Vascular Endothelial Growth Factor Upregulates the Expression of Matrix Metalloproteinases in Vascular Smooth Muscle Cells

Role of flt-1

From the Department of Vascular and Cardiac Disease, Therapeutics, Parke-Davis Pharmaceutical Research, Ann Arbor, Mich.

	Abstract

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Abstract—Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain–containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase–polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.

Key Words: vascular endothelial growth factor • matrix metalloproteinase • vascular smooth muscle cell • angiogenesis • flt-1

	Introduction

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Vascular endothelial growth factor (VEGF) in cells is a homodimeric glycoprotein that is best known for its capacity to induce angiogenesis, to increase vascular permeability, and to stimulate production of the thrombogenic protein tissue factor.¹ First isolated from a tumor-conditioned medium, it is now clear that nonmalignant cells including vascular smooth muscle cells (SMCs), monocytes, mesangial cells, and megakarocytes² also synthesize VEGF. VEGF exerts its biological function through high-affinity tyrosine kinase receptors, ie, vascular endothelial growth factor receptors (VEGFRs), on the cellular membrane, namely, the kinase insert domain–containing receptor (KDR) and the fms-like tyrosine kinase-1 receptor (Flt-1).³ The murine homologue of KDR is also known as fetal liver kinase-1 receptor (Flk-1). On ligand binding, the VEGFR undergoes autophosphorylation with subsequent intracellular calcium mobilization, activation of phospholipase C, and upregulation of cGMP.⁴ A novel VEGF isoform-specific receptor, neuropilin-1, has been described recently.⁵

Most of our understanding about VEGF biology and its receptors derives from studies carried out in endothelial cells (ECs). Emerging evidence, however, suggests the expression of VEGFRs in cell types other than ECs. For example, flt-1 expression in monocytes has recently been confirmed; this receptor mediates chemotaxis and tissue factor production.⁶ The expression of KDR in melanoma cell lines mediates the proliferative response to exogenous VEGF.⁷ Expression of flt-1 has also been reported in retinal pericytes, trophoblasts, and renal glomerular mesangial cells.⁸ These studies indicate the influence VEGF may have on activities other than those promoting EC proliferation and migration.

We examined the effect of VEGF on vascular SMCs, another major cell type in the vascular wall. While ECs are the obvious cell type involved in angiogenesis, new vessels are soon covered by nonendothelial, desmin-positive cells.⁹ These cells are pericytes in nature, but their origin is currently unclear. Using tissue culture methods, Nicosia and Villaschi¹⁰ showed a transformation of rat aortic SMCs into pericytes. Morphologically, the SMC-derived cells shared basement membrane with ECs, had cytoplasmic processes wrapped around endothelium, and exhibited phagocytic activity. On the other hand, by using quail-chick chimeras, Stein et al¹¹ showed that microvascular pericytes could also evolve from perivascular fibroblasts. These investigators have proposed that in angiogenesis the resident mesenchymal cells are activated, migrate through extracellular matrix (ECM) barriers, undergo phenotypic alteration, and abluminally cover endothelial sprouts.^{9 10 12} Many details of this integrated process remain to be explored. It is unclear how vascular SMCs become activated and migrate from roots to tips of newly formed capillaries. However, the major importance of VEGF in angiogenesis makes it a potential candidate for mediating vascular SMC functions during angiogenesis.

A widely accepted feature of SMC activation is enhanced production of matrix metalloproteinases (MMPs), a family of zinc-dependent proteinases with maximal activity at neutral environment.¹³ MMPs can digest various ECM components, and ECM breakdown seems to be a prerequirement for most, if not all, cellular migration processes. For example, Kenagy et al¹⁴ revealed that MMP-9 activity is involved in SMC migration. Pauly et al¹⁵ reported that a neutralizing antibody to MMP-2 inhibited the migration of SMCs across a synthetic ECM membrane, and overexpression of an endogenous MMP inhibitor, a tissue inhibitor of metalloproteinase-1 (TIMP-1), reduces the migration and metastasis of tumor cell lines.¹⁶ At least 18 MMP members have been identified and are divided into interstitial collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and -9), stromelysins (MMP-3, -7, and -10), and novel MMP members (MMP-11 and -14).¹⁷ In the arterial wall, contractile medial SMCs express MMP-2 and minor amounts of MMP-14, a membrane-type MMP-1.¹⁸ However, activated SMCs, such as cells in atherosclerotic plaque, express large amounts of MMP-1, -3, -9, and -14, and activated MMP-2.^{18 19} In vitro, cultured SMCs constitutively express MMP-2, and their production of MMP-1, MMP-3, and MMP-9 is stimulated by a host of growth factors and cytokines, including interleukin-1, transforming growth factor-ß, and tumor necrosis factor-.²⁰ Considering the possible role of MMP in vascular SMC migration, it is tempting to postulate that growth factors such as VEGF could exert their function on SMC MMP expression during angiogenesis. Several reports indicate that VEGF stimulates MMP-1 expression by ECs^{6 21} ; however, the effects of VEGF on vascular SMC MMP expression are unexplored.

The purpose of the present study was to examine the effect of VEGF on vascular SMC MMP production. Using cultured SMCs, we illustrated that VEGF upregulates MMP-1, -3, and -9 expression in these cells. This effect was not accompanied by enhanced cellular proliferation. Furthermore, we showed the expression of the high-affinity VEGF receptor flt-1 in human SMCs and its phosphorylation on VEGF treatment, suggesting its role in mediating VEGF action. The upregulation of MMPs is concomitant with accelerated migration of SMCs through synthetic ECM barriers. Taken together, our data suggest a novel role for VEGFs in angiogenesis, ie, stimulating MMP production by vascular SMCs. The MMPs may facilitate SMC migration and play a role in maturation and stabilization of newly formed vessels in angiogenesis.

	Materials and Methods

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Cell Culture and Proliferation Assay
Human aortic SMCs were purchased from Clonetics, and their homogenous nature was confirmed by immunostaining with anti–smooth muscle (SM) -actin (Sigma). The cells were maintained in SM2 BulletKits (Clonetics). Normal human umbilical vein endothelial cells (HUVECs) were also purchased from Clonetics and maintained in EGM-2 BulletKits (Clonetics), and their endothelial nature was confirmed by anti–factor VIII staining (Sigma). Cultures of both cell types were maintained at 37°C in 5% CO₂ and 95% ambient air, and the culture media were changed every 2 days. During VEGF treatment protocols (R&D System), cells were grown in FBS to a 90% confluent state in 6-well polystyrene plates (Becton-Dickinson) and rinsed 3 times with a serum-free medium before arresting with serum-free medium containing 0.2% bovine albumin for 24 hours. The cells were then incubated with VEGF in specified conditions. Similar treatment protocols were used to examine the effect of placenta growth factor (PLGF, R&D System) on SMCs.

Proliferation of human SMC and HUVEC was measured using a cell proliferation kit II (XTT, Boehringer Mannheim). Cells were grown in 96-well microtiter plates with 100 µL of culture medium (described above) for 36 hours. An aliquot of 50 µL XTT labeling mixture was added into each well, and the mixture was incubated for another 12 hours. The spectrophotometric absorbance at 492 and at 690 nm was measured for each well, and the ratio (A₄₉₂/A₆₉₀) was used to indicate the number of living cells.

Measurement of MMP Proteins by ELISA and Zymography
Expression of MMP-1 and MMP-3 protein by human SMCs was measured using BioTrak Human MMP ELISA kits (Amersham). Briefly, 100 µL of standard or tested samples (cultured medium) was added into appropriate antibody-coated wells of the microplate and incubated at 25°C for 2 hours. Samples were then aspirated, and the wells were washed 4 times with wash buffer. An aliquot of 100 µL of peroxidase conjugate was added to the wells and incubated at 25°C for 1 hour. Wells were washed another 4 times with wash buffer, and 100 µL of 3,3',5,5'-tetramethylbenzidine substrate was dispensed into all wells. After further incubation for 30 minutes at room temperature, 100 µL of sulfuric acid was added, and MMP protein was calculated with spectrophotometric readings at 450 nm.

Enzymatic activity of MMP-9 was evaluated using zymography analysis as reported previously.¹⁸ Aliquots of culture media (containing 50 µg of protein) from human SMCs, with or without VEGF treatment, were mixed with 2x sample buffer (Novex) and loaded on a 10% polyacrylamide gel incorporated with 0.1% gelatin for electrophoresis. The culture medium of phorbol ester–treated human fibroblasts and MMP-2/MMP-9 zymographic standards (Chemicon) were used as standards for gelatinases. Gels were renatured for 30 minutes, developed overnight, stained with 0.25% Coomassie brilliant blue (Sigma), and destained to visualize the MMP bands.

RNA Extraction and cDNA Synthesis
Cytoplasmic RNA was extracted from cultured cells using TRIzol reagent (GIBCO) as described earlier.¹⁸ Digesting with DNase (Promega) for 60 minutes at 37°C further purified the RNA that was obtained. The purified RNA was used for cDNA synthesis and Northern blot analysis. For cDNA synthesis, an aliquot of 15 µg of purified RNA was added into 50 µL of aqueous solution containing 300 U SuperScript II reverse transcripts (GIBCO), 60 U RNasin (Promega), 25 µg random hexanucleotide primers (Amersham), 5 µg 10x reverse transcription buffer (GIBCO), and 1.25 mmol/L dNTP (Promega). The synthetic reaction was carried out for 60 minutes at 42°C.

Analysis of mRNA Expression for MMPs
The expression of mRNA for MMP-1, MMP-3, and MMP-9 before and after VEGF treatment was determined using Northern blot analysis. Total RNA from phorbol 12-myristrate 13-acetate (PMA)–treated human fibroblasts was used as a positive control. Cytoplasmic RNA was separated on a 1% agarose gel and then transferred onto a nylon membrane (Hybond-N+, Amersham). The membrane, with immobilized RNA (by UV light cross-linking), was hybridized to digoxigenin-conjugated antisense MMP RNA probes overnight. The MMP probes were synthesized by an in vitro transcription reaction with Riboprobe in vitro Transcription Systems (Promega) and DIG-UTP (Amersham). Segments for human MMP-1, MMP-3, and MMP-9 were amplified using the reverse transcriptase (RT)–polymerase chain reaction (PCR) technique, and primers were designed according to published sequences¹⁷ (MMP-1: sense 5'-tttgggctgaaagtgactgggaa, antisense 5'-ttcccagtcactttcagcccaaa; MMP-3: sense 5'-aaggatccggtctgtttcactcggccaa, antisense 5'-ttgaattcactgaagaaatagaaaaacc; and MMP-9: sense 5'-aaggatccgactatgacac cgaccgtcg, antisense 5'-aagaattcggcgccggtagggctggta). The amplified cDNA segments were separated in agarose gel, isolated, and inserted into pGEM-3, as described previously.¹⁸ Linearized plasmids with appropriate restriction enzymes were used as templates for probe synthesis. After hybridization, the membrane was washed first in 2x SSC for 30 minutes followed by a blocking buffer (Boehringer Mannheim) for another 30 minutes. The membrane was incubated in a solution containing 75 mU/mL anti–DIG-AP conjugate (Boehringer Mannheim) for 20 minutes. After a thorough washing with a solution of 0.1 mol/L maleic acid, 0.15 mol/L NaCl, and 0.3% (vol/vol) Tween 20, the membrane was incubated with chemiluminescent substrate CSPD (Boehringer Mannheim) solution for 5 minutes. The membrane was dried at 37°C, exposed for 1 to 5 minutes at room temperature to x-ray film (Kodak), and developed. The exposed film was scanned into a computer program (Adobe Photoshop, v. 3.0.4, Adobe Systems Inc), and densitometry analysis was performed using the NIH Image Analysis software to measure the MMP mRNA signal with 28S rRNA as a reference for relative RNA loading. Quantity of 28S rRNA was measured by densitometry.

SMC Invasion Assay Using Matrigel and Vitrogen
Different in vitro and ex vivo methods have been developed for measuring cellular migration.¹⁴ Migration of human SMCs was assayed using 24-well Transwell cell culture chambers with 8.0-µm-pore polycarbonate filter inserts (Costar). The filters were coated with either Matrigel (Becton Dickinson) or Vitrogen (Collagen Corp). The stock solution of Matrigel was diluted to 300 µg/mL using serum-free SM2 medium, and that of Vitrogen was diluted to 100 µg/mL using sterile 0.05 mol/L acetic acid. An aliquot of 75 µL Matrigel (22.5 µg/well) or Vitrogen (7.5 µg/well) was added into each filter insert and incubated overnight at room temperature under a laminar flow hood. The next day, Vitrogen-coated filters were rinsed twice with 0.5 mL PBS to remove residual acid; coated inserts of either Matrigel or Vitrogen were rehydrated with 0.5 mL of SM2 for at least 2 hours. Cultured SMCs were trypsinized and suspended in SM2 at a concentration of either 300 000 cells/mL (Matrigel) or 200 000 cells/mL (Vitrogen) in serum-free SM2, and a volume of 100 µL SM2 containing suspended cells was applied to coated insert filters. In certain experiments, 100 ng/mL of BB-94 (batimastat, [4(N-hydroxylamino)-2R-isobutyl-3S-(thiomethyl)-succinyl]-L-phenylalanine-N-methylamide, MW 478, British Biotechnology) was mixed with the cell suspensions. In the lower chamber, 500 µL of SM2 was added with increasing concentrations of VEGF. The chambers were incubated for 20 hours at 37°C, the inserts were removed, and the SMCs on the upper side of the filters were scraped off. To rule out potential contribution of cellular proliferation within a set of experiments, the number of cells was determined after 20 hours of incubation. Culture media were collected from both upper and lower chambers; cells attached to the filters were collected by trypsinization. Cell number was measured by counting total mitochondrial dehydroxygenase activity (as detailed previously) and by analyzing total DNA content.¹⁸ The SMCs that migrated into the lower side of the chamber were incubated for 60 minutes at 37°C with calcein-AM (15 µg/mL, Molecular Probes). Quantification of the fluorescent-labeled SMCs was made with an inverted microscope equipped with epifluorescence and attached to an Apple power PC 8100 using Image Analysis 1.56 (NIH software). Migration was quantified by converting the fluorescent light emitted by cells into pixels and measuring the number of pixels in the whole filters.

Characterization of flt-1 by RT-PCR, Immunoprecipitation, and Immunoblotting
To study the possible expression of 2 high-affinity VEGFRs, KDR and flt-1, in human SMCs, segments of either receptor were amplified using RT-PCR with oligonucleotide primers. A typical PCR cycle consisted of denaturation (1 minute at 94°C), annealing (1 minute at 55°C), and extension (3 minutes at 72°C). Thirty-five cycles were carried out in the presence of 2.0 U Taq DNA polymerase (Promega) to get sufficient cDNA. The primers were designed according to published data²² (flt-1: sense 5'-aaggatccgcac cttggttgtggctgac, antisense 5'-aagaattccgtgctgcttcctggtcc; flk-1: sense 5'-ccaagcttacgtttgagaacctc, antisense 5'-ccggatccattggcccgct taac). The PCR products were separated in agarose gel and put into pGEM-T vector (Promega) for sequencing using the dideoxy chain termination method.

The expression and phosphorylation of flt-1 protein in human SMCs after VEGF treatment was examined using immunoprecipitation followed by immunoblotting. Cells were washed twice with a phosphate buffer after VEGF treatment and scraped into a lysis buffer (50 mmol/L HEPES, 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L PMSF, 1 mmol/L sodium orthovanadate, and 50 mmol/L sodium fluoride, pH 7.5). The cell lysate was transferred into Eppendorf tubes and precipitated at 14 000 rpm for 10 minutes. The supernatant was incubated with anti–flt-1 antibody (Santa Cruz, 1:100) and rotated for 2 hours; 50 µL of protein A–Sepharose (Sigma, 150 mg/mL) was added and rotated for 2 hours at 4°C. The mixture was precipitated, and supernatant was discarded. The immunoprecipitate was washed (50 mmol/L HEPES, 150 mmol/L NaCl, 10% glycerol, and 1% Triton X-100, pH 7.5) and dissolved into 30 µL SDS sample buffer. Immunoblotting was performed with a monoclonal anti–phosphotyrosine antibody (Upstate Biotech, 1:1000), and signals were detected with an ECL kit (Amersham). Blot stripping and reprobing were carried out as recommended by the manufacturer.

Expression of KDR protein in human SMCs was also determined with immunoblotting, as described above for flt-1. The monoclonal antibody to human KDR was purchased from Santa Cruz.

Statistics
All data were expressed as mean±SE. Throughout the results and figure legends, the term n=x is used to indicate the number of independent experiments (x) performed. Statistical significance was determined with an ANOVA; a weighted Bonferroni analysis (stringency adjusted to allow for multiple comparisons) was performed to determine the differences between groups within an ANOVA. A value of P<0.05 was considered significant.

	Results

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Proliferation Studies
The effect of exogenous VEGF on human SMCs and HUVEC proliferation was measured using total mitochondrial dehydroxygenase activity to reflect the living cell number. As shown in Figure 1, VEGF (10 to 40 ng/mL) treatment had no significant effect on human SMC proliferation. In contrast, over the same dose range, VEGF stimulated HUVEC proliferation up to 3-fold above baseline (Figure 1). This differential effect of VEGF on SMC and EC proliferation correlates well with published reports.^{21 23}

View larger version (33K): [in this window] [in a new window]   Figure 1. Effect of VEGF on SMC and EC proliferation. VEGF stimulates the proliferation of ECs but not vascular SMCs. Data are expressed as fold values above control and presented as mean±SE (n=4). *P<0.05 vs control.

Effect of VEGF on SMC MMP Protein Expression
Enhanced MMP-1 and MMP-3 secretion after VEGF treatment was measured with a human MMP ELISA kit. As shown in Figure 2, VEGF significantly enhanced MMP-1 secretion from human SMCs. MMP-1 concentrations increased 2-fold at the lower VEGF concentration (10 ng/mL) and >5-fold at higher concentrations (20 and 40 ng/mL). The difference in MMP-1 secretion between stimulated and unstimulated cells was significant at all of the VEGF concentrations tested. The effect of VEGF on MMP-3 secretion was less prominent. The MMP-3 secretion increased 2-fold at 10 ng/mL and 3-fold at 20 ng/mL (Figure 2). There was a trend for MMP-3 secretion to decrease at 40 ng/mL compared with 20 ng/mL, although this was not statistically significant.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of VEGF on MMP-1 and MMP-3 secretion. VEGF upregulates MMP-1 (A) and MMP-3 (B) secretion from human SMCs. Human MMP-1 and MMP-3 were measured using an ELISA kit, and data are represented as fold values above control. Data are presented as mean±SE (MMP-1 [n=6], MMP-3 [n=5]). *P<0.05 vs control.

MMP-9 production by human and rabbit SMCs was analyzed using zymography. The induction of MMP-9 in human SMCs is maximal at 20 ng/mL of VEGF, and MMP-9 production increased 5-fold (Figure 3). To confirm that the MMP upregulation observed was due to VEGF activity, VEGF preparations were either heat-inactivated (100°C, 30 minutes) or antibody-neutralized (neutralizing anti-hVEGF, R&D System). Zymographic analysis was performed in both human and rabbit SMCs after stimulation with these inactivated VEGF preparations with concentrations equivalent to 20 ng/mL of VEGF. As anticipated, both treatments abolished the MMP stimulation observed with VEGF (data not shown).

View larger version (47K): [in this window] [in a new window]   Figure 3. Effect of VEGF on human MMP-9 secretion. VEGF enhances MMP-9 secretion from human SMCs. A, Enzymatic activity of human MMP-9 was analyzed using SDS-PAGE gelatin zymography, and gelatin lysis bands were measured using a densitometer. B, MMP-9 activities are shown after stimulation with VEGF (n=4). Data are presented as mean±SE. *P<0.05 vs control.

Effect of VEGF on SMC MMP mRNA Expression
Northern blot analysis was used to examine steady-state MMP mRNA alteration before and after VEGF treatment. Approximately 15 µg of cytoplasmic RNA was applied into each well; the integrity of samples was confirmed by ethidium bromide staining of 18S and 28S rRNA. As shown in Figure 4, there was minimal expression of MMP-1, -3, and -9 in unstimulated human SMCs. However, marked elevation of mRNA for all 3 MMPs was observed after VEGF treatment. The mRNA levels for MMP-1, -3, and -9 were significantly upregulated after stimulation with 20 and 40 ng/mL of VEGF.

View larger version (75K): [in this window] [in a new window]   Figure 4. Effect of VEGF on human MMP mRNA alteration. Northern blot analysis of human MMPs after VEGF treatment is shown. Equal loading of RNA was confirmed by the ethidium bromide staining of 28S and 18S rRNA (n=3).

Expression and Phosphorylation of flt-1 in Human SMCs
To explore possible signal transduction pathways for VEGF, expression of the VEGF receptors KDR and flt-1 was analyzed in human SMCs. The expression of flt-1 in human SMCs was observed using PCR amplification of a human flt-1–specific cDNA segment (flt-1, 1808 to 2394 nt, GenBank accession No. x51602). The 500-bp cDNA was observed in RT-PCR products of VEGF-stimulated SMCs (Figure 5). This cDNA was further confirmed as flt-1 by DNA sequencing with an automated sequencer (data not shown). The protein level of flt-1 was examined using immunoprecipitation with an anti–flt-1 antibody followed by immunoblotting with an anti–phosphotyrosine antibody. We precipitated cellular proteins with the anti–flt-1 antibody, which also illustrated the phosphorylation of flt-1 after VEGF stimulation. As shown in Figure 6, flt-1 was expressed in SMCs, and its phosphorylation was significantly enhanced after VEGF stimulation (Figure 6).

View larger version (82K): [in this window] [in a new window]   Figure 5. PCR amplification of human flt-1 and KDR from cultured SMCs. The PCR products in agarose gel were stained with ethidium bromide; these products were further purified from the gel, ligated into pGEM-T vector, and sequenced. Human ECs were used as a positive control.

View larger version (54K): [in this window] [in a new window]   Figure 6. Expression and phosphorylation of flt-1 and KDR. A, Proteins from human SMCs, with or without VEGF stimulation, were immunoprecipitated with anti–flt-1 antibody, separated in SDS-PAGE gel, and detected with anti–phosphotyrosine antibody. Proteins from VEGF-treated ECs were used as a positive control. B, Proteins from human SMCs were separated by SDS-PAGE electrophoresis, and expression of KDR protein was detected with anti–KDR antibody. Proteins from human ECs were used as a control.

In contrast to flt-1, we could not detect specific cDNA fragments for KDR (KDR, 1721 to 2373 nt, GenBank accession No. x61656) (Figure 5). Lack of KDR protein expression in human SMCs was further confirmed by immunoblot analysis with human endothelial cell lysates as a positive control (Figure 6).

To confirm the role of flt-1 in mediating upregulation of MMP secretion, human SMCs were treated with an flt-1–specific ligand, PLGF. As shown in Figure 7, PLGF, at a concentration comparable to that of VEGF, stimulated MMP-1 and MMP-9 secretion from SMCs. MMP stimulation is significant in both 20 and 40 ng/mL PLGF–treated cells; the magnitude of the response was similar to that observed with VEGF. These data strongly support the role of active flt-1 in mediating enhanced MMP secretion from human SMCs.

View larger version (34K): [in this window] [in a new window]   Figure 7. Effect of PLGF on MMP secretion. PLGF stimulates MMP-1 and MMP-9 secretion from human SMCs. Data are expressed as fold values above control (n=4). *P<0.05 vs control.

Effect of VEGF on SMC Invasion Into ECM
A biological activity associated with MMP upregulation in SMCs was examined using a migration assay with modified Boyden chambers. The polycarbonate filter was coated with either Matrigel (tumor stromal matrix) or Vitrogen (a mixture of 95% type I collagen and 5% type III collagen). As shown in Figure 8, VEGF significantly enhanced human SMC invasion into both matrix barriers. Furthermore, the increased invasion in Matrigel was inhibited by the MMP inhibitor BB-94 (100 ng/mL), supporting a role of MMPs in the invasive process into a "thick" matrix layer (Figure 8). In contrast, BB-94 at 100 ng/mL had no significant effect on invasion into Vitrogen, an observation also found in previous publications.

View larger version (27K): [in this window] [in a new window]   Figure 8. VEGF and SMC invasion into ECM. VEGF enhances human SMC invasion into Matrigel and Vitrogen. A, VEGF-enhanced invasion of human SMCs into Matrigel that was inhibited by an MMP inhibitor (n=6). B, VEGF-enhanced invasion of human SMCs into Vitrogen and the effect of an MMP inhibitor (n=6). Data are presented as mean±SE. *P<0.05 vs control.

The migrated SMCs in the lower side of filters coated with matrix have the potential to proliferate and that could interfere with interpretation of cellular migration data. To address this issue, total cells in each well of a Transwell chamber were collected; cell number and DNA content were measured. As expected, 20 hours of incubation with VEGF in the presence of Matrigel or Vitrogen did not significantly increase cell number (data not shown); this observation is consistent with our data on VEGF and SMC proliferation (Figure 1).

	Discussion

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Our study, for the first time, reveals that VEGF enhances MMP expression in vascular SMCs. This effect is most pronounced for MMP-1 and MMP-9; the effect on MMP-3 is less prominent. Upregulated MMP expression is not accompanied by increased cellular proliferation, indicating unique signal transduction pathways for these 2 events in SMCs. By PCR amplification and immunoprecipitation, we illustrated flt-1 expression in human SMCs and its phosphorylation after VEGF stimulation, suggesting a role for this receptor in mediating VEGF action. The role of flt-1 was further confirmed by stimulation of MMP secretion from SMCs by PLGF, an flt-1–specific ligand. In contrast, similar approaches failed to detect expression of another high-affinity VEGF receptor, KDR, in vascular SMCs. VEGF stimulation also accelerated SMC migration through the "thick" Matrigel in our experiment, an effect partially inhibited by BB94; these data are in accordance with a permissive role of MMP in cellular migration.¹⁷

Angiogenesis is defined as formation of new blood vessels by a process of sprouting from preexisting vessels.²⁴ Although studies using isolated ECs suggest that ECs possess all information for new vessel development, in vivo data clearly indicate that vessels arising after an angiogenic stimulus are composed of both ECs and mural cells (SMCs/pericytes).^{9 25} Moreover, evidence suggests that SMC/pericytes take an active part in regulating EC function during new vessel formation. Using a coculture system, pericytes and SMCs are capable of suppressing EC proliferation and migration; activation of transforming growth factor-ß has been proposed as a mediator for this inhibition.²⁶ The same factor could also induce the differentiation of perivascular mesenchymal cells to SMC/pericyte lineage.²⁷ In addition to diffusible factors, in vivo and in vitro studies show the existence of gap junctions between EC and SMC/pericytes.²⁸ The junction may be important in mediating EC/pericyte interaction as cellular contact is necessary for inhibition of EC proliferation.^{12 26 28} ECM components synthesized by SMC/pericytes, such as fibronectin, are also important in regulating EC activity.^{3 29} Besides its role in stabilization and maturation of EC in a relatively late stage of angiogenesis, the SMC/pericyte may also take part in the initial stage of neovascularization. For example, Nehls et al⁹ showed the presence of pericyte-like cells in the early stage of mesenteric endothelial sprouting (angiogenesis) in situ. These cells showed plump cell bodies and increased cell organelles near angiogenic stimuli that suggests cellular "activation." Schlingemann et al³⁰ demonstrated that these cells overexpress a chondroitin sulfate proteoglycan (HMW-MAA), a molecule previously implicated in melanoma cell migration. Little information is available about the mechanism of SMC/pericyte activation in angiogenesis even though platelet-derived growth factor and basic fibroblast growth factor have been suggested to induce directed migration of perivascular mesenchymal cells and SMC/pericytes.^{1 12} Our data indicate a prominent growth factor in angiogenesis, eg, VEGF, that stimulates secretion of MMPs from arterial SMCs and also accelerates SMC migration through synthetic ECM barriers. Because both ECs and SMCs are capable of synthesizing VEGF at sites of angiogenesis,²³ we propose that autocrine and paracrine actions contribute to SMC activation during neovascularization.

An important finding of our study is that VEGF not only causes flt-1 phosphorylation but also upregulates MMP secretion from SMCs. Our observation that VEGF-induced autophosphorylation of flt-1 in SMCs and the lack of KDR expression supports an active role of flt-1 in mediating MMP upregulation. This conclusion is further supported by stimulation of MMP secretion from SMCs by PLGF, an flt-1–specific ligand. Flt-1 has high binding affinity for VEGF but does not appear to have a major role in mediating cellular mitogenic effects in ECs.³¹ However, evidence is clear that flt-1 is able to mediate the biological functions of VEGF. In contrast to ECs, monocytes exclusively express flt-1 on their cell surface.⁶ Stimulation of monocytes with VEGF or PLGF, another ligand for flt-1, results in tissue factor production and chemotaxis of monocytes.⁶ Treatment of flt-1–transfected porcine aortic endothelial cells, which lack endogenous VEGFRs, results in phosphorylation of Src family members such as Fyn and Yes.⁴ NIH 3T3 cells transfected with flt-1 undergo tyrosine phosphorylation of phospholipase C and GTPase-activating protein complex, and to a lesser extent, they undergo the expression of c-fos³¹ after VEGF treatment. These downstream events of flt-1 phosphorylation could contribute to the MMP gene regulation observed in our study. Promoter analysis of human MMP-1, -3, and -9 reveals that all 3 genes have classical TATA elements at position -30±2 and activator protein-1 (AP-1) elements at position -70±4. The transcription factor AP-1, which binds the AP-1 elements in the gene promoters, is composed of a protein complex including members of the c-fos family.³² The MMP upregulation observed in our study might be through the VEGF–flt-1–c-fos signal transduction pathway. A recently reported isoform-specific VEGF receptor has been cloned from HUVEC and tumor cell lines.⁵ This receptor is neuropilin-1, a transmembrane protein initially described in developing neural systems.³³ Functional studies indicate that neuropilin enhanced the binding of VEGF to KDR resulting in VEGF-mediated chemotaxis. The expression of this receptor in SMCs has not been reported; however, tissue distribution and functional characterization of this novel receptor as a coreceptor for KDR do not support a role of neuropilin in SMCs.

We also demonstrated a potential physiological role of enhanced MMP expression by arterial SMCs after VEGF stimulation. MMPs are an important family of proteases that have been implicated in migration of SMCs, keratinocytes, and metastasis of various tumor cells.^{16 18 34} Although ECM remodeling is primarily responsible for promoting cellular migration, enhanced MMP activity also induces the release of growth factors and the generation of ECM degradation products with chemotactic properties.^{17 35} Previous studies suggest a causal relationship between MMP activity and angiogenesis. For example, angiogenic factors including basic and acidic fibroblast growth factor, tumor necrosis factor-, transforming growth factor-ß, and VEGF induce the synthesis of MMPs,³⁶ and MMP inhibitors like TIMP-1 and BB-94 block angiogenesis in vivo.^{37 38} However, the studies conducted to date have been targeted primarily at EC production of MMPs. Results of our studies indicate that the angiogenic factor VEGF could stimulate MMP secretion from the SMCs present in the vessel wall and provide an additional source of MMPs in the process of angiogenesis. Vascular localization of SMC-derived MMPs may permit their spatially controlled role in degradation of subendothelial basement membrane, and that is a crucial step in the initial stage of angiogenesis. Upregulated MMPs may also promote SMC migration in a later stage of angiogenesis. The result of our findings that VEGF accelerates SMC invasion into ECM barriers strongly supports this conclusion. In the present study, we did not specifically address the nature of various MMPs, ie, their active or latent forms. However, consistent with our experimental protocols and previous reports, we believe that our assays measured both active and latent forms of these proteases.³⁹ BB-94 is a potent inhibitor of active MMPs.^{39 40} Inhibition of VEGF-stimulated SMC migration through Matrigel by this compound strongly suggests that at least part of the VEGF-enhanced MMP production is in an active form.

The effect of VEGF on MMP production and vascular SMC migration may also contribute to other pathologies in the vessel wall, eg, neointimal formation and atherogenesis. Various stimuli such as balloon catheter deendothelialization, accumulation of lipid peroxidation products, and arterial reconstruction using venous conduits result in local production of VEGF in the arterial wall.^{41 42} We have shown that VEGF enhances MMP secretion from SMCs possibly by way of flt-1 activation, and it is well documented that MMPs contribute to SMC migration^{39 40 43} and proliferation⁴⁴ both in vivo and in vitro. Thus, it is possible that VEGF contributes to neointimal formation and/or atherogenesis by promoting SMC migration. Lazarous et al⁴⁵ demonstrated that a daily arterial bolus injection of VEGF peptide resulted in exacerbated neointimal thickening in the iliofemoral artery. Couper et al⁴⁶ illustrated that high-level flt-1 expression in carotid artery SMCs appeared only 7 days after balloon catheter deendothelialization and was restricted to the neointima. VEGF could also exert a beneficial effect to inhibit intimal hyperplasia by promoting reendothelialization after arterial injury.^{47 48} Thus, there may be a balance between the beneficial and the deleterious effects of VEGF in various vascular pathologies.

Our study extends the function of VEGF to arterial SMCs. VEGF upregulates MMP production by SMCs possibly through its binding with flt-1 in the SMCs. SMC-derived MMPs may be critical in degradation of basement membrane and SMC migration in angiogenesis and in atherogenesis.

	Acknowledgments

 
The authors thank Dr Robert Panek for helpful discussion; Gina Lu and Tawny Dahring are acknowledged for excellent technical assistance in migration assay and immunoprecipitation, respectively. We also wish to express our gratitude to Wendy Rosebury and Xiaokang Lu for immunostaining of SM -actin and factor VIII and Brian Batley for help in NIH Image analysis.

	Footnotes

 
Reprint requests to Dr Joan A. Keiser, Senior Director, Vascular and Cardiac Disease, Therapeutics, Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd, Ann Arbor, MI 48105.

Received February 2, 1998; accepted July 8, 1998.

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