Responsiveness to growth factors in aortic vascular smooth muscle cells from rats with cirrhosis

Khalid A. Tazi, Concetta Trombino, Richard Moreau, and Didier Lebrec

Laboratoire d'Hémodynamique Splanchnique et de Biologie Vasculaire, Unité de Recherches de Physiopathologie Hépatique, Institut National de la Santé et de la Recherche Médicale, Hôpital Beaujon, 92118 Clichy, France

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Hemodynamic changes in cirrhosis may be associated with alterations in aortic vascular smooth muscle cell (AVSMC) function. The present study compared the proliferative response to serum and growth factors in cirrhotic and control AVSMC. Serum from cirrhotic rats, cirrhotic cell lysates, and the conditioned medium of cultured cirrhotic AVSMC induced an increase in [3H]thymidine incorporation in control but not in cirrhotic AVSMC. Platelet-derived growth factor-beta (PDGF-BB) induced a greater increase in [3H]thymidine incorporation in cirrhotic than in control cells. [3H]thymidine incorporation induced by cirrhotic conditioned medium was blocked by anti-PDGF antibody. Immunoblot studies showed that the anti-PDGF antibody recognized a 30-kDa protein in the conditioned medium of cirrhotic AVSMC culture, a protein corresponding to PDGF. Binding studies of PDGF-BB indicated a twofold increase in receptor density in cirrhotic AVSMC with no alteration in affinity for PDGF-BB. We conclude that an increased responsiveness of cirrhotic AVSMC to the PDGF could contribute to alterations in AVSMC and muscle cell tone that may play a role in the hemodynamic changes in cirrhosis.

platelet-derived growth factor; fibroblast growth factor; hemodynamic changes

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CIRRHOSIS IS ASSOCIATED with hyperdynamic circulation, characterized by increased cardiac output and splanchnic blood flow and reduced systemic and splanchnic vascular resistances (25). Moreover, vascular hyporeactivity to vasoconstrictors occurred in cirrhosis (27). The mechanisms that cause the hemodynamic alterations in cirrhosis remain unclear. Functional changes in aortic vascular smooth muscle cell (AVSMC) contractility, growth, and responsiveness to vasoactive agents might contribute to these alterations (15, 40, 41). Although the primary function of AVSMC is contraction and maintenance of vascular tone, in cirrhosis, AVSMC appear to react abnormally.

Vascular tone is regulated by a variety of extracellular factors, including circulating cytokines and vasoactive hormones (11, 20), which play important roles in AVSMC function (33). In cirrhosis the production and circulating levels of cytokines and vasoactive substances are increased (21, 26); this may alter AVSMC contraction and growth (1, 17, 28).

Platelet-derived growth factor (PDGF), the most potent mitogenic cytokine for AVSMC (14, 34), induces smooth muscle cell contraction (2). Hence, regulated, local secretion of PDGF within the vasculature could play a role in maintaining smooth muscle tone (6). PDGF may be implicated in controlling AVSMC phenotype and function (38) as well as in regulating the expression of smooth muscle actin (4). Disturbances in PDGF ligand production and/or PDGF receptor (PDGFR) expression have been associated with pathological conditions (35). For example, overexpression of the PDGFR has been implicated in cirrhosis; moreover, PDGFR-beta mRNA and protein were overexpressed in cultured lipocytes from rats with cirrhosis (44).

The present study compared the proliferative response to serum and growth factors [basic fibroblast growth factor (bFGF), PDGF-AA, PDGF-AB, and PDGF-BB] in AVSMC from cirrhotic and control rats. Moreover, binding of PDGF-BB and the expression of its receptor on the surface of cirrhotic and control AVSMC were also investigated.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Induction of cirrhosis. Cirrhosis was induced by bile duct ligation, as previously described (23). Briefly, under ether anesthesia, the common bile duct was exposed by median laparotomy and occluded by double ligature with a nonresorbable suture (7-0 silk). The first tie was made below the junction of the hepatic ducts, and the second was made above the entrance of the pancreatic ducts. The common bile duct was then resected between the two ligatures, and the abdominal incision was closed. Thoracic aortae were resected from rats 4-5 wk after bile duct ligation. This delay is necessary for the development of secondary bilary cirrhosis.

Preparation and culture of AVSMC. Rat AVSMC were isolated and cultured as previously described with minor modifications (39). Briefly, under sterile conditions, thoracic aortae were resected from cirrhotic and normal adult male Sprague-Dawley rats (Charles River Laboratories, Saint-Aubin-Lès-Elbeuf, France). Aortae were cleaned of adherent and adventitial tissue and incubated for 10 min at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 130 U/ml collagenase (type IV) and 1 mg/ml elastase (type IV). The intact and emptied tunica adventitia tube was removed and rinsed thoroughly. The medial tubes were minced with scissors and reincubated at 37°C in the original digestion medium for 1-2 h until single cell suspension was obtained. Suspended cells were centrifuged at 400 g for 5 min, washed with DMEM, and resuspended in DMEM culture medium containing 2 mM L-glutamine, 2 g/l NaHCO3, 100 U/ml penicillin, and 100 µg/ml streptomycin. Fetal calf serum (FCS, 10%) was then added, and cells were cultured in the above medium at 37°C in a humidified atmosphere of 5% CO2-95% air. For the cell subcultures, cells were rinsed once with phosphate-buffered saline (PBS) and treated briefly with trypsin.

AVSMC from cirrhotic and control rats were characterized as a homogeneous population of smooth muscle cells. The cells exhibited typical "hill-and-valley" growth of AVSMC, as viewed under phase-contrast microscopy (9). Furthermore, identification of AVSMC was verified by smooth muscle alpha -actin staining (12).

Cell viability was checked by the exclusion of trypan blue and always exceeded 95%. All experiments were performed with cultured AVSMC from 12 normal and 12 cirrhotic rats. Cells were studied between passages 4 and 16, during which the investigated parameters remained stable. There were no significant differences in cell number and [3H]thymidine incorporation between AVSMC from control and sham-operated rats (data not shown).

To obtain quiescent cells, the cells were seeded on day 0 in 10% FCS-DMEM. After attachment (on day 2), the cells were washed with DMEM to remove residual FCS and placed in 0.5% FCS for 2 days. Incubation of cells in DMEM-0.5% FCS has been shown to induce growth arrest, maintaining AVSMC in a quiescent, noncatabolic state, and to promote expression of AVSMC-specific contractile protein. A decrease in the mitotic index and [3H]thymidine incorporation could be attained at 0.5% FCS. This concentration yielded the best combination of cell viability and quiescence (12, 30).

AVSMC proliferation in culture. AVSMC were seeded at a density of 104 cells/ml in 24-well dishes in 10% FCS-DMEM; medium was changed every 2nd day. Cells were counted by hemocytometer after they were harvested by gentle trypsinization.

Determination of [3H]thymidine incorporation. On day 0, cells were seeded at an initial density of 104 cells/ml in 24-well dishes and allowed to grow in 10% FCS-DMEM. On day 2, cells were placed in 0.5% FCS for 2 days to render them quiescent, then washed successively twice with 10 mg/ml bovine serum albumin (BSA) in PBS and twice with PBS alone. Cells were incubated for 24 h in triplicate with fresh medium containing 1 µCi/ml [3H]thymidine in DMEM containing increasing concentrations of growth factors. After incubation, dishes were rinsed twice with PBS. DNA was precipitated by addition of 1 ml of 10% trichloroacetic acid per well for 18 h at 4°C. Cells were digested with 1 ml of 1 M NaOH per well at room temperature. Aliquots were assayed for [3H]thymidine radioactivity and protein content. [3H]thymidine radioactivity incorporation was determined by liquid scintillation spectrometry, and protein concentrations were determined by the Bradford method using the Bio-Rad assay reagent and BSA as a standard (8).

Determination of cell number in the presence of growth factors. Cells were seeded on day 0 in 10% FCS-DMEM. After attachment (on day 2), cells were washed with DMEM to remove residual FCS and placed in 0.5% FCS for 2 days to render them quiescent. On day 4, cells in triplicate were incubated in medium containing increasing concentrations of growth factors. Cells were counted on day 8 by hemocytometer after they were harvested by gentle trypsinization.

Collection of conditioned medium. Control and cirrhotic AVSMC were grown in 10% FCS-DMEM. Conditioned media of cirrhotic and control AVSMC subconfluent cultures were collected, centrifuged at 300 g for 10 min, and used or frozen at -20°C for subsequent assays of mitogenic activity (10).

Assay for mitogenesis of conditioned medium. Equal volumes (1 ml) of conditioned medium of cultures of control and cirrhotic AVSMC cultures were added to quiescent AVSMC cultures grown in 24-well plates (10). Each well was pulsed with 1 µCi/ml [3H]thymidine. After 24 h of incubation, [3H]thymidine incorporation and cell number were determined as described above.

Assay for mitogenesis of cell lysates. Cells were scraped from plates and homogenized in PBS (pH 7.4) containing protease inhibitors (1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 1 µM leupeptin). The lysates were centrifuged. The samples were diluted in DMEM (1 ml/well) and used directly for assay of mitogenic activity on quiescent cultures (22).

To evaluate the effects of tyrphostin (tyrphostin 25: [3,4,5-trihydroxybenzylidene]malononitrile), an inhibitor of tyrosine kinase (3), and the anti-PDGF antibody on FCS-induced [3H]thymidine incorporation of AVSMC, quiescent AVSMC from cirrhotic and control rats were incubated in DMEM supplemented with 10% FCS alone or with increasing concentrations of tyrphostin (or anti-PDGF) and 1 µCi/ml [3H]thymidine. After 24 h, [3H]thymidine incorporation was measured as described above. The results are expressed as percentages of corresponding values in the absence (100%) of tyrphostin or in the absence (100%) of anti-PDGF antibody.

The neutralizing capability of anti-PDGF antibody (that recognizes all 3 forms of PDGF) has been verified in control experiments: the effect of PDGF-AA, PDGF-AB, and PDGF-BB on proliferation of quiescent AVSMC was neutralized by the preincubation (for 2 h in DMEM) of each growth factor (10 ng/ml) in the presence of anti-PDGF antibody (10 µg/ml). At this concentration, anti-PDGF antibody was not toxic for quiescent AVSMC (as evidenced by normal cell appearance under contrast microscopy and by trypan blue staining) and did not induce a significant change in basal [3H]thymidine incorporation and cell number (data not shown).

To determine the effects of anti-PDGF antibody on conditioned medium-induced [3H]thymidine incorporation, the conditioned medium of AVSMC cultures was preincubated with anti-PDGF antibody for 2 h at 37°C, then the treated conditioned medium was added in the presence of 1 µCi/ml [3H]thymidine to cultured AVSMC, and [3H]thymidine incorporation was determined after 24 h (10).

To determine the effect of tyrphostin on PDGF, conditioned medium, cell lysates, and serum-induced [3H]thymidine incorporation, quiescent AVSMC were treated with 100 µM tyrphostin. After 24 h, 10 µM PDGF, conditioned medium, cell lysates, 10% FCS, or 10% serum from cirrhotic rats was added in the presence of 1 µCi/ml [3H]thymidine, and [3H]thymidine incorporation was measured after 24 h.

Western blot conditioned medium. Conditioned medium of cirrhotic and control AVSMC cultures was filtered through a 0.2-µm syringe filter, centrifuged (4,000 g) for 1 h, and diluted in an electophoresis buffer [3% sodium dodecyl sulfate (SDS), 62.5 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, 10% glycerol, 0.001% bromphenol blue, pH 6.8], then boiled at 100°C for 10 min and subjected to 12% SDS-gel electrophoresis (24). Gels were calibrated using prestained molecular mass standards. After electrophoresis, proteins were transferred to nitrocellulose filters (0.45 µm). Nitrocellulose papers were then blocked by incubation in 5% BSA, 0.15 M NaCl, and 0.01 M Tris · HCl, pH 7.4, overnight at 4°C. The blots were washed twice for 10 min in 0.15 M NaCl and 0.01 M Tris · HCl, pH 7.4, twice in the same buffer supplemented with 0.05% Triton X-100, and finally three times for 5 min with Triton-free wash buffer. The blots were then incubated in the presence of a primary antibody that recognizes all forms of PDGF in 0.1% BSA, 0.15 M NaCl, and 0.01 M Tris · HCl, pH 7.4. The blots were washed as described above and incubated with a secondary antibody (anti-mouse immunoglobulin, peroxidase-linked species-specific whole antibody). The blots were then washed three times for 15 min with 0.15 M NaCl, 0.01 M Tris · HCl, and 0.05% Triton X-100, pH 7.4, and finally three times for 5 min with Triton-free wash buffer. The immunoreactivity was assessed using an enhanced chemiluminescent (ECL) Western blot detection system according to the manufacturer's instructions.

PDGF binding assays. Binding assays were performed in cultured AVSMC as described previously (7). Cells were plated in 24-well dishes in DMEM supplemented with FCS and grown to confluence. For saturation binding, the cells were rinsed with PBS containing 1 mM CaCl2 and 1 mg/ml BSA, then incubated at 4°C for 3 h in DMEM with 0.25% BSA with increasing concentrations of 125I-PDGF-BB. Nonspecific binding was determined using 500 ng/ml unlabeled PDGF-BB. After incubation the cells were washed three times with ice-cold PBS buffer. Cells were then solubilized with 1 N NaOH. Radioactivity in an aliquot of solubilized cells was measured using a gamma counter. Results were corrected to total cellular protein concentrations determined by the method of Bradford with BSA as a standard. Saturation binding was calculated by subtracting the amount of bound 125I-PDGF-BB obtained in the presence of 500 ng/ml excess unlabeled PDGF-BB (nonspecific binding) from that obtained in the absence of unlabeled PDGF-BB (total binding). The dissociation constant (Kd) and the capacity of binding sites (Bmax) were determined after transformation of high-affinity binding by Scatchard analysis.

Western blot of PDGFR. Western blot studies of AVSMC lysates were performed using polyclonal rat PDGFR-beta antibody, as described previously (44). AVSMC from cirrhotic or control rats were lysed in an electrophoresis buffer (3% SDS, 62.5 mM Tris · HCl, 10% glycerol, 2% beta -mercaptoethanol, pH 6.8). The protein content (100 µg/lane) was subjected to 5% SDS-gel electrophoresis. After electrophoresis, proteins were transferred to nitrocellulose filters (0.45 µm), then nitrocellulose filters were blocked by incubation in 5% BSA, 0.15 M NaCl, and 0.01 M Tris · HCl, pH 7.4, overnight at 4°C. The blots were washed twice for 10 min in 0.15 M NaCl and 0.01 M Tris · HCl, pH 7.4, twice in the same buffer supplemented with 0.05% Triton X-100, and finally three times for 5 min with Triton-free wash buffer. The blots were then incubated in the presence of primary antibody (antibody to rat PDGFR-beta ) in 0.1% BSA, 0.15 M NaCl, and 0.01 M Tris · HCl, pH 7.4. The blots were washed as described above and incubated with secondary antibody (anti-mouse immunoglobulin, peroxidase-linked species-specific whole antibody). The blots were washed three times for 15 min with 0.15 M NaCl, 0.01 M Tris · HCl, and 0.05% Triton X-100, pH 7.4, and finally three times for 5 min with Triton-free wash buffer. The immunoreactivity was assessed using an ECL Western blot detection system according to the manufacturer's instructions.

Statistical analysis. Triplicates were used for each experimental protocol. Each experiment was repeated three to six times. Values are means ± SD. Results were compared by Student's t-test.

Materials. DMEM, FCS, collagenase, elastase, penicillin, and streptomycin were purchased from GIBCO (Grand Island, NY); FGF, PDGF-AA, PDGF-BB, PDGF-A, antirat alpha -actin antibody, and tyrphostin 25 from Sigma Chemical (St. Louis, MO); [3H]thymidine, 125I-PDGF-BB, the Western blot detection system, and Hyperfilm-ECL from Amersham International; and anti-PDGF and anti-PDGFR-BB from Genzyme (Boston, MA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AVSMC proliferation in culture. When control and cirrhotic AVSMC were cultured in 10% FCS-DMEM, control AVSMC grew to a significantly higher number than cirrhotic AVSMC. At confluence the difference between control and cirrhotic AVSMC was 32 ± 7% (n = 12); it did not vary statistically between passages 4 and 16.

In contrast to cell number, protein content per cell was significantly higher in cirrhotic than in control AVSMC (437 ± 22 vs. 335 ± 27 pg/cell, P < 0.05, n = 4).

Effect of serum on [3H]thymidine incorporation in cultured AVSMC. The effects of FCS and rat serum on [3H]thymidine incorporation in cirrhotic and control AVSMC are shown in Fig. 1. [3H]thymidine incorporation was significantly higher in cirrhotic than in control AVSMC regardless of the serum used (Fig. 1). Control AVSMC incorporated more [3H]thymidine in the presence of rat serum (vs. FCS), and cirrhotic rat serum was more mitogenic than control rat serum (86.2 ± 9.1 and 68.8 ± 8.5 × 104 counts/min (cpm)/mg protein, respectively, P < 0.05). However, there was no significant difference in [3H]thymidine incorporation when cirrhotic AVSMC were incubated in the presence of FCS, cirrhotic serum, or control rat serum.


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Fig. 1.   Stimulation of [3H]thymidine incorporation in rat aortic vascular smooth muscle cells (AVSMC) by fetal calf serum (FCS) and rat serum. Quiescent AVSMC from cirrhotic (hatched bars) and control rats (open bars) were incubated in DMEM alone, DMEM supplemented with 10% FCS, 10% control rat serum (control serum), or 10% cirrhotic rat serum (cirrhotic serum) and 1 µCi/ml [3H]thymidine. After 24 h, [3H]thymidine incorporation was measured. Values are means ± SD of 6 experiments. *Significantly different (P < 0.05) from control AVSMC. dagger  Significantly different (P < 0.05) from corresponding value of FCS.

Effect of conditioned medium and cell lysates on AVSMC growth. Cirrhotic AVSMC lysates and the conditioned medium of cirrhotic AVSMC cultures induced stimulation of [3H]thymidine incorporation in DNA of control AVSMC. However, neither the conditioned medium of control AVSMC cultures nor control cell lysates were mitogenic for cirrhotic AVSMC (Table 1).

                              
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Table 1.   [3H]thymidine incorporation of AVSMC in response to conditioned medium and cell lysates

The conditioned medium of cirrhotic AVSMC cultures and cirrhotic AVSMC lysates also induced an increase in control cell number (27 ± 5 and 31 ± 8%, respectively).

When the conditioned medium of cirrhotic AVSMC cultures was collected in the absence of serum, it failed to stimulate the proliferation of control AVSMC (data not shown).

Effects of growth factors on AVSMC growth. Stimulation of [3H]thymidine incorporation obtained by bFGF and PDGF-AA was not significantly different between cirrhotic and control AVSMC (Fig. 2). bFGF and PDGF-AA induced 3.0- and 1.6-fold stimulation of [3H]thymidine incorporation, respectively (Fig. 2, A and B). PDGF-BB induced a dose-dependent stimulation of [3H]thymidine incorporation in cirrhotic and control AVSMC (Fig. 2C). The increased stimulation of [3H]thymidine incorporation in cirrhotic AVSMC (11-fold) was approximately twice that in control AVSMC (6-fold). PDGF-AB (Fig. 2D) also increased [3H]thymidine incorporation in cirrhotic AVSMC more than in control AVSMC (8- and 5-fold, respectively).


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Fig. 2.   Effects of growth factors on [3H]thymidine incorporation in rat AVSMC. [3H]thymidine incorporation was determined in presence of increasing concentrations of basic fibroblast growth factor (bFGF) and platelet-derived growth factors (PDGF). black-square, Cirrhotic AVSMC; square , control AVSMC. Values are means ± SD of 6 experiments. * Significantly different (P < 0.05) from control AVSMC.

All three isoforms of PDGF and bFGF caused a dose-dependent increase in cell number in cirrhotic and control AVSMC (data not shown), with PDGF-BB being more potent than PDGF-AA, PDGF-AB, and FGF (Table 2). The 10 ng/ml concentration of PDGF-BB led to a 1.9- and a 2.1-fold increase in cell number in cirrhotic and control AVSMC, respectively.

                              
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Table 2.   Effect of growth factors on proliferation of AVSMC

Effects of anti-PDGF antibody on [3H]thymidine incorporation in AVSMC. The PDGF antibody caused a dose-dependent inhibition of [3H]thymidine incorporation induced by FCS. The inhibition rate of [3H]thymidine incorporation was significantly more marked in cirrhotic than in control AVSMC: 41 ± 8 and 19 ± 5%, respectively (Fig. 3).


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Fig. 3.   Effects of anti-PDGF antibody on FCS-induced [3H]thymidine incorporation in AVSMC. Quiescent AVSMC from cirrhotic (bullet ) and control rats (open circle ) were incubated in DMEM supplemented with 10% FCS (100%) alone or with increasing concentrations of anti-PDGF antibody and 1 µCi/ml [3H]thymidine. After 24 h, [3H]thymidine incorporation was measured. Results are expressed as percentages of corresponding values in absence of anti-PDGF antibody. Values are means ± SD of 6 experiments. * Significantly different (P < 0.05) from control AVSMC.

[3H]thymidine incorporation in control AVSMC induced by conditioned medium of cirrhotic AVSMC cultures was blocked by the anti-PDGF antibody. Incubation of control AVSMC with the anti-PDGF antibody caused an inhibition of 87 ± 7 and 70 ± 10% of [3H]thymidine incorporation induced by cirrhotic cell lysates and serum from cirrhotic rats, respectively (Table 3).

                              
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Table 3.   Effect of anti-PDGF antibody on [3H]thymidine incorporation induced by conditioned medium, cell lysates, and cirrhotic rat serum in cultured AVSMC

Effects of tyrphostin on [3H]thymidine incorporation of AVSMC. [3H]thymidine incorporation in cirrhotic and control AVSMC was inhibited by tyrphostin. The maximal inhibition rate of [3H]thymidine incorporation in cirrhotic and control AVSMC was 80 ± 7 and 74 ± 8%, respectively (Fig. 4). Preincubation of AVSMC with tyrphostin prevented PDGF-induced stimulation of [3H]thymidine incorporation in cirrhotic and control AVSMC and inhibited [3H]thymidine incorporation in cultured control AVSMC induced by conditioned medium of cirrhotic AVSMC cultures (Table 4).


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Fig. 4.   Effects tyrphostin on FCS-induced [3H]thymidine incorporation in AVSMC. Quiescent AVSMC from cirrhotic (black-square) and control rats (square ) were incubated in DMEM supplemented with 10% FCS (100%) alone or with increasing concentrations of tyrphostin and 1 µCi/ml [3H]thymidine. After 24 h, [3H]thymidine incorporation was measured. Results are expressed as percentages of corresponding values in absence of tyrphostin. Values are means ± SD of 6 experiments. * Significantly different (P < 0.05) from control AVSMC.

                              
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Table 4.   Effect of tyrphostin on [3H]thymidine incorporation in cultured AVSMC

PDGF expression in conditioned medium. Figure 5 shows Western blots of unconditioned media and conditioned media of control and cirrhotic AVSMC cultures. The anti-PDGF antibody recognized a protein with an apparent molecular mass of ~30 kDa in unconditioned medium and cirrhotic conditioned medium. No band corresponding to 30 kDa was seen in the conditioned medium of control cultured AVSMC. However, there are two lower-molecular-mass bands of ~35 and 65 kDa.


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Fig. 5.   Western blot of PDGF in conditioned medium. Conditioned medium of cultured AVSMC was prepared and applied to 12% SDS-polyacrylamide gels. Proteins were resolved by electrophoresis and transferred to nitrocellulose. Blots were probed with anti-PDGF antibody that recognizes all forms of PDGF. Immunoreactivity was visualized by autoradiography after incubation with secondary antibody. Lane M, unconditioned medium; lane 1, culture medium from control AVSMC; lane 2, culture medium from cirrhotic AVSMC. Approximate sizes of prestained molecular mass standards are indicated on right.

PDGF binding assay. Figure 6 shows the binding of increasing amounts of 125I-PDGF-BB on cultured AVSMC. The maximal level of 125I-PDGF-BB binding to cirrhotic and control AVSMC was 53.2 ± 11.5 and 28.5 ± 5.5 fmol/106 cells, respectively (P < 0.05). The Scatchard plot obtained by transformation of saturation binding (Fig. 6) showed that PDGF-BB had similar affinities to the PDGFR in cirrhotic and control AVSMC (Kd = 980 ± 70 and 1,060 ± 90 pM, respectively), but there were more receptors on the surface of cirrhotic AVSMC than on control AVSMC (3.3 and 1.8 × 104/cell, respectively).


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Fig. 6.   Binding of 125I-PDGF to intact AVSMC from cirrhotic (black-square) and control (square ) AVSMC at 4°C. A: saturation profiles. Values are means ± SE from 6 separate experiments. B: Scatchard plot obtained by transformation of saturation binding data.

PDGFR-beta expression in AVSMC. The presence of the PDGFR-beta protein was confirmed by Western blot analysis of AVSMC lysates. Representative autoradiographs (Fig. 7) revealed single bands with an apparent molecular mass of ~180 kDa in control and cirrhotic AVSMC. However, PDGFR-beta was more expressed in cirrhotic than in control AVSMC (Fig. 7). Densitometry of autoradiographs from three individual experiments showed a 280 ± 40% increase in PDGFR-beta signal intensity in cirrhotic AVSMC in comparison to control AVSMC.


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Fig. 7.   Western blot of PDGF-beta receptors (PDGFR-beta ) in lysates of AVSMC. Lysate protein was measured, and equal amounts were applied to 5% SDS-polyacrylamide gels. Proteins were resolved by electrophoresis and transferred to nitrocellulose. Blots were probed with anti-PDGFR-beta antibody. Immunoreactivity was visualized by autoradiography after incubation with secondary antibody. Lane 1, lysate proteins from control AVSMC; lane 2, lysate proteins from cirrhotic AVSMC. Approximate sizes of prestained molecular mass standards are indicated on right. Arrow, migration position of PDGFR-beta .

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results of the present study show that, under identical culture conditions, proliferation rates were significantly higher for control than for cirrhotic AVSMC. At confluence there were significantly fewer cells in cultured cirrhotic AVSMC than in cultured control AVSMC. However, protein content per cell was significantly greater in cirrhotic than in control AVSMC. These results suggest that cirrhotic AVSMC were significantly larger than control AVSMC.

The rate of [3H]thymidine incorporation in AVSMC was higher in rats with cirrhosis than in control rats. This difference was observed regardless of the serum tested (FCS or cirrhotic and control rat serum). In addition, rat serum was more mitogenic than FCS in control AVSMC, with cirrhotic rat serum being more potent than control rat serum. However, the rate of [3H]thymidine incorporation in cirrhotic AVSMC was not stimulated by rat serum. The differences in [3H]thymidine incorporation and cell numbers observed between cirrhotic and control AVSMC may reflect differences in responsiveness to growth factors in serum. Previous studies have demonstrated that the increase in AVSMC size was associated with an increase in proliferative response to PDGF (32).

PDGF has been identified as the major mitogen in serum for AVSMC (34). Experiments using anti-PDGF antibody have shown that the [3H]thymidine incorporation activity in serum due to PDGF is twofold higher in cirrhotic than in control AVSMC. The three possible isoforms of the two chains, PDGF-AA, PDGF-AB, and PDGF-BB, have been found to differ in functional activities, probably because of different binding specificities to two separate receptor classes (16). The present results show that all three isoforms stimulate growth in control and cirrhotic AVSMC, with PDGF-BB being the most potent. Moreover, [3H]thymidine incorporation induced by PDGF-BB in cirrhotic AVSMC was approximately twice that in control AVSMC. This could be explained by increased PDGFR density on cirrhotic AVSMC or by an increased affinity of these receptors for the growth factor. These hypotheses were tested by radiolabeled ligand binding assays on intact AVSMC and by Western blot studies using anti-PDGFR-beta . Results showed a twofold increase in receptor density in cirrhotic AVSMC and no alterations in the affinity for the growth factor. Western blot analysis demonstrated that cirrhotic AVSMC expressed more PDGFR-beta protein than control AVSMC. These results provide evidence of an increased proliferative response to PDGF-BB associated with increased expression of PDGFR-beta in cultured AVSMC from rats with cirrhosis.

Previous studies have demonstrated that disturbances in PDGFR expression may be associated with different pathological conditions (19, 32, 35). For example, cultured AVSMC from spontaneously hypertensive rats overexpress PDGFR-alpha and demonstrate an enhanced proliferative response to PDGF-AA (32). AVSMC from diabetic rabbits express higher levels of PDGFR-beta mRNA and show an enhanced proliferative response to PDGFR-beta in vitro compared with AVSMC in control rabbits (19). The increased expression of PDGFR has also been implicated in cirrhosis, since PDGFR-beta mRNA and protein were overexpressed in cultured lipocytes from cirrhotic rats (44). On the other hand, the expression of PDGF-beta has been shown to depend on the phenotype of AVSMC: synthetic AVSMC express more PDGFR than contractile AVSMC (42). One possible explanation for the increased expression of PDGFR-beta in cirrhotic AVSMC is that, in vivo, they are converted to the synthetic phenotype with alterations in function, whereas control AVSMC are known to retain the contractile phenotype.

The increased expression of PDGFR-beta by cirrhotic AVSMC may play a role in the hemodynamic changes observed in cirrhosis, since PDGF is known to be implicated in cytoskeletal reorganization by modulating the expression of smooth muscle actin (13, 18).

The medium of cultured cirrhotic AVSMC (conditioned in the presence of FCS) was shown to contain one or more factors that stimulate [3H]thymidine incorporation of cultured AVSMC from control rats. However, when medium was conditioned in the absence of serum, it failed to stimulate the proliferation of control AVSMC. These results suggest that a serum factor(s) was essential for the induction of mitogen factor from cirrhotic AVSMC. The medium of cultured control AVSMC conditioned in the absence or in the presence of FCS was not mitogenic for cirrhotic AVSMC. The mitogenic activity of this factor was neutralized by the anti-PDGF antibody. Immunoblot studies of AVSMC culture media using a polyclonal antibody that recognizes all forms of PDGF showed a major band of 30 kDa in the conditioned medium of cultured cirrhotic AVSMC. This pattern is consistent with the size of mature PDGF (29-31 kDa) (16). No band corresponding to PDGF was seen in the conditioned medium of cultured control AVSMC. PDGF may have been present in the control conditioned medium but at concentrations below the threshold that we could detect by Western blot analysis. The PDGF concentration may be lower because it is consumed but not produced by control AVSMC. The anti-PDGF antibody recognized two lower bands (35 and 65 kDa) in control conditioned medium that may correspond to higher-molecular-mass forms of PDGF, resulting from modification during conditioning, or represent nonspecific binding by anti-PDGF antibody.

Production of PDGF-like proteins has been also observed in cultured AVSMC from young but not adult rats (37). It has been suggested that autocrine stimulation of AVSMC growth involving production of PDGF-like mitogens may be, in certain cases, part of the transformed phenotype (29). These results suggest phenotype changes in cirrhotic AVSMC.

Our results suggested that the PDGF was more markedly expressed in cirrhotic than in control rat serum, since the former was more mitogenic for control AVSMC than the latter. Moreover, [3H]thymidine incorporation induced by serum from cirrhotic rats was inhibited but not entirely blocked by the anti-PDGF antibody. The inability of the antibody to entirely inhibit mitogenic activity suggests the presence of other mitogenic factor(s) in serum from cirrhotic rats. This hypothesis is supported by the fact that tyrphostin, a potent tyrosine kinase-dependent PDGF inhibitor (36), inhibited conditioned medium and PDGF-induced stimulation of [3H]thymidine incorporation but not [3H]thymidine incorporation induced by cirrhotic rat serum.

This study shows clear differences in PDGFR-beta densities between cirrhotic and control AVSMC associated with differences in the rates of [3H]thymidine incorporation and PDGF-like protein release. We do not know whether the differences found in cultured AVSMC reflect the in vivo situation. It has been shown that PDGF mRNA expression may be stimulated by vasoactive substances such as angiotensin II (5). In addition, PDGF and vasoconstrictors may regulate smooth muscle alpha -actin of AVSMC (31, 43). On the basis of these findings, it has been hypothesized that the marked stimulation of vasoactive systems that occurs in cirrhosis may induce an increased expression of PDGF in cirrhotic AVSMC, which retain this phenotype in vitro. Thus, in cirrhosis, one consequence of PDGF ligand and receptor induction is AVSMC phenotype changes, which may be important in the hemodynamic impairment observed in cirrhosis (5).

In conclusion, this study demonstrates abnormal growth of cultured cirrhotic AVSMC in response to serum and growth factors and an increased expression of PDGFR. This suggests that cirrhosis alters AVSMC function.

    FOOTNOTES

Address for reprint requests: K. A. Tazi, INSERM U-24, Hôpital Beaujon, 92118 Clichy, France.

Received 27 August 1996; accepted in final form 25 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 273(4):G883-G890
0193-1857/97 $5.00 Copyright © 1997 the American Physiological Society




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