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(Circulation. 1998;97:181-187.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Different Proliferative Properties of Smooth Muscle Cells of Human Arterial and Venous Bypass Vessels

Role of PDGF Receptors, Mitogen-Activated Protein Kinase, and Cyclin-Dependent Kinase Inhibitors

Zhihong Yang, MD; Barry S. Oemar, MD; Thiery Carrel, MD; Beat Kipfer, MD; Friedgard Julmy; ; Thomas F. Lüscher, MD

From Cardiovascular Research (Z.Y., B.S.O., T.F.L.), Institute for Physiology, University Zürich-Irchel, Switzerland; Cardiology (F.J.), Cardiovascular and Thoracic Surgery (T.C., B.K.), Inselspital Bern, Switzerland; and Cardiology (Z.Y., B.S.O., T.F.L.), University Hospital Zürich, Switzerland.

Correspondence to Thomas F. Lüscher, MD, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland.

	Abstract

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Background—Internal mammary artery (IMA) bypass grafts have a higher patency than saphenous vein (SV) grafts. Intimal hyperplasia of SV grafts is due to smooth muscle cell (SMC) proliferation and migration. We hypothesized that different SMC growth activity exists in IMA and SV, which may explain the different patencies of arterial and venous grafts.

Methods and Results—SMCs were isolated from IMA and SV by explant culture and stimulated with serum or platelet-derived growth factor-BB (PDGF-BB). Cell growth was analyzed by explant outgrowth rate, ³H-thymidine incorporation, or cell counting. PDGF receptor expression and autophosphorylation, regulation of mitogen-activated protein kinases (MAPKs), and cyclin-dependent kinase inhibitors (p27^Kip1 and p21^Cip1) were analyzed by molecular techniques. SMC outgrowth from explants by serum (20%) over a 20-day period was more pronounced in SV (37±5%) than in IMA (4±3%; P<.001) of the same patients. Serum (10%) increased cell number more rapidly in SV (2x10⁴/well to 18±4x10⁴/well; P<.05) than in IMA (2x10⁴/well to 9±4x10⁴/well; P<.05) over an 8-day period. PDGF-BB (0.01 to 10 ng/mL) stimulated ³H-thymidine incorporation (1347±470% above control levels) and increased cell number in SV (2x10⁴/well to 5±1x10⁴/well; P<.05) but not in IMA. PDGF - and ß-receptors were similarly expressed and were activated in both SV and IMA. PDGF-BB induced a similar MAPK activation (kinetics and maximal activity) in both SV and IMA cells but increased MAPK protein level only in SV. Furthermore, PDGF-BB markedly downregulated the cell cycle inhibitor p27^Kip1 in SV, but this was much less pronounced in IMA.

Conclusions—SMCs from SVs exhibit enhanced proliferation compared with IMA in spite of functional growth factor receptor expression and MAPK activation. However, PDGF increased MAPK protein level only in SV and downregulated cell cycle inhibitor (p27^Kip1) more potently in SV than in IMA. This may explain the resistance to growth stimuli of IMA SMCs and may contribute to the longer patency of arterial versus venous grafts.

Key Words: muscle, smooth • vessels • cells • growth substances • enzymes

	Introduction

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In patients with coronary artery disease, bypass grafting is performed with autologous SV or IMA. Arterial grafts have a higher patency and lower patient mortality than venous grafts; up to 50% of venous grafts occlude within 10 years after implantation.^{1 2} The decreased ability of venous endothelial cells to release nitric oxide, a potent vasodilator and platelet inhibitor, and in turn the increased platelet–vessel wall interaction may contribute to early vein graft occlusion,^{3 4} whereas late occlusions are secondary to graft attrition due to intimal thickening,^{5 6 7} which occurs frequently in SVs and coronary arteries but very rarely in IMAs.⁷

SMC proliferation and migration play an important role in intimal thickening in atherosclerosis and restenosis^{8 9} and are crucial for long-term patency of bypass grafts in particular venous grafts.^{6 10 11} Growth factors such as PDGF released from activated platelets, SMCs, and monocytes⁹ are an important mediator of SMC proliferation and migration.^{9 12 13} After implantation of veins, platelet deposition occurs, particularly in the absence of antiplatelet therapy.¹⁴ Moreover, the randomized, double-blind STARC study demonstrated that trapidil, a PDGF antagonist, reduces restenosis after percutaneous transluminal coronary angioplasty and improves clinical outcome in patients,¹⁵ and inhibition of PDGF ß-receptor by antisense oligonucleotide suppresses intimal thickening in the rat carotid artery.¹⁶ This indicates that PDGF released from platelets and vascular cells may precipitate proliferative vascular disease, including venous graft disease. On the other hand, different growth properties of SMCs in response to growth factors may also determine vascular disease. It is well documented^{17 18 19 20} that different phenotypic SMCs are present in different blood vessels and even in the same vessels from different origins, have different growth activity, and may determine heterogeneity of biological properties of blood vessels to develop vascular disease.

Although the mechanisms of cell growth regulation are not completely understood, recent evidence^{21 22 23 24} indicates that the protein kinase cascade of c-Raf/MEK/MAPK, which is activated by tyrosine kinase receptors (PDGF receptors) or G-protein–coupled receptors (ie, PAR1, the thrombin receptor), is an important mechanism of transmitting extracellular growth signaling into the cell nucleus, thereby activating transcription factors (eg, c-Myc, c-Fos, and p62^TCF) and regulating downstream gene expression and cell cycle progression.

Cell cycle progression is regulated at several phase transition points by the activity of protein kinases, namely, CDKs that are bound and activated by the different cyclins D, E, and A.^{25 26} CDK activity is also regulated by association with several cell cycle inhibitor proteins known as CKIs.^{27 28} Two major classes of CKIs have been identified recently in mammalian cells: those specific for CDK4 or CDK6 (INK4s: p16^INK4a, p15^INK4b, p18^INK4c, and p19^INK4d) and those able to inhibit all the CDKs (p21^Cip1, p27^Kip1, and p57^Kip2).^{27 28 29 30 31 32 33 34} It seems that p27^Kip1 is most directly involved in cell cycle restriction control.^{33 35} In quiescent cells, the p27^Kip1 level is high and decreases as cells enter the cell cycle.^{36 37} Moreover, constitutive expression of p27^Kip1 in cultured cells causes cell cycle arrest in G1,^{33 35} demonstrating the critical role of p27^Kip1 in cell cycle progression.

In the present study, we demonstrated the different growth properties of SMCs from human IMA and SV. The different regulation of MAPK and cell cycle regulatory factors, namely, p27^Kip1 and p21^Cip1, in response to growth factors such as PDGF has also been analyzed.

	Methods

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Chemicals and Materials
BSA (7.5%), monoclonal antibody against -smooth muscle actin, and all chemicals for Northern and Western blots as well as protein kinase assay were from Sigma Chemical Co; recombinant PDGF-BB and all tissue culture materials were from Gibco; ³H-methyl-thymidine and (r-³²P)ATP (6000 Ci/mmol) were from Amersham; the rabbit anti-PDGF ß-receptor antibody was a kind gift from Dr M. Pech (Hoffman La-Roche, Basel, Switzerland); trichloroacetic acid was from Fluka; and anti-p42^mapk antibodies (C14), anti-p27^Kip1 (C19), and p21^Cip1187 were from Santa Cruz Biotechnol Inc. The PDGF -receptor cDNA probe of 618 base pairs was a gift from Dr M. Roth (Basel, Switzerland), and a human ß-actin cDNA probe (catalog No. 4230) and PDGF ß-receptor cDNA probe (catalog No. 59735) were purchased from the American Type Culture Collections.

Cell Isolation and Cultivation
SVs from 16 patients (age, 62±2 years; 10 men and 6 women) and IMAs from 11 patients (age, 61±3 years; 9 men and 2 women) were used for cell culture or explant culture with 20% FCS DMEM after removal of endothelium and adventitia under a dissection microscope. The cells were characterized as previously described.^{38 39} In addition, SVs from 8 patients (age, 58±5 years; 7 men and 1 woman) and IMAs from 12 patients (age, 54±3 years; all men) were collected for PDGF receptor gene expression (see below). The medium was changed every 2 to 3 days. All experiments were performed between passages 5 and 7.

Measurement of Cell Growth
Cell Outgrowth From Explants
To avoid individual variations, outgrowth of cells from explants was analyzed in IMAs and SVs from the same patients (n=5; four men and one woman; age, 61±8 years), and the explant culture was performed as described above. At 20 days after culture, total explants and explants with cell outgrowth were counted. Cell outgrowth rate was calculated as follows: Cell Outgrowth Rate=N(+)/N(t)x100%, where N(+) is the number of explants with cell outgrowth (regardless of cell number) and N(t) is the total number of explants seeded.

DNA Synthesis and Cell Division
Cultured cells were harvested and used for further studies and exposed to PDGF-BB or FCS. Cells were seeded on 12-well plates at a density of 2x10⁴/well, and DNA synthesis was measured by ³H-thymidine (0.5 µCi/mL; 70 to 85 Ci/mol) incorporation after 24 hours.^{38 39} In other experiments, quiescent SMCs were stimulated by PDGF-BB (10 ng/mL) or FCS (10%) over an 8-day period; cell number was counted every 2 days (Coulter counter).

PDGF Receptor Analysis
Northern Blot
SVs (n=8 from 7 men and 1 woman; age, 58±5 years) and IMAs (n=12, all from men aged 54±3 years) were collected. After dissection, medium was frozen in liquid nitrogen and kept at -70°C. Total RNA was isolated from tissues with the use of TRIzol reagent (Gibco BRL). Twenty micrograms of RNA from each sample was separated by electrophoresis through 1.2% agarose/1.4 mol/L formaldehyde gel containing 0.35 µg/mL ethidium bromide. Northern blotting and hybridization were performed.⁴⁰ After high-stringency washing, blots were exposed to a roentgenogram (X-Omat, Eastman Kodak Co) for autoradiography. After autoradiography, blots were stripped by hot 0.1% SDS solution for 1 hour for hybridization with other cDNA probes.

Immunoprecipitation and Western Blot
PDGF ß-receptor autophosphorylation was studied. Quiescent SMCs were stimulated with PDGF-BB (10 ng/mL) at different times. After stimulation, cells were washed with ice-cold PBS and harvested with cold extraction buffer (120 mmol/L sodium chloride, 50 mmol/L Tris, 20 mmol/L sodium fluoride, 1 mmol/L benzamidine, 1 mmol/L DTT, 1 mmol/L EDTA, 6 mmol/L EGTA, 15 mmol/L sodium pyrophosphate, 0.8 µg/mL leupeptin, 30 mmol/L p-nitrophenyl phosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 1% Nonidet P40). Cell debris was removed by centrifugation (12 000g for 10 minutes at 4°C). Cell lysates (100 µg) were subjected to agarose-conjugated monoclonal anti-phosphotyrosine antibody (4G10, Upstate Biotechnology Inc) and agitated overnight at 4°C. Immune complexes were washed three times with extraction buffer, eluted at 95°C for 10 minutes in Laemmli's SDS-PAGE sample buffer (50 mmol/L Tris-Cl, 100 mmol/L DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol), and subjected to 8% SDS-PAGE gels for electrophoresis. Proteins were then transferred onto Immobilon-P filter papers (Millipore AG) with a semidry transfer unit (Hoefer Scientific Instruments). Membranes were then blocked by 5% skim milk in PBS-Tween buffer (0.1% Tween 20; pH 7.5) for 1 hour and incubated with rabbit anti-PDGF ß-receptor antibodies (1:2000). The immunoreactive bands were detected by use of an enhanced chemiluminescence (ECL) system (Amersham).

MAPK Activation
Western Blot
MAPK (p42^mapk) activation (phosphorylation) was analyzed by mobility shift on Western blots as described previously.⁴¹ The cell lysates (20 µg) were treated with 5x Laemmli's SDS-PAGE sample buffer and subjected to 10% SDS-PAGE gel for electrophoresis. Western blot was performed as above except that anti-p42^mapk antibody (1:2000) was used as the primary antibody.

In-Gel MAPK Assay
Samples (20 µg) treated with 5x Laemmli's SDS-PAGE sample buffer were electrophoresed on 10% SDS-PAGE containing 0.5 mg/mL MBP (Sigma) as described previously.⁴² After electrophoresis, SDS was removed by washing the gel with two changes of 20% 2-propanol in 50 mmol/L Tris (pH 8.0) for 1 hour at room temperature and then with two changes of 50 mmol/L Tris (pH 8.0) containing 5 mmol/L 2-mercaptoethanol for 1 hour. The enzyme was denatured by incubating the gel with two changes of 6 mol/L guanidine-HCl for 1 hour and renatured with five changes of 50 mmol/L Tris (pH 8.0) containing 0.04% Tween 40 and 5 mmol/L 2-mercaptoethanol for 1 hour (5 times for 12 minutes each). The gel was then incubated with 40 mmol/L HEPES (pH 8.0) containing 2 mmol/L DTT and 10 mmol/L magnesium chloride. The kinase reaction was performed in conditions inhibitory to cyclic nucleotide-dependent protein kinases and Ca²⁺-dependent protein kinases by incubating the gel at 25°C for 1 hour with 40 mmol/L HEPES (pH 8.0) containing 0.5 mmol/L EGTA, 10 mmol/L magnesium chloride, 2 µmol/L protein kinase inhibitor peptide (rabbit sequence; Sigma), 40 µmol/L ATP, and 2.5 µCi/mL (r-³²P)ATP (6000 Ci/mmol). After incubation, the gel was washed with 5% trichloracetic acid solution containing 1% sodium pyrophosphate until the radioactivity of the solution became negligible. The gel was stained with rapid Coomassie blue solution (10% acetic acid, 0.006% Coomassie brilliant blue), destained with 10% acetic acid, dried routinely, and then subjected to autoradiography. MAPK activity was measured as total activity of p44^mapk and p42^mapk.

Regulation of p42^mapk Protein Level
To study the regulatory effects of PDGF-BB on MAPK protein levels, subconfluent quiescent SMCs (80%) from either IMAs or SVs were stimulated by PDGF-BB (10 ng/mL) for a longer period (from 3 to 24 hours). Cells were then harvested with cold extraction buffer as described above. Twenty micrograms of cell extracts from each sample were used for 12% SDS-PAGE as described above.

Regulation of CKIs
To study regulation of CKIs (p27^Kip1 and p21^Cip1) by PDGF-BB, the same blots as described above were stripped by stripping buffer (100 mmol/L 2-mercaptoethanol, 2% SDS, 62.5 mmol/L Tris-Cl, pH=6.7) at 50°C for 30 minutes, extensively washed by PBS-Tween buffer (0.1% Tween 20, twice for 10 minutes each time), and then incubated with antibodies against p27^Kip1 and p21^Cip1 for Western blotting.

Statistics
Data are presented as mean±SEM. ³H-thymidine incorporation and MAPK activity were expressed as percent increase above control. In all experiments, n equals the number of patients from which vessels were obtained. Student's t test for paired observations and ANOVA followed by Scheffé's test for repeated measurements were used. A two-tailed probability value .05 was considered significant.

	Results

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Serum Growth Factor
Proliferative properties of SMCs from tissue explants of IMAs and SVs of the same patients were analyzed in medium containing 20% FCS over a 20-day period. At this stage, fewer cells grew out of IMA than SV explants (Fig 1; n=5). Frequently, cells from IMA ceased to proliferate further. Cell outgrowth rate (regardless of cell number) was much higher in SV explants (37±5%) at 20 days than in IMA (Fig 2, left; 4±3%; P<.001; n=5). SMCs growing out of IMA and SV explants were harvested and stimulated with 10% FCS. Cells from SVs proliferate much faster (from 2x10⁴/well to 18±4x10⁴/well) than those from IMAs (from 2x10⁴/well to 9±4x10⁴/well; n=5 to 6; P<.05; Fig 2, right).

View larger version (70K): [in this window] [in a new window]   Figure 1. Outgrowth of SMCs from SV and IMA tissue explants. Explants of the same patients were cultured in DMEM containing 20% FCS over a 20-day period (n=5). Many fewer cells grew out of IMA than SV. Cell outgrowth rate (regardless of cell number) was much higher in SV than IMA.

View larger version (14K): [in this window] [in a new window]   Figure 2. Cell outgrowth rate from explants of SV and IMA. Left, After 20 days in culture medium containing 20% FCS, cell outgrowth from SV explants was greater than from IMA explants of the same patients (n=5; P<.001). Right, Proliferation of isolated IMA and SV SMCs in response to FCS (10%) over an 8-day period. Cell number increased much faster in SV than in IMA (n=5 to 6). *P<.05.

Platelet-Derived Growth Factor
Differences were even more obvious when the cells were stimulated with PDGF. In cultured SV SMCs, PDGF-BB (0.05 to 10 ng/mL) stimulated ³H-thymidine incorporation, with a maximal effect at 10 ng/mL (Fig 3, left; 1347±470% above control after 24 hours, P<.05, n=6). In contrast, IMA cells did not respond to PDGF-BB significantly (Fig 3, left; n=6). PDGF-BB (10 ng/mL) increased cell number in SVs (Fig 3, right; from 2x10⁴/to 5±1x10⁴/well, n=5, P<.05) over an 8-day period but had no significant effects in IMAs (Fig 3, right; n=3, P=NS).

View larger version (16K): [in this window] [in a new window]   Figure 3. Proliferative responses to PDGF of SMCs from IMA and SV. Left, After 24 hours of stimulation with PDGF-BB, 3H-thymidine incorporation increased in SV (; n=6) but not IMA (; n=6). Right, PDGF-BB (10 ng/mL for 8 days) also increased cell number of SV but not IMA SMCs (n=3 to 5).

PDGF Receptor Expression and Activation
Northern blot demonstrated abundant PDGF ß-receptor mRNA expression in media of IMAs (n=12), SVs (n=8), and aorta (n=1); -receptor mRNA was less expressed in all the blood vessels (Fig 4A). PDGF ß-receptor tyrosine autophosphorylation occurred rapidly after PDGF-BB (10 ng/mL) stimulation in cultured cells of both IMA and SV within 2 minutes (Fig 4B; n=3).

View larger version (33K): [in this window] [in a new window]   Figure 4. PDGF receptor expression and activation in SV and IMA SMCs. A, Northern blot showing abundant PDGF ß-receptor (R) mRNA expression in IMA (n=12), SV (n=8), and aorta (n=1) but less -receptor expression. B, PDGF ß-receptor tyrosine autophosphorylation by PDGF-BB occurred in SMCs from both IMA and SV (n=3). Anti-PY indicates anti-phospho-tyrosine.

Activation of MAPKs
In cultured SMCs of SV and IMA, PDGF-BB (10 ng/mL) stimulated p42^mapk phosphorylation, as demonstrated by a slower mobility of the phosphorylated form (pp42^mapk) on Western blot (Fig 5A; n=3). In-gel kinase assay with cell lysates, as measured by phosphorylation of MBP, demonstrated that in both SV and IMA cells, two isoforms of MAPKs (p42^mapk and p44^mapk) were activated, but p42^mapk was dominant (Fig 5B, n=3). Maximal stimulation occurred at 10 to 15 minutes. The maximal activity as well as kinetics of MAPKs (p42^mapk and p44^mapk) after PDGF-BB (10 ng/mL) stimulation was comparable in SV (maximum activity: 1413±100% above control) and IMA (Fig 5C; 1216±162%; n=3; P=NS versus SV).

View larger version (39K): [in this window] [in a new window]   Figure 5. MAPK activation in SV and IMA SMCs. A, Western blot demonstrating activation of p42mapk (phosphorylation) by PDGF-BB in a time-dependent manner in both IMA and SV, as shown by slower mobility of pp42mapk on the blot (n=3). B, Autoradiography of in-gel kinase assay showed similar activation of p42/44mapk (measured by phosphorylation of MBP) by PDGF-BB (10 ng/mL) in SV and IMA SMCs, with a dominance of p42mapk isoform (n=3). C, Kinetics of in-gel MAPK (p42/44mapk) activity by PDGF-BB (measured by scintillation counting of 32P in MBP in the gel slices obtained from Fig 5B as shown above) was similar in both SV and IMA cells (n=3).

Regulation of MAPK (p42^mapk) Protein Level by PDGF-BB
Interestingly, after prolonged exposure of SMCs to the growth factor PDGF-BB (10 ng/mL for 24 hours), the major isoform of MAPK (p42^mapk) protein level was enhanced in the SV in a time-dependent manner, whereas the p42^mapk protein level remained unchanged in SMCs of IMAs (Fig 6; n=3), although MAPK could be activated by PDGF-BB in a short time period in these cells, as demonstrated above.

View larger version (13K): [in this window] [in a new window]   Figure 6. Regulation of MAPK (p42mapk) protein level by PDGF in SMCs of SV and IMA. Western blot demonstrated that prolonged exposure of SMCs to PDGF-BB (10 ng/mL) time dependently increased p42mapk protein level in SV but not in IMA during 24 hours' stimulation (n=3).

Regulation of CKIs (p27^Kip1/p21^Cip1) by PDGF-BB
At the same time, the more downstream mechanism of cell growth, such as regulation of cell cycle inhibitors, namely, p27^Kip1 and p21^Cip1, was studied. In cultured SMCs from SVs, PDGF-BB (10 ng/mL) markedly downregulated the CKI p27^Kip1 in a time-dependent manner (from 3 to 24 hours) (Fig 7A, n=3). Downregulation of p27^Kip1 was much more pronounced in the SV than in the IMA (Fig 7A, n=3). In contrast to p27^Kip1, p21^Cip1 protein level was much lower than p27^Kip1 in both SV and IMA cells. Only a transient, weak induction of p21^Cip1 was observed by PDGF-BB (10 ng/mL) in both SV and IMA cells up to 24 hours (Fig 7B, n=3).

View larger version (22K): [in this window] [in a new window]   Figure 7. Regulation of CKI (p27Kip1 and p21Cip1) protein level by PDGF in SMCs of SV and IMA. A, Western blot demonstrated that PDGF-BB (10 ng/mL) markedly downregulated p27Kip1 protein level in SMCs of SV in a time-dependent manner, which was more pronounced than in IMA cells (n=3). B, In contrast, protein level of p21Cip1 was weakly and transiently induced by PDGF-BB under this condition in both SV and IMA (n=3).

	Discussion

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This study demonstrates that SMCs of human SV have much more pronounced growth activity in response to serum growth factor or PDGF than do those of IMA. This difference was obvious both when intact pieces of media or cultured cells were used. Interestingly, SMCs from both SV and IMA expressed functional PDGF receptors and similar MAPK activation (either kinetics or maximal activity) to PDGF in SV and IMA. However, MAPK protein level was increased readily by prolonged exposure of SMCs to PDGF in SV but not in IMA. Furthermore, in SMCs of SV, the CKI p27^Kip1 protein level was markedly downregulated by PDGF but was less so in IMA.

PDGF is composed of two disulfide-linked polypeptide chains, denoted A and B. Three dimeric forms of PDGF (AA, AB, and BB) occur and are released by different cells, in particular by activated platelets.^{43 44} The three isoforms of PDGF interact with two different PDGF receptor subunits, - and ß-receptors.^{43 44 45} PDGF-AA binds only -receptor, whereas BB activates all the receptors, which is why this isoform was used in the present study.^{45 46} The observation that SMCs of IMA but not those of SV are resistant to serum or PDGF-BB is most interesting. Importantly, this was apparent both in intact tissues as well as in cultured cells and hence is likely to also be relevant in vivo in patients. This difference is not due to a defect of PDGF receptor expression or function in IMA cells because mRNA as well as PDGF receptor protein was expressed in the artery, with a dominance of ß-receptors, and the receptor could be activated by exposure to PDGF, as demonstrated by the receptor tyrosine autophosphorylation, which is in agreement with our previous study with another growth factor, thrombin.⁴¹ A difference in spontaneous cell death between SMCs from IMA and SV seems unlikely because no differences in cell detachment were observed in the arterial and venous cell cultures. Possible differences in induced cell death (apoptosis) between SV and IMA cells in response to agents such as nitric oxide⁴⁷ need further investigation. It is also unlikely that SMCs from IMA have delayed entry into the S phase in response to the growth factor PDGF because no significant ³H-thymidine uptake was observed and no significant increase in cell number was shown even after 8 days of PDGF stimulation, as demonstrated in Fig 3. Confirmation of this would require cell cycle analysis by fluorescence-activated cell sorter study. It is obvious that IMA cells have intrinsic resistance to growth stimuli, because SV SMCs proliferate much faster than IMA cells in response to PDGF, serum, and thrombin, as demonstrated in this and previous studies.⁴¹ Although we did not address the question whether migration might also differ in SV and IMA SMCs, the results of the outgrowth experiments highly support such a notion (see Fig 1).

Different growth properties of SMCs can be characterized by phenotypes of the cells, as shown by many studies.^{17 18 19 20} In cultured SMCs of rat carotid arteries, cells isolated from intima have normal PDGF receptor expression and function but have very weak mitogenic activity compared with cells from the media.¹⁸ The different proliferative properties shown in the present study cannot be due to the possibility that SV cells are only from media and IMA cells only from intima because SVs often develop intimas even before they are used as grafts, whereas IMAs very rarely develop intimas.^{6 7 41} Different phenotypes of SMCs, as proposed by several groups,^{17 18 19 20} may explain the different proliferative properties, but the underlying biochemical or molecular mechanisms were not analyzed in those studies. Therefore, the question is raised as to whether the receptor-mediated signal transduction mechanism(s), such as the c-Raf/MEK/MAPK pathway, might be different in the two vessels.

The two best-characterized isoforms of MAPKs p42^mapk (ERK-2) and p44^mapk (ERK-1) are activated through tyrosine kinase receptors or G-protein–coupled receptors such as the thrombin receptor.^{21 22 23 24 41 48} MAPKs are activated via phosphorylation on threonine and tyrosine residues by MEK, the dual specific protein kinase. MEK itself is activated by upstream kinase Raf or MEKK. Activation of p42^mapk and p44^mapk can be translocated into the nucleus and phosphorylate an array of transcription factors involved in regulation of cell growth.²¹ Again, the c-Raf/MEK/MAPK pathway activated by PDGF is very rapid and has similar kinetics and maximal activity in both SV and IMA cells, implying that MAPK activation is intact in IMA cells, which is in agreement with our previous observation with other growth factors such as thrombin.⁴¹ The dissociation of MAPK activity from cell growth has been reported in many studies. For instance, in human dermal fibroblasts, PDGF-AA and -BB have similar kinetics and activity of MAPK activation, but only PDGF-BB stimulated cell growth.⁴⁹ It is possible that growth factor–mediated signal transduction cascades other than Raf/MEK/MAPK are needed together with MAPK to stimulate cell growth. PLD activated by mitogens has recently been suggested to be one of these signaling transduction pathways.^{50 51} Indeed, blockade of either PLD or MAPK could inhibit cell growth.⁵⁰ Furthermore, agonists such as phorbol ester, which only activates the MAPK pathway and not PLD, have been shown to be insufficient to stimulate cell growth.⁵⁰ Whether such signal transduction pathways are defective or different in IMA than in SV cells requires further investigation.

A very interesting observation in the present study is the fact that an increase in MAPK protein level but not MAPK activity stimulated by PDGF is associated with cell growth. Indeed, prolonged exposure of SMCs to PDGF increased the p42^mapk protein level in SV but not in IMA in a time-dependent manner, although PDGF activates MAPK in IMA cells comparably to SV. To the best of our knowledge, this is the first study demonstrating a correlation of an increase in MAPK protein level, but not activity, with cell proliferation. This may explain the dissociation of MAPK activity from cell growth shown in many studies.^{52 53 54} The exact mechanisms or pathways leading to an increase in MAPK protein level by PDGF in SV but not IMA require further investigation.

Alternatively, a growth inhibitory mechanism that blocks the cell cycle progression in IMA may also be dominant in spite of activation of early signal transduction pathways. Furthermore, we have shown in the present study that CKIs, in particular p27^Kip1, an important inhibitor of cell cycle transition from G1 into the S phase,^{33 35} were markedly downregulated in SV SMCs by PDGF-BB; such downregulation was more pronounced in SV than in IMA cells. This must also contribute to the resistance of IMA cells to growth stimuli. However, another CKI, p21^Cip1, was much less expressed, and a transient, weak induction of p21^Cip1 during PDGF stimulation in both SV and IMA cells was observed. The meaning of this phenomenon is not yet clear. The above results imply that p27^Kip1 may be crucial in the regulation of cell cycle progression in human bypass vessels. Indeed, p27^Kip1 accumulates in growth-arrested cells either by mitogen-deprivation or antiproliferative agents^{36 37 55 56} and is decreased by growth factor stimulation in various cell lines.^{36 37 55 56} Constitutive expression of p27^Kip1 in cultured cells causes cell cycle arrest in G1^{33 35} , and inhibition of p27^Kip1 by antisense caused cells to proliferate independently on mitogens in Balb/3T3 fibroblasts.⁵⁷ Those results strongly support the critical role of p27^Kip1 in control of cell cycle progression. Downregulation of p27^Kip1 could be due to the inhibition of protein synthesis or an increase in protein degradation or regulation at the transcriptional level.^{36 37 56 58 59} Further clarification of the molecular mechanisms of p27^Kip1 downregulation by PDGF in SV and determination of the reason this downregulation was less pronounced in IMA cells should provide us with important clues for understanding the pathogenesis of proliferative vascular disease such as venous bypass graft disease and restenosis after percutaneous transluminal coronary angioplasty.

The findings of the present study may explain venous bypass graft disease. Platelets have been implicated in this condition, both as mediators of thrombotic occlusions and of chronic proliferative changes after implantation.^{4 14} The importance of platelet activation and adhesion is reinforced by clinical studies demonstrating that platelet inhibitors improve (although they do not normalize) graft function and patient survival.¹⁴ A further understanding of why SMCs of IMA are resistant to growth stimuli may have great biological and clinical impact. This could provide the basis for new approaches to effectively activate such inhibitory mechanisms in venous grafts and to pharmacologically prevent coronary bypass graft disease in patients.

	Selected Abbreviations and Acronyms

 

CDK = cyclin-dependent kinase CKI = cyclin-dependent kinase inhibitor ERK = extracellular signal-regulated kinase IMA = internal mammary artery MAPK = mitogen-activated protein kinase MBP = myelin basic protein MEK = MAP kinase kinase MEKK = MEK kinase PDGF = platelet-derived growth factor PLD = phospholipase D SMC = smooth muscle cell SV = saphenous vein

	Acknowledgments

 
This study was supported by the Swiss National Research Foundation (32–32541.91/2), the Swiss Heart Foundation, a grant-in-aid from Mobiliar Insurance, and a grant from Sulzer Biomedical Technology, Winterthur, Switzerland.

Received June 2, 1997; revision received September 17, 1997; accepted September 25, 1997.

	References

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