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(Circulation Research. 1997;81:289-296.)
© 1997 American Heart Association, Inc.

Articles

Growth Factor and Cytokine-Regulated Hyaluronan-Binding Protein TSG-6 Is Localized to the Injury-Induced Rat Neointima and Confers Enhanced Growth in Vascular Smooth Muscle Cells

Li Ye, Rosalia Mora, Nahid Akhayani, Christian C. Haudenschild, , Gene Liau

From the Department of Molecular Biology (L.Y., R.M., N.A., G.L.) and Experimental Pathology (C.C.H.), Jerome H. Holland Laboratory, American Red Cross, Rockville, Md, and the Department of Anatomy and Cell Biology (G.L.), The George Washington University Medical Center, Washington, DC.

Correspondence to Dr Gene Liau, Department of Molecular Biology, Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail Liau{at}usa.redcross.org

	Abstract

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Abstract Hyaluronan (HA) and HA-binding proteins have been implicated in a diverse array of biological processes, including development, tissue repair, and tumor invasion. However, the role of HA and HA-binding proteins in atherosclerosis and restenosis is poorly understood. PS4 (TSG-6) is a HA-binding protein expressed by cultured vascular smooth muscle cells (SMCs) in response to serum and growth factor stimulation. To delineate a possible role for TSG-6 in vascular disease progression, we have characterized its expression in cultured SMCs and in a rat vascular injury model, and we have studied the effect of constitutive overexpression of TSG-6 on SMC behavior. We found that interleukin-1 (IL-1) but not tumor necrosis factor or interleukin-6 was able to stimulate TSG-6 expression in SMCs. The IL-1 pathway could be distinguished from the growth factor pathway by its insensitivity to protein synthesis inhibitors. Furthermore, epidermal growth factor, fibroblast growth factor-1, and transforming growth factor-ß1 were all capable of augmenting maximum IL-1–induced expression of TSG-6. To gain further insight into the function of TSG-6 in SMCs, we examined the effect of constitutive overexpression of TSG-6 on these cells. We found that TSG-6–overexpressing cells grew >50% faster than control cells. Furthermore, this growth advantage became more evident in the absence of serum growth factors, with an average increase in cell number of 118% over control cells after 6 days. Consistent with these in vitro data, we observed intense immunostaining for TSG-6 in proliferating SMCs in the rat neointima after injury, whereas only an occasional cell was positive for TSG-6 in the medial layer and in nonballooned arteries. We conclude that the expression of TSG-6 is tightly controlled by growth factors and cytokines via two distinct pathways in SMCs and that overexpression of TSG-6 confers a growth advantage to these cells.

Key Words: hyaluronan • smooth muscle cell • extracellular matrix • cell proliferation • restenosis

	Introduction

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Atherosclerosis and restenosis are vascular diseases initiated by injury to the blood vessel wall with subsequent macrophage and lymphocyte infiltration.^{1 2} One consequence of this inflammatory response is the migration and proliferation of vascular SMCs accompanied by increased ECM synthesis. This process plays an important role in disease progression, and growth factors and cytokines are essential mediators of the SMC response. We previously used subtractive cloning to isolate a cDNA encoding a serum-inducible and growth factor–inducible protein (PS4) in SMCs.^{3 4} PS4 encodes a 276–amino acid secreted protein that exhibits a 94% sequence identity with the translated sequence of a cDNA (TSG-6) cloned from human fibroblasts on the basis of responsiveness to TNF.⁵ TSG-6 expression in fibroblasts, unlike that in SMCs, is not regulated by growth factors but is specifically induced by the inflammatory cytokines IL-1 and TNF.⁵ Elevated TSG-6 protein levels have been found in the synovial fluid of arthritic patients,⁶ and recombinant TSG-6 has potent anti-inflammatory activity in vivo.⁷ Based on these results, it was suggested that TSG-6 may constitute part of a cytokine-initiated feedback loop that operates to downregulate the inflammatory response.⁷

The NH2-terminal proximal domain of TSG-6 contains an HA-binding module with homology to cartilage link protein, aggrecan, versican, and the adhesion receptor CD44.^{8 9} HA and HA-binding proteins are involved in normal development and tissue remodeling, events characterized by rapid cell proliferation and migration.^{10 11} HA has recently been found to be a specific constituent of the ECM associated with human restenotic arteries and of the neointima in experimentally injured arteries.¹² We postulate that HA and HA-binding proteins may significantly influence the vascular wound repair process associated with atherogenesis and restenosis. In the present study, we have examined the regulation of the HA-binding protein TSG-6 in cultured SMCs and determined the in vivo expression of this protein in an animal model of vascular injury. To begin to elucidate the function of this protein, we have also determined the effect of constitutive overexpression of TSG-6 on SMC growth.

	Materials and Methods

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Cell Culture
SMCs were isolated from the aortas of New Zealand White rabbits by enzymatic digestion as previously described.¹³ They were routinely cultured in medium 199 with 10% FBS, 4 mmol/L L-glutamine, 100 U/mL penicillin G sodium, 100 µg/mL streptomycin sulfate, and 0.25 µg/mL amphotericin B. Rat pulmonary artery SMCs (PAC1) were cultured in the same medium. SMCs were made quiescent by changing to medium containing 0.5% FBS, 10 µmol/L insulin, and 5 µg/mL transferrin (low-serum medium) for 48 to 72 hours. Subsequently, the cells were stimulated either by feeding with medium containing 20% FBS or by adding the appropriate concentration of EGF, IL-6, or TNF (Upstate Biotechnology, Inc), TGF-ß1 (R & D System), FGF-1 (provided by Dr W. Burgess, American Red Cross, Rockville, Md), or IL-1 (provided by Dr T. Maciag, American Red Cross, Rockville, Md) to the low-serum medium. Cycloheximide (Boehringer-Mannheim Corp) and puromycin (Sigma Chemical Co) were used at a final concentration of 10 and 100 µg/mL, respectively.

RNA Preparation and Northern Blot Hybridization
Total cellular RNA was isolated from cultured SMCs by acid guanidinium isothiocynate–phenol–chloroform extraction (TRI-reagent LS, Molecular Research Center, Inc).¹⁴ Total RNA (10 µg) was size-fractionated on a 1% agarose gel and transferred to nitrocellulose (Schleicher & Schuell) or Zetabind nylon membranes (Cuno Inc). The immobilized RNA was hybridized overnight with 3 to 5x10⁶ cpm/mL of ³²P-labeled cDNA probe prepared by random primer synthesis (Boehringer-Mannheim). A 1.4-kb PS4 (TSG-6) cDNA was used as a probe.⁴ An 800-bp human GAPDH cDNA was used as a control probe.¹⁵ Hybridization and membrane wash conditions have been described previously.^{15 16}

Preparation of TSG-6 cDNA Expression Vector and Transfection Into SMCs
The full-length (1430-bp) rabbit TSG-6 cDNA⁴ was inserted into the eukaryotic expression vector pcDNA3, which was under the control of the human cytomegalovirus promoter (A-type construct) and also contained the neomycin resistance gene for positive selection (Invitrogen). A second TSG-6 expression plasmid (B type) was generated with a 950-bp EcoRV fragment containing 66 bp of the 5' untranslated sequence, the coding domain, and 51 bp of the 3' untranslated sequence. The TSG-6–pcDNA3 plasmids and the control pcDNA3 plasmid were transfected into rabbit vascular SMCs (SMC112)¹⁵ and rat pulmonary artery SMCs (PAC1).¹⁷ Cells were plated at a density of 1 to 2x10⁵ cells per 100-mm culture dish, and the plasmids were introduced into the cells the following day via a standard CaPO₄ transfection protocol (Stratagene). SMCs that have incorporated the recombinant DNA were selected for resistance to G418 (300 to 500 µg/mL) (GIBCO-BRL). Single antibiotic-resistant colonies were isolated with the aid of cloning rings and propagated in the presence of 300 µg/mL G418.

SMC Proliferation Assay
TSG-6–overexpressing SMCs and control cells were seeded in 10% FBS–containing medium at a density of 2x10³ cells/cm² in 12- or 6-well plates. The medium was replaced every 3 days for the duration of the experiment. For determination of cell number, SMCs were washed with PBS and harvested by trypsin-EDTA, and the cell number was determined using a Coulter Counter (Coulter Electronics). To determine SMC growth in serum-free medium, cells were seeded in 10% FBS–containing medium for 17 hours, and the cells were then washed twice with PBS and subsequently incubated in medium 199 containing 1% Nutridoma NS (Boehringer-Mannheim). Cell numbers were determined as above.

Generation of TSG-6 Antisera and Its Characterization
Polyclonal antiserum was generated against a 17-residue peptide (YCGDELPEDIISTGNVM, residue 209 to 225) derived from the predicted amino acid sequence of the rabbit TSG-6 cDNA.^{3 4} This sequence is conserved between rabbit and human except for the substitution of Asp for Glu²¹⁶. The peptide was synthesized on a branching lysine core using the MAP system¹⁸ and inoculated into rabbits, and the antiserum was characterized by enzyme-linked immunosorbent assay. Briefly, 96-well plates were coated overnight at 4°C with 10 µg/mL of MAP–PS4-B peptide in buffer containing 100 mmol/L Na₂CO₃-NaHCO₃, pH 9.6. Subsequently, nonspecific binding sites were blocked with 1% BSA in PBS for 2 hours at 37°C. The plates were then washed three times with PBS containing 0.05% Tween 20 and 0.2% BSA (wash buffer). Rabbit sera were added in varying dilutions and incubated for 1 hour at 37°C and washed three times with wash buffer. Goat anti-rabbit IgG conjugated to horseradish peroxidase (Sigma), diluted 1:30 000 in PBS with 1% BSA, was then added to the wells and incubated for 30 minutes at 37°C, followed by five washes. ABTS peroxidase substrate (Kirkegaard & Perry Laboratories, Inc) was used for color development, and the results were quantified using a microplate reader at 410 nm. We examined three unrelated MAP peptides and found that the antiserum recognized only the MAP–PS4-B peptide. In addition, preimmune serum and two antisera directed against other MAP peptides did not react with MAP–PS4-B.

Tissue Extraction and Western Blot Analysis
Crude protein extracts were prepared from rabbit and mouse tissues by homogenizing in 0.15 mol/L NaCl and 0.05 mol/L Tris-HCl, pH 8, containing 1% Triton X-100 and 0.02% sodium azide in the presence of proteinase inhibitors (1 µg/mL aprotinin, 10 µg/mL each of leupeptin and pepstatin, and 1 mmol/L each of phenylmethylsulfonyl fluoride and EDTA). After the extract was incubated for 2 hours at 4°C on a rocking platform, the samples were sedimented twice for 10 minutes in a microfuge at 13 000 rpm, and the supernatant fluid was used for subsequent analysis. For Western blot analysis, 250 µg of tissue lysates or 150 ng of recombinant TSG-6 (provided by Dr J. Vilcek and Dr H-G. Wisniewski, Department of Microbiology and Kaplan Cancer Center, New York University Medical Center) were electrophoresed on a 12.5% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane (Schleicher & Schuell). The filters were incubated with PS4-B antisera (1:20 000) and subsequently with goat anti-rabbit IgG coupled to horseradish peroxidase (1:30 000). Blocking buffer containing 0.2% BSA in 100 mmol/L Tris-HCl, pH 7.4, 0.14 mol/L NaCl, and 0.05% Tween 20 was used for all the incubations. The reactive protein bands were visualized with a commercial enhanced chemiluminescent detection kit (NEN Dupont).

Tissue Collection, Preparation, and Immunohistochemistry
Balloon injury was performed on rat iliac arteries according to established procedures.¹⁹ Two weeks after injury, the entire vasculature was perfused with 4% buffered formaldehyde via the ascending aorta, and multiple vascular rings were embedded and stained as described below. Serial paraffin-embedded sections (5 µm thick) were reacted for 1 hour at room temperature with PS4-B antiserum or preimmune serum (diluted 1:1200). In addition, serial sections of the rat iliac artery were stained for PCNA using a monoclonal PCNA antibody (diluted 1:50) (DAKO Corp). Primary antibodies were detected with biotin-conjugated secondary antibody and avidin–horseradish peroxidase using a commercially available ABC kit (Vector Laboratories). All antibodies were diluted in PBS containing 10% normal goat serum and 1% BSA. Controls include omission of the primary antibody and utilization of preimmune serum.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TSG-6 Expression Is Regulated by the Inflammatory Cytokine IL-1 in Cultured Rabbit Vascular SMCs via a Protein Synthesis–Independent Manner
We previously demonstrated that TSG-6 mRNA expression in vascular SMCs is upregulated by the growth factors FGF-1, EGF, and TGF-ß1 and that this regulation is dependent on de novo protein synthesis.^{3 4} However, in fibroblasts, TSG-6 expression is not regulated by these growth factors but is specifically induced by the inflammatory cytokines, IL-1 and TNF, via NF–IL-6 binding sites.^{5 20} Therefore, we examined whether IL-1, IL-6, and TNF were also capable of stimulating TSG-6 expression in SMCs. As shown in Fig 1, quiescent SMCs did not express detectable TSG-6 mRNA, but an increase in TSG-6 mRNA level was observed 2 hours after IL-1 addition. After 4 hours, there was a substantial elevation in TSG-6 expression in IL-1–treated cells that was comparable to that in SMCs stimulated with EGF but less than that in cells stimulated with serum. Treatment with IL-6 or TNF had no effect on the expression of TSG-6 (Fig 1). Additional studies using between 5 and 100 ng/mL of TNF for up to 6 hours also had no effect on TSG-6 expression (results not shown).

View larger version (79K): [in this window] [in a new window]   Figure 1. IL-1 stimulates TSG-6 mRNA expression, and the induction was potentiated by treatment with CHX. Quiescent cells were treated with 1 ng/mL of IL-1, 10 ng/mL of IL-6, and 20 ng/mL of TNF for the indicated times in the presence or absence of CHX. The cells were harvested for RNA isolation, and the mRNA level of TSG-6 was analyzed by Northern blotting. GAPDH mRNA level was also determined and served as a control. CHX was used at a concentration of 10 µg/mL.

Given that the protein synthesis inhibitor, CHX, abrogated the FGF-1– and TGF-ß1–mediated increase in TSG-6 mRNA expression,^{3 4} it was of interest to determine whether CHX similarly inhibited TSG-6 mRNA induction by IL-1. SMCs were incubated with IL-1 in the presence of CHX for 4 hours, and as shown in Fig 1, CHX did not block the IL-1–mediated induction of TSG-6 mRNA expression but enhanced this expression. One explanation for this finding is that a labile protein is responsible for the FGF-1– and TGF-ß1–mediated but not the IL-1–mediated increase in TSG-6 mRNA expression. It is also possible that polysome stabilization by CHX could account for the observed increase in TSG-6 mRNA levels in cells treated with IL-1 and CHX.²¹ To distinguish between these possibilities, we examined the effect of puromycin, a protein synthesis inhibitor that promotes polysome dissociation, on FGF-1– and IL-1–induced TSG-6 expression. As shown in Fig 2, puromycin inhibited the FGF-1–mediated increase in TSG-6 expression but, like CHX, was unable to inhibit IL-1–mediated TSG-6 induction. Indeed, both puromycin and CHX appear to enhance the IL-1–stimulated increase in TSG-6 expression (Fig 2). We conclude that TSG-6 expression is upregulated by the inflammatory cytokine IL-1 in SMCs and that unlike the response to growth factors, stimulation of TSG-6 expression by IL-1 does not require de novo protein synthesis.

View larger version (49K): [in this window] [in a new window]   Figure 2. The protein synthesis inhibitor puromycin exerts differential effects on IL-1–mediated versus FGF-mediated induction of TSG-6 expression. SMCs were growth-arrested for 48 hours and stimulated with FGF-1 (10 ng/mL) or IL-1 (2 ng/mL) for 4 hours in the presence and absence of the protein synthesis inhibitor puromycin. The cells were harvested for RNA isolation, and the mRNA level of TSG-6 was analyzed by Northern blotting. GAPDH mRNA level and ethidium bromide–stained 18S and 28S ribosomal RNA served as controls. Puromycin was used at a concentration of 100 µg/mL.

TSG-6 mRNA Expression Is Stimulated by IL-1 Through a Pathway Independent of That Used by EGF, FGF-1, and TGF-ß1
One possible explanation for the de novo protein synthesis requirement of TSG-6 expression by growth factors is that they could induce IL-1 expression with subsequent autocrine stimulation of TSG-6 expression. However, we did not observe increased expression of IL-1 in SMCs treated with TGF-ß1, FGF-1, EGF, or serum (results not shown). We then examined whether these growth factors were capable of acting independently of IL-1–mediated induction of TSG-6 expression. We first determined that maximum induction of TSG-6 expression was achieved in the presence of 1 ng/mL of IL-1 (Fig 3). The time course of induction by IL-1 was prolonged with a strong level of expression still evident by 9 hours, but by 16 hours, TSG-6 mRNA became undetectable (Fig 3). We next used an amount of IL-1 (2 ng/mL) that was more than sufficient to elicit a maximum TSG-6 response and determined whether the addition of growth factors could further increase the TSG-6 mRNA level. The data, shown in Fig 3, indicated that FGF-1, EGF, and TGF-ß1 were all capable of increasing the expression of TSG-6 mRNA beyond that of IL-1 alone. These results and the lack of requirement for de novo protein synthesis indicate that IL-1 induction of TSG-6 expression in SMCs may occur via a pathway distinct from that used by the other growth factors.

View larger version (34K): [in this window] [in a new window]   Figure 3. Dose and time-course response of TSG-6 to IL-1 stimulation and the effect of EGF, FGF-1, and TGF-ß1. Quiescent SMCs were treated with the indicated amount of IL-1 for 4 hours and analyzed for TSG-6 RNA expression by Northern blotting (top). Quiescent SMCs were treated with 2 ng/mL of IL-1 for the indicated time and similarly analyzed for TSG-6 expression (middle). Cells were treated with TGF-ß1, FGF-1, and EGF with or without 2 ng/mL of IL-1 and analyzed for TSG-6 expression. Growth factor concentrations were 10 ng/mL, except for TGF-ß1, which was at 5 ng/mL. Cells were treated for 4 hours, except as indicated for TGF-ß1 (bottom). GAPDH mRNA level served as a control in all three panels.

SMCs Overexpressing TSG-6 Exhibit Enhanced Cell Proliferation
As a first approach toward understanding the function of TSG-6 in SMCs, we generated a number of cell lines that constitutively overexpressed TSG-6. Northern blot analysis indicated that the individual transfected cell lines expressed elevated levels of TSG-6 RNA (Fig 4). By contrast, we did not detect TSG-6 mRNA expression in SMCs transfected with the control vector (Fig 4). During the process of characterizing these cell lines, we noted that TSG-6–overexpressing cell lines consistently grew faster than control cells. Therefore, we performed cell proliferation assays on control and TSG-6 transfectants. The results, shown in Fig 5, confirmed that SMCs overexpressing TSG-6 grew at a faster rate than did control cells. Both rabbit aortic SMCs (SMC112) and rat pulmonary SMCs (PAC1) overexpressing TSG-6 exhibited a higher proliferative capacity. The mean increase, based on four independent experiments, for the rabbit SMC lines RSHA and RSHB was 50±22% and 66±46%, respectively. Analysis of the remaining SMC112 cell lines indicated that there was an overall correlation between the level of TSG-6 expression and cell proliferation. SMC112 cells that expressed the highest TSG-6 mRNA level (A1, B1, and B3) grew at the fastest rate; the low expressors (A4 and B5) grew the slowest (Fig 5 and results not shown). We conclude that high-level constitutive overexpression of TSG-6 confers an increase in cell growth.

View larger version (60K): [in this window] [in a new window]   Figure 4. TSG-6 mRNA levels in SMCs transfected with a TSG-6 cDNA–containing plasmid or with a control plasmid. Individual G418-resistant colonies, transfected with control or rabbit TSG-6 cDNA–containing plasmids, were grown to confluence, and TSG-6 expression was determined by Northern blotting. Shown in panel A are cell lines derived from the SMC112 parental cell line. A1 and A4 lines were transfected with the A-type TSG-6 construct, and lines B1 through B8 were transfected with the B-type construct. Shown in panel B are cell lines derived from the PAC1 parental cell line. The A3 line was transfected with the A-type expression construct, and lines B1 through B8 were transfected with the B-type construct. Total RNA (10 µg) was loaded per lane, and GAPDH mRNA expression was used as a control for RNA loading.

View larger version (14K): [in this window] [in a new window]   Figure 5. SMCs overexpressing TSG-6 exhibit enhanced cell growth. SMCs were seeded in 6- or 12-well plates at a density of 2x103 cells/cm2 and cultured in the presence of 10% FBS–containing medium. After 1 and 4 days, the cells were harvested, and the cell number was determined. Shown in the left panel are the cell number of rabbit aortic SMCs (SMC112) transfected with the control plasmid () and two cell lines transfected with plasmids containing the rabbit TSG-6 cDNA, RSHA1 (RSHA, ) and RSHB3 (RSHB, ). Shown in the right panel are the cell number of rat pulmonary SMCs (PAC1) transfected with the control plasmid () and plasmids containing the TSG-6 cDNA, PA3 (PA, ) and PB8 (PB, ). The plotted values are mean±SD of triplicate determinations and are representative of four experiments.

The Increase in Growth Rate in TSG-6–Overexpressing SMCs Is Maintained in the Absence of Serum
We next examined whether the increase in growth observed in TSG-6–overexpressing cell lines was maintained in the absence of serum factors. SMCs were cultured in normal medium containing 10% FBS for 17 hours to ensure appropriate cell attachment and then switched to serum-free medium, and the cell number was assessed after 1, 3, and 6 days. The results, shown in Fig 6, indicate that whereas control cells became quiescent under serum-free conditions, the TSG-6–overexpressing cells were able to undergo additional rounds of cell division. All four of the TSG-6–overexpressing cell lines achieved cell numbers that were approximately twice that of control cells by the sixth day in culture. A mean of 118±11% increase was found for these cell lines. Cell growth of the TSG-6 transfectants did not reach the level of cells grown in the presence of 10% serum, indicating that continued proliferation of these cells remained serum growth factor dependent (compare Fig 5 with Fig 6). We conclude that constitutive TSG-6 overexpression confers a growth advantage over control cells even under serum-free conditions.

View larger version (14K): [in this window] [in a new window]   Figure 6. The increased growth in TSG-6–overexpressing SMCs is maintained in the absence of serum. SMCs were plated at a density of 2x103 cells/cm2 and cultured in the presence of 10% FBS medium for 17 hours. Subsequently, the serum-containing medium was removed, and the cells were washed twice with PBS. The cells were subsequently cultured in a serum-free medium and harvested for cell number determination at the indicated times. Shown in the left panel are the cell number of rabbit aortic SMCs (SMC112) transfected with the control plasmid () and two cell lines transfected with plasmids containing the rabbit TSG-6 cDNA, RSHA1 (RSHA, ) and RSHB3 (RSHB, ). Shown in the right panel are the cell number of rat pulmonary SMCs (PAC1) transfected with the control plasmid () and plasmids containing the TSG-6 cDNA, PA3 (PA, ) and PB8 (PB, ). The plotted values are mean±SD of duplicate determinations.

TSG-6 Expression Is Mainly Restricted to the Neointima in the Injured Rat Blood Vessel
Given that TSG-6 confers a growth advantage and is tightly regulated by growth factors and cytokines in cultured SMCs, it was of interest to examine the pattern of TSG-6 expression in the blood vessel. We generated an antibody against a 17-residue peptide (amino acids 209 to 225) derived from the predicted amino acid sequence of the TSG-6 cDNA. We determined the specificity of the TSG-6 antiserum by Western blot analysis and found that the antiserum was able to recognize the synthetic MAP–PS4-B peptide used as the immunogen as well as the glycosylated human recombinant TSG-6 produced in insect cells²² (Fig 7). Furthermore, the antiserum specifically detected a single polypeptide with an approximate molecular mass of 30 kD in mouse tissue (Fig 7). We conclude that the antiserum specifically recognizes the TSG-6 protein in several species.

View larger version (98K): [in this window] [in a new window]   Figure 7. Demonstration of specific recognition of the TSG-6 protein by the polyclonal antiserum generated against a 17-residue peptide based on the predicted amino acid sequence of the PS4 (rabbit TSG-6) cDNA. Western blotting analysis was performed with preimmune (P) or immune (I) serum against the immunogen (MAP–PS4-B peptide [MAP-PS4]), recombinant human TSG-6 (rTSG-6), and extracts of mouse skeletal (sk.) muscle.

We used this antiserum to examine the expression of TSG-6 in a well-established rat vascular injury model.^{19 23} In this model, injury of the artery with a balloon catheter induces maximal SMC proliferation within the first week and proliferation declines to basal levels by 8 weeks. Neointimal SMC proliferation is maximal at 2 weeks, and these cells form a distinct layer that is easily distinguishable from the media. As shown in Fig 8A, the neointima of a rat iliac artery 2 weeks after injury stained intensely for TSG-6. By contrast, only an occasional SMC in the media as well as in the uninjured artery stained positively for TSG-6 (Fig 8A and 8B). In addition, some of the endothelial cells of the capillaries also stained for TSG-6. It was interesting that although the majority of the neointima stained for TSG-6, the region closest to the lumen showed the most intense staining. This pattern of TSG-6 expression was similar to that observed for PCNA, a marker for cell proliferation (Fig 8C). Preimmune serum did not stain the injured blood vessel (Fig 8D). Our results indicate that the majority of medial SMCs did not express detectable levels of TSG-6, whereas this protein was expressed at high levels by neointima SMCs in the injured aorta. In addition, we find that the highest level of TSG-6 expression was associated with the region of greatest proliferative activity.

View larger version (114K): [in this window] [in a new window]   Figure 8. TSG-6 is specifically expressed by neointimal SMCs in the balloon catheter–injured blood vessel. A, Rat iliac artery immunostained with TSG-6 antiserum. Arrow marks the position of the internal elastic lamina. Note that the majority of SMCs in the neointima were positively stained for TSG-6, whereas the medial SMCs were not immunoreactive for TSG-6. B, Normal uninjured iliac stained with TSG-6 antiserum. C, A serial section of the injured iliac artery immunostained for proliferation of cell nuclear antigen. D, A serial section of the injured iliac artery stained with the preimmune antiserum.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
HA and HA-binding proteins have been implicated in key biological processes including cell growth, migration, and differentiation.^{10 11} These events are hallmarks of blood vessel wall diseases,¹ yet the possible role of HA and HA-binding proteins in modifying these cellular behaviors during vascular disease progression remains sparsely explored. In the present study, we have shown that overexpression of the HA-binding protein, TSG-6, confers an enhanced growth rate in SMCs. This growth advantage was particularly evident in the absence of serum growth factors, but the increase in cell number remained less than that of control transfectants exposed to serum. The HA receptor CD44 is expressed in vivo by SMCs after injury and mediates HA stimulation of DNA synthesis in cultured SMCs.²⁴ Overexpression of a second HA receptor, RHAMM, in 10T1/2 fibroblasts also causes an increase in cell number.²⁵ RHAMM is essential for injury-induced SMC migration in vitro,²⁶ but its role in SMC proliferation has not been examined. However, this receptor is reported to regulate the expression of Cdc2 and, therefore, entry into mitosis in 10T1/2 cells.²⁷ Furthermore, HA synthesis increases in proliferating cells, and this increase may be required for mitosis.^{28 29} Future studies will focus on whether the ability of TSG-6 to bind HA is essential for the growth-stimulatory activity of this protein. It will also be of interest to explore the possibility that TSG-6–mediated increase in SMC growth may involve the HA receptors RHAMM and/or CD44.

The presence of HA in diseased human arteries has recently been examined, and intense staining for HA was identified in the loose myxoid ECM surrounding stellate-shaped SMCs.^{12 30} It was postulated that similar to other biological processes, such as morphogenesis, wound healing, and tumorigenesis, HA may be a marker for a transitional ECM during the early phase of vascular lesion development.¹² The expression of HA has also been examined during closure of the ductus arteriosus, a developmental process that may serve as an useful model for intimal thickening.³¹ It was found that closure of the ductus arteriosus is preceded by an increase in HA and that in animals with a genetic defect in the ductus arteriosus, HA fails to accumulate.³¹ These studies implicate HA in the development of an intimal thickening. We have found that TSG-6 is localized specifically to the neointima 2 weeks after balloon injury. The localization is very similar to that reported for HA¹² and coincides with the presence of PCNA-positive SMCs. Given that TSG-6 has been demonstrated to interact with HA,^{8 9} this protein may be involved in the formation of the HA-containing transitional matrix that facilitates cellular migratory and proliferative activity. However, although some extracellular staining was detectable, the majority of the immunopositive staining was found to be cell associated. Several scenarios can be envisioned for this observation. First, given that TSG-6 is only a 30-kD protein, it is possible that extracellular TSG-6 may be poorly fixed. We have examined the effect of other fixation methods, including Bouin's fixative and methacarn, and have not observed increased extracellular staining (R. Mora and G. Liau, unpublished data, 1997). A second possibility is that the anti–MAP peptide antibody may recognize epitopes that are conformationally altered or are masked once TSG-6 is secreted. For example, two antibodies made against the first 30 amino acids of TGF-ß1 recognize distinct intracellular versus extracellular pools of TGF-ß1.³² Finally, it is possible that secreted TSG-6 may remain cell surface–associated.

The lack of TSG-6 in the normal vessel and its appearance in the neointima after blood vessel injury indicate that the expression of this protein is tightly controlled by SMCs in vivo. We have shown that in cultured SMCs, TSG-6 expression is regulated by the growth factors TGF-ß1 and FGF-1^{3 4} as well as by the inflammatory cytokine IL-1. Our results suggest that induction of TSG-6 expression by IL-1 is mediated by a pathway distinct from that used by FGF-1 and TGF-ß1. Consistent with this hypothesis is the finding that in human foreskin fibroblasts the growth factor–mediated TSG-6 induction pathway is not functional, yet the TSG-6 gene remains highly responsive to TNF and IL-1.⁵ Our data are more reminiscent of data recently reported for chondrocytes in which both growth factors and inflammatory cytokines can induce TSG-6 expression.³³ Similar to what we have observed in SMCs,^{3 4} TGF-ß1 induced TSG-6 mRNA expression with delayed kinetics, whereas IL-6 had a minimal effect.³³ However, unlike SMCs, FGF was a poor stimulator of TSG-6 expression in chondrocytes.³³ TNF is the most potent inducer of TSG-6 expression in fibroblasts⁵ as well as chondrocytes,³³ and it was surprising that this cytokine had no detectable effect on TSG-6 mRNA expression in SMCs. However, TSG-6 expression in endothelial cells as well as in a number of transformed cell lines is also not affected by TNF treatment,⁸ and differences in IL-1–mediated and TNF-mediated TSG-6 induction in fibroblasts have been observed.³⁴ Although IL-1, IL-6, and TNF are all believed to act via NF–IL-6 binding sites on the TSG-6 promoter,^{20 34} additional complexities, such as activator and inhibitory isoforms of NF–IL-6,³⁵ and interaction with other transcriptional factors, such as NF-B,^{36 37} C/ATF,³⁸ the glucocorticoid receptor,³⁹ and the activator protein-1 complex,⁴⁰ likely account for the differential regulation observed with these cytokines. A clearer understanding of the regulation of TSG-6 in SMCs may provide important clues toward determining its role in vascular disease progression.

The mechanism by which TSG-6 overexpression results in increased SMC growth is unclear. Two factors (IL-1 and FGF-1) that stimulate TSG-6 expression are SMC mitogens.^{41 42} However, depending on parameters such as cell density, TGF-ß1 can be either a positive or a negative regulator of SMC growth.⁴³ Furthermore, TNF and IL-6 have also been reported to enhance SMC growth, yet these cytokines do not induce TSG-6 expression.^{44 45} Thus, TSG-6 may not be essential for SMC proliferation but may facilitate the action of other SMC mitogens. One possibility is that this HA-binding protein could modulate HA synthesis or the interaction of HA with the cell surface. TSG-6 also has been shown to potentiate the plasmin inhibitory activity of the serine protease inhibitor, inter--inhibitor.⁷ Although altered plasmin activity is usually considered in the context of tissue remodeling and cell migration, it is possible that TSG-6–mediated change in extracellular protease and protease inhibitor balance is responsible for the observed increase in cell growth. TSG-6 could also alter cell adhesion or facilitate release of sequestered serum mitogens, thereby enhancing cell growth. Overall, our data support a significant role for TSG-6 in the vascular injury response, but further studies are required to clarify the role of HA and this HA-binding protein in blood vessel diseases.

	Selected Abbreviations and Acronyms

 

CD = cluster determinant CHX = cycloheximide ECM = extracellular matrix EGF = epidermal growth factor FGF = fibroblast growth factor HA = hyaluronan IL = interleukin MAP = multiple antigenic peptide NF = nuclear factor PCNA = proliferating cell nuclear antigen RHAMM = receptor for HA-mediated motility SMC = smooth muscle cell TGF = transforming growth factor TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported in part by a grant from the National Institutes of Health (HL-37510 to Dr Liau). We would like to acknowledge the expert photographic assistance of D. Norman. We would also like to thank E. Smith and P. Feng for expert technical assistance.

Received January 16, 1997; accepted May 12, 1997.

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