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Published ahead of print on July 10, 2003, doi:10.1165/rcmb.2003-0050OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 101-108, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0050OC

Regulation of Proteoglycan Synthesis by Leukotriene D4 and Epidermal Growth Factor in Bronchial Smooth Muscle Cells

Susan Potter-Perigo, Coralie Baker, Christina Tsoi, Kathleen R. Braun, Scott Isenhath, Gaylene M. Altman, Leonard C. Altman and Thomas N. Wight

The Hope Heart Institute and Departments of Pathology and Medicine, Division of Allergy and Infectious Diseases, and School of Nursing, University of Washington, School of Medicine, Seattle, Washington

Address correspondence to: Thomas N. Wight, Ph.D., Department of Vascular Biology, The Hope Heart Institute, 1124 Columbia Street, Seattle, WA 98104. E-mail: twight{at}hopeheart.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Extracellular matrix (ECM) expansion contributes to airway remodeling in asthma. This study examines the effect of leukotriene D4 (LTD4), combined with epidermal growth factor (EGF), on proteoglycan synthesis by cultured human bronchial smooth muscle cells (BSMCs). LTD4 plus EGF stimulated proliferation of BSMCs with increased versican synthesis. Further, versican mRNA splice variants, V0 and V1, were differently regulated in BSMCs by LTD4 plus EGF. Synthesis of [35S]-methionine labeled versican V0, as a percentage of total versican, was doubled. This upregulation was confirmed by Western analysis. Synthetic changes were paralleled by alterations in versican V0 mRNA. The effects of LTD4 and EGF on proteoglycan synthesis were inhibited by montelukast. Similar upregulation of versican V0 was observed in arterial smooth muscle cells (ASMCs) stimulated with LTD4 plus EGF as measured by western and reverse transcriptase–polymerase chain reaction analyses. Changes in ECM in the asthmatic airway may parallel those in atherosclerotic lesions where proliferating ASMCs synthesize a versican-rich expanded ECM. Inhibition of these processes could lead to reduced tissue expansion in the early phases of asthma progression.

Abbreviations: arterial smooth muscle cell, ASMC • bronchial smooth muscle cell, BSMC • Dulbecco's modified Eagle's medium, DMEM • extracellular matrix, ECM • epidermal growth factor, EGF • epidermal growth factor receptor, EGF-R • fetal calf serum, FCS • fibroblast growth factor, FGF • leukotriene D4, LTD4 • reverse transcriptase–polymerase chain reaction, RT-PCR • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is characterized by airway wall thickening and obstruction. Part of this thickening is due to airway smooth muscle hyperplasia and hypertrophy (1, 2) and part is due to deposits of connective tissue in the extracellular matrix (ECM) (3–12). Recent studies have shown an increase in lung proteoglycans and hyaluronan, two components of the ECM, in lung fibrosis (13–16) and in mild forms of asthma (3, 12, 16, 17). Also, bronchial fibroblasts from patients with asthma synthesize increased amounts of proteoglycans (18), suggesting that the deposition of these molecules may be an early remodeling response. These events precede the deposition of collagens in later stages of fibrosis (8, 9). ECM enriched in proteoglycans tends to favor cellular proliferative responses, whereas ECM enriched in collagen tends to suppress proliferation (19). It may be that targeting processes that are involved in early matrix remodeling can prevent those processes involved in late fibrotic events.

Epidermal growth factor (EGF) has been identified as a significant contributor to changes in the airway wall in asthma (20). EGF and the EGF receptor (EGF-R) have been found in bronchial smooth muscle cells (BSMCs) in the developing rat lung (21), and they are markedly increased in BSMCs in the asthmatic human airway (22). Leukotriene D4 (LTD4) and other leukotrienes are potent bronchoconstrictors that contribute significantly to acute and chronic components of asthma (23, 24), and these mediators have been shown to stimulate the proliferation of smooth muscle cells (25–27). EGF alone stimulates the proliferation of cultured BSMC, and this stimulation is synergistically increased by the addition of LTD4 (28); however, the effects of LTD4 and EGF on ECM synthesis have not been examined. In other cell types, including arterial smooth muscle cells (ASMCs) and fibroblasts, proliferative stimuli cause qualitative and quantitative alterations in ECM composition (19). In addition, BSMCs cultured in atopic serum show increased deposition of ECM components (29). Our goal was to determine if the proliferative effects of EGF and LTD4 on BSMCs were correlated with changes in ECM synthesis. Increased expression and synthesis of versican concomitant with stimulation of proliferation in other cell types is well known (30, 31); however, a causal link between the two phenomena has not been proved. In this article we have characterized the major proteoglycans synthesized by cultured BSMCs and compared them to the well-characterized proteoglycans synthesized by ASMCs. These studies show that BSMCs synthesize the same major proteoglycans as ASMCs but in different relative amounts. In addition, we have studied the proliferation of BSMCs induced by LTD4 and EGF, and found that this event is temporally correlated with specific changes in the synthesis of isoforms of versican as well as other ECM components. Finally, we examined the effect of montelukast, an antagonist of CysLT1, an LTD4 receptor, (32) on these events and found that it partially inhibited the response of BSMCs to LTD4 plus EGF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Proteoglycan Isolation
Human BSMCs were obtained from Clonetics (BioWhittaker, Walkersville, MD). Samples from each cell line were immunostained by Clonetics and found to be negative for Factor VIII, a marker for endothelial cells, and positive for {alpha}-actin, a marker for smooth muscle cells. Because the phenotypes of smooth muscle cells and myofibroblasts are not distinct (33), the identification of these cells as smooth muscle also relies on their tissue source, the normal bronchial wall. The cells were maintained and grown in Clonetics proprietary medium (Smooth Muscle Cell Basal Medium) supplemented with 0.5 ng/ml human EGF, 5 µg/ml insulin, 2 ng/ml human fibroblast growth factor (FGF), 50 µg/ml Gentamicin, 50 ng/ml Amphotericin-B, and 5% fetal bovine serum (FBS). For experiments, cells were seeded at 6,300 cells per cm2 in 100-mm-diameter dishes or 15,000 cells per cm2 in 24-well tissue culture dishes and maintained in growth medium for 3–4 d until determined by visual inspection to be 80–90% confluent. They were then cultured for another 48 h in growth arrest medium, which was basal medium with FBS reduced to 0.1% and no added EGF or FGF. Treatments, including controls, were then added in fresh growth arrest medium. Montelukast was added 10 min before the addition of LTD4 and EGF. Montelukast was a generous gift from Merck and Co. (Whitehouse Station, NJ). EGF was purchased from Sigma (St. Louis, MO) and LTD4 from Biomol (Plymouth Meeting, PA). To investigate the effect of EGF and LTD4 on proteoglycan synthesis by BSMC, we first determined the optimal concentrations for interaction of these two agents for stimulation of total proteoglycan synthesis (data not shown). These were 1 ng/ml EGF and 0.5 µM LTD4, and all of the subsequent experiments reported here were performed using these concentrations. Human ASMCs (a generous gift from Elaine Raines, Department of Pathology, University of Washington, Seattle, WA) were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and growth-arrested in DMEM with 0.1% FBS (34).

To obtain radiolabeled proteoglycans, cells were metabolically labeled with 100 µCi/ml Na2[35S]-sulfate or 40 µCi/ml [35S]-methionine. The medium was then combined with protease inhibitors (5 mM benzamidine, 100 mM 6-aminohexanoic acid, and 50 mM phenylmethylsulfonyl fluoride). The cell layer was rinsed with phosphate-buffered saline and solubilized in 8 M urea buffer (8M urea, 2 mM EDTA, 0.25 M NaCl, 50 mM Tris-HCl, and 0.5% Triton-X 100 detergent, pH 7.4) containing protease inhibitors (31, 35).

Aliquots were taken for determination of total [35S]-sulfate incorporation into proteoglycans by cetylpryidinium chloride precipitation (36). Medium and cell layer extracts were concentrated and purified by ion exchange chromatography on diethylaminoethyl Sephacel in 8 M urea buffer and eluted with 8 M urea buffer containing 3 M NaCl (31, 35). Aliquots containing 30,000 dpm [35S]-sulfate or 100,000 dpm [35S]-methionine were precipitated in 80% ethanol and 1.3% potassium acetate before sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and some were digested with 2.3 U/ml chondroitin ABC lyase (Sigma) in 300 mM TRIS-buffered solution (300 mM TRIS, 0.06 mg/ml bovine serum albumin, 18 mM sodium acetate, pH 8.0) for 3 h at 37°C (37). SDS-PAGE was performed using a 4–12% gradient resolving gel and a 3.5% stacking gel.

Northern Blot Analysis
Total RNA was isolated from cultured BSMCs and ASMCs using the RNeasy Mini Kit with optional on-column DNase digestion with the RNase-Free DNase set (Qiagen, Valencia, CA). Purified samples were fractionated on 0.8% formaldehyde-agarose gels, alkali-denatured in 50 mM NaOH and 10 mM NaCl, and transferred to nylon blotting membranes (Zeta Probe; Bio-Rad, Richmond, CA). Blots were hybridized with [32P]-dCTP labeled cDNA probes to the following proteoglycans: versican, a mixture of clones F1 and C7 for base-pairs 1–1373 and 2607–6092 specific for the N-terminal and the ß-glycosaminoglycan regions, respectively (38), which recognize both splice variants, V0 and V1; full-length human biglycan, clone p16 (39); full-length bovine decorin (40); and a 1.34-kb portion of human perlecan, (41). These were generous gifts from Erkki Ruoslahti (The Burnham Institute, La Jolla, CA); Larry Fisher and Marian Young (both of the Craniofacial and Skeletal Disease Branch of the National Institute of Health, Bethesda, MD); and Renato Iozzo (Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA), respectively. In addition, probes for human collagen type I (42) and fibronectin (43) were generous gifts from F. Ramirez (Department of Microbiology and Immunology, State University of New York, Brooklyn, NY), and Jens Fisher (Department of Pathology, The University of Washington, Seattle, WA), respectively. For quantitation of mRNA levels, autoradiograms were normalized to the amount of 28S ribosomal RNA as revealed by ethidium bromide staining.

Reverse Transcriptase–Polymerase Chain Reaction
Expression of the two versican splice variants, V0 and V1, was examined by reverse transcriptase–polymerase chain reaction (RT-PCR) (44). Single-strand cDNA synthesis was performed in 50-µl reactions using 1–2 µg total RNA with random primers, 1 µl Rnase inhibitor (RNA-SIN; Ambion, Austin, TX), and 2.5 µl ImProm-II Reverse Transcriptase (Promega, Madison, WI) for 1 h at 42°C. The following versican primers were designed from Genbank sequences: versican V0 (U16306): forward primer 3959GAA ACC ACC TCT CAT CGA CAG G, reverse primer 4662GCA CAG CAG TAG ACA ATT CCG to give an expected product of 706 bp; versican V1 (X15998): forward 1135GCT TTG ACC AGT GCG ATT ACG, reverse 1701GCA CAG CAG TAG ACA ATT CCG to give an expected PCR product of 569 bp.

Conditions for PCR amplification were optimized for MgCl2 concentration and temperature using the PTC-200 gradient thermal cycler (MJ Research, Waltham, MA). PCR reactions were performed using 1–2 µl single-strand synthesis product, 250 mM dNTP, 1x Amplitaq Gold Buffer, 5 pmol of each primer, 0.75 U Amplitaq Gold Enzyme (PE Biosystems, Foster City, CA), and 1.5 mM MgCl2 in a 20-µl reaction volume.

After a 10-min initial enzyme activation step at 95°C for the Amplitaq Gold, denaturation was done for 1 min at 95°C, annealing at 61° for 1 min, and extension at 72°C for 50 s. Identical samples were taken out at different cycles to ensure linearity. Linearity was obtained at 35 and 37 cycles. For comparison of expression levels of the two different splice variants, PCR was performed on samples of reverse-transcribed RNA incubated in parallel under conditions resulting in a linear time course. They were then scanned and quantitated and expressed as a ratio of the two splice variant bands. A single band was obtained with each primer set.

Western Blotting
Proteoglycans were digested with chondroitin ABC lyase, applied to SDS-PAGE, and electrophoretically transferred to 0.2-µm nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using a BioRad (Hercules, CA) Transblot SD Semi-Dry Transfer Cell. The transferred proteins were then detected with primary antibodies and enhanced chemiluminescence (Western-Light Chemiluminescent Detection System with CSPD proprietary luminescent substrate; Tropix, Bedford, MA). Decorin was detected using the antibody, LF 136 (39), a generous gift from Dr. Larry Fisher (Craniofacial and Skeletal Disease Branch of the National Institutes of Health, Bethesda, MD). Antibodies recognizing versican V0 and versican V1, prepared against the {alpha} and ß glycosaminoglycan regions of versican splice variants by affinity purification (38) were a generous gift from Dieter Zimmermann (University of Zurich, Department of Pathology, Zurich, Switzerland).

DNA Assay
Cells were rinsed with phosphate-buffered saline, solubilized with 10 mM EDTA pH 12.3, neutralized with 1 M KH2PO4, combined with 100 mM NaCl-10 mM TRIS pH 7.0 buffer containing 200 ng/ml Hoescht 33,258 dye, and read with a flourimeter with excitation and emission wavelengths of 350 and 455 nm (45).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To characterize proteoglycan expression, BSMCs and ASMCs were grown to confluence, arrested in low serum medium for 48 h, and total RNA was harvested for Northern blot analysis. BSMCs and ASMCs contain mRNA for the matrix molecules decorin, collagen type I, versican, and perlecan, but in different relative amounts. In BSMCs, decorin, and collagen type I transcripts accounted for a larger portion of the total mRNA than in ASMCs. ASMCs, on the other hand, contained relatively more mRNA for versican and perlecan (Figure 1). Little or no versican message was found in growth-arrested BSMCs. Longer exposure of autoradiograms from growth-arrested cells was required to reveal mRNA for versican in the BSMCs and for decorin in the ASMCs.



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  		Figure 1. A comparison of ECM mRNA levels in growth-arrested cultured human BSMCs and ASMCs. Each cell type was represented by 15 µg total RNA on a single blot so that relative proportions of the different mRNAs can be compared. Northern blots reveal that both cell types contain mRNA for versican, perlecan, collagen type I, and decorin but in different relative amounts. 28S ribosomal RNA stained with ethidium bromide is included as a control for loading (A). Versican mRNA in BSMCs and decorin mRNA in ASMCs could not be detected. Autoradiograms which have been exposed for a sufficient time to detect versican and decorin mRNA in arrested BSMC and ASMC preparations, respectively, are shown in B.

 
To characterize the proteoglycans synthesized by these cells, BSMCs and ASMCs were grown to confluence, arrested in low serum medium for 48 h, and labeled for another 24 h with [35S]-sulfate in fresh growth-arrest medium. Proteoglycans in the cell culture medium were isolated and purified by ion exchange chromatography and applied to SDS-PAGE. It was observed that BSMCs and ASMCs synthesized and secreted the same proteoglycans but in different relative amounts. Versican and decorin were identified in medium from BSMCs by analogy to previous SDS-PAGE analysis of ASMCs (46). The predominant proteoglycan in medium from BSMCs was decorin, whereas that found in ASMC medium was versican (Figure 2A). These differences are consistent with those found by mRNA analysis (Figure 1). The identities of versican and decorin in BSMC cultures were also confirmed by Western blot analysis (Figure 2B) (38, 39).



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  		Figure 2. A comparison of [35S]-sulfate labeled proteoglycans synthesized by growth-arrested cultured BSMCs and ASMCs. Confluent cells were metabolically labeled with [35S]-sulfate for 24 h. Conditioned medium was purified by ion exchange, concentrated, and applied to SDS-PAGE. The two cell types secrete predominantly versican and decorin proteoglycans but in different relative amounts (A). Proteoglycans from proliferating BSMCs were also digested with chondroitin ABC lyase, applied to SDS-PAGE and processed for Western analysis to confirm the presence of versican and decorin (B).

 
It has previously been shown that EGF and LTD4 stimulate [3H]-thymidine incorporation in BSMCs (28). We obtained similar results. To determine the time course of the proliferative response of BSMCs to LTD4 plus EGF, cells were grown to confluence, growth arrested, and stimulated with the combination of these two activators. DNA was harvested from parallel wells on each day from Days 0–4 after stimulation (Figure 3A). The maximum effect was observed on Day 3. Controls had grown 42% above Day 0, whereas EGF plus LTD4–treated cells had grown 88% above the Day 0 level, or about twice as much as control (P <= 0.05). When the cells were grown to a greater, postconfluent density before addition of EGF and LTD4, they did not respond to these stimuli (not shown). Thus, cell density is clearly an important determinant of BSMC growth regulation by EGF and LTD4.



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  		Figure 3. The effect of EGF and LTD4 on proliferation of cultured BSMCs as measured by accumulation of DNA. (A) Cells were plated at subconfluent density (1.5 x 104/cm2) and grown for 3 d in complete growth medium; changed into growth-arrest medium for 2 d, and then stimulated in fresh growth arrest medium with or without 1 ng/ml EGF and 0.5 µM LTD4. Open circles, control; closed circles, stimulated. Cells from parallel wells were harvested for DNA analysis each day beginning on the day of stimulation through 4 d of growth. (B) Cells were seeded, grown, arrested, and stimulated as in A. Wells were harvested for DNA analysis on Day 3. Growth is expressed as the increase in DNA between Days 0 and 3 and normalized to controls. Additions included: 1, none (control); 2, 100 nM montelukast; 3, 0.5 µM LTD4; 4, 1 ng/ml EGF; 5, EGF and LTD4; and 6, EGF, LTD4, and montelukast. Differences at the 5% level were observed between the pairs: 1 and 4, 1 and 5, 1 and 6, 4 and 5.

 
A study to determine the effect of montelukast on EGF plus LTD4–stimulated proliferation of BSMCs demonstrated partial inhibition. To first determine appropriate concentrations of montelukast for these studies, growth-arrested cells were stimulated to proliferate by addition of 10% FBS with different added concentrations of montelukast. Cell growth was not altered by 100 nM montelukast, although it was reduced 20% by 500 nM montelukast (data not shown). It was therefore concluded that any effect of montelukast at 100 nM would be specific and mediated through the LTD4 receptor. Arrested BSMC were then grown in the presence of EGF and LTD4 with or without 100 nM montelukast and harvested for DNA analysis after 3 d in culture (Figure 3B). EGF alone stimulated proliferation above control, whereas LTD4 did not. EGF plus LTD4 stimulated proliferation more than EGF alone, and addition of montelukast produced a small reduction in the amount of DNA that was not statistically significant.

To determine if stimulation by EGF and LTD4 had an effect on proteoglycan mRNA expression, growth-arrested BSMCs were cultured with EGF plus LTD4 and total RNA was harvested at 1, 2, and 3 d after stimulation. Maximal effects were observed at Day 3. Figure 4A shows representative Northern blots for ECM components on Day 3. Both control and LTD4 plus EGF media were made in fresh growth-arrest medium, which by itself provides some stimulation of proliferation (Figure 3A) and causes an increase in versican mRNA levels in comparison to growth-arrested cells (Figures 1 and 4A). In Figure 4B, mRNA for each day is expressed as a percent of control level for each matrix molecule. The average of two different experiments is presented. On Day 3, mRNA for versican V0 from EGF plus LTD4–stimulated cells was increased to 1.90-fold control, and that for decorin was reduced to 0.38-fold control. Fibronectin transcripts increased to 1.29-fold control and collagen type I mRNA decreased to 0.57-fold control. Transcripts for versican V1, perlecan, and biglycan showed little change in comparison to control levels. In other experiments, versican V1 was slightly upregulated in comparison to controls, but not to the same extent as versican V0 (data not shown). As a result of the marked increase observed in V0 mRNA levels at Day 3, all further studies focused on versican expression and synthesis at this time point after stimulation with EGF and LTD4.



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  		Figure 4. A comparison of ECM mRNA levels in control and activated BSMCs. BSMCs were growth arrested as in Figure 3, treated with 1 ng/ml EGF and 0.5 µM LTD4 in fresh growth-arrest medium, and total RNA was harvested for Northern analysis at 24, 48, and 72 h. RNA was hybridized to cDNA for the matrix molecules, versican, decorin, biglycan, perlecan, collagen type I, and fibronectin. (A) Representative bands obtained on Day 3. (B) Results from two experiments which are normalized to 28S RNA levels to control for loading and then expressed as fold control. Error bars represent the range of two observations. Solid bars, versican V0; open bars, versican V1; bars with vertical lines, fibronectin; bars with dots, biglycan; bars with fine horizontal lines, perlecan; bars with cross-hatching, collagen type I; bars with wide horizontal lines, decorin. This analysis shows a major decrease in relative levels of decorin and collagen type I message levels on Days 2 and 3, and a doubling of versican V0 message on Day 3.

 
To examine the effect of LTD4 plus EGF on the synthesis and secretion of versican or decorin, growth-arrested BSMCs were cultured with the activators, labeled with [35S]-sulfate between 48 and 72 h thereafter, and proteoglycan production determined on SDS-PAGE. In EGF plus LTD4–activated cells, [35S]-sulfate labeled versican, which includes versican V0 and versican V1, was increased (Figure 5A). Western analysis, using an antibody specific for the {alpha}-GAG domain of versican V0, demonstrated a 46% increase in synthesis of versican V0 above control when determined on a per-volume basis (Figure 5B). In cells treated with EGF and LTD4, SDS-PAGE analysis showed that decorin synthesis was reduced when expressed as a percent of the total synthesized (by analysis with NIH Image) from 62–52%. Although the synthesis of 35S-sulfate–labeled decorin was reduced as a percent of the total proteoglycans made, overall proteoglycan synthesis was increased. Moreover, the decorin band ran at a larger apparent molecular weight than the control, indicating that there may be longer glycosaminoglycan chains and hence more sulfation per decorin molecule. Previous studies in ASMC have shown increased glycosaminoglycan chain size to be responsible for such shifts in response to proliferative stimuli (35). Consequently, there was no net change in the 35S-sulfate–labeled decorin production on a per-cell basis.



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  		Figure 5. Effects of EGF and LTD4 on proteoglycan synthesis by human BSMCs as measured by incorporation of [35S]-sulfate into glycosaminoglycans, incorporation of [35S]-methionine into core proteins, and Western analysis. Cells were growth arrested and then stimulated with 1 ng/ml EGF and 0.5 µM LTD4, as for Figure 3. (A) Cells were metabolically labeled with [35S]-sulfate from 48–72 h after stimulation. Conditioned medium was harvested for SDS-PAGE analysis with equal counts per lane. Treatment with the activators caused a relative increase in the proportion of total versican (V0 plus V1) in comparison to decorin and increased the apparent molecular weight of decorin. (B) Conditioned medium was prepared for SDS-PAGE as in A and then subjected to Western blotting with antibody to V0 in lanes loaded with equal volumes of medium. V0 secretion was increased by treatment with the activators. (C) Cells were metabolically labeled with [35S]-methionine during the last 24 h of a 72-h stimulation period, purified with ion-exchange chromatography, digested with chondroitin ABC lyase, and subjected to SDS-PAGE. The gel was loaded with equal volumes of labeled medium. Lanes 1–6 are media from cells cultured with these additions: 1, none (control); 2, EGF and LTD4; 3, 100 nM montelukast; 4, 500 nM montelukast; 5, EGF, LTD4, and 100 nM montelukast; and 6, EGF, LTD4, and 500 nM montelukast. Stimulation by the activators caused a reduction in the amount of decorin core protein relative to versican core proteins, and an increase in the ratio of versican V0 to versican V1 core proteins as well as an increase in the total amount of labeled material. The increase in versican V0 secretion relative to versican V1 was partially inhibited by the addition of montelukast. (D) Cells were treated as in C. Different concentrations of montelukast were used to demonstrate the dose-dependent nature of its inhibition of the increase in versican V0 relative to V1. Lane 1, control; lane 2, 1 ng/ml EGF alone; lanes 3–7, EGF and 0.5 µM LTD4. Montelukast doses were: 0, 50, 100, 200, and 500 nM in lanes 3–7, respectively. The protein bands were scanned and the relative density of the two core protein bands was determined. At 50 nM, montelukast had no effect; 100 nM decreased the versican V0/versican V1 ratio by 30% and 200 nM montelukast returned the ratio to control levels.

 
Next, core protein synthesis was measured. Growth-arrested BSMCs were labeled with [35S]-methionine from 48–72 h after stimulation with fresh growth-arrest medium with or without EGF and LTD4. The assumption was made that the specific activity of the methionine pool was identical in the two experimental conditions. Proteoglycans secreted into the culture medium were isolated by ion exchange, digested with chondroitin ABC lyase, and applied to SDS-PAGE (Figure 5C). Lanes were loaded with material from equal volumes of medium to demonstrate the net change in proteoglycan synthesis. Total [35S]-material was increased by 100% in the LTD4 plus EGF–stimulated cells in comparison to the control. The increase in total synthesis was partially a consequence of an increase in cell number (Figure 3A). When normalized to cell number, the increase is ~ 50% above control. Labeled material in cells treated only with montelukast was similar to that from controls; however, cells treated with LTD4, EGF, and montelukast contained an intermediate amount of material, indicating that montelukast partially inhibited the combined effect of the two activators.

The lanes were scanned vertically with NIH Image software to quantify the relative synthesis of the two versican splice variants, versican V0 and versican V1, and decorin resulting from each treatment. [35S]-methionine–labeled decorin comprised 49% of the total label in the control cells but only 36% of the total in the LTD4 plus EGF–stimulated cells (Figure 5C). This reduction in decorin core protein as a percent of the total labeled material is consistent with the reduction we observed in decorin mRNA from similarly treated cultures (Figure 4). On the other hand, there was no change in the amount of decorin core synthesis on a per-cell basis. Wells cultured with EGF, LTD4, and 100 or 500 nM montelukast contained 43 and 42% decorin, values intermediate between the control and stimulated values of 49 and 36%, respectively. Thus, montelukast partially inhibited the change in decorin synthesis as a percent of the total proteoglycans due to stimulation with EGF and LTD4.

The relative intensity of versican V0 and versican V1 bands was also determined within each lane (Figure 5C). EGF and LTD4 altered synthesis of the proteoglycan core proteins, revealing differential regulation of the versican splice variants V0 and V1. Versican V0 as a percent of the total versican was 8.4% in the control cells and more than doubled at 20.3% in the LTD4 plus EGF–stimulated cells. When normalized to cell number, V0 production was increased 180% above controls, whereas V1 was increased 50% above control cells. This preferential increase in versican V0 relative to versican V1 after stimulation with these activators is consistent with the changes observed in mRNA levels for those two splice variants (Figure 4). In cells treated with 100 and 500 nM montelukast, versican V0 comprised 12.1 and 10.0% of the total versican, whereas in cells treated with LTD4 plus EGF and 100 or 500 nM montelukast, it was 18.9 and 15.6% of the total versican, a level intermediate between control and activated cells.

Montelukast inhibits the increase in versican V0 core protein synthesis relative to versican V1 in a dose-dependent fashion. In these experiments, cells were treated as for Figure 5C. Arrested cells were stimulated with EGF plus LTD4, and different concentrations of montelukast from 0–500 nM were added to the stimulated cells. [35S]-methionine was added after 48 h and the medium was collected at 72 h after stimulation. The ratio of versican V0 to versican V1, as revealed by SDS-PAGE, was more than doubled by treatment with EGF plus LTD4. No reduction was detected with the addition of 50 nM montelukast; 100 nM montelukast reduced the effect by ~ 30%; and 200 nM montelukast returned the V0/V1 ratio to the control level (Figure 5D). These results confirm the differential regulation of the two splice variants shown in Figure 5C.

To determine if the selective upregulation of versican V0 in stimulated BSMCs was unique to those cells or if it is a more general phenomenon in smooth muscle cells, low serum arrested ASMCs were stimulated with LTD4 and EGF for 24 h and assayed for versican V0 mRNA and protein synthesis. Cell number did not change over 24 h, so that normalization of results to cell number was not necessary. Western analysis of medium proteoglycans demonstrated increased versican V0 synthesis in comparison to controls (Figure 6A). Total RNA was subjected to RT-PCR. This analysis showed that versican V0 mRNA was increased with respect to versican V1 mRNA, from a versican V0 to versican V1 ratio of 0.40 in unstimulated control cells to 0.72 in LTD4 and EGF–treated cells (P <= 0.05) (Figures 6B and 6C).



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  		Figure 6. Analyses of versican V0 and V1 expression in ASMCs after treatment with EGF and LTD4 by western blotting and RT-PCR. Subconfluent ASMCs were growth arrested in DMEM with 0.1% fetal calf serum for 48 h. They were then fed fresh medium alone or with 1 ng/ml EGF and 0.5 µM LTD4, and harvested at 24 h. (A) Equal volumes of conditioned media were subjected to Western analysis for the V0 variant of versican. Quantitation of versican expression by RT-PCR of mRNA from cells incubated with EGF and LTD4 or control. (B) Representative lanes were probed for versican V0 and versican V1. (C) Lanes, as in B, were scanned and quantitated. The ratio of versican V0 to versican V1 was determined. Versican V0 message was increased with respect to V1 message by treatment with EGF and LTD4 (P <= 0.05, n = 3). Bars indicate SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Airway remodeling in chronic asthma may be viewed as a consequence of inflammation, resulting in release of cytokines and growth factors causing, in turn, activation of fibroblasts and smooth muscle cells in the vicinity of the injury. Activated smooth muscle cells can change into a proliferative and/or migratory state (47) with the elaboration of increased levels of matrix metalloproteinases, other proteases (48), and an altered complement of ECM molecules (49, 50). The presence of large amounts of versican in fibroproliferative lesions is a constant feature of all of the major fibrotic lung diseases and has been observed in fatal asthma (16). Also, the spatial association of versican with proliferating and migrating myofibroblasts in the diseased lung suggests a significant contribution of this molecule to the phenomenon of remodeling in chronic lung diseases (16). However, the regulation of BSMCs' expression and synthesis of versican splice variants in the lung has not been studied. Increased proliferation and versican V0 synthesis by BSMCs in response to EGF and LTD4, as we describe, are consistent with previous in vivo (16) and in vitro observations (28). To our knowledge, the preferential increase of the V0 splice variant, in comparison to V1, has not previously been shown.

Our studies show increased synthesis of total proteoglycans and versican V0 by BSMCs in response to EGF and LTD4. Although versican V0 mRNA and protein synthesis were increased by the combined addition of LTD4 and EGF, versican V1 production showed little or no change. This differential regulation of the two splice variants was also found in ASMCs, suggesting that it occurs in various smooth muscle cell types. Variation of versican V0 and V1 expression with cell proliferation has also been observed in tissue from aortic aneurisms where downregulation of versican V0 in comparison to versican V1 has been found along with loss of ASMCs (51).

In our studies of LTD4- and EGF-induced BSMC proliferation, 100 nM montelukast reduced the effect of these activators but did not reduce it to the effect of EGF alone. This is not surprising because the affinity of LTD4 for the LTD4 receptor, CysLT1, is greater than that of montelukast (IC50 = 0.9–1.0 nM versus 2.3–4.5nM [32]) and LTD4 was added in a 5-fold excess, on a molar basis, above the concentration of montelukast. In addition, CysLT2, LTD4 receptors, are present on BSMCs, and these receptors are not inhibited by montelukast (52). LTD4 plus EGF stimulation of proteoglycan synthesis was completely inhibited by 200 nM montelukast. The success of this inhibition may be partly due to the fact that montelukast was added to the cultures before addition of LTD4.

mRNA for collagen type I was reduced and that for fibronectin was increased in BSMCs exposed to EGF and LTD4. Decorin expression was reduced as a percentage of the total proteoglycans at the protein and mRNA levels, suggesting that it is regulated differently from the other proteoglycans by EGF and LTD4. Co-regulation of decorin and collagen type I synthesis has been described in other cell systems (53) where decorin has been implicated in the regulation of size and abundance of collagen fibrils (54). In addition, conditions that stimulate proliferation of fibroblasts, epithelial cells, and endothelial cells induce downregulation of collagen type I synthesis and upregulation of fibronectin synthesis (55).

In studies similar to ours with ASMCs, versican synthesis increased in response to proliferative stimuli, and it is also known that versican accumulates in atherosclerotic plaque where the population of ASMCs has increased (50). Consistent with these phenomena, our data show that changes in proteoglycan synthesis by BSMCs are temporally coordinated with increased levels of DNA. In addition, increased expression of glycosaminoglycan synthases in LTD4 and EGF–activated BSMCs is suggested by the greater apparent molecular weight of decorin made by these cells. This increase is also found in ASMCs, which have been stimulated to proliferate by platelet-derived growth factor (31).

Growth-arrested BSMCs and ASMCs express collagen type I and predominant proteoglycans versican and decorin but in different relative amounts, corresponding to differences in the ECM found in the airway wall and in the human aorta. Collagen type I and decorin are expressed at a higher level in BSMCs than in ASMCs and are major components in the normal airway wall and in lung fibrosis (3, 4, 7–9). Versican, the predominant proteoglycan found in ASMCs, is also highly expressed in normal arterial tissue and especially in atherosclerotic intima (49, 50). The molecular composition of proteoglycans secreted by BSMCs is similar to that found in fibroblast cultures (56) in that relatively less versican is produced than in ASMCs. This suggests that the BSMCs used in our study may be similar to myofibroblasts. Myofibroblasts have been shown to originate from both fibroblasts and smooth muscle cells and cannot be distinguished from smooth muscle cells by any exclusive set of characteristics (33). Therefore, the {alpha}-actin–positive smooth muscle cells derived from the bronchial wall used in this study may be physiologically similar to myofibroblasts and thus the progenitors of occlusive fibrotic airway myofibroblasts (57).

Cell migration and elaboration of ECM in atherosclerosis has been explained by the "Response to Injury" model, in which an early response includes ASMC migration, proliferation, and proteoglycan secretion. Versican and hyaluronan are major ECM components involved in vascular remodeling in atherosclerosis. These molecules have been shown to accompany cell proliferation and result in expansion of the extracellular space (58). The original function of this expansion may be the provision of a loose, hydrated matrix in which cells can migrate and divide. Collagen type I and decorin are usually coordinately regulated and their synthesis is inversely regulated with proliferation (19) as we have observed in these studies. Later stages of the atherosclerotic response can include deposition of collagen in this space to form a strong and antiproliferative matrix that is similar to that found in the fibrotic lung ECM (7–9). The proliferative, versican-synthesizing phase of BSMC activity may be chronically maintained in severe pulmonary fibrosis, where high levels of versican are found (13), whereas in asthma it may occur briefly after each asthmatic event, followed by a return to the normal, nonproliferating cell type that synthesizes primarily decorin and collagen components of the ECM. An understanding of the sequence of BSMC responses to activation by inflammatory agents will help to illuminate the asthmatic sequellae in vivo. Each asthmatic event could result in a small increase in the total number of BSMCs and amount of ECM. The greater tendency of BSMCs to synthesize an ECM rich in collagen and decorin and with less versican than ASMCs may explain the different consequence of chronic injury in the lung, where gradual fibrosis is predominant, and in the aorta, where accumulation of a proteoglycan-rich luminal ECM leads to accumulation of lipids and thrombus formation.


   Acknowledgments
 
The authors thank the Merck Medical School Grant Program for financial assistance and Ellen Briggs for editing the manuscript.

Received in original form February 11, 2003

Received in final form June 25, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Redington, A. E., and P. H. Howarth. 1997. Airway wall remodeling in asthma. Thorax 52:310–312.[Medline]
  2. Chetta, A., A. Foresi, M. DelDonno, G. Bertorelli, A. Pesci, and D. Olivieri. 1997. Airways remodeling is a distinctive feature of asthma and is related to severity of disease. Chest 111:852–857.[Abstract/Free Full Text]
  3. Roberts, C. R. 1995. Is asthma a fibrotic disease? Chest 107:111S–117S.[Free Full Text]
  4. Roberts, C. R., T. N. Wight, and V. C. Hascall. 1997. Proteoglycans. In The Lung: Scientific Foundations, 2nd ed. R. G. Crystal and J. B. West, editors. Lippincott-Raven Publishers, Philadelphia, PA. 757–767.
  5. Altraja, A., A. Laitinen, I. Virtanen, M. Kämpe, B. G. Simonsson, S. Karlsson, L. Häkansson, P. Venge, H. Sillastu, and L. A. Laininen. 1996. Expression of laminins in the airways in various types of asthmatic patients: a morphometric study. Am. J. Respir. Cell Mol. Biol. 15:482–488.[Abstract]
  6. Laitinen, A., A. Altraja, M. Kampe, M. Linden, I. Virtanen, and L. A. Laitinen. 1997. Tenascin is increased in airway basement membrane of asthmatics and decreased by an inhaled steroid. Am. J. Respir. Crit. Care Med. 156:951–958.[Abstract/Free Full Text]
  7. Pare, P. D., J. R. Bai, and C. R. Roberts. 1996. The structural and functional consequences of chronic allergic inflammation of the airways. Ciba Found. Symp. 206:71–86.
  8. Roche, W. R., R. Beasley, J. H. Williams, and S. T. Holgate. 1989. Superepithelial fibrosis in the bronchi of asthmatics. Lancet 1:520–524.[CrossRef][Medline]
  9. Brewster, C. E. P., P. H. Howarth, J. Djukanovic, J. Wilson, S. T. Holgate, and W. R. Roche. 1990. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 3:507–511.
  10. Holgate, S. T., D. E. Davies, P. M. Lackie, S. J. Wilson, S. M. Puddicombe, and J. L. Lordan. 2000. Epithelial-mesenchymal interactions in the pathogenesis of asthma. J. Allergy Clin. Immunol. 105:193–204.[CrossRef][Medline]
  11. Page, C. 2000. The role of proteoglycans in the regulation of airways inflammation and airways remodeling. J. Allergy Clin. Immunol. 105:S518–S521.[CrossRef][Medline]
  12. Huang, J., R. Olivenstein, R. Taha, Q. Hamid, and M. Ludwig. 1999. Enhanced proteoglycan deposition in the airway wall of atopic asthmatics. Am. J. Respir. Crit. Care Med. 160:725–729.[Abstract/Free Full Text]
  13. Bensadoun, E. S., A. K. Burke, J. C. Hogg, and C. R. Roberts. 1996. Proteoglycan deposition in pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 154:1819–1828.[Abstract]
  14. Karlinsky, J. B. 1982. Glycosaminoglycans in emphysematous and fibrotic hamster lungs. Am. Rev. Respir. Dis. 125:85–88.
  15. Cantor, J. O., M. Cerrata, S. H. Mott, I. Mandl, and G. M. Turino. 1983. Glycosaminoglycan synthesis in bleomycin-induced pulmonary fibrosis: biochemistry and autoradiography. Proc. Soc. Exp. Biol. Med. 174:172–181.[CrossRef][Medline]
  16. Roberts, C. 2002. Versican in the cell biology of pulmonary fibrosis. In Proteoglycans in Lung Disease, Lung Biology in Health and Disease. H. A. Garg, P. J. Roughly, and C. N. Hales, editors. Marcel Dekker, NY. 168.
  17. Shute, J. K., J. Criss, P. Hockey, and P. H. Howarth. 1998. Increased proteoglycans in sputum in asthma and cystic fibrosis (CF) and increased GAG levels in CF [C48; Poster: L63; Abstract page: A607]. ALA/ATS International Conference, April 24–29, 1998.
  18. Tremblay, G. M., M. J. Lafreniere-Allard, G. Westergren-Thorsson, A. Malmstrom, J. Chakir, J. Dube, and L. P. Boulet. In vitro production of proteoglycans by asthmatic bronchial fibroblasts correlates with airway hyperresponsiveness [A91; Poster: 513; Abstract page: A247]. ALA/ATS International Conference, April 24–29, 1998.
  19. Wight, T. N., M. G. Kinsella, and E. Qwarnström. 1993. The role of proteoglycans in cell adhesion, migration and proliferation. Curr. Opin. Cell Biol. 4:793–801.
  20. Vignola, A. M., G. Chiappara, P. Chanez, A. M. Merendino, E. Pace, M. Spatafora, J. Bousquet, and G. Bonsignore. 1997. Growth factors in asthma. Monaldi Arch. Chest Dis. 52:159–169.[Medline]
  21. Strandjord, T. P., J. G. Clark, and D. K. Madtes. 1994. Expression of TGF-alpha, EGF, and EGF receptor in fetal rat lung. Am. J. Physiol. 267:L384–L389.
  22. Amishima, M., M. Munakata, Y. Nasuhara, A. Sato, T. Takahashi, Y. Homma, and Y. Kawakami. 1998. Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway. Am. J. Respir. Crit. Care Med. 157:1907–1912.
  23. Wenzel, S. E. 1997. Arachadonic acid metabolites: mediators of inflammation in asthma. Pharmacotherapy 17:3S–12S.[Medline]
  24. Leff, A. R. 2001. Regulation of leukotrienes in the management of asthma: biology and clinical therapy. Annu. Rev. Med. 52:1–14.[CrossRef][Medline]
  25. Porreca, E., C. Di Febbo, M. Reale, R. Barbacane, A. Mezzetti, F. Cuccurullo, and P. Conti. 1995. Modulation of rat vascular smooth muscle cell (VSMC) proliferation by cysteinyl leukotriene D4: a role for mediation of interleukin 1. Atherosclerosis 113:11–18.[CrossRef][Medline]
  26. Palmberg, L., H. E. Claesson, and J. Thyberg. 1989. Effects of leukotrienes on phenotypic properties and growth of arterial smooth muscle cells in primary culture. J. Cell Sci. 93:391–399.[Abstract/Free Full Text]
  27. Palmberg, L., J. A. Lindgren, J. Thyberg, and H. E. Claesson. 1991. On the mechanism of induction of DNA synthesis in cultured arterial smooth muscle cells by leukotrienes: possible role of prostaglandin endoperoxide synthase products and platelet-derived growth factor. J. Cell Sci. 98:141–149.[Abstract/Free Full Text]
  28. Panettieri, R. A., Jr., E. M. L. Tan, V. Ciocca, M. A. Luttmann, T. B. Leonard, and D. W. P. Hay. 1998. Effects of LTD4 on human airway smooth muscle cell proliferation, matrix expression, and contraction in vitro: differential sensitivity to cysteinyl leukotriene receptor antagonists. Am. J. Respir. Cell Mol. Biol. 19:453–461.[Abstract/Free Full Text]
  29. Johnson, P. R. A., J. L. Black, S. Carlin, Q. Ge, and P. A. Underwood. 2000. The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture. The effect of beclomethasone. Am. J. Respir. Crit. Care Med. 162:2145–2151.[Abstract/Free Full Text]
  30. Wight, T. N., S. Potter-Perigo, and T. Aulinskas. 1989. Proteoglycans and Vascular Cell Proliferation. Am. Rev. Respir. Dis. 140:1132–1135.[Medline]
  31. Schönherr, E., H. T. Järveläinen, L. J. Sandell, and T. N. Wight. 1991. Effects of platelet-derived growth factor and transforming growth factor-ß1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J. Biol. Chem. 266:17640–17647.[Abstract/Free Full Text]
  32. Lynch, K. R., G. P. O'Neil, Q. Liu, D. S. Im, N. Sawyer, K. M. Metters, N. Coulombe, M. Abramovitz, D. J. Figueroa, Z. Zeng, B. M. Connolly, C. Bai, C. P. Austin, A. Chateauneuf, R. Stocco, G. M. Greig, S. Kargman, S. B. Hooks, E. Hosfield, D. L. Williajms, Jr., A. W. Ford-Hutchinson, C. T. Caskey, and J. F. Evans. 1999. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399:789–793.[CrossRef][Medline]
  33. Desmouliere, A., and G. Gabbiani. 1996. The role of the myofibroblast in wound healing and fibrocontractive diseases. In The Molecular and Cellular Biology of Wound Repair. R. A. Clark, editor. Plenum Press, New York. 311–338.
  34. Ross, R., J. Glomset, B. Kariya, and L. Harker. 1974. A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Natl. Acad. Sci. USA 71:1207–1210.[Abstract/Free Full Text]
  35. Schönherr, E., H. T. Järveläinen, M. G. Kinsella, L. J. Sandell, and T. N. Wight. 1993. Platelet-derived growth factor and transforming growth factor-ß1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 13:1026–1036.[Abstract/Free Full Text]
  36. Wasteson, A., K. Uthne, and B. Westermark. 1973. A novel assay for the biosynthesis of sulphated polysaccharide and its application to studies on the effects of somatomedin on cultured cells. Biochem. J. 136:1069–1074.[Medline]
  37. Saito, H., T. Yamagata, and S. Suzuki. 1968. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243:1536–1542.[Abstract/Free Full Text]
  38. Zimmerman, D. R., and E. Ruoslahti. 1989. Multiple domains of the large fibroblast proteoglycan, versican. EMBO J. 8:2975–2981.[Medline]
  39. Fisher, L. W., J. D. Termine, and M. F. Young. 1989. Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species. J. Biol. Chem. 264:4571–4576.[Abstract/Free Full Text]
  40. Day, A. A., C. I. McQuillan, J. D. Termine, and M. R. Young. 1987. Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone. Biochem. J. 248:801–805.[Medline]
  41. Dodge, G. F., I. Kovalszky, M. L. Chu, J. R. Hassell, O. W. McBride, H. F. Yi, and R. V. Iozzo. 1991. Heparan sulfate proteoglycan of human colon: partial molecular cloning, cellular expression, and mapping of the gene (HSPG2) to the short arm of human chromosome #1. 10:673–680.
  42. Chu, M. L., J. C. Myers, M. P. Bernard, J. F. Ding, and F. Ramirez. 1982. Cloning and characterization of five overlapping cDNAs specific for the human pro {alpha} 1 (I) collagen chain. Nucleic Acids Res. 10:5925–5934.[Abstract/Free Full Text]
  43. Baley, P., I. Lutzelschwab, T. Scott-Burden, W. Kung, and U. Eppenberger. 1990. Modulation of extracellular-matrix synthesized by cultured stromal cells from normal human breast tissue by epidermal growth factor. J. Cell. Biochem. 43:111–125.[CrossRef][Medline]
  44. Rappolee, D. A., D. Mark, M. J. Banda, and Z. Werb. 1988. Wound macrophage express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science 241:708–712.[Abstract/Free Full Text]
  45. West, D. C., A. Sattar, and S. Kumar. 1985. A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells. Anal. Biochem. 147:289–295.[CrossRef][Medline]
  46. Olin, K. L., S. Potter-Perigo, P. H. Barrett, T. N. Wight, and A. Chait. 1999. Lipoprotein lipase enhances the binding of native and oxidized low density lipoproteins to versican and biglycan synthesized by cultured arterial smooth muscle cells. J. Biol. Chem. 274:34629–34636.[Abstract/Free Full Text]
  47. Chamley-Campbell, J. H., R. G. Campbell, and R. Ross. 1981. Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. J. Cell Biol. 89:379–383.[Abstract/Free Full Text]
  48. Nikkari, S. T., H. T. Järveläinen, T. N. Wight, M. Ferguson, and A. W. Clowes. 1994. Smooth muscle cell expression of extracellular matrix genes after arterial injury. Am. J. Pathol. 144:1348–1356.[Abstract]
  49. Wight, T. N. 1996. The vascular extracellular matrix. In Atherosclerosis and Coronary Artery Disease. V. Fuster, R. Ross, and E. J. Topol, editors. Raven Press, New York. 421–440.
  50. Wight, T. N., S. Lara, R. Riessen, R. Le Baron, and J. Isner. 1997. Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries. Am. J. Pathol. 151:963–973.[Abstract]
  51. Theocharis, A. D., I. Tsolakis, A. Hjerpe, and N. K. Karamanos. 2001. Human abdominal aortic aneurysm is characterized by decreased versican concentration and specific downregulation of versican isoform V0. Atherosclerosis 154:367–376.[CrossRef][Medline]
  52. Heise, C. E., B. F. O'Dowd, D. J. Figueroa, N. Sawyer, T. Nguyen, D. S. Im, R. Stocco, J. N. Bellefeuille, M. Abramovitz, R. Cheng, D. L. Williams, Z. Zeng, Q. Liu, L. Ma, M. K. Clements, N. Coulombe, Y. Liu, C. P. Austin, S. R. George, G. P. O'Neill, K. M. Metters, K. R. Lynch, and J. F. Evans. 2000. Characterization of the human cysteinyl leukotriene 2 receptor. J. Biol. Chem. 275:30531–30536.[Abstract/Free Full Text]
  53. Järveläinen, H. T., M. L. Iruela-Arispe, M. G. Kinsella, L. J. Sandell, E. H. Sage, and T. N. Wight. 1992. Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro. Exp. Cell Res. 203:395–401.[CrossRef][Medline]
  54. Pogany, G., D. J. Hernandez, and K. G. Vogel. 1994. The in vitro interaction of proteoglycans with type 1 collagen is modulated by phosphate. Arch. Biochem. Biophys. 31113:102–111.
  55. Danen, E. H., and K. M. Yamada. 2001. Fibronectin, integrins, and growth control. J. Cell. Physiol. 189:1–13.[CrossRef][Medline]
  56. Qwarnström, E., H. Järveläinen, M. Kinsella, C. Ostberg, L. Sandell, R. Page, and T. Wight. 1993. Interleukin-1 beta regulation of fibroblast proteoglycan synthesis involves a decrease in versican steady-state mRNA levels. Biochem. J. 294:613–620.
  57. Roberts, C. R., and A. K. Burke. 1998. Remodeling of the extracellular matrix in asthma: proteolgycans synthesis and degradation. Can. Respir. J. 5:48–50.[Medline]
  58. Evanko, S. P., J. C. Angello, and T. N. Wight. 1999. Formation of versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 19:213–225.



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