This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pickering, J. G. Articles by Chow, L. H. Search for Related Content PubMed PubMed Citation Articles by Pickering, J. G. Articles by Chow, L. H.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:475-482.)
© 1997 American Heart Association, Inc.

Articles

Coordinated Effects of Fibroblast Growth Factor-2 on Expression of Fibrillar Collagens, Matrix Metalloproteinases, and Tissue Inhibitors of Matrix Metalloproteinases by Human Vascular Smooth Muscle Cells

Evidence for Repressed Collagen Production and Activated Degradative Capacity

J. Geoffrey Pickering; Carol M. Ford; Bao Tang; ; Lawrence H. Chow

From the John P. Robarts Research Institute and London Health Sciences Centre, Department of Medicine (Cardiology), Medical Biophysics, and Biochemistry, University of Western Ontario, London, Canada.

Correspondence to J. Geoffrey Pickering, London Health Sciences Centre, 339 Windermere Rd, London, Ontario N6A 5A5, Canada. E-mail gpickrng{at}rri.uwo.ca

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract Fibroblast growth factor-2 (FGF-2) is an established mediator of smooth muscle cell (SMC) proliferation after vascular injury. However, the influence of FGF-2 on collagen fiber remodeling, which may be a prerequisite for vascular SMC accumulation, is not well understood. We determined that FGF-2 almost completely abrogated the formation of immunodetectable type I collagen fibers in the extracellular matrix of cultured human vascular SMCs. This was associated with reduced expression of pro-chains for types I and III collagen, as assessed by Western blot analysis, and a corresponding reduction in collagen synthesis. Densitometry of Northern blots indicated a potent reduction of mRNA encoding pro-chains for types I and III collagen and a minor reduction in mRNA for pro-chains for type V collagen. Interstitial collagenase (MMP-1), which is required for degradation of collagen types I and III, was not expressed by SMCs under basal culture conditions, but expression was induced by FGF-2, with a potent, dose-dependent increase in MMP-1 protein in conditioned medium. Metalloproteinase inhibitors TIMP-1, TIMP-2, and TIMP-3 were expressed by unstimulated SMCs and were differentially regulated by FGF-2. TIMP-1 expression increased modestly, TIMP-2 expression was repressed, and TIMP-3 was relatively unaffected. The net effect on substrate degradation, as assessed by zymography of conditioned media, was induction of MMP-1 lytic activity by FGF-2, with no effect on the activity of MMP-2, MMP-3, or MMP-9. These data indicate that stimulation of human SMCs with FGF-2 establishes a phenotype in which collagen fiber production is repressed and the capacity for fiber degradation activated. This coordinated response may be critical for SMC accumulation during vascular remodeling as well as atherosclerotic plaque destabilization.

Key Words: smooth muscle cells • fibroblast growth factor • collagen • metalloproteinase

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The elaboration of collagen is one of the important functions of vascular SMCs.^{1 2 3} Collagen is a key component of the extracellular framework of the arterial media and a major constituent of human atherosclerotic plaque, comprising as much as 60% of the total protein content.^{4 5} Most abundant are the fiber-forming collagens types I and III, although type V, a minor fibrillar collagen, is also present and may be proportionately increased in atherosclerosis.^{6 7}

In addition to their capacity for collagen production, SMCs can degrade extracellular collagen. This is accomplished by the elaboration of MMPs, a family of zinc-containing endopeptidases. Within this family, MMP-1, or interstitial collagenase, is particularly critical to collagen turnover, as it has the ability to hydrolyze triple-helical collagen. The action of MMP-1 is to cleave the triple helix at a single site to produce a three-quarter-length fragment and a one-quarter-length fragment.^{8 9} These products can denature under physiological conditions and undergo extensive degradation by other enzymes, including the gelatinases MMP-2 and MMP-9 and the stromelysin MMP-3.^{10 11} Another characteristic of MMPs is their susceptibility to inhibition by TIMPs, which form stoichiometric 1:1 complexes with the active form of MMPs.¹² TIMP-1 and TIMP-2 have recently been shown to be secreted by SMCs.¹¹ TIMP-3 is a more recently described member of the TIMP family and has MMP inhibitory activity similar to that of the other TIMPs.¹³ The distribution of TIMP-3 in tissue, however, differs from that of TIMP-1,¹⁴ and its expression by human vascular SMCs is unknown.

FGF-2 (ie, basic FGF) is a member of the FGF family of heparin-binding proteins that regulate diverse cellular functions. In vitro, FGF-2 is a potent mitogen for SMCs.¹⁵ When administered in vivo, FGF-2 can induce angiogenesis¹⁶ and can augment SMC and endothelial cell proliferation and migration in the injured rat carotid artery.^{17 18 19} Lindner and Reidy²⁰ demonstrated that injection of a neutralizing antibody against FGF-2 significantly decreased injury-induced SMC proliferation, strongly implicating a role for this growth factor in vascular repair. Antibody delivered 6 days after injury did not have an antiproliferative effect, suggesting that the role of FGF-2 in this model is primarily related to early cellular events.

Rapid remodeling of the extensive collagen framework of the artery wall may be a prerequisite for SMC accumulation in vascular disease, and the mitogenic and migratory effects of FGF-2 might therefore need to be accompanied by altered collagen fiber metabolism. FGF-2 has been reported to inhibit collagen synthesis by SMCs²¹ ; however, details of the effect on collagen expression are unclear. One report suggested that the effect was specific to type III collagen,²¹ while another showed repression of type I collagen.²² The effect on the expression of type V collagen remains unknown. The effect of FGF-2 on mediators of collagen fiber degradation is also incompletely understood, and there is no information of the effect on TIMPs. Because SMCs have the capacity to mediate both collagen production and degradation, regulation by growth factors may be critical in determining whether the net effect will be collagen accumulation, degradation, or a steady state. Our results suggest that FGF-2 induces a unique SMC phenotype characterized by repressed collagen fiber production and heightened collagen degradative capacity.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
SMC Culture
Primary cultures of human arterial SMCs were initiated by explant outgrowth of unused segments of internal mammary artery retrieved at the time of coronary artery bypass surgery.^{23 24} The identity of vascular SMCs was confirmed morphologically and by positive immunostaining with a monoclonal antibody to smooth muscle -actin (clone 1A4, Sigma Chemical Co). Cells were grown in medium (M199, GIBCO/BRL) unless indicated otherwise and supplemented with the designated concentration of FBS. All experiments were performed with SMCs in the third or fourth subculture.

Immunofluorescence Microscopy for Type I Collagen Fibers
SMCs were seeded onto multiwell slides in M199 with 10% FBS. Twenty-four hours after seeding, the medium was replaced with M199 supplemented with 1% FBS, 10 µg/mL sodium ascorbate, and 0 to 50 ng/mL recombinant FGF-2 (GIBCO/BRL). Cells were maintained under these conditions for 9 days, with exchange of the respective incubation media on days 3 and 6. We found in preliminary experiments that this protocol allowed optimal accumulation of immunodetectable collagen fibers in the extracellular matrix. Washed cells were fixed in cold acetone, blocked with 3% BSA, and incubated with rabbit antiserum to the C-telopeptide region of the 1(I) chain of human type I collagen (LF67, a gift from Dr L.W. Fisher, National Institute of Dental Research, Bethesda, Md). Bound primary antibody was detected with TRITC-conjugated goat anti-rabbit IgG F(ab)' dimer (Jackson ImmunoResearch) at 1:200 dilution. Cells were coverslipped with glycerol/PBS (9:1, vol/vol) containing Hoechst 33258 (2.5 µg/mL, Sigma) to identify cell nuclei and evaluated by fluorescence microscopy.

Western Blot Analysis
Cells at near-confluence were incubated with 10 µg/mL sodium ascorbate, 50 µg/mL ß-aminoproprionitrile, and the designated concentration of FGF-2 (0 to 100 ng/mL) for 72 hours. The medium was harvested with the protease inhibitors PMSF (0.1 mmol/L) and leupeptin (10 µg/mL), and cells were harvested in lysis solution (PBS, pH 7.4; 1% SDS; 1% sodium deoxycholate; 0.1% Triton X-100; 0.1 mmol/L EDTA; 0.1 mmol/L PMSF; and 10 µg/mL leupeptin). The total protein content of the conditioned media and cell lysate was measured by Lowry assay (Bio-Rad). Samples were mixed with an equal volume of 2x SDS gel loading buffer (100 mmol/L Tris chloride, pH 6.8; 4% SDS; 20% glycerol; 200 mmol/L DTT; and 0.2% bromphenol blue), and equal amounts of protein were resolved on 6% (for collagens) or 15% (for MMP-1 and TIMPs) polyacrylamide gels under reducing conditions. Proteins were electrophoretically transferred to a polyvinylidine difluoride membrane (Immobilon P, Millipore). After the membranes were blocked overnight, they were incubated with the designated primary antibody for 3 to 15 hours at room temperature. The primary antibodies employed were the following: LF67 (see above); a monoclonal antibody to the triple-helical domain of human type III collagen (clone 3G4, GIBCO/BRL); and monoclonal antibodies to human MMP-1, TIMP-1, and TIMP-2 (Oncogene Science). Bound primary antibody was reacted with anti-rabbit peroxidase-conjugated IgG or anti-mouse peroxidase-conjugated Fab fragments for 1 hour and detected by chemiluminescence according to the manufacturer's recommendations (Boehringer Mannheim). Washed blots were exposed to x-ray film (Kodak XAR-5). Purified rat type I collagen (a gift from Dr B.M.C. Chan, John P. Robarts Research Institute, London, Canada) and human type III collagen (GIBCO/BRL) were run as standards. These were also used to confirm that the two antibodies were specific for their respective collagens and did not cross-react.

Measurement of Collagen Synthesis
SMCs at near-confluence were incubated with 10 µg/mL sodium ascorbate, 50 µg/mL ß-aminoproprionitrile, and the designated concentration of FGF-2 (0 to 25 ng/mL) in Dulbecco's modified Eagle's medium (Pro-free) with 1% dialyzed FBS. After 48 hours the cells were labeled for 24 hours with [³H]Pro (10 µCi/mL, 100 mCi/mmol; DuPont-NEN). Media were harvested as described above, and the proteins were precipitated overnight at 4°C with 24% (NH₄)₂SO₄. The precipitate was dissolved in 1x SDS gel loading buffer, and the proteins were resolved on a 6% polyacrylamide gel under reducing conditions. Fixed gels were subjected to fluorography with EN³HANCE (DuPont-NEN) as per the manufacturer's recommendations. In some experiments, labeled proteins were digested with pepsin (1 µg/mL) for 24 hours at 4°C before precipitation to eliminate fibronectin from the Pro-labeled proteins and convert the procollagen species to their mature forms.²⁵ The position of the various collagen species was confirmed by running ³H-labeled rat type I collagen (DuPont-NEN) and comparing our results with previously established collagen and procollagen migration patterns.^{26 27}

RNA Isolation and Northern Blot Analysis
Cells incubated in M199 with 1% FBS and the designated concentrations of FGF-2 for 72 hours were lysed in a solution of 4 mol/L guanidinium isothiocyanate; 25 mmol/L sodium citrate, pH 7.0; 0.1 mol/L 2-mercaptoethanol; and 0.5% sarkosyl. Total RNA was isolated with the acid-phenol technique.²⁸ Ten micrograms of RNA was electrophoresed through agarose (1.2%)-formaldehyde (0.6 mol/L) and transferred to a nylon membrane (Zetaprobe GT, Bio-Rad), which was then baked at 80°C. Blots were prehybridized for at least 2 hours at 42°C in 50% formamide, 7% SDS, 1x Denhardt's solution, 5x SSPE, 100 µg/mL tRNA, and 40 µg/mL heat-denatured herring sperm DNA. Hybridization was performed for 8 to 24 hours in an identical solution containing 100 ng of cDNA probes labeled with [-³²P]dCTP (specific activity, 10⁸ to 10⁹ counts per minute per microgram) and random-hexamer priming. Membranes were washed several times, with a final stringency wash at 50°C in 0.1x SSC containing 0.1% SDS. Blots were then exposed to Kodak XAR-5 film with intensifying screens at -80°C. Band density was quantified by digital videodensitometry.

The probes included cDNA fragments for human pro1(I) collagen (pSP3, a gift from Dr C. Farrell, Amgen, Thousand Oaks, Calif), human pro1(III) collagen (Hf934, ATCC), human pro2(V) collagen (N3-6, kindly provided by Dr J. Meyers, University of Pennsylvania, Philadelphia), human MMP-1 (pCllase 1, ATCC), human TIMP-2 (pSS38, ATCC), and human TIMP-1 and mouse TIMP-3 (phTIMPGEM2 and pmTIMP-3, respectively, gifts from Dr D. Edwards, University of Calgary, Calgary, Alberta). A cDNA probe for human GAPDH (pHcGAP, ATCC) was used as an internal control for mRNA levels.

Zymography
Media from control and FGF-2–treated cultures were harvested, and equal amounts of total protein were separated under nonreducing conditions on 10% SDS polyacrylamide gels impregnated with gelatin or casein (1 mg/mL). Gels were washed in 2.5% Triton X-100 and incubated at 37°C for 48 hours in buffer containing 50 mmol/L Tris chloride, 5 mmol/L CaCl₂, and 40 mmol/L NaN₃. Gels were stained with Coomassie brilliant blue (Sigma). To confirm MMP activity as the cause of substrate lysis, duplicate gels were prepared in which zymography was performed in the presence of 10 mmol/L EDTA. In some experiments an aliquot of conditioned medium was incubated with APMA (1.0 mmol/L) at 37°C for 1 hour before gel electrophoresis to convert proMMPs to their activated forms.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Formation of a Collagen Fiber Matrix by Human SMCs Is Prevented by FGF-2
To determine whether FGF-2 affected the formation of an extracellular collagen fiber matrix, human SMC cultures were immunostained with LF67, an antibody that recognizes the C-telopeptide region of the 1(I) chain of human type I collagen. As shown in Fig 1, control SMCs showed an elaborate network of type I collagen fibers after 9 days of culture. In contrast, FGF-2–treated cultures at 9 days contained scant collagen fibers in the extracellular matrix. The inhibitory effect was evident with FGF-2 concentrations as low as 10 ng/mL.

View larger version (83K): [in this window] [in a new window]   Figure 1. Immunofluorescence photomicrographs of human SMC cultures immunostained for type I collagen fibers. Collagen fibers were detected with TRITC-labeled secondary antibody and appear red; nuclei were counterstained with Hoechst 33258 and appear blue. After 9 days in culture an extensive network of collagen fibers is detectable (left). Treatment with 25 ng/mL FGF-2 (right) inhibits the accumulation of a collagen fiber matrix by the same time. Most fields showed even fewer fibers than depicted here.

FGF-2 Inhibits Production of pro-Chains of Collagen Types I and III by Human SMCs
Expression of fibrillar collagen by human SMCs was studied further by Western blot analysis. Component -chains for collagen types I and III, the major fibrillar collagens of the arterial wall, were studied by use of polyclonal and monoclonal antibodies, respectively. Antisera against pro1(I) collagen (LF67) detected three major bands. The smallest of these comigrated with the mature 1(I) collagen chain purified from type I rat tail collagen. The heaviest band corresponded in size to that previously established for full-length pro1(I) collagen chain [45 kD larger than the mature 1(I) collagen chain].^{26 27} The intermediate band corresponded in size to partially processed pro1(I) collagen from which either the N-terminal or C-terminal propeptide (but not both) had been cleaved. As shown in Fig 2, the amount of each of these species in the culture media fell after exposure to FGF-2.

View larger version (60K): [in this window] [in a new window]   Figure 2. Effect of FGF-2 on expression of collagen -chains by human SMCs. A, Western blot analysis depicting effect of FGF-2 on extracellular accumulation of pro1(I) and 1(I) collagen. Three bands were detected by antiserum LF67, which identified the full-length pro1(I) chain, partially processed pro1(I) collagen chains lacking either the N- or C-terminal propeptide, and mature 1(I) collagen lacking both terminal propeptides. All were reduced by FGF-2. CI denotes control 1(I) collagen chain from rat tail tendon. B, Western blot analysis depicting effect of FGF-2 on accumulation of pro1(III) collagen. Full-length pro1(111) collagen was detected and showed concentration-dependent inhibition by FGF-2. Partially processed pro1(III) collagen was only faintly detected but showed a similar response to FGF-2. CIII denotes control mature 1(III) chain from type III collagen. C, Northern blot analysis of FGF-2–treated human SMCs, showing concentration-dependent inhibition of pro1(I) and pro1(III) collagen mRNAs. FGF-2 also reduced the abundance of the 6.3-kb transcript of pro2(V) collagen but not the 5.0-kb transcript. This differential transcript response became more apparent after longer exposure, as shown in Fig 6. Results are representative of 3-6 experiments.

The antibody against 1(III) collagen was directed to the triple-helical domain and detected a major band corresponding to full-length pro1(III) collagen chain and faint bands of either partially processed procollagen species or the mature 1(III) collagen chain (confirmed by assessing purified type III collagen). As shown in Fig 2, FGF-2 induced a potent, dose-dependent inhibition of pro1(III) collagen chains in the culture media. A reduction in intracellular procollagen accumulation was also found when cell lysates were studied (data not shown). Selectivity was confirmed by probing the transfer membranes for fibronectin (polyclonal rabbit anti-human fibronectin antibody, GIBCO/BRL), which did not decline in response to FGF-2 (data not shown).

FGF-2 Inhibits Expression of pro1(I), pro1(III), and pro2(V) Collagen mRNAs
To further clarify the mechanism by which FGF-2 reduced fibrillar collagen production, we measured steady-state mRNA levels by Northern blot analysis and quantified band intensity by videodensitometry. The characteristic transcript polymorphism for pro1(I), pro1(III), and pro2(V) collagen mRNAs was observed, with two bands detected for each procollagen type. FGF-2 induced a concentration-dependent reduction in the level of mRNA encoding pro1(I) collagen (Fig 2c). After 72 hours of treatment with 50 ng/mL FGF-2, pro1(I) mRNA levels (normalized to GAPDH mRNA) fell to 0.2 of basal level. A concentration-dependent reduction in mRNA encoding pro1(III) collagen was also observed, and 50 ng/mL produced a reduction in mRNA abundance of 0.25 of basal level. In contrast to pro1(I) and pro1(III) collagen mRNAs, the effect on mRNA expression for the pro2(V) chain was very mild and was detected for the heavier 6.3-kb transcript only (Figs 2c and 6a). The 5.0-kb transcript showed no change over the entire range of concentrations (0 to 100 ng/mL).

View larger version (59K): [in this window] [in a new window]   Figure 6. A, Northern blot showing time course of steady-state mRNA levels in FGF-2–treated (25 ng/mL) human SMCs for pro1(I) collagen, pro1(III) collagen, pro2(V) collagen, MMP-1, TIMP-1, and TIMP-2. B, Quantitative time-course data for fibrillar collagens and MMP-1 (top) and TIMP-1 and TIMP-2 (bottom). Band density is expressed relative to that of GAPDH mRNA. Not shown is TIMP-3, which remained essentially constant. Results are representative of 2 experiments.

FGF-2 Inhibits Collagen Synthesis by Human SMCs
To determine whether FGF-2 inhibits biosynthesis of collagen, SMCs were metabolically labeled with [³H]Pro and the products analyzed by gel electrophoresis. As shown in Fig 3, collagen synthesis was inhibited by FGF-2. In contrast, TGFß increased fibrillar collagen synthesis, consistent with previous reports.²⁹ Inhibition by FGF-2 was seen when SMCs were labeled for either 4 or 24 hours.

View larger version (50K): [in this window] [in a new window]   Figure 3. Effects of FGF-2 on biosynthesis of collagen by human SMCs. A, Gel electrophoresis fluorogram of [3H]Pro-labeled proteins secreted into the culture medium. Synthesis of pro1() and 1() collagen was reduced by FGF-2 (25 ng/mL) and increased by TGFß (10 ng/mL). Autoradiogram is representative of 4 experiments. B, Gel electrophoresis fluorogram of pepsin-resistant [3H]Pro-labeled proteins, showing an inhibitory effect of FGF-2 on synthesis of -chains incorporated into triple-helical collagen. C denotes control, vehicle-treated cultures.

FGF-2 Induces MMP-1 Protein and mRNA Expression
MMP-1 is critical to collagen turnover because it can degrade triple-helical collagen, which is otherwise highly resistant to proteolysis.³⁰ By Western blot analysis, no MMP-1 was detected in the media of untreated SMCs cultured in 1% FBS, and a barely detectable level of MMP-1 was present when SMCs were cultured in 10% FBS (Fig 4). However, treatment with FGF-2 induced a marked concentration-dependent increase in MMP-1 in the medium (Fig 4a). In contrast, exposure to TGFß (10 to 25 ng/mL) did not induce expression of MMP-1 (Fig 4b). Gene transcripts for MMP-1 were not detected by Northern blot analysis of SMCs cultured under basal conditions, but MMP-1 mRNA expression was induced by treatment with FGF-2 (Fig 4c).

View larger version (21K): [in this window] [in a new window]   Figure 4. Induction and concentration-dependent increase of MMP-1 protein and mRNA expression in FGF-2–treated human SMCs. A, Western blot analysis of proteins secreted from SMCs cultured with 10% FBS and designated concentrations of FGF-2. B, Western blot analysis of proteins secreted from SMCs cultured with 1% FBS and either FGF-2 (10 ng/mL), TGFß (10 ng/mL), or angiotensin II (AII, 10-7 mol/L) C, Northern blot analysis of SMCs, showing effect of FGF-2. results are representative of 3 experiments.

TIMP-1, TIMP-2, and TIMP-3 Are Differentially Regulated by FGF-2
Under basal conditions, human SMCs expressed transcripts for TIMP-1, TIMP-2, and TIMP-3 (Figs 5 and 6). The actions of FGF-2 on mRNA abundance differed among the three TIMPs, however. TIMP-1 mRNA was increased 2.8-fold by 50 ng/mL FGF-2. An increase in secreted TIMP-1 protein was also found by Western blot analysis, although to a minor degree (data not shown). In contrast, there was a concentration-dependent decrease in TIMP-2 mRNA expression of 50% at 50 ng/mL FGF-2. This repression was evident for the 3.5- and 1.0-kb TIMP-2 transcripts. A decrease in TIMP-2 protein level in conditioned media was also observed (data not shown). A major TIMP-3 transcript of 4.5 kb was detected, similar in size to that recently detected in mouse fibroblasts.¹⁴ TIMP-3 mRNA levels were relatively unaffected by FGF-2.

View larger version (67K): [in this window] [in a new window]   Figure 5. Northern blot analysis showing differential influence of FGF-2 on expression of TIMP-1, TIMP-2, and TIMP-3 in human SMCs. Results are representative of 2 experiments.

Kinetics of FGF-2–Induced Changes in Fibrillar Collagen, MMP-1, and TIMP mRNAs
To compare the kinetics of FGF-2–induced effects on fibrillar collagen, MMP-1, and TIMPs, the time course of gene expression was assessed by Northern blot analysis. SMCs were incubated with 25 ng/mL FGF-2, and specific mRNA levels were determined between 0 and 49 hours. Levels of mRNAs encoding pro1(I) collagen and pro1(III) collagen had begun to decline after 6 hours of treatment (Fig 6). As noted above, an effect on pro2(V) collagen mRNA was observed for the 6.3-kb transcript only, and this was also evident by 6 hours. No effect on the 5.0-kb pro2(V) collagen mRNA level was observed during the 49-hour period, and net mRNA levels did not clearly decline until 35 hours and afterward(Fig 6b). mRNA for MMP-1 was not detected at 0 and 6 hours of treatment but was evident by 12 hours. The kinetics of TIMP changes were similar for that of collagen, although the magnitude of the change was less.

Effect of FGF-2 on Gelatinolytic and Caseinolytic Activity of Conditioned Media
Under basal conditions (1% FBS), there was a major zone of gelatinolytic activity at 70 kD and a fainter zone 10 kD smaller (Fig 7a). Both zones were abolished by incubation with EDTA, and the intensity of the fainter was enhanced after protein exposure to APMA (Fig 7b). This pattern of activity is consistent with that of secreted MMP-2 in its precursor and activated forms.³¹ There was no detectable effect of FGF-2 on this gelatinase. There was evidence for low-level MMP-9 activity (94 kD) under basal conditions but no detectable influence of FGF-2. FGF-2 did induce faint gelatinolytic activity that migrated as a doublet, with an apparent mass of 50 kD. This zone was not present in gels incubated with EDTA and is characteristic of pro-MMP-1.^{31 32} Activity was faint, as is typical for MMP-1 in gelatin,^{11 31 32} likely reflecting the single cleavage in the 1(I) or 2(I) collagen chain that would only mildly reduce Coomassie blue staining. Nonetheless, it was a consistent finding in four experiments and was not observed in SMCs exposed to vehicle or SMCs treated with TGFß (data not shown). Casein zymography of conditioned media showed no detectable activity compatible with MMP-3 after 48 hours of incubation in either control or FGF-2–treated cultures.

View larger version (31K): [in this window] [in a new window]   Figure 7. Gelatinolytic activity of conditioned medium. A, SMCs were incubated for 48 hours with or without FGF-2 (25 ng/mL), and media were harvested and analyzed by gelatin zymography. B, Media were harvested from untreated SMC cultures and incubated with or without APMA. Arrows depict bands that were intensified by APMA and are consistent with MMP-9 (upper arrow) and MMP-2 (lower arrow) activity. Zymograms are representative of 5 experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results presented here demonstrate that FGF-2 influences collagen fiber metabolism by human SMCs at several levels. FGF-2 repressed expression of type I and type III collagen protein and the mRNA encoding pro-chains for these collagens, with a corresponding inhibition of collagen synthesis. As well, FGF-2 induced MMP-1 mRNA and protein expression, with modest and differential effects on the expression of TIMPs. These results suggest a coordinated effect of FGF-2 on human SMCs, leading to a phenotype of suppressed collagen production and enhanced degradative capacity.

Although the best-studied cellular effects of FGF-2 pertain to its mitogenic and promigratory properties,¹⁵ a few studies have indicated an effect on collagen metabolism. Basic FGF was shown to repress collagen synthesis in osteoblastic cells³³ and keloid fibroblasts.³⁴ As well, two studies of SMCs demonstrated that FGF-2 induced a net decrease in total collagen synthesis.^{21 22} The effect of FGF-2 on the expression of specific fibrillar collagens, however, differed in these two reports. Using rabbit SMCs, Majors and Ehrhart²¹ observed that FGF-2 coated on culture dishes had no effect on pro1(I) collagen mRNA levels but repressed expression of pro1(III) collagen mRNA. In contrast, Kennedy and coworkers²² reported that FGF-2 inhibited expression of pro-chains for type I collagen by human SMCs; other fibrillar collagens were not evaluated in their study. The basis for these differences in the published results is not known, although they could reflect differences in the method of FGF-2 delivery (soluble versus substrate attached). The present study of soluble FGF-2 supports the finding that FGF-2 decreases type I collagen expression, with a concentration-dependent repression of both mRNA and protein chains and a nearly complete abrogation of the formation of a type I collagen fiber matrix.

The rate of decline of pro1(I) collagen mRNA was fairly brisk (detected at 6 hours), suggesting a direct effect of FGF-2 on mRNA abundance. This finding differs substantially from that of Kennedy and coworkers,²² who did not detect suppression of pro1(I) collagen mRNA until 72 to 96 hours of treatment. The basis for this difference is not clear; however, studies in osteoblastic cells have shown kinetics very similar to ours, with a reduction in pro1(I) collagen mRNA detectable between 4 and 8 hours.³³

Our analysis also included the two other fibrillar collagens known to be relevant to vascular disease. pro1(III) collagen mRNA and protein levels declined in response to FGF-2; these changes paralleled in magnitude and time course those of pro1(I) collagen mRNA and protein. In contrast, the effect on pro2(V) collagen mRNA was unique in two respects. Like pro1(I) and pro1(III) collagen mRNA, two transcripts for pro2(V) were expressed. However, unlike pro1(I) and pro1(III) collagen, levels of the heavier pro2(V) transcript only fell after FGF-2 stimulation. The basis for transcript polymorphism among collagens is not definitively established, although utilization of different polyadenylation sites has been implicated for the 1(I) gene.³⁵ It is conceivable that the 3' untranslated segment influences transcript properties (such as stability) and that differences in this region could account for the differential response to FGF-2. Alternative promoters and/or transcription start sites are also possible, although neither have been identified for the 2(V) collagen gene. Notwithstanding the decline in the 6.3-kb transcript, the decline in total pro2(V) mRNA abundance in response to FGF-2 was small relative to that for either pro1(I) or pro1(III) collagen mRNA. Of note, differences in the regulation of type V collagen versus collagen types I and III have also been observed for TGFß. Lawrence et al³⁶ observed that elevation in type V collagen production due to TGFß was greater than that for types I and III.

The extracellular degradation of the major fibrillar collagens (types I and III) is dependent on the action of MMP-1 by virtue of its ability to cleave triple-helical collagen. It was therefore significant that FGF-2 not only repressed fibrillar collagen production but also induced MMP-1 expression. Induction of MMP-1 mRNA expression by FGF-2 was also observed by Kennedy and coworkers,²² although they did not assess protein levels and substrate degradation. Although one in vivo study failed to detect MMP-1 mRNA in the injured rat carotid artery,³⁷ several growth factors that have been implicated in vascular disease have been found to induce MMP-1 gene expression in SMCs, including PDGF,³⁸ TNF, and IL-1.¹¹ It is noteworthy, however, that PDGF and to a lesser extent IL-1 and TNF can enhance collagen synthesis,²⁹ whereas FGF-2 is clearly inhibitory. FGF-2 might therefore be expected to modify the collagen environment to a greater extent than other mediators by virtue of its reciprocal effects on expression of collagen and collagenase.

After primary cleavage of collagen by MMP-1, the products denature and undergo further proteolysis by several enzymes, including gelatinases (MMP-2 and MMP-9) and stromelysin (MMP-3). FGF-2 would appear to be neutral in this latter phase of degradation, as it had little effect on expression of MMP-2, -3, or -9. MMP-2 was expressed constitutively, consistent with previous in vitro and in vivo studies,^{11 31 37} and is thus potentially available for proteolysis of collagen fragments. As well, in vitro expression and activity of gelatinases and stromelysin have been found to be augmented by TNF and IL-1,¹¹ and these factors are thus candidates for regulating the distal events in the collagen degradation cascade.

Expression of TIMPs by SMCs must also be considered in the control of collagen turnover, given the role of TIMPs in modulating MMP function. TIMP-1 and TIMP-2 were found to be expressed in unstimulated cultures of human SMCs, consistent with a report by Galis and coworkers.¹¹ TIMP-3, which has only recently been cloned and identified as a distinct member of the TIMP family,^{13 14} was also expressed by these cells. Interestingly, tissue localization studies have shown that TIMP-3 transcripts do not typically collocalize with those of TIMP-1.¹⁴ The presence of all three TIMPs in SMCs thus suggests a complex system for the control of matrix metabolism by human vascular SMCs.

Our finding of enhanced TIMP-1 expression by FGF-2 is consistent with a general observation of TIMP-1 inducibility in other cell types.³⁹ Because TIMP-1 can inhibit MMP-1, however, this raises the possibility that induction of MMP-1 expression may not correlate with MMP-1 activity. While the capacity for collagen degradation may be tempered by increased TIMP-1 expression, we suspect that the net effect remains degradative for several reasons. First, TIMPs interact with MMPs with 1:1 stoichiometry, and the magnitude of increase found in MMP-1 expression after FGF-2 stimulation was far more striking than that for TIMP-1. Second, the rise in TIMP-1 was not accompanied by a rise in either TIMP-2 or TIMP-3; indeed, there was a decline in TIMP-2 and no notable change in TIMP-3. Because all three TIMPs have similar in vitro inhibitory activity against MMP-1,^{13 40} the aggregate effect may in fact be neutral. It should also be recognized that in addition to controlling the activity of MMPs, TIMPs possess growth-promoting activities,^{41 42} and the lack of uniformity in TIMP expression may reflect this diversity in function. Finally, net substrate degradation attributable to MMP-1 was detected by zymography in conditioned media of FGF-2–treated cultures but not in control cultures. This observation indicates that after FGF-2-treatment, MMP-1 was indeed present in excess of all three TIMPs. Taken together, our findings indicate that FGF-2 imparts a collagen-degradative phenotype to human SMCs.

In summary, FGF-2 potently inhibits collagen fiber production by human SMCs and induces MMP-1 expression, with modest and differential effects on expression of TIMPs. These changes may represent a mechanism for thinning the local collagen environment during vascular remodeling, which in turn may be critical for intimal accumulation of SMCs. Because FGF-2 has been identified in coronary lesions of patients with unstable angina,⁴³ the current findings may also be relevant to the mechanism of destabilization of atherosclerotic plaque.

	Selected Abbreviations and Acronyms

 

APMA = aminophenylmercuric acetate ATCC = American Type Culture Collection FBS = fetal bovine serum FGF = fibroblast growth factor IL = interleukin MMP(s) = matrix metalloproteinase(s) PDGF = platelet-derived growth factor SMC(s) = smooth muscle cell(s) TGF = transforming growth factor TIMP(s) = tissue inhibitor(s) of matrix metalloproteinases TNF = tumor necrosis factor

	Acknowledgments

 
This work was supported by a grant from the Medical Research Council of Canada (MRC) and an MRC Scholarship to J.G.P.

Received September 28, 1995; accepted May 20, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Ross R, Klebanoff SJ. The smooth muscle cell, I: in vivo synthesis of connective tissue proteins. J Cell Biol. 1971;50:159-171. [Abstract/Free Full Text]

2. Campbell G, Campbell J, Manderson J, Horrigan S, Rennick R. Arterial smooth muscle: a multifunctional mesenchymal cell. Arch Pathol Lab Med. 1988;112:977-986. [Medline] [Order article via Infotrieve]

3. Ang A, Tachas G, Campbell J, Bateman J, Campbell G. Collagen synthesis by cultured rabbit aortic smooth-muscle cells: alteration with phenotype. Biochem J. 1990;265:461-469. [Medline] [Order article via Infotrieve]

4. Barnes MJ. Collagens in atherosclerosis. Collagen Rel Res. 1985;5:65-97.

5. Shekhonin BV, Domagatsky SP, Muzykantof VR, Idelson GY, Rukosuev VS. Distribution of type 1, III, IV and V collagen in normal and atherosclerotic human arterial wall: immunomorphologic characteristics. Collagen Rel Res. 1985;5:355-368.

6. Murata K, Motayama T, Kotake C. Collagen types in various layers of the human aorta and their changes with the atherosclerotic process. Atherosclerosis. 1986;60:251-262. [Medline] [Order article via Infotrieve]

7. Morton LF, Barnes MJ. Collagen polymorphism in the normal and diseased blood vessel wall: investigation of collagen types I, III, and V. Atherosclerosis. 1982;42:41-51. [Medline] [Order article via Infotrieve]

8. Gross J, Nagai Y. Specific degradation of the collagen molecule by tadpole collagenolytic enzyme. Proc Natl Acad Sci U S A. 1965;54:1197-1204. [Free Full Text]

9. Netzel-Arnett S, Salari A, Goli UB, Van Wart HE. Evidence for a triple helix recognition site in the hemopexin-like domain of human fibroblast and neutrophil interstitial collagenases. Ann N Y Acad Sci. 1994;732:22-30. [Medline] [Order article via Infotrieve]

10. Weiss JB. Enzymatic degradation of collagen. In: Hall DA, Jackson DS, eds. International Review of Connective Tissue Research. New York, NY: Academic Press; 1976:102-149.

11. Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemore EN, Lark MW, Amento E, Libby P. Cytokine-stimulated human vascular smooth muscle cells synthesize a compliment of enzymes required for extracellular matrix digestion. Circ Res. 1994;75:181-189. [Abstract/Free Full Text]

12. Woessner JF. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991;5:2145-2154. [Abstract]

13. Apte SS, Olsen BR, Murphy G. The gene structure of tissue inhibitor of metalloproteinases (TIMP)-3 and its inhibitory activities define the distinct TIMP gene family. J Biol Chem. 1995;270:14313-14318. [Abstract/Free Full Text]

14. Leco KJ, Khokha R, Pavloff N, Hawkes SP, Edwards DR. Tissue inhibitor of metalloproteinase-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J Biol Chem. 1994;269:9352-9360. [Abstract/Free Full Text]

15. Gospodarowicz D. Fibroblast growth factor. Crit Rev Oncog. 1989;1:1-26. [Medline] [Order article via Infotrieve]

16. Folkman J, Klagsbrun M. Angiogenic factors. Science. 1987;235:442-447. [Abstract/Free Full Text]

17. Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest. 1990;85:2004-2008.

18. Lindner V, Lappi DA, Baird A, Majack RA, Reidy MA. Role of basic fibroblast growth factor in vascular lesion formation. Circ Res. 1991;68:106-113. [Abstract/Free Full Text]

19. Jackson CL, Reidy MA. Basic fibroblast growth factor: its role in the control of smooth muscle cell migration. Am J Pathol. 1993;143:1024-1031. [Abstract]

20. Lindner V, Reidy M. Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1991;88:3739-3743. [Abstract/Free Full Text]

21. Majors A, Ehrhart LA. Basic fibroblast growth factor in the extracellular matrix suppresses collagen synthesis and type III procollagen mRNA levels in arterial smooth muscle cells. Arterioscler Thromb. 1993;13:680-686. [Abstract/Free Full Text]

22. Kennedy SH, Qin H, Lin L, Tan EM. Basic fibroblast growth factor regulates type I collagen and collagenase gene expression in human smooth muscle cells. Am J Pathol. 1995;146:764-771. [Abstract]

23. Pickering JG, Weir L, Rosenfield K, Stetz J, Jekanowski J, Isner JM. Smooth muscle cell outgrowth from human atherosclerotic plaque: implications for the assessment of lesion biology. J Am Coll Cardiol. 1992;20:1430-1439. [Abstract]

24. Pickering JG, Bacha P, Weir L, Jekanowski J, Nichols JC, Isner JM. Prevention of smooth muscle cell outgrowth from human atherosclerotic plaque by a recombinant fusion protein specific for the epidermal growth factor receptor. J Clin Invest. 1993;91:724-729.

25. Sykes B, Puddle B, Francis M, Smith R. The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun. 1976;72:1472-1480. [Medline] [Order article via Infotrieve]

26. Franceschi RT, Iyer BS, Cui Y. Effects of ascorbic acid on collagen matrix formation and osteoblast differentiation in murine MC3T3-E1 cells. J Bone Min Res. 1994;9:843-854. [Medline] [Order article via Infotrieve]

27. Bellows CG, Sodek J, Yao K-L, Aubin JE. Phenotypic differences in subclones and long-term cultures of clonally derived rat bone cell lines. J Cell Biochem. 1986;31:153-169. [Medline] [Order article via Infotrieve]

28. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

29. Amento EP, Ehsani N, Palmer H, Libby P. Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells. Arterioscler Thromb. 1991;11:1223-1230. [Abstract/Free Full Text]

30. Jeffrey J. The biological regulation of collagenase activity. In: Mecham RP, ed. Regulation of Matrix Accumulation. Toronto, Canada: Academic Press Inc; 1986:53-98.

31. Kenagy RD, Nikkari ST, Welgus HG, Clowes AW. Heparin inhibits the production of three matrix metalloproteinases (stromelysin, 92 kD gelatinase, and collagenase) in primate arterial smooth muscle cells. J Clin Invest. 1994;93:1987-1993.

32. Werb Z, Tremble PM, Behrendsten O, Crowley E, Damsky CH. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol. 1989;109:877-889. [Abstract/Free Full Text]

33. Hurley MM, Abreu A, Harrison JR, Lichtler AC, Raisz LG, Kream BE. Basic fibroblast growth factor inhibits type I collagen gene expression in osteoblastic MC3T3-E1 cells. J Biol Chem. 1993;268:5588-5593. [Abstract/Free Full Text]

34. Tan EML, Rouda S, Greenbaum SS, Moore JH, Fox JW, Sollberg S. Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts. Am J Pathol. 1993;142:463-470. [Abstract]

35. Bornstein P, Sage H. Regulation of collagen gene expression. Prog Nucleic Acid Res Mol Biol. 1989;37:67-105. [Medline] [Order article via Infotrieve]

36. Lawrence R, J HD, Sonenshein G. Transforming growth factor ß1 stimulates type V collagen expression in bovine vascular smooth muscle cells. J Biol Chem. 1994;269:9603-9609. [Abstract/Free Full Text]

37. Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res. 1994;75:539-545. [Abstract/Free Full Text]

38. Yanagi H, Sasaguri Y, Sugama K, Morimatsu M, Nagase H. Production of tissue collagenase (matrix metalloproteinase 1) by human aortic smooth muscle cells in response to platelet-derived growth factor. Atherosclerosis. 1992;91:207-216.

39. Overall C. Regulation of tissue inhibitor of metalloproteinase expression. Ann N Y Acad Sci. 1994;732:51-64. [Medline] [Order article via Infotrieve]

40. Ward RV, Hembry RM, Reynolds JJ, Murphy G. The purification of tissue inhibitor of metalloproteinase-2 from its 72 kDa progelatinase complex. Biochem J. 1991;278:179-187.

41. Hayakawa T, Yamasita K, Ohuchi E, Shinagawa A. Cell growth-promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2). J Cell Sci. 1994;107:2373-2379. [Abstract]

42. Stetler-Stevenson WG, Bersch N, Golde DW. Tissue inhibitor of metalloproteinase-2 (TIMP-2) has erythroid-potentiating activity. FEBS Lett. 1992;296:231-234. [Medline] [Order article via Infotrieve]

43. Flugelman MY, Virmani R, Correa R, Yu Z-X, Farb AF, Leon MB, Elami A, Fu Y-M, Casscells W, Epstein SE. Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina. Circulation. 1993;88:2493-2500.[Abstract/Free Full Text]

This article has been cited by other articles:

				A. C. Newby Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates Cardiovasc Res, February 15, 2006; 69(3): 614 - 624. [Abstract] [Full Text] [PDF]

				C. M. Dollery and P. Libby Atherosclerosis and proteinase activation Cardiovasc Res, February 15, 2006; 69(3): 625 - 635. [Abstract] [Full Text] [PDF]

				J. T. Peterson The importance of estimating the therapeutic index in the development of matrix metalloproteinase inhibitors Cardiovasc Res, February 15, 2006; 69(3): 677 - 687. [Abstract] [Full Text] [PDF]

				A. C. Newby Dual Role of Matrix Metalloproteinases (Matrixins) in Intimal Thickening and Atherosclerotic Plaque Rupture Physiol Rev, January 1, 2005; 85(1): 1 - 31. [Abstract] [Full Text] [PDF]

				E. Fera, C. O'Neil, W. Lee, S. Li, and J. G. Pickering Fibroblast Growth Factor-2 and Remodeled Type I Collagen Control Membrane Protrusion in Human Vascular Smooth Muscle Cells: BIPHASIC ACTIVATION OF Rac1 J. Biol. Chem., August 20, 2004; 279(34): 35573 - 35582. [Abstract] [Full Text] [PDF]

				K. A. Detillieux, F. Sheikh, E. Kardami, and P. A. Cattini Biological activities of fibroblast growth factor-2 in the adult myocardium Cardiovasc Res, January 1, 2003; 57(1): 8 - 19. [Abstract] [Full Text] [PDF]

				A. R. Kranenburg, W. I. de Boer, J. H. J.M. van Krieken, W. J. Mooi, J. E. Walters, P. R. Saxena, P. J. Sterk, and H. S. Sharma Enhanced Expression of Fibroblast Growth Factors and Receptor FGFR-1 during Vascular Remodeling in Chronic Obstructive Pulmonary Disease Am. J. Respir. Cell Mol. Biol., November 1, 2002; 27(5): 517 - 525. [Abstract] [Full Text] [PDF]

				B. C. Berk Vascular Smooth Muscle Growth: Autocrine Growth Mechanisms Physiol Rev, July 1, 2001; 81(3): 999 - 1030. [Abstract] [Full Text] [PDF]

				E. Rocnik, L. Saward, and J. G. Pickering HSP47 Expression by Smooth Muscle Cells Is Increased During Arterial Development and Lesion Formation and Is Inhibited by Fibrillar Collagen Arterioscler Thromb Vasc Biol, January 1, 2001; 21(1): 40 - 46. [Abstract] [Full Text] [PDF]

				F. Tronc, Z. Mallat, S. Lehoux, M. Wassef, B. Esposito, and A. Tedgui Role of Matrix Metalloproteinases in Blood Flow-Induced Arterial Enlargement : Interaction With NO Arterioscler Thromb Vasc Biol, December 1, 2000; 20 (12): e120 - e126. [Abstract] [Full Text] [PDF]

				U. Ikeda, M. Shimpo, R. Ohki, H. Inaba, M. Takahashi, K. Yamamoto, and K. Shimada Fluvastatin Inhibits Matrix Metalloproteinase-1 Expression in Human Vascular Endothelial Cells Hypertension, September 1, 2000; 36(3): 325 - 329. [Abstract] [Full Text] [PDF]

				E. Rocnik, L. H. Chow, and J. G. Pickering Heat Shock Protein 47 Is Expressed in Fibrous Regions of Human Atheroma and Is Regulated by Growth Factors and Oxidized Low-Density Lipoprotein Circulation, March 21, 2000; 101(11): 1229 - 1233. [Abstract] [Full Text] [PDF]

				R. M. Korah, V. Sysounthone, Y. Golowa, and R. Wieder Basic Fibroblast Growth Factor Confers a Less Malignant Phenotype in MDA-MB-231 Human Breast Cancer Cells Cancer Res., February 1, 2000; 60(3): 733 - 740. [Abstract] [Full Text]

				C. M. Ford, S. Li, and J. G. Pickering Angiotensin II Stimulates Collagen Synthesis in Human Vascular Smooth Muscle Cells : Involvement of the AT1 Receptor, Transforming Growth Factor-{beta}, and Tyrosine Phosphorylation Arterioscler Thromb Vasc Biol, August 1, 1999; 19(8): 1843 - 1851. [Abstract] [Full Text] [PDF]

				C. Becerril, A. Pardo, M. Montaño, C. Ramos, R. Ramírez, and M. Selman Acidic Fibroblast Growth Factor Induces an Antifibrogenic Phenotype in Human Lung Fibroblasts Am. J. Respir. Cell Mol. Biol., May 1, 1999; 20(5): 1020 - 1027. [Abstract] [Full Text]

				M. D. Rekhter Collagen synthesis in atherosclerosis: too much and not enough Cardiovasc Res, February 1, 1999; 41(2): 376 - 384. [Abstract] [Full Text] [PDF]

				N. L’Heureux, S. Pâquet, R. Labbé, L. Germain, and F. A. Auger A completely biological tissue-engineered human blood vessel FASEB J, January 1, 1998; 12(1): 47 - 56. [Abstract] [Full Text]

				J. G. Pickering, S. Uniyal, C. M. Ford, T. Chau, M. A. Laurin, L. H. Chow, C. G. Ellis, J. Fish, and B. M. C. Chan Fibroblast Growth Factor-2 Potentiates Vascular Smooth Muscle Cell Migration to Platelet-Derived Growth Factor : Upregulation of {alpha}2ß1 Integrin and Disassembly of Actin Filaments Circ. Res., May 19, 1997; 80(5): 627 - 637. [Abstract] [Full Text]

				S. Li, L. H. Chow, and J. G. Pickering Cell Surface-bound Collagenase-1 and Focal Substrate Degradation Stimulate the Rear Release of Motile Vascular Smooth Muscle Cells J. Biol. Chem., November 3, 2000; 275(45): 35384 - 35392. [Abstract] [Full Text] [PDF]

This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pickering, J. G. Articles by Chow, L. H. Search for Related Content PubMed PubMed Citation Articles by Pickering, J. G. Articles by Chow, L. H.