominantly found sequestered in the extracellular matrix (ECM) via HSPG (11).

The extracellular matrix surrounding the vascular endothelium is comprised of two compartments, the vascular basement membrane that lies between the endothelial cells and the vascular smooth muscle cells and the interstitial matrix. The interstitial matrix is comprised primarily of fibrillar collagens and glycoproteins such as fibronectin. Fibronectin is suggested to play an important role in endothelial cell recognition and response to ECM during angiogenesis (3). Fibronectin consists of three repeating domains, fibronectin type I, type II, and type III and interacts with many molecules including: fibrin, collagen, integrins, and HSPG. Fibronectin contains a heparin-binding domain and has been found to interact with syndecan-4 and glypican-1 (12). Therefore, HSPG have been proposed to function as receptors for anchoring heparin-binding ECM proteins, like fibronectin, to endothelial cells (13, 14). Recently fibronectin has also been shown to bind VEGF, suggesting an additional role for this ECM protein in the storage of VEGF (15). Thus, in addition to its important roles in modulating endothelial cell adhesion and migration, fibronectin is also likely involved in regulating VEGF interactions with the endothelium during angiogenesis.

New vasculature is usually recruited to regions of tissue where demand for blood circulation is high. One principle indicator of undervascularization is low pO2 levels (hypoxia). Indeed, hypoxia has been found to be a major inducer of angiogenesis in many physiological and pathological processes, such as wound healing and tumor metastasis (16, 17). Hypoxia has been shown to effect the expression of a number of proteins, including VEGF (18-20). One characteristic of a hypoxic environment is reduced extracellular pH. Wound beds have been shown to have an extracellular pH ~6, whereas tumors have been measured to have decreased extracellular pH ranging from pH 7.0 to as low as pH 5.8 (21, 22). One might expect that acidic pH would have detrimental effects on endothelial cells. However, endothelial cells survive fairly well under these conditions (23). Indeed, it was found that the rate of VEGF-stimulated microvessel growth is increased at acidic pH in an endothelial cell culture model system (23). 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. Extracellular pH could impact extracellular protein structure and interactions, which may ultimately influence cell activity. It was discovered that low extracellular pH increases cell surface binding and nuclear localization of IGF-1 in the presence of its binding protein IGFBP-3 (24). Moreover, we have found that VEGF binding to cells and the ECM is increased under acidic conditions and these interactions were dependent on HSPG (25). It is possible that the increased binding of VEGF to the ECM at low pH reflects changes in VEGF binding to fibronectin. Interestingly, hypoxic conditions have been shown to stabilize fibronectin and increase its secretion from cells (26). In addition, fibronectin has been associated with being highly expressed in several tumors (27-30). Thus, fibronectin might participate in directing angiogenesis via pH-sensitive binding of VEGF.

In the present study, we evaluated the effects of pH on VEGF-fibronectin interactions. We found that as the pH decreased, VEGF interactions with fibronectin increased. We also found that heparin enhanced both VEGF165 and VEGF121 interactions with fibronectin at acidic pH. In addition, the increased binding at low pH was reversed when the pH was returned to neutral. We further found that VEGF that had released from fibronectin at neutral pH retained its ability to stimulate extracellular-regulated kinases 1/2 (Erk1/2) phosphorylation in endothelial cells. Thus, acidic extracellular pH may serve to enhance VEGF storage in the ECM at sites of high vascular demand.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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). Heparinase II and Heparinase I from F. heparinum were from Seikagaku America, Inc. (Ijamsville, MD). Heparin, phenylmethylsulfonyl fluoride, sodium orthovanadate, fibronectin, and secondary antibody raised against rabbit IgG and conjugated with horseradish peroxidase were obtained from Sigma. 125I-Bolton-Hunter reagent was obtained from PerkinElmer (Boston, MA). Bovine serum albumin was obtained from American Bioanalytical (Natick, MA). Phosphate-buffered saline, Dulbecco's Modified Eagle's Medium (DMEM), penicillin/streptomycin, L-glutamine, and HEPES buffer were from Invitrogen. Collagen I was purchased from BD Biosciences. Calf Serum (CS) was from Hyclone (Logan, UT). Primary antibodies for phospho-extracellular-regulated kinase 1/2 (Erk1/2), and total Erk1/2 was purchased from New England Biolabs (Beverly, MA). ECL detection kit was purchased from Amersham Biosciences (Uppsala, Sweden). 125I-VEGF165 and 125I-VEGF121 were prepared by a modified Bolton-Hunter procedure and retained their ability to stimulate endothelial cells (25).

Cell Culture—Bovine aortic endothelial cells (BAEC) were a gift from Dr. Elazer Edelman at MIT (Cambridge, MA). BAEC were maintained in low glucose DMEM supplemented with 10% calf serum, 5 mM glutamine, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate. For experiments, BAEC were used between passages 8 and 16. Cell number was determined with a Coulter Counter (Miami, FL).

Preparation ECM-coated Dishes—ECM protein-coated dishes were prepared as previously described (31). Non-treated tissue culture dishes were coated with either fibronectin, collagen I, or bovine serum albumin (BSA) at 20 µg/ml in ion free phosphate-buffered saline overnight at 4 °C. To maintain consistent coating between dishes, 250 µl per 1 cm2 was used to coat the dishes (31). Protein assays reveal ~70% adsorption of proteins under these conditions.

125I-VEGF Binding—Equilibrium binding assays were carried out on fibronectin, collagen I, or BSA-coated dishes. Binding assays conducted on BAEC were carried out as previously described (25). Binding assays conducted at various pH values were carried out in binding buffer containing 25 mM HEPES adjusted to the indicated pH (7.5-5.5) in DMEM (without bicarbonate) containing 0.1% BSA. Matrices were washed once with ice-cold binding buffer. Binding buffer was added to the matrices and incubated at 4 °C for 10 min. 125I-VEGF165 (0.12 nM) or 125I-VEGF121 (0.14 nM) was added to matrices and the binding reaction was allowed to proceed for 2.5 h at 4 °C. After the binding period, unbound 125I-VEGF was removed by washing the matrices three times with ice-cold binding buffer. Matrices were then solubilized with 1 N NaOH to extract the bound 125I-VEGF. 125I-VEGF binding was quantified by counting in a Cobra Auto-Gamma 5005 {gamma}-counter (Packard Instruments, Meridian, CT). Nonspecific binding was measured using a 500-fold excess of unlabeled VEGF and this value was subtracted from each sample. To determine the effects of removing heparan sulfate proteoglycans, fibronectin-coated plates were treated with 0.5 units/ml of heparinase III for 1 h at 37 °C prior to conducting the binding studies. Heparinase III and digestion products were removed by washing two times with binding buffer. To determine the effects of heparin, various concentrations were added to the binding buffer prior to the addition of 125I-VEGF. All conditions were conducted in triplicate, and each experiment was repeated at least three separate times.

Dissociation of 125I-VEGF from Fibronectin—Fibronectin-coated dishes were prepared and binding assays were conducted at pH 7.5 or pH 5.5 as described. 125I-VEGF165 (0.6 nM) or 125I-VEGF121 (0.7 nM) was added to fibronectin for 2.5 h at 4 °C. After the binding incubation, unbound VEGF was removed by washing the fibronectin-coated dishes three times with binding buffer. Fresh binding buffer (pH 7.5, 6.5, 5.5) was added (500 µl/well) and the plates were allowed to incubate at 4 °C. At various time points, the buffer containing released VEGF was collected, the wells were washed once with 500 µl of the corresponding buffer, fresh buffer was added, and the incubation continued. After the final time point, the fibronectin was solubilized in 1 N NaOH to account for the remaining 125I-VEGF bound. The effect of heparin was also evaluated on VEGF dissociation from fibronectin. Heparin (1 µg/ml) was added to fibronectin prior to the addition of 125I-VEGF. After the binding incubation, fresh buffer without heparin was added to the well.

Activation of Erk1/2 in BAEC—Erk12/activation in BAEC was evaluated in response to VEGF that had been pre-bound to BAEC at pH 5.5. BAEC were plated at 20,000 cells per well in 6-well dishes. After 24 h, the medium was replaced with DMEM containing 0.5% CS for 24 h, to quiesce the cells. Cells were incubated with binding buffer at pH 5.5 for 10 min at 37 °C. VEGF165 (0.6 nM) was added to the cells at pH 5.5 for 60 min at 37 °C. Unbound VEGF165 was removed. Cells were incubated for 10 min in buffer at pH 7.5, 7.0, 6.5, 6.0, or 5.5. Erk1/2 activation in BAEC was evaluated in response to VEGF that had been dissociated from fibronectin. Quiescent endothelial cells were generated as stated above. Binding buffer (1 ml) was added to cells at pH 7.5 or pH 5.5, for 90 min. VEGF165 (3.0 nM) and VEGF121 (3.5 nM) were allowed to bind to fibronectin-coated dishes in the presence of 1 µg/ml of heparin for 1 h at 37 °C as described above. After binding, fibronectin-coated dishes were washed three times to remove any unbound VEGF. New binding buffer (1 ml) was added to the fibronectin-coated dishes at pH 7.5 or pH 5.5 for 20 min. The buffer containing released VEGF was collected and added to BAEC. Cell lysates were collected after 2, 5, 10, 20, and 30 min stimulation. 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 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. Blots were stripped of antibodies and reprobed with anti-Erk1/2 antibody. Immunoreactive bands were visualized with chemiluminescence using horseradish peroxidase-conjugated anti-rabbit IgG and ECL reagent. Membranes were stained with Ponceau S to evaluate total protein loaded. Experiments were repeated at least three times.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Heparinase Decreases VEGF165 and VEGF121 Binding to BAEC—We previously found that VEGF165 and VEGF121 interactions with BAEC and ECM are enhanced by acidic pH (25). In addition, VEGF165 and VEGF121 binding to BAEC and ECM at pH 7.5 and pH 5.5 was reduced by pretreating the cells with heparinase III to digest HS chains (25). These results suggested that VEGF interactions with endothelial cells at acidic pH are dependent on HS. However, heparinase III treatment only reduced binding by ~35% suggesting that other binding components exist for VEGF at reduced pH, or that heparinase III digestion did not completely remove all of the HS chains involved. To explore the possibility that the VEGF binding sites on HS were not fully degraded by heparinase III alone, we conducted binding assays on BAEC that were treated with various heparinases (Fig. 1). BAEC were treated with heparinase I, heparinase II, heparinase III, or a combination of the enzymes for 1 h at 37 °C. Binding assays were conducted at pH 5.5 with VEGF165 and VEGF121. It was observed that heparinase I, II, and III reduced VEGF165 and VEGF121 binding by ~40%. Combining the various heparinases did not further reduce binding. These results indicate that VEGF binding at acidic pH involves components in addition to HS.



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  		FIG. 1.
Effects of heparinase treatments on 125I-VEGF165 and 125I-VEGF121 binding to BAEC at pH 5.5. BAEC were treated with 0.5 units/ml of heparinase II or III; or 1 µg/ml of heparinase I or various combinations of each for 1 h at 37 °C prior to conducting binding assays with 125I-VEGF165 (0.16 nM) and 125I-VEGF121 (0.14 nM) at pH 5.5. After 125I-VEGF165 (black bars) and 125I-VEGF121 (white bars) binding occurred, the cells were washed with a high salt, neutral pH buffer to release bound VEGF. Samples were quantitated in a {gamma} counter. Treatments are labeled as: NT, no treatment; I, heparinase I; II, heparinase II; III, heparinase III. Representative data are presented as the mean of triplicate determinations ± S.E., and % Bound was defined as: (VEGF bound in heparinase-treated/VEGF-bound in untreated) x 100. An ANOVA was conducted and revealed that the enzyme treatments caused a statistically significant reduction in VEGF165 and VEGF121 binding (p < 0.01). However, ANOVA of the various enzyme treatments (I, II, III, or combinations) revealed no significant differences between treatments (p = NS).

 
VEGF Binding to Fibronectin Increases with Decreasing pH—Previously we found that the increased binding of VEGF to cells at low pH was not dependent on the expression of VEGF receptors (25). It has also recently been shown that VEGF directly binds to fibronectin near the N- and C- terminal domains. Therefore, altered VEGF-fibronectin interactions may be responsible for the HS-independent binding of VEGF at low pH. To test this possibility, we conducted binding assays on fibronectin-coated dishes with VEGF165 and VEGF121 at pH 7.5, 6.5, and 5.5 (Fig. 2). It was found that as the pH decreased, VEGF165 and VEGF121 binding to fibronectin increased. VEGF165 binding at pH 5.5 was increased by ~4-fold and VEGF121 binding was increased by ~9-fold compared with that at pH 7.5. Therefore, VEGF interactions with fibronectin appear to be sensitive to pH. The fibronectin used was purified from bovine plasma, and fibronectin is known to bind heparin and HS. Thus, it is possible that these preparations might contain contaminating HS, which could be responsible for the pH-sensitive binding of VEGF to the fibronectin-coated dishes. To determine if endogenous HS in the fibronectin matrix is contributing to VEGF binding at acidic pH, we treated fibronectin-coated dishes with heparinase III to remove any contaminating HS chains. We observed that heparinase treatment had no effect on VEGF165 or VEGF121 binding to fibronectin at neutral or acidic pH (Fig. 3, A and B). Therefore, we conclude that the increased binding of VEGF to fibronectin at acidic pH is not the result of contaminating HS.



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  		FIG. 2.
Effects of pH on 125I-VEGF165 and 125I-VEGF121 binding to fibronectin. Fibronectin-coated dishes were prepared. Binding assays were conducted using 0.12 nM 125I-VEGF165 (black bars) and 0.14 nM 125I-VEGF121 (white bars). Total binding was determined by extraction in 1 N NaOH. Samples were quantitated using a {gamma} counter. Representative data are presented as the mean of triplicate ± S.E. ANOVA followed by the Newman-Keul's multiple comparison t test was run revealing that VEGF165 and VEGF121 binding was significantly different at the three pHs tested (ANOVA, p < 0.001 for both VEGF165 and VEGF121; Newman-Keul's, p < 0.001 for all comparisons).

 



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  		FIG. 3.
Heparinase III treatment of fibronectin. Fibronectin-coated dishes were treated with 0.5 units/ml of heparinase III. Binding assays were conducted using 0.12 nM 125I-VEGF165 (A) and 0.14 nM 125I-VEGF121 (B). Total binding was determined by extraction in 1 N NaOH. Samples were quantitated using a {gamma} counter. Representative data are presented as the mean of triplicate ± S.E. Heparinase III treatment showed no statistically significant effects on VEGF165 or VEGF121 binding (based on paired Student's t test).

 
Fibronectin is just one component of the ECM. Therefore, we wanted to investigate whether the ability to bind VEGF at low pH was a general property of protein-coated surfaces or if it was specific to fibronectin. Toward this end, we conducted binding assays on collagen type I-coated dishes and bovine serum albumin (BSA)-coated dishes in comparison with binding to fibronectin-coated dishes (Fig. 4). It was observed that there was no significant binding of VEGF165 (Fig. 4A) or VEGF121 (Fig. 4B) to collagen type I or to BSA at pH 7.5 or pH 5.5. Therefore, the binding observed for fibronectin was not a general nonspecific interaction that occurs with all proteins absorbed to tissue culture plastic.



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  		FIG. 4.
VEGF165 and VEGF121 binding to fibronectin, collagen, and BSA at neutral and acidic pH. Binding assays were conducted on fibronectin (FN), collagen type I (Col), and BSA. 125I-VEGF165 (0.12 nM) (A) and 125I-VEGF121 (0.14 nM) (B) were added to dishes for 2.5 h at 4 °C at pH 7.5 and pH 5.5. Total binding was determined by extraction in 1 N NaOH. Samples were quantitated using a {gamma} counter. Representative data are presented as the mean of triplicate ± S.E. Binding of VEGF165 and VEGF121 were significantly different at the two pHs tested (p < 0.01), while there was no significant effects of pH on binding to collagen or BSA (based on paired Student's t test).

 
Heparin Potentiates VEGF Binding to Fibronectin at Acidic pH—Fibronectin is a complex ECM protein with many protein-binding domains. HSPG have been found to interact with fibronectin in two domains, one near the N terminus and the other near the C terminus. Certain isoforms of VEGF also interact with HS. It has been shown that VEGF interactions with heparin are enhanced by decreased pH. Therefore, we wanted to determine if VEGF-heparin-fibronectin interactions are affected by changes in pH. Also, collagen type I interacts with HS, therefore, HS may serve to bridge VEGF to collagen type I. We conducted binding assays with VEGF165 and VEGF121 on fibronectin- and collagen type I-coated dishes in the presence of various concentrations of heparin at pH 7.5 and 5.5. Heparin had no effect on VEGF165 or VEGF121 binding to collagen type I at either pH (Fig. 5). However, low concentrations (1 µg/ml) of heparin potentiated VEGF165 binding to fibronectin at pH 7.5, while high concentrations of heparin (100 µg/ml) decreased VEGF165 binding. At pH 7.5, heparin had no effect on VEGF121 binding to fibronectin, consistent with the fact that VEGF121 is unable to interact with heparin at neutral pH. However, at pH 5.5, low concentrations of heparin (1 µg/ml) increased both VEGF165 and VEGF121 binding to fibronectin, while high concentrations of heparin (100 µg/ml) decreased binding (Fig. 5B). These effects are consistent with the effects of heparin on VEGF binding to endothelial cells (10, 25). Interestingly, these data support our previous finding that VEGF121 is converted from a non-heparin binding growth factor at pH 7.5 to one that binds heparin at acidic pH. Therefore, heparin and heparan sulfate might participate with fibronectin to modulate VEGF165 and VEGF121 binding to the ECM at acidic pH.



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  		FIG. 5.
Effects of heparin on VEGF binding to fibronectin and collagen. Binding assays were conducted on fibronectin. 125I-VEGF165 (0.12 nM) was added to fibronectin ({diamondsuit}) and collagen ({blacktriangleup}); and 125I-VEGF121 (0.14 nM) was added to fibronectin ({square}) and collagen ({circ}) for 2.5 h at 4 °C in the presence of various concentrations of heparin at pH 7.5 (A) and pH 5.5 (B). Total binding was determined by extraction in 1 N NaOH. Samples were quantitated using a {gamma} counter. Representative data are presented as the mean of triplicate ± S.E.

 
Dissociation of VEGF from Fibronectin—A fibronectin-heparin matrix at acidic pH may be acting to store VEGF165 and VEGF121 at acidic pH, trapping VEGF at sites of high vascular demand to be released to stimulate endothelial cell invasion. Therefore, we evaluated if VEGF that was bound to fibronectin and fibronectin-heparin matrices at acidic pH, could be stimulated to be released from the matrix by increasing the pH. Experiments were conducted where VEGF165 and VEGF121 were allowed to bind to fibronectin or fibronectin-heparin dishes at pH 5.5. After binding, unbound VEGF was removed and fresh buffer, adjusted to pH 7.5, 6.5, or 5.5, was added to the wells. Samples were collected at various times and dissociated VEGF was measured. It was found that dissociation of VEGF165 and VEGF121 from fibronectin and fibronectin-heparin was slow at pH 5.5 compared with pH values 6.5 and 7.5 (Fig. 6). Correspondingly, the calculated rates of VEGF dissociation increased significantly as the pH was increased (Table I). Consistent with the conditional heparin-binding characteristics of VEGF121, heparin caused a significant reduction of VEGF121 dissociation at pH 5.5 (Fig. 6, C and D). However, heparin did not reduce VEGF165 dissociation at any of the pH values tested. In fact, heparin caused a slight increase in VEGF165 release at pH 7.5 suggesting that the VEGF165-heparin-fibronectin complexes dissociate faster than the VEGF165-fibronectin complexes (Fig. 6, A and B). These results suggest that the presence of the native heparin-binding domain contributes to the overall dissociation of VEGF165 from fibronectin-heparin matrices. Thus, low pH appears to stabilize VEGF-fibronectin complexes, yet these tight interactions can readily be reversed by exposure to neutral pH.



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  		FIG. 6.
VEGF dissociation from fibronectin in the presence or absence of heparin with increasing pH. Binding assays were conducted on fibronectin at pH 5.5 with 125I-VEGF165 (0.6 nM) in the absence of heparin (A) and in the presence of heparin (B); and 125I-VEGF121 (0.7 nM) in the absence of heparin (C) and in the presence of heparin (D). After binding, fresh buffer was added to fibronectin at pH 7.5 ({diamondsuit}), pH 6.5 ({square}), and pH 5.5 ({triangleup}) without heparin and collected at various time points. Samples were quantitated using a {gamma} counter. Representative data are presented as the mean of triplicate ± S.E.

 


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  		TABLE I
Rates of dissociation of VEGF from fibronectin

VEGF165 and VEGF121 bound to fibronectin-coated dishes in the presence (+) and absence (-) of heparin was allowed to dissociate at pH 7.5, 6.5 or 5.5 as described in Fig. 6. The data generated were analyzed using KaleidaGraph version 3.51 to determine observed dissociation rates based on a reversible bi-molecular process, as

bound to fibronectin, VEGFr is free VEGF, and FN is fibronectin, such that the rate of dissociation (V) relates to the appearance of free VEGF

 
Erk1/2 Activation by VEGF after Dissociation from Fibronectin—Since we have established that VEGF bound to fibronectin at acidic pH can be released by increasing the pH, we wanted to determine if the dissociated VEGF was active. We used Erk1/2 activation as a marker for VEGF activity on BAEC. VEGF was bound to fibronectin in the presence of heparin for 1 h at 37 °C at pH 7.5 and pH 5.5. Unbound VEGF was removed and bound VEGF was allowed to dissociate for 20 min in pH 7.5 or pH 5.5 buffer. Dissociated VEGF was then added to quiescent BAEC. Erk1/2 activation was analyzed after 10 min treatment of the cells. It was found that VEGF165 that was bound at pH 7.5 and dissociated at pH 7.5 stimulated a small degree of Erk1/2 phosphorylation (Fig. 7A). In contrast, VEGF165 bound at pH 5.5 and dissociated at pH 5.5 displayed no activation of Erk1/2. VEGF165 bound at pH 5.5 and dissociated from fibronectin at pH 7.5 showed the highest degree of Erk1/2 phosphorylation. Similar results were observed for VEGF121 binding to fibronectin at pH 5.5 and dissociated at pH 7.5 (Fig. 7B). These results indicate that VEGF bound at pH 5.5 and released at pH 7.5 retains its biological activity. Moreover, the relative activities of the various samples are consistent with the VEGF binding and release data (Figs. 2, 3, 4, 5, 6), which predicts that samples bound at pH 5.5 and released at pH 7.5 would have the highest levels of VEGF. Thus, fibronectin and heparin may act to store VEGF in the ECM at acidic pH in a dormant state, and once the pH is returned to neutral, VEGF is released from these storage sites to stimulate target cells.



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  		FIG. 7.
VEGF activity after binding to fibronectin. Binding assays were conducted on fibronectin-coated dishes with 3.0 nM of VEGF165 (A) and 3.5 nM of VEGF121 (B) in the presence of heparin (1 µg/ml) at 37 °C for 1 h. Meanwhile, subconfluent BAEC were treated with pH 7.5 or pH 5.5 buffers for 90 min at 37 °C. After binding occurred on fibronectin, unbound VEGF was removed and fresh buffer was added to the fibronectin-coated dishes and the incubation continued for 20 min. Media containing released VEGF was collected and added to BAEC for 10 min. Control experiments were conducted on quiescent BAEC by adding 0.6 nM VEGF165 (A) or 0.7 nM VEGF121 to cells for 10 min at pH 7.5 or pH 5.5. Cells were extracted and SDS-PAGE (12%) was conducted and transferred to Immobilon membranes. Blots were probed with antiphospho-Erk1/2 antibody and visualized with ECL reagent. Blots were stripped of antibodies and reprobed with anti-Erk1/2 antibody.

 
VEGF Activation of Erk1/2 at pH 7.5 after Prebound to BAEC at pH 5.5—VEGF binding to endothelial cells increased under acidic conditions but this did not translate into increased activity at the level of VEGFR-2 and Erk1/2 phosphorylation (25). Thus, the increased binding might represent a mechanism whereby VEGF is stored in the ECM where it cannot activate cells. At higher pH the stored VEGF might be released and become available to stimulate cells. To evaluate this model, we conducted experiments to determine if VEGF bound to cells at pH 5.5 can be induced to activate Erk1/2 in endothelial cells when the pH is raised. VEGF165 was bound to endothelial cells at pH 5.5 for 1 h at 37 °C. Unbound VEGF was removed, and new media was added to cells at pH 7.5, 7.0, 6.5, 6.0 or pH 5.5. Cell lysates were collected at 10 min (Fig. 8). It was observed that VEGF165 bound to cells at pH 5.5 could activate Erk1/2 at pH 7.5, 7.0, and 6.5 but not at pH 6.0 or 5.5. In particular, between pH 6.5 and 6.0 there appears to be a critical switch in activation potential and or release of bound VEGF. Therefore, VEGF165 that bound to BAEC at acidic pH was able to stimulate Erk1/2 activation once the extracellular pH was increased toward pH 7.5.



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  		FIG. 8.
VEGF165 binding to BAEC at pH 5.5 followed by activation at various pH values. VEGF165 (0.6 nM) was added to cells at pH 5.5 for 60 min at 37 °C. Unbound VEGF165 was removed. Cells were incubated for 10 min at pH 7.5, 7.0, 6.5, 6.0, or 5.5 (top panel). The bottom panel represents the stimulation of subconfluent BAEC by directly adding 0.6 nM VEGF165 (no prior binding occurred) to the cells at the indicated pH (7.5-5.5). Cells were extracted and subjected to SDS-PAGE (12%) and transferred to Immobilon membranes. Blots were probed with antiphospho-Erk1/2 antibody and visualized with ECL reagent. Blots were stripped of antibodies and reprobed with anti-Erk1/2 antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Angiogenesis is an important process that complex organisms use to provide nutrients to cells that are unable to acquire them under their present conditions. One of the major signals that elicits an angiogenic response is hypoxia. Hypoxia has been shown to upregulate VEGF expression (20). Interestingly, fibronectin has been shown to be highly expressed in several tumors (27-30). In addition, hypoxic conditions have been shown to stabilize fibronectin and increase its secretion from cells (26). Hypoxic conditions also lead to decreases in extracellular pH. We have previously demonstrated that extracellular pH is an important factor in regulating VEGF interactions with cells and the ECM (25). In particular, we found that VEGF binding to HS is enhanced as the extracellular pH decreases. However, when cells were treated with heparinase III or a combination of heparinase I, II, and III (Fig. 1) to remove HS chains, VEGF binding only decreased by ~40%, suggesting that there are other VEGF binding components involved at low pH. Here, we report that VEGF interactions with fibronectin increased with decreased pH. Moreover, the addition of exogenous heparin, which interacts with fibronectin and VEGF, enhanced VEGF165 and VEGF121 binding to fibronectin at acidic pH. In addition, heparin had no effect on VEGF121 binding at neutral pH, supporting the concept that VEGF121 is converted into a heparin-binding protein at acidic pH. Thus, the regulation of VEGF binding to fibronectin by pH could play an important role in localizing VEGF within the ECM in tissues that are in need of vascularization.

Interestingly, VEGF165 and VEGF121 that bound to fibronectin at acidic pH, are capable of being rapidly dissociated by increasing the pH to neutral. In addition, when VEGF165 and VEGF121 were bound to fibronectin in the presence of heparin at pH 5.5, their rates of dissociation were different. The VEGF121-heparin-fibronectin complexes that formed at pH 5.5 dissociated significantly slower than VEGF121-fibronectin complexes at pH 5.5 and pH 6.5. This suggests that a tight complex forms between VEGF121, heparin, and fibronectin that is not easily dissociated at low pH, but once returned to pH 7.5, this complex is destabilized and dissociates at the same rate as VEGF121 in the absence of heparin. In contrast, VEGF165-heparin-fibronectin complexes formed at pH 5.5 actually dissociated slightly faster than those formed in the absence of heparin at pH 7.5 and 6.5. At 5.5, the presence of heparin had little effect on VEGF165 dissociation. These data suggest that VEGF121 and VEGF165 interact with heparin and fibronectin in different manners, which would contribute to their overall dissociation rate. While VEGF165 interacts with heparin through its well-characterized traditional heparin-binding domain (32), both VEGF165 and VEGF121 may also bind heparin at low pH via a cryptic site that is only competent to bind heparin under acidic conditions. Thus, heparin binding of VEGF121 shows complete dependence on pH, while VEGF165 retains the ability to bind heparin at neutral pH via its traditional heparin-binding domain, which is required for full biological activity at neutral pH (10, 33). Each of these VEGF-heparin interactions would be governed by its own characteristic binding kinetics such that the overall rate of VEGF release from a fibronectin-heparin matrix would represent a composite of all interactions involved. Taken together, these data indicate that the high level of binding of VEGF to fibronectin-heparin matrices can be rapidly reversed as the pH increases toward neutrality. Interestingly, we found that VEGF that was bound to a fibronectin-heparin matrix at pH 5.5, and dissociated at pH 7.5, was able to stimulate Erk1/2 activation in endothelial cells. Therefore, VEGF that is bound to matrices at low pH and dissociated at neutral pH retains its biological activity toward endothelial cells.

Hypoxia is one of the major inducers of angiogenesis and has been found to upregulate VEGF expression (20, 34). In addition, acidic pH up-regulates VEGF mRNA in human glioblastoma cells (35). HSPG, which modulate VEGF activity, are up-regulated at sites of active angiogenesis, primarily those under hypoxic conditions, such as tumors (36-40). Also, decreased extracellular pH increases the secretion of fibronectin isoforms in trophoblasts (26). Based on these previous findings and those reported here, we propose that hypoxic conditions result in the generation of an acidic extracellular environment, which leads to the storage of VEGF in the extracellular matrix via fibronectin and HSPG (Fig. 9). These acidic locations, by definition, would not be adjacent to the existing vasculature; hence there would be no target endothelial cells in the immediate environment of the secreted VEGF. However, the matrix storage system would predict the generation of a gradient of VEGF through the reversible binding of VEGF to immobile sites (fibronectin and HSPG) in the matrix (41). While the total amount of VEGF in the ECM would decrease with proximity to existing vessels, the relative proportion of VEGF in an active versus a stored state would be expected to increase. Thus, threshold concentrations of active VEGF could initiate new blood vessel sprouting and growth toward regions containing high VEGF levels. As new vessels move into regions of low pH and high VEGF levels, the corresponding extracellular pH would rise resulting in conversion of stored VEGF to the active form, further stimulating the directed growth and migration of the new vessel. Therefore, a dynamic system of reversible VEGF storage and activation within the ECM could contribute to the positional guidance of new blood vessels to undercirculated/hypoxic/acidic regions of tissue via pH-sensitive matrix binding of VEGF.



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  		FIG. 9.
Model of pH effects on VEGF interactions. A, schematic representation of VEGF interactions with fibronectin and HSPG at acidic and neutral pH in relation to its ability to stimulate VEGF receptors on endothelial cells. At low pH, there are high levels of VEGF bound to fibronectin and HSPG in the ECM with little receptor activation as indicated by Erk1/2 activation. The switch to high pH (6.5-7.5) would be proposed to be accompanied by the release of VEGF from the ECM and stimulation of VEGF receptors leading to Erk1/2 activation. B, schematic representation of how pH-sensitive binding of VEGF could contribute to directed angiogenesis into a hypoxic tissue. VEGF would be predicted to be distributed such that the area of the tissue that is most hypoxic (lowest pH) would contain the most VEGF because of the high binding to fibronectin and HSPG as well as the increased expression of VEGF. Thus, variable matrix binding of VEGF would allow a stable VEGF gradient to be established from the most acidic to the least acidic region of the tissue. Hence, the region of the tissue that is closest to the existing vasculature would contain the least amount of deposited VEGF ({circ}) yet this VEGF would be in the active state (•) such that it could participate in initiating angiogenesis. As the blood vessel grows into the acidic tissue the extracellular pH would increase causing the release of high levels of stored VEGF, further propagating blood vessel growth.

 
In addition to understanding the basic process of angiogenesis, these data suggest that a fibronectin-heparin matrix formulated under acidic pH might have therapeutic applications. For example, a fibronectin-heparin-VEGF matrix might provide a means for the controlled delivery of VEGF to places where new vasculature is required. Alternatively, using a fibronectin-heparin matrix at acidic pH might also provide a means of depleting VEGF in situations where it is detrimental, such as in certain pathological conditions. While the detailed process by which VEGF regulates angiogenesis remains unclear, it will be critical to consider the role of local changes in pH in modulating this complex process.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL56200 and by Training Grant AG00115. 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. Back

{ddagger} 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; BAEC, bovine aortic endothelial cells; BSA, bovine serum albumin; CS, calf serum; Col, collagen type I; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; Erk1/2, extracellular-regulated kinases 1 and 2; FGF, fibroblast growth factor; FN, fibronectin; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MAP kinase, mitogen-activated protein kinase; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Elizabeth Denholm at Biomarin Pharmaceuticals for generously providing heparinase III.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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