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Comparison of gene expression patterns and migration capability at quiescent and proliferating vascular smooth muscle cells stimulated by cytokines
Life Sciences 70 (2002) 799–807
Comparison of gene expression patterns and migration capability at quiescent and proliferating vascular smooth muscle cells stimulated by cytokines Jin-kun Wen*, Mei Han, Bin Zheng, Shu-Li Yang Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, People’s Republic of China Received 23 April 2001; accepted 27 June 2001
Abstract The changes of the gene expression patterns and the migration capability of vascular smooth muscle cells (VSMC) at the quiescent and proliferating states were investigated after VSMC was stimulated by cytokines. VSMC migration was measured using a model of wounding injury of confluent cultured cells and a Boyden chamber assay. The migration distance of the quiescent VSMC induced by cytokines bFGF, IL-1b and TNF-a was 5.8, 4.7, and 4.2 times as long as that of the control group, respectively. However, the migration distance of the proliferating VSMC was only one-fourth of the quiescent cells under the same effects. The results of Boyden chamber assay indicated that VSMC was capable of degrading collagen and traversed the pores of membrane barriers in response to bFGF as a chemoattractant, and VSMC number across the membrane was markedly increased as concentration gradient of bFGF ascended. Northern blot and nuclear run-off assay showed that bFGF not only stimulated the expression of migration-related matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) but also enhanced the transcription of proliferation-related osteopontin (OPN) and c-jun genes in the quiescent VSMC. bFGF could moderately increase the expression of OPN and c-jun, but had no significant effect on the expression of MMP-2 and TIMP-2 in the proliferating cells under the same conditions. These findings suggest that VSMC migration capability and the expression activity of migration- and proliferation-related genes were significantly distinct for the quiescent and proliferating VSMC. The cellular migration capability and the expression activity of migrationrelated genes in the quiescent VSMC were much higher than those in the proliferating cells in response to cytokines. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Vascular smooth muscle cell; Migration; Proliferation; Cytokines; Gene expression
* Corresponding author. No.361, Zhongshan Road, Shijiazhuang, 050017. Tel.: 186-311-6044121-5563; fax: 186-311-6048177. E-mail address: [email protected] (J.-k. Wen) 0024-3205/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 4 6 -1
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Introduction The phenotypic modulation and inappropriate proliferation of vascular smooth muscle cells (VSMCs) are major components of atherosclerosis and the restenosis that can occur after balloon angioplasty. It has been suggested that VSMCs in vivo or freshly plated in vitro have a high volume fraction of myofilaments and few biosynthetic organelles and retain the ability to contract in response to vasoconstrictors, whereas VSMCs grown in vitro have a low volume fraction of myofibrils and a high proportion of biosynthetic organelles and may lose their ability to contract[1,2]. These two phenotypes were originally defined as “contractile” (differentiated) or”synthetic” (dedifferentiated). There is evidence that VSMCs in restenosis and atheromatous plaques have undergone phenotypic modulation. Furthermore, there is also evidence that passaged VSMCs are synthetic phenotype[1]. Recent in vitro studies have revealed that passaged VSMCs remain quiescent (stationary phase) when they were seeded in a serum-free medium or reached confluence, and freshly passaged VSMCs in a serum-containing medium were proliferating[3,4]. Currently, passaged VSMCs have been used as a major tool to study the regulatory mechanisms of the phenotypic modulation of VSMCs. However, to date, it has not been paid close attention to whether migration capability and gene expression pattern are identical between the proliferating and quiescent VSMCs[5,6]. In the present study, rat VSMCs were synchronized to the quiescent state (G0) or were left proliferating by differently conditioned culture. Migration capability and gene expression patterns in both quiescent and proliferating VSMCs were tested. Materials and methods Vascular smooth muscle cell isolation and culture Rat aortic VSMCs were isolated from the thoracic and abdominal aortas of SpragueDawley rats (weighting 100 to 130 g) as described previously[7]. Isolated cells were seeded in M199 containing 15% fetal calf serum (FCS) and passaged after reaching confluence using 0.125% trypsin. Cells for the experiments described in this study were used between passaged 4z9. The quiescent VSMCs were obtained by allowing the cells to achieve contact inhibition and to grow in a serum-free medium for 72 h. The degree of VSMC synchronization was monitored by flow cytometry. After the cells were cultured in serum-free medium for 72 h, approximately 85% of the cells were in the G0/G1 phase. The logarithmic phase cells grown to 90% confluence in M199 containing 15% FCS were acted as a proliferating state. VSMC migration assay Two types of migration assays were used. In the first, migration was measured using a monolayer-wounding protocol[8] in which cells migrated from a confluent area into an area that was mechanically denuded of cells. In the second type of assay, collagen-coated transpore tissue culture inserts composed of a polycarbonate membrane with pores of 8mm diameter were used. For monolayer-wounding cell migration assay, the quiescent and proliferating VSMCs were scraped with a sterile single-edged razor blade, respectively. Cultures were then exposed to
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the experimental medium (bFGF 4 U/ml, IL-1b 3000 U/ml, or TNF-a 3000 U/ml in M199 containing 1% FCS, respectively) for 5 h. After this period of time, the cells were washed twice with PBS, fixed with absolute ethanol, and stained with H.E. Five microscopic fields were evaluated for each experiment. The maximum distance migrated (wound edge to nucleus of the farthest cell) was determined in each field under 3200 magnification and compared with VSMCs grown in M199 supplemented with 1% FCS to calculate the opposite value. Migration assay across membrane was performed with a modified Boyden chamber as previously described[9]. PVPF filters (8 mm pore diameter) were first coated with a solution containing 0.1% (v/v) acetic acid and 8 mg/ml collagen from rat tail, and constituted an artificial basement membrane (BM, 10 mm in thickness). The membrane was then rehydrated with M199 before use. Boyden chambers were assembled by adding 4 U/ml or 1 U/ml bFGF in M199 containing 1% FCS to the lower (chemoattractant) chamber, respectively. The quiescent VSMCs suspended in M199 containing 1% FCS were then added to the upper chamber. The chambers were incubated for 24 h at 378C. At the end of the incubation period, the filter in the Boyden apparatus was divided into four parts. The cells on the upper surface of three parts of the filter were mechanically removed. The cells on the underside of the filter were fixed and stained. Four fields were counted per part under high-power field in a microscope (3200). The other part of the filter was scanned with an electron microscope. All experiments were run in triplicate. Data are expressed as mean6S.D. Student’s t test for paired data was used for statistical analysis. Total RNA isolation and northern blotting After the quiescent and proliferating VSMCs or VSMCs grown on the collagen-coated dish were incubated with M199 containing bFGF (4 U/ml) and 1% FCS for 24h, total cellular RNA was prepared from these cells as described[10] and quantified by absorbance at 260 nm. RNA (30 mg) was fractionated on 1% agarose-formaldehyde gels and transferred on to Hybond-N membranes by capillary blotting using 103SSC. RNA was then cross-linked to the membrane by baking at 808C for 2 h. After prehybridization, the blots were hybridized with [a-32P] dCTP-labelled cDNA probes for 20z24 h at 428C. The filters were then washed twice with 23SSC in 0.2% SDS at room temperature, and again at 558C as needed. The filters were exposed to Kodak XAR-5 film at –708C for 24z72 h. Nuclear run-off assay Nuclear run-off assay was performed as described previously[11]. Briefly, nuclei were harvested, as follows, from the quiescent and proliferating VSMCs treated with bFGF (4 U/ml) and 1% FCS for 24 h. The cells were rinsed twice with ice-cold PBS, scraped and pelleted by centrifugation at 1500 rpm for 5 min at 48C. The cell pellet was resuspended in 1 ml NP-40 lysis buffer (10 mmol/L Tris·Cl, pH 7.5, 10 mmol/L NaCl, 3 mmol/L MgCl2 and 1% (v/v) NP-40), incubated for 2z3 min on ice, and centrifuged at 3000 rpm for 5 min. Nuclei were resuspended in 100 ml of 20% glycerol, 50 mmol/L Tris·Cl, pH 8.0, 5 mmol/L MgCl2, 0.1 mmol/L EDTA and stored at –708C. Frozen nuclei were thawed to 258C and reacted in 100 ml reaction buffer consisting of 10 mmol/L Tris·Cl, pH 8.0, 5 mmol/L MgCl2, 150 mmol/L KCl, 2 mmol/L each of ATP, CTP, GTP (sigma), 100 mCi[a-32P] UTP and 100 units of RNA-
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guard. The reaction was carried out at 308C for 30 min. RNA transcripts were purified with phenol/chloroform extraction. Target fragments (1mg) were alkali-denatured and spotted on to nylon membranes using a dot blot apparatus. The membranes were hybridized with transcribed RNAs containing an equal amount of radioactivity, and then autoradiographed with film. Results Quiescent and proliferating VSMCs exhibit markedly distinct migration capabilities As shown in Fig. 1, migration activities were significantly increased when VSMCs at the quiescent state were stimulated by cytokines. After this phenotypic cells were exposed to bFGF, IL-1b or TNF-a for 5 h, the migration distance at the various groups was 5.8-fold, 4.7fold, and 4.2-fold greater than that of the control group, respectively. VSMCs in the control group migrated 42611.4 mm under the same conditions. When times stimulated by these factors were prolonged for another 7 h, migration distance at the various cells was 2-fold longer than that of 5 h. In contrast, migration distance of the proliferating VSMCs was only one-fourth of the quiescent cells following stimulated by the cytokines under the same effects. VSMC migration induced by the cytokines was also demonstrated by migration assay in a modified Boyden chamber. When concentration gradient of bFGF in the upper and lower chamber was adjusted to 0 U/ml, 1 U/ml, and 4 U/ml, the number of VSMCs migrating across PVPF membrane was 4.9161.97, 8.0862.50, and 21.0964.16, respectively. It is thus evident that the number of cell migration across the membrane was markedly increased as concentration gradient of bFGF ascended (Fig. 2). Significant differences in expression activities of genes related to cell migration and proliferation exist between the quiescent and proliferating VSMCs In order to compare whether effect of the cytokines and collagen on expression of cell migration- and proliferation-associated genes was identical in both quiescent and proliferating VSMCs, these two cellular subtypes were cultured in M199 containing bFGF (4 U/ml) and 1% FCS or in tissue culture Petri dish coated with the collagen. Northern blots showed that the genes related to cell migration and proliferation, including MMP-2, TIMP-2, OPN, and c-jun, were upregulated by bFGF, to a certain extent, in the quiescent VSMCs. Of the four genes, MMP-2 and c-jun were strongly expressed in these cells. Their expression in the cells grown on Petri dish coated with the collagen was lower than that in the cells stimulated by bFGF. Only a small increase in expression of OPN and c-jun was detected in the proliferating VSMCs treated with bFGF under the same conditions. The expression activity of these four genes was very low in the two cells treated without bFGF (Fig. 3). Similar results were obtained using nuclear run-off assay. Only the expression of OPN and c-jun genes could be detected in the proliferating VSMCs treated with bFGF. In contrast, these four genes were simultaneously transcripted when the quiescent VSMCs were stimulated by bFGF (Fig. 4). Whether the quiescent VSMCs or proliferating VSMCs possessed no detectable MMP-2, TIMP-2, c-jun and OPN mRNA by nuclear run-off assay when they were not stimulated by bFGF (results not shown).
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Fig. 1. VSMC migration after treatment with cytokines. A: Representative photomicrographs illustrating migration of the quiescent VSMCs. 1. Before VSMC migration; 2. Migration of VSMCs treated with 1% FCS for 5 hours; 3. Migration of VSMCs treated with bFGF11% FCS for 5 hours; 4. Migration of VSMCs treated with IL-1b11% FCS for 5 hours; 5. Migration of VSMCs treated with TNF-a11% FCS for 5 hours. B: Bar graph illustrating VSMC relative migrating activity. * P,0.01 versus 1% FCS; m P,0.01 versus the proliferating VSMC.
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Fig. 2. Bar graph showing the effect of concentration gradient of bFGF on VSMC transpore migration. 1. bFGFgradient at 4 U/ml; 2. bFGF-gradient at 1 U/ml; 3. bFGF-no gradient; 4. 1% FCS-no gradient. Each bar represents the mean 6 S.D. of three separate experiments. * P,0.01 versus bFGF-no gradient ; m P,0.01 versus 1% FCS-no gradient.
Fig. 3. Northern analysis of MMP-2, TIMP-2, OPN and c-jun mRNA in the quiescent and proliferating VSMCs. Panels A and B represent the different VSMCs treated with or without bFGF for 24 hours, respectively. Lanes are as follows: 1. Quiescent VSMCs; 2. VSMCs grown on Petri dish coated with collagen; 3. Proliferating VSMCs. The different mRNAs are shown on the right of each panel.
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Fig. 4. Nuclear run-off assay for transcription activity of MMP-2, TIMP-2, OPN and c-jun genes. Panels A and B are the transcription products of the nuclei from the quiescent and proliferating VSMCs, respectively. The transcription products are shown on the top of Panel A.
The degradation of extracellular matrix (ECM) is required for VSMC migration through the reconstituted BM To determine the relationship between ECM degradation and VSMC migration across the membrane, we reconstituted BM barrier by coating PVPF filters with collagen from rat tail, and observed changes in ECM degradation during migration of VSMCs. Scanning electron micrograph showed that the collagen coated on the filter was first degraded, and the pores adjacent to the cells were exposed when VSMCs migrated across the BM barrier. VSMCs on the upside of the filter changed from the extending shape to spherical or elliptical shape, and traversed the pores of the filter under the action of the chemoattractant bFGF. It could be seen from the underside of the filter that VSMC passing through the pore extended cell foot processes, while the cell body remained in the pore (Fig. 5).
Fig. 5. Scanning electron micrograph for transpore cell migration assay. A: VSMC attaching on PVPF filter coated with collagen; B: the exposed pores after degradation of collagen; C: Entire cell body passing through the pore of filter; D: Cell foot processes extending through the 8-mm diameter pores of PVPF filter.
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Discussion We show in the present study that passaged VSMCs in the quiescent state migrated rapidly in response to cytokines bFGF, IL-1b and TNF-a, whereas migration that occurred in the proliferating VSMCs was much slower than that of the quiescent cells under the same conditions. It has been demonstrated that VSMCs in the arterial media belong to the quiescent cells. The different migration responses to cytokine stimulation suggest that the quiescent VSMCs existing in the arterial media could easily migrate into the vascular intima and initiate their proliferation when the vessel intima is injured. It seems likely that the functional capabilities and morphological characteristics of the quiescent cells favored their migration compared with the proliferating cells. The relative rank order of potency stimulating VSMC migration is bFGF. IL-1b. TNF-a among the factors observed in the experiments. Therefore, we used bFGF as an inducer to study the relationship between VSMC migration ability and gene expression of migration related genes. The degradation of ECM is the biochemical basis of VSMC migration and tumor metastasis[12]. The results of cell migration assay across the filter indicate that VSMC migration is accompanied by local degradation of the matrix in the pathway of migration. It has been demonstrated that ECM degradation in vivo is dependent on activity of proteolytic enzyme[13]. MMP-2 may efficiently degrade type IV collagen, the major structural component of basement membranes, and is very important in VSMC migration from the arterial media to the neointima after injury to the vessel. TIMP-2 is a tissue inhibitor of MMP-2, MMPs and their inhibitors coregulate ECM turnover, and maintain vessel wall integrity[14]. The results of Northern blot and nuclear run-off assay revealed that the expression of MMP-2 and TIMP-2 in the quiescent VSMCs could significantly be induced by bFGF. We speculate that these two proteins might modify the way cells interact with surrounding matrix by enhancing ECM turnover, and overcome the inhibitory effect exerted by the normal ECM. This is probably another reason why migration activity of the quiescent cells was much higher than that of the proliferating VSMCs following stimulated by cytokines. Moreover, our results demonstrated that bFGF could also markedly induce the expression of OPN and c-jun genes in both quiescent and proliferating VSMCs. It has been known that OPN is a protein marker associated with the synthetic phenotype[1]. OPN could be detected in both quiescent and proliferating VSMCs, suggesting that these two VSMCs might be the synthetic phenotype. OPN contains an Arg-Gly-Asp motif that binds integrin, and thus can mediate VSMC adhesion and migration by interaction with this receptor[15]. The expression product of c-jun gene, which is an important transcription factor that promotes the expression of OPN and MMP-2, participates in the regulation processes of VSMC proliferation[16,17]. Accordingly, regardless of the quiescent or proliferating state, the expression of c-jun and OPN genes is necessary to maintaining the synthetic phenotype of VSMCs. However, the causality between the expression of these genes and the phenotypic modulation of VSMCs remains to be studied. Acknowledgments This study was supported by National Natural Science Foundation of China.
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References 1. Shanahan CM, Weissberg PL, Metcalfe JC. Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circulation Research 1993; 73(1):193–204. 2. Hultgardh-Nilsson A, Lovdahl C, Blomgren K, Kallin B, Thyberg J. Expression of phenotype- and proliferationrelated genes in rat aortic smooth muscle cells in primary culture. Cardiovascular Research 1997; 34(2):418–30. 3. Han M, Wen JK, Li XY, Zhou XX. Comparison of .gene expression pattern and migration at different phase of cell cycle in vascular smooth muscle cell stimulated by cytokines. Chinese Journal of Biochemistry and Molecular Biology 2000;16(6):809–13. 4. Suzuki T, Nagai R, Yazaki Y. Mechanisms of transcriptional regulation of gene expression in smooth muscle cells. Circulation Research 1998;82(12):1238–42. 5. Zhou XX, Wen JK, Han M. The change of MMP-2 gene expression and collage turnover during vascular restenosis development. Chinese Journal of Biochemistry and Molecular Biology 1999;15(6):997–1001. 6. Frid MG, Aldashev AA, Dempsey EC, Stenmark KR. Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities. Circulation Research 1997;81(6):940–52. 7. Gunther S, Alexander RW, Atkinson WJ, Gimbrone MA Jr. Functional angiotensin II receptors in cultured vascular smooth muscle cells. Journal of Cellular Biology 1982 ;92(2):289–98. 8. Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circulation Research 1996 ;78(2):225–30. 9. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Research 1987;47(12):3239–45. 10. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Analytical Biochemistry 1987;162(1):156–9. 11. Cowan DB, Lye SJ, Langille BL. Regulation of vascular connexin43 gene expression by mechanical loads. Circulation Research 1998;82(7):786–93. 12. Strauss BH, Robinson R, Batchelor WB, Chisholm RJ, Ravi G, Natarajan MK, Logan RA, Mehta SR, Levy DE, Ezrin AM, Keeley FW. In vivo collagen turnover following experimental balloon angioplasty injury and the role of matrix metalloproteinases. Circulation Research 1996;79(3):541–50. 13. Southgate KM, Fisher M, Banning AP, Thurston VJ, Baker AH, Fabunmi RP, Groves PH, Davies M, Newby AC. Upregulation of basement membrane-degrading metalloproteinase secretion after balloon injury of pig carotid arteries. Circulation Research 1996;79(6):1177–87. 14. Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circulation Research 1995;77(5):863–8. 15. Xuan JW, Hota C, Shigeyama Y, D’Errico JA, Somerman MJ, Chambers AF. Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions. Journal of Cellular Biochemistry 1995;57(4):680–90. 16. Yamamoto S, Hijiya N, Setoguchi M, Matsuura K, Ishida T, Higuchi Y, Akizuki S. Structure of the osteopontin gene and its promoter. Annual of New York Academic Science 1995;760:44–58. 17. Han M, Wen JK. Study on expression mechanisms of MMP-2 gene induced by cytokines in vascular smooth muscle cells. Chinese Journal of Biochemistry and Molecular Biology 2000;16(2):249–53.
Comparison of gene expression patterns and migration capability at quiescent and proliferating vascular smooth muscle cells stimulated by cytokines Jin-kun Wen*, Mei Han, Bin Zheng, Shu-Li Yang Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang, People’s Republic of China Received 23 April 2001; accepted 27 June 2001
Abstract The changes of the gene expression patterns and the migration capability of vascular smooth muscle cells (VSMC) at the quiescent and proliferating states were investigated after VSMC was stimulated by cytokines. VSMC migration was measured using a model of wounding injury of confluent cultured cells and a Boyden chamber assay. The migration distance of the quiescent VSMC induced by cytokines bFGF, IL-1b and TNF-a was 5.8, 4.7, and 4.2 times as long as that of the control group, respectively. However, the migration distance of the proliferating VSMC was only one-fourth of the quiescent cells under the same effects. The results of Boyden chamber assay indicated that VSMC was capable of degrading collagen and traversed the pores of membrane barriers in response to bFGF as a chemoattractant, and VSMC number across the membrane was markedly increased as concentration gradient of bFGF ascended. Northern blot and nuclear run-off assay showed that bFGF not only stimulated the expression of migration-related matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) but also enhanced the transcription of proliferation-related osteopontin (OPN) and c-jun genes in the quiescent VSMC. bFGF could moderately increase the expression of OPN and c-jun, but had no significant effect on the expression of MMP-2 and TIMP-2 in the proliferating cells under the same conditions. These findings suggest that VSMC migration capability and the expression activity of migration- and proliferation-related genes were significantly distinct for the quiescent and proliferating VSMC. The cellular migration capability and the expression activity of migrationrelated genes in the quiescent VSMC were much higher than those in the proliferating cells in response to cytokines. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Vascular smooth muscle cell; Migration; Proliferation; Cytokines; Gene expression
* Corresponding author. No.361, Zhongshan Road, Shijiazhuang, 050017. Tel.: 186-311-6044121-5563; fax: 186-311-6048177. E-mail address: [email protected] (J.-k. Wen) 0024-3205/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 4 6 -1
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Introduction The phenotypic modulation and inappropriate proliferation of vascular smooth muscle cells (VSMCs) are major components of atherosclerosis and the restenosis that can occur after balloon angioplasty. It has been suggested that VSMCs in vivo or freshly plated in vitro have a high volume fraction of myofilaments and few biosynthetic organelles and retain the ability to contract in response to vasoconstrictors, whereas VSMCs grown in vitro have a low volume fraction of myofibrils and a high proportion of biosynthetic organelles and may lose their ability to contract[1,2]. These two phenotypes were originally defined as “contractile” (differentiated) or”synthetic” (dedifferentiated). There is evidence that VSMCs in restenosis and atheromatous plaques have undergone phenotypic modulation. Furthermore, there is also evidence that passaged VSMCs are synthetic phenotype[1]. Recent in vitro studies have revealed that passaged VSMCs remain quiescent (stationary phase) when they were seeded in a serum-free medium or reached confluence, and freshly passaged VSMCs in a serum-containing medium were proliferating[3,4]. Currently, passaged VSMCs have been used as a major tool to study the regulatory mechanisms of the phenotypic modulation of VSMCs. However, to date, it has not been paid close attention to whether migration capability and gene expression pattern are identical between the proliferating and quiescent VSMCs[5,6]. In the present study, rat VSMCs were synchronized to the quiescent state (G0) or were left proliferating by differently conditioned culture. Migration capability and gene expression patterns in both quiescent and proliferating VSMCs were tested. Materials and methods Vascular smooth muscle cell isolation and culture Rat aortic VSMCs were isolated from the thoracic and abdominal aortas of SpragueDawley rats (weighting 100 to 130 g) as described previously[7]. Isolated cells were seeded in M199 containing 15% fetal calf serum (FCS) and passaged after reaching confluence using 0.125% trypsin. Cells for the experiments described in this study were used between passaged 4z9. The quiescent VSMCs were obtained by allowing the cells to achieve contact inhibition and to grow in a serum-free medium for 72 h. The degree of VSMC synchronization was monitored by flow cytometry. After the cells were cultured in serum-free medium for 72 h, approximately 85% of the cells were in the G0/G1 phase. The logarithmic phase cells grown to 90% confluence in M199 containing 15% FCS were acted as a proliferating state. VSMC migration assay Two types of migration assays were used. In the first, migration was measured using a monolayer-wounding protocol[8] in which cells migrated from a confluent area into an area that was mechanically denuded of cells. In the second type of assay, collagen-coated transpore tissue culture inserts composed of a polycarbonate membrane with pores of 8mm diameter were used. For monolayer-wounding cell migration assay, the quiescent and proliferating VSMCs were scraped with a sterile single-edged razor blade, respectively. Cultures were then exposed to
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the experimental medium (bFGF 4 U/ml, IL-1b 3000 U/ml, or TNF-a 3000 U/ml in M199 containing 1% FCS, respectively) for 5 h. After this period of time, the cells were washed twice with PBS, fixed with absolute ethanol, and stained with H.E. Five microscopic fields were evaluated for each experiment. The maximum distance migrated (wound edge to nucleus of the farthest cell) was determined in each field under 3200 magnification and compared with VSMCs grown in M199 supplemented with 1% FCS to calculate the opposite value. Migration assay across membrane was performed with a modified Boyden chamber as previously described[9]. PVPF filters (8 mm pore diameter) were first coated with a solution containing 0.1% (v/v) acetic acid and 8 mg/ml collagen from rat tail, and constituted an artificial basement membrane (BM, 10 mm in thickness). The membrane was then rehydrated with M199 before use. Boyden chambers were assembled by adding 4 U/ml or 1 U/ml bFGF in M199 containing 1% FCS to the lower (chemoattractant) chamber, respectively. The quiescent VSMCs suspended in M199 containing 1% FCS were then added to the upper chamber. The chambers were incubated for 24 h at 378C. At the end of the incubation period, the filter in the Boyden apparatus was divided into four parts. The cells on the upper surface of three parts of the filter were mechanically removed. The cells on the underside of the filter were fixed and stained. Four fields were counted per part under high-power field in a microscope (3200). The other part of the filter was scanned with an electron microscope. All experiments were run in triplicate. Data are expressed as mean6S.D. Student’s t test for paired data was used for statistical analysis. Total RNA isolation and northern blotting After the quiescent and proliferating VSMCs or VSMCs grown on the collagen-coated dish were incubated with M199 containing bFGF (4 U/ml) and 1% FCS for 24h, total cellular RNA was prepared from these cells as described[10] and quantified by absorbance at 260 nm. RNA (30 mg) was fractionated on 1% agarose-formaldehyde gels and transferred on to Hybond-N membranes by capillary blotting using 103SSC. RNA was then cross-linked to the membrane by baking at 808C for 2 h. After prehybridization, the blots were hybridized with [a-32P] dCTP-labelled cDNA probes for 20z24 h at 428C. The filters were then washed twice with 23SSC in 0.2% SDS at room temperature, and again at 558C as needed. The filters were exposed to Kodak XAR-5 film at –708C for 24z72 h. Nuclear run-off assay Nuclear run-off assay was performed as described previously[11]. Briefly, nuclei were harvested, as follows, from the quiescent and proliferating VSMCs treated with bFGF (4 U/ml) and 1% FCS for 24 h. The cells were rinsed twice with ice-cold PBS, scraped and pelleted by centrifugation at 1500 rpm for 5 min at 48C. The cell pellet was resuspended in 1 ml NP-40 lysis buffer (10 mmol/L Tris·Cl, pH 7.5, 10 mmol/L NaCl, 3 mmol/L MgCl2 and 1% (v/v) NP-40), incubated for 2z3 min on ice, and centrifuged at 3000 rpm for 5 min. Nuclei were resuspended in 100 ml of 20% glycerol, 50 mmol/L Tris·Cl, pH 8.0, 5 mmol/L MgCl2, 0.1 mmol/L EDTA and stored at –708C. Frozen nuclei were thawed to 258C and reacted in 100 ml reaction buffer consisting of 10 mmol/L Tris·Cl, pH 8.0, 5 mmol/L MgCl2, 150 mmol/L KCl, 2 mmol/L each of ATP, CTP, GTP (sigma), 100 mCi[a-32P] UTP and 100 units of RNA-
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guard. The reaction was carried out at 308C for 30 min. RNA transcripts were purified with phenol/chloroform extraction. Target fragments (1mg) were alkali-denatured and spotted on to nylon membranes using a dot blot apparatus. The membranes were hybridized with transcribed RNAs containing an equal amount of radioactivity, and then autoradiographed with film. Results Quiescent and proliferating VSMCs exhibit markedly distinct migration capabilities As shown in Fig. 1, migration activities were significantly increased when VSMCs at the quiescent state were stimulated by cytokines. After this phenotypic cells were exposed to bFGF, IL-1b or TNF-a for 5 h, the migration distance at the various groups was 5.8-fold, 4.7fold, and 4.2-fold greater than that of the control group, respectively. VSMCs in the control group migrated 42611.4 mm under the same conditions. When times stimulated by these factors were prolonged for another 7 h, migration distance at the various cells was 2-fold longer than that of 5 h. In contrast, migration distance of the proliferating VSMCs was only one-fourth of the quiescent cells following stimulated by the cytokines under the same effects. VSMC migration induced by the cytokines was also demonstrated by migration assay in a modified Boyden chamber. When concentration gradient of bFGF in the upper and lower chamber was adjusted to 0 U/ml, 1 U/ml, and 4 U/ml, the number of VSMCs migrating across PVPF membrane was 4.9161.97, 8.0862.50, and 21.0964.16, respectively. It is thus evident that the number of cell migration across the membrane was markedly increased as concentration gradient of bFGF ascended (Fig. 2). Significant differences in expression activities of genes related to cell migration and proliferation exist between the quiescent and proliferating VSMCs In order to compare whether effect of the cytokines and collagen on expression of cell migration- and proliferation-associated genes was identical in both quiescent and proliferating VSMCs, these two cellular subtypes were cultured in M199 containing bFGF (4 U/ml) and 1% FCS or in tissue culture Petri dish coated with the collagen. Northern blots showed that the genes related to cell migration and proliferation, including MMP-2, TIMP-2, OPN, and c-jun, were upregulated by bFGF, to a certain extent, in the quiescent VSMCs. Of the four genes, MMP-2 and c-jun were strongly expressed in these cells. Their expression in the cells grown on Petri dish coated with the collagen was lower than that in the cells stimulated by bFGF. Only a small increase in expression of OPN and c-jun was detected in the proliferating VSMCs treated with bFGF under the same conditions. The expression activity of these four genes was very low in the two cells treated without bFGF (Fig. 3). Similar results were obtained using nuclear run-off assay. Only the expression of OPN and c-jun genes could be detected in the proliferating VSMCs treated with bFGF. In contrast, these four genes were simultaneously transcripted when the quiescent VSMCs were stimulated by bFGF (Fig. 4). Whether the quiescent VSMCs or proliferating VSMCs possessed no detectable MMP-2, TIMP-2, c-jun and OPN mRNA by nuclear run-off assay when they were not stimulated by bFGF (results not shown).
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Fig. 1. VSMC migration after treatment with cytokines. A: Representative photomicrographs illustrating migration of the quiescent VSMCs. 1. Before VSMC migration; 2. Migration of VSMCs treated with 1% FCS for 5 hours; 3. Migration of VSMCs treated with bFGF11% FCS for 5 hours; 4. Migration of VSMCs treated with IL-1b11% FCS for 5 hours; 5. Migration of VSMCs treated with TNF-a11% FCS for 5 hours. B: Bar graph illustrating VSMC relative migrating activity. * P,0.01 versus 1% FCS; m P,0.01 versus the proliferating VSMC.
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Fig. 2. Bar graph showing the effect of concentration gradient of bFGF on VSMC transpore migration. 1. bFGFgradient at 4 U/ml; 2. bFGF-gradient at 1 U/ml; 3. bFGF-no gradient; 4. 1% FCS-no gradient. Each bar represents the mean 6 S.D. of three separate experiments. * P,0.01 versus bFGF-no gradient ; m P,0.01 versus 1% FCS-no gradient.
Fig. 3. Northern analysis of MMP-2, TIMP-2, OPN and c-jun mRNA in the quiescent and proliferating VSMCs. Panels A and B represent the different VSMCs treated with or without bFGF for 24 hours, respectively. Lanes are as follows: 1. Quiescent VSMCs; 2. VSMCs grown on Petri dish coated with collagen; 3. Proliferating VSMCs. The different mRNAs are shown on the right of each panel.
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Fig. 4. Nuclear run-off assay for transcription activity of MMP-2, TIMP-2, OPN and c-jun genes. Panels A and B are the transcription products of the nuclei from the quiescent and proliferating VSMCs, respectively. The transcription products are shown on the top of Panel A.
The degradation of extracellular matrix (ECM) is required for VSMC migration through the reconstituted BM To determine the relationship between ECM degradation and VSMC migration across the membrane, we reconstituted BM barrier by coating PVPF filters with collagen from rat tail, and observed changes in ECM degradation during migration of VSMCs. Scanning electron micrograph showed that the collagen coated on the filter was first degraded, and the pores adjacent to the cells were exposed when VSMCs migrated across the BM barrier. VSMCs on the upside of the filter changed from the extending shape to spherical or elliptical shape, and traversed the pores of the filter under the action of the chemoattractant bFGF. It could be seen from the underside of the filter that VSMC passing through the pore extended cell foot processes, while the cell body remained in the pore (Fig. 5).
Fig. 5. Scanning electron micrograph for transpore cell migration assay. A: VSMC attaching on PVPF filter coated with collagen; B: the exposed pores after degradation of collagen; C: Entire cell body passing through the pore of filter; D: Cell foot processes extending through the 8-mm diameter pores of PVPF filter.
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Discussion We show in the present study that passaged VSMCs in the quiescent state migrated rapidly in response to cytokines bFGF, IL-1b and TNF-a, whereas migration that occurred in the proliferating VSMCs was much slower than that of the quiescent cells under the same conditions. It has been demonstrated that VSMCs in the arterial media belong to the quiescent cells. The different migration responses to cytokine stimulation suggest that the quiescent VSMCs existing in the arterial media could easily migrate into the vascular intima and initiate their proliferation when the vessel intima is injured. It seems likely that the functional capabilities and morphological characteristics of the quiescent cells favored their migration compared with the proliferating cells. The relative rank order of potency stimulating VSMC migration is bFGF. IL-1b. TNF-a among the factors observed in the experiments. Therefore, we used bFGF as an inducer to study the relationship between VSMC migration ability and gene expression of migration related genes. The degradation of ECM is the biochemical basis of VSMC migration and tumor metastasis[12]. The results of cell migration assay across the filter indicate that VSMC migration is accompanied by local degradation of the matrix in the pathway of migration. It has been demonstrated that ECM degradation in vivo is dependent on activity of proteolytic enzyme[13]. MMP-2 may efficiently degrade type IV collagen, the major structural component of basement membranes, and is very important in VSMC migration from the arterial media to the neointima after injury to the vessel. TIMP-2 is a tissue inhibitor of MMP-2, MMPs and their inhibitors coregulate ECM turnover, and maintain vessel wall integrity[14]. The results of Northern blot and nuclear run-off assay revealed that the expression of MMP-2 and TIMP-2 in the quiescent VSMCs could significantly be induced by bFGF. We speculate that these two proteins might modify the way cells interact with surrounding matrix by enhancing ECM turnover, and overcome the inhibitory effect exerted by the normal ECM. This is probably another reason why migration activity of the quiescent cells was much higher than that of the proliferating VSMCs following stimulated by cytokines. Moreover, our results demonstrated that bFGF could also markedly induce the expression of OPN and c-jun genes in both quiescent and proliferating VSMCs. It has been known that OPN is a protein marker associated with the synthetic phenotype[1]. OPN could be detected in both quiescent and proliferating VSMCs, suggesting that these two VSMCs might be the synthetic phenotype. OPN contains an Arg-Gly-Asp motif that binds integrin, and thus can mediate VSMC adhesion and migration by interaction with this receptor[15]. The expression product of c-jun gene, which is an important transcription factor that promotes the expression of OPN and MMP-2, participates in the regulation processes of VSMC proliferation[16,17]. Accordingly, regardless of the quiescent or proliferating state, the expression of c-jun and OPN genes is necessary to maintaining the synthetic phenotype of VSMCs. However, the causality between the expression of these genes and the phenotypic modulation of VSMCs remains to be studied. Acknowledgments This study was supported by National Natural Science Foundation of China.
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