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Abstract
Abstract The effect of the three platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on migration was investigated in cultured human saphenous vein smooth muscle cells. The modified Boyden chamber technique yielded efficacies BB>>AB, AA=0. However, the BB concentration-response relationship displayed a pronounced peak, occurring between 1 and 10 ng/mL, with no response above this range. Checkerboard analysis showed that the promotion of migration at low concentrations was chemotactic in nature but that the downturn was independent of gradient. Furthermore, at high concentrations BB was able to prevent chemotaxis induced by fetal calf serum and epidermal growth factor (EGF). Experiments using low concentrations of BB in combination with high concentrations of AA to saturate PDGF α-receptors in the presence and absence of a neutralizing antibody to α-receptors revealed that α-receptor activation induced partial inhibition of chemotaxis but this did not account for the inhibition of migration by high concentrations of BB. Despite possessing no significant chemotactic action itself, high concentrations of the AB isoform completely inhibited BB induced chemotaxis. Taken together these results suggest that the chemotactic signal induced by PDGF is dominated by PDGF β-receptors and switches from positive at low concentrations to negative at higher concentrations. Stimulation of DNA synthesis by the three isoforms (as measured by [3H] thymidine incorporation) yielded saturable responses for the AB and BB isoforms, with similar efficacy and weak or no response for the AA isoform. Concentration-dependent patterns of tyrosine phosphorylation of certain proteins mirrored the form of the chemotactic response and suggest one possible underlying regulatory mechanism to account for the disparity between PDGF-induced chemotaxis and DNA synthesis.
- Received August 7, 1996.
- Accepted April 3, 1997.
Platelet-derived growth factor (PDGF) acts as a mitogen and chemoattractant for vascular smooth muscle cells (SMCs).1 The active growth factor exists as a disulfide-linked dimer composed of two types of peptide chains termed A and B, which give rise to three isoforms denoted PDGF-AA, -AB, and -BB. In addition, there are two types of PDGF receptors termed PDGF-Rα and PDGF-Rβ, which dimerize and become active on binding the growth factor to initiate an intracellular signaling cascade via tyrosine phosphorylation.2 The A chain can bind only to PDGF-Rα but the B chain can bind both receptor subtypes. This means that PDGF-AA activates only αα-dimers, PDGF-AB activates αα- and αβ- dimers (See Reference 33 ), and PDGF-BB activates αα-, αβ-, and ββ-dimers. Thus the three isoforms of PDGF can elicit a diverse range of signals in SMCs.4 5
Although PDGF is one among many molecules involved in normal and pathological processes such as wound healing, angiogenesis, atherosclerosis, and restenosis, there is evidence that it does have particularly important roles to play, especially with regard to SMC migration. Studies using animal injury models showed that infusion of PDGF-BB promoted intimal thickening and promoted SMC migration from the media to intima.6 Furthermore, an antibody against PDGF reduced myointimal hyperplasia in response to injury.7 Similarly, PDGF-BB production has been associated with intimally directed SMC migration and proliferation in organ cultured human saphenous vein.8 The actions of PDGF on saphenous vein cells is of particular interest because it is the most widely used conduit for coronary artery and infrainguinal bypasses, and myointimal hyperplasia as a result of SMC migration and proliferation accounts for ≈80% of failures of saphenous vein grafts.9
Knowledge of the actions of PDGF on saphenous vein–derived SMC proliferation and migration would be useful in interpreting the events observed in these more complex in vivo situations. In this study we have therefore investigated the effect of PDGF-AA, -AB, and -BB on SMC derived from human saphenous vein. These studies show marked differences between the chemotactic and early proliferative responses to a given isoform and major differences between the isoforms themselves. We further show that the chemotactic signal switches from a positive signal at low concentrations of PDGF-BB to a negative signal at higher concentrations. The dominant signals at all concentrations and to all isoforms appear to be mediated by β-receptors, indicating a dual role for these receptors in saphenous vein SMC chemotaxis. Regulation of tyrosine phosphorylation of certain key signaling molecules may underlie some of the observed behavior.
Methods
Cell Culture
Human vascular smooth muscle was obtained from saphenous vein from patients undergoing cardiovascular surgery. Tissues were surplus to requirements, and their use conformed to the guidelines of our local Ethics Committee. SMCs were cultured using an explant technique described previously10 in a culture medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal calf serum (FCS, 15% vol/vol), l-alanyl-l-glutamine (4 mmol/L), penicillin (100 U/mL), streptomycin (100 μg/mL), gentamicin (25 μg/mL), and buffered with HEPES (25 mmol/L). Cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C. When confluence was reached, the cells were subcultured under the same conditions described above and were used for experiments when they reached confluence at passage 2. The identity of the cells as SMCs was regularly confirmed by immunocytochemistry in which positive staining for α-actin (a marker for smooth muscle11 ) was demonstrated. All repeat experiments were carried out on cells derived from different individuals.
Migration Assays
Confluent monolayers of SMCs were washed twice in PBS and briefly exposed to a trypsin solution. Neutralization of the trypsin was achieved by addition of 8 mL of culture medium. The resultant cell suspension was then centrifuged (1000 rpm, 10 minutes) and resuspended in serum-free medium (SFM) supplemented with bovine serum albumin (1 mg/mL) at a density of 2.25×105 cells/mL.
Migration assays were carried out in blind well chemotaxis chambers. The upper and lower compartments were separated by 13-mm-diameter, gelatin-coated polycarbonate filters with 8-μm pores; 0.4 mL of cell suspension was added to the upper compartment and 0.3 mL of the relevant control or chemoattractant (in SFM) was added to the lower compartment unless otherwise stated. Separate control experiments were performed for each cell line in each experiment. Migration was allowed to proceed for 5 hours in a humidified atmosphere of 5% CO2 at 37°C. Cells were then fixed in absolute alcohol and stained for 15 minutes with toluidine blue (65 mmol/L). Cells on the upper side of the filters were removed, leaving the cells on the underside of the filters for counting. Cells in four fields of view (×200 magnification) on duplicate filters were counted.
Measurement of DNA Synthesis
Stimulation of DNA synthesis was measured by using the incorporation of [methyl 3H]-thymidine.12 SMCs were seeded at a density of 9×104 cells/mL, 200 μL/well in 96 well plates and allowed to attach overnight. The 15% FCS containing medium was removed and the cells were washed twice with phosphate-buffered saline (PBS, without calcium and magnesium). The cells were maintained in DMEM supplemented with bovine serum albumin (1 mg/mL), insulin (1 μmol/L), transferrin (5 mg/mL), sodium selenite (0.1 μmol/L), and ascorbic acid (0.5 mmol/L) for 72 hours to growth arrest the cells in the G0 phase of the cell cycle. Growth stimulation was then commenced with medium containing the different PDGF isoforms. After 24 hours of incubation, [methyl 3H]-thymidine was added (1 μCi/well, 40 μCi/mL) for a further 6 hours (determined from time course experiments13 ). The experiment was terminated by washing the cells twice with trichloroacetic acid (10%, wt/vol) and then solubilizing the cells with 200 μL NaOH (1 mol/L) at 37°C for 12 hours. To precipitate solubilized DNA, the pH was neutralized with 50 μL hydrochloric acid (4 mol/L). Precipitated DNA was harvested onto glass fiber filter mats and the radioactivity counted using a Matrix 96 direct beta counter (Canberra Packard).
Measurement of Tyrosine Phosphorylation
Cells were plated onto Petri dishes (1×106 per dish) in FCS and allowed to attach overnight, then growth arrested in SFM for 3 to 4 days. Cells were then stimulated with PDGF-BB (0.1 to 100 ng/mL) for 60 minutes, washed twice in ice-cold PBS, and lysed with 1 mL lysis buffer Tris-HCl (50 mmol/L, pH 7.4), NaCl (150 mmol/L), EGTA (100 nmol/L), NP-40 (1%), and Na deoxycholate (0.25%) containing PMSF, NaF, Na3VO4 (all 1 mmol/L) and aprotinin, pepstatin, and leupeptin (1 μg mL). Samples containing 15 μg cell protein were then run on a 10% polyacrylamide gel, transferred to nitrocellulose, and probed with an antiphosphotyrosine antibody (PY20).
Materials
Recombinant human PDGF-AB was obtained from Boehringer Mannheim. Recombinant human PDGF-AA, -BB, and all cell culture materials were from Gibco Life Technologies. Anti-PDGF-Rα antibody was purchased from Genzyme. PY20 was obtained from Affiniti Research Products. Polycarbonate filters were from Poretics. All other chemicals were purchased from Sigma.
Statistics and Data Analysis
Data are presented as mean±SEM, and results were analyzed using Friedman’s nonparametric test for repeated-measures ANOVA followed by Conover’s multiple-range test for individual differences if the results of ANOVA were significant. A value of P<.05 was considered significant. EC50 values for DNA synthesis were calculated by nonlinear regression on Excel (Microsoft) using a macro (written by A.D. Hughes).
Results
Chemotaxis
The three PDGF isoforms had markedly distinct effects on human SMC migration. The concentration-response relationships to each isoform are shown in Fig 1⇓. PDGF-AA failed to elicit migration over the range 0.1 to 100 ng/mL in four cell lines and surprisingly, the AB isoform produced only a small increase that did not reach overall statistical significance. In these experiments the response to 15% FCS and SFM were used as positive and negative controls, respectively. Peak cell counts were SFM, 29±11; PDGF-AA, 20±6; PDGF-AB, 68±37; PDGF-BB, 225±65; and FCS, 213±24 (mean±SEM, n=4 to 11). All the cell lines used in this study responded to FCS, indicating that the lack of migration in response to PDGF-AA and PDGF-AB was not due to a lack of viability of the cells.
Effect of platelet-derived growth factor (PDGF)-AA, -AB, and BB on migration in cultured human saphenous vein smooth muscle cells determined using a modified Boyden chamber assay. Data are mean±SEM for four separate cell lines and are expressed in percentage terms relative to the response to 2 ng/mL PDGF-BB. *P<.05 by Friedman and Conover.
In contrast, PDGF-BB was able to potently stimulate migration to a peak level comparable to FCS. However, the response to this isoform consisted of a biphasic curve with a peak that reached a maximum value between 1 and 10 ng/mL (Fig 1⇑). The responses at 0.1 and 1 ng/mL were 29±11% and 76±17% (mean±SEM) of the peak response for a given cell line, which means that the concentration of PDGF-BB required to stimulate half maximal migration was in the range 0.1 to 1 ng/mL. Concentrations of PDGF-BB >10 ng/mL produced responses comparable to or less than that to SFM, indicating that there was little or no migration occurring across 90% of the experimentally tested range. For cells incubated in 100 ng/mL PDGF-BB, trypan blue exclusion levels at t=0 and 5 hours were not significantly different (data not shown), which was used as a verification of cell viability. In addition, thymidine incorporation at this concentration was not impaired (see below).
To test the nature of the induced migration to PDGF-BB and to investigate the downturn, an extended checkerboard analysis was performed for both low and high concentrations of this isoform (Fig 2⇓). Again, BB (2 ng/mL) strongly induced migration relative to SFM but only when the cells were exposed to an appropriately directed gradient. When there was no gradient or a reverse gradient, present migration was abolished (Fig 2⇓ [III and IV]). Thus the migration was chemotactic in nature. However, at the high BB concentration (100 ng/mL), all combinations failed to elicit migration (Fig 2⇓ [V, VI, and VII]). Migration to high BB was in fact reduced to levels significantly below that of unstimulated cells.
Checkerboard analysis of the response of human saphenous vein smooth muscle cells to platelet-derived growth factor (PDGF)-BB. SFM denotes serum-free medium; numbers denote the concentrations of PDGF-BB in ng/mL. Schematic representations of the modified Boyden chambers indicate to which compartment the growth factors were added. Data are mean±SEM for four separate cell lines expressed as the percentage of the response to SFM. *P<.05 by Friedman and Conover.
The next step therefore was to establish whether the high concentrations of BB were simply nonstimulatory or actively inhibitory for chemotaxis. To test this, cells were stimulated to migrate by FCS (15%) with or without added PDGF-BB (100 ng/mL). Fig 3⇓ shows that this concentration of PDGF-BB was able to severely inhibit FCS-induced migration. A similar experiment was performed (Fig 4a⇓) in which cells were stimulated by epidermal growth factor (EGF, 0.1 ng/mL), another member of the family of receptor tyrosine kinase–linked growth factors that potently stimulates migration in these cells (mean cell count, 324±134, n=8). In this experiment, PDGF-BB (100 ng/mL) was added to the upper and lower chambers to remove the effect of any PDGF gradient. The presence of a high concentration of BB also abolished the stimulatory effect of EGF. A similar effect was observed using the AB isoform (Fig 4b⇓).
Effect of platelet-derived growth factor (PDGF)-BB on fetal calf serum (15% vol/vol) (FCS)-induced chemotaxis in human saphenous vein smooth muscle cells. SFM and BB100 denote serum-free medium and PDGF-BB (100 ng/mL), respectively. Schematic representations of the modified Boyden chambers indicate to which compartment the chemotactic agents were added. Data are mean±SEM for four separate cell lines expressed as the percentage of the response to FCS alone. *P<.05 by Friedman and Conover.
a, Effect of platelet-derived growth factor (PDGF)-BB on epidermal growth factor (EGF)-induced chemotaxis in human saphenous vein smooth muscle cells. SFM denotes serum-free medium; numbers denote concentrations of the growth factors (ng/mL). Schematic representations of the modified Boyden chambers indicate to which compartment the chemotactic agents were added. Data are mean±SEM for four separate cell lines expressed as the percentage of the response to EGF alone. *P<.05 by Friedman and Conover. b, Effect of PDGF-AB on EGF-induced chemotaxis. *P<.05 by Friedman and Conover.
Having established the presence of a negative influence on chemotaxis at high concentrations of PDGF-BB and -AB, the next step was to determine which receptor subtype was responsible. The BB isoform binds to both α- and β-receptors.2 There is evidence that activation of α-receptors inhibits migration in some SMC types.14 15 It was therefore appropriate to investigate whether activation of PDGF-Rα could account for the downturn in the PDGF-BB concentration-response curve. In these experiments, cells stimulated by PDGF-BB (2 ng/mL) were also exposed to PDGF-AA (1 and 100 ng/mL) in both the upper and lower compartments, following the protocol of Koyama et al15 to separate chemotactic from chemokinetic effects. The results are summarized in Fig 5⇓. PDGF-AA at a concentration of 1 ng/mL had no effect on PDGF-BB–induced chemotaxis. At a concentration of 100 ng/mL PDGF-AA, the mean response to PDGF-BB was reduced by 35±17% (n=4) and 26±14% (n=4) when the AA isoform was in the lower and upper compartment, respectively. PDGF-BB alone, however, at a concentration of 100 ng/mL, produced a response significantly lower than unstimulated cells (n=4). These results suggest that active PDGF-Rα may display some inhibitory properties in these cells, but they do not account for the abolition of the chemotactic response at higher concentrations of PDGF-BB.
Effect of platelet-derived growth factor (PDGF)-AA on PDGF-BB–induced chemotaxis in human saphenous vein smooth muscle cells. SFM, AA, and BB denote serum-free medium, PDGF-AA, and PDGF-BB, respectively; numbers denote concentrations (ng/mL). Schematic representations of the modified Boyden chambers indicate to which compartment the growth factors were added. Data are mean±SEM for four separate cell lines expressed as the percentage of the response to PDGF-BB (2 ng/mL) alone. *P<.05 by Friedman and Conover.
The data above receive further support from studies conducted with an anti–PDGF-Rα neutralizing antibody (which is specific for primate α-receptors and does not recognize β-receptors).16 Fig 6a⇓ shows that the antibody had no effect on chemotaxis induced by PDGF-BB at 2 ng/mL (n=4), indicating that α-receptors played no role in the chemotactic response to low concentrations of PDGF-BB. However, application of the antibody did recover the inhibition of chemotaxis by PDGF-AA (100 ng/mL). This result specifically confirmed the data in Fig 5⇑ in establishing a minor inhibitory role for PDGF-Rα in PDGF-BB–induced chemotaxis via α-receptor activation at high concentrations of PDGF-BB. Fig 6b⇓ shows the effect of the same antibody on the high BB concentration. Clearly, the antibody had no effect on the inhibition of chemotaxis at high concentrations of BB (n=4). This indicates that activation of PDGF-Rα by high concentrations of BB was not necessary for the observed reduction in chemotaxis and suggests that the downturn could be wholly accounted for by activation of PDGF-Rβ.
a, Effect of a neutralizing anti–platelet derived growth factor (PDGF) α-receptor antibody (αAb) on the interaction between PDGF-AA–and PDGF-BB–induced chemotactic signals. SFM denotes serum-free medium; numbers denote the concentrations of the isoforms used (ng/mL). Schematic representations of the modified Boyden chambers indicate to which compartment the growth factors and antibodies were added. Data are mean±SEM for four separate cell lines expressed as the percentage of the response to PDGF-BB (2 ng/mL) alone. *P<.05 by Friedman and Conover. b, Effect of αAb on PDGF-BB–induced chemotactic signals at high levels of PDGF β-receptor activation. Data are mean±SEM for four separate cell lines expressed as the percentage of the response to PDGF-BB (2 ng/mL) alone.
A final piece of supportive evidence for the above proposal could be demonstrated by testing the effect of the AB isoform on the ability of the BB isoform to induce chemotaxis. PDGF-AB had no significant chemotactic stimulatory effect on these cells (Fig 1⇑). Cells stimulated by PDGF-BB (2 ng/mL) were incubated with or without PDGF-AB (100 ng/mL). As can be seen from Fig 7⇓, PDGF-AB completely inhibited PDGF-BB–induced chemotaxis. Since PDGF-AB is thought to cause activation of αα- and αβ-dimers, this inhibitory effect of PDGF-AB on PDGF-BB–induced chemotaxis was presumably due to activation of αβ-dimers, since activation of αα-dimers alone by PDGF-AA (Figs 5⇑ and 6⇑) produced only a modest inhibition.
Effect of platelet-derived growth factor (PDGF)-AB on PDGF-BB–induced chemotaxis in human saphenous vein smooth muscle cells. SFM, AB, and BB denote serum-free medium, PDGF-AB, and PDGF-BB, respectively; numbers denote the concentrations used (ng/mL). Schematic representations of the modified Boyden chambers indicate to which compartment the growth factors were added. Data are mean±SEM for four separate cell lines expressed as the percentage of the mean response to PDGF-BB (2 ng/mL) alone. *P<.05 by Friedman and Conover.
DNA Synthesis
To complement the migration data, the effect of the PDGF isoforms on DNA synthesis was determined by [3H]-thymidine incorporation. The results agree well with our previously reported results on the cellular uptake of thymidine in cultured saphenous vein cells exposed to PDGF.17 Representative incorporation data (Fig 8⇓) show that both the AB and BB isoforms of PDGF were able to concentration-dependently stimulate DNA synthesis in a saturable manner, but there was no significant response to the AA isoform. The AB and BB isoforms were approximately equally efficacious and also had similar EC50 values of 7±1 and 11±1 ng/mL, respectively.
Effect of platelet-derived growth factor (PDGF)-AA, AB, and BB on DNA synthesis in cultured human saphenous vein smooth muscle cells determined by [3H]-thymidine incorporation. Graph shows typical concentration-response relationships for individual cell lines expressed as a percentage of maximal [3H]-thymidine incorporation in response to PDGF-BB and are representative of experiments carried out on six to eight separate cell lines.
Tyrosine Phosphorylation
To explore possible mechanisms to account for the downturn in the chemotactic response, the pattern of tyrosine phosphorylation elicited by a range of concentrations of PDGF-BB was measured using the Western blotting technique. Fig 9⇓ shows that PDGF-BB induced tyrosine phosphorylation of a number of cellular proteins at concentrations as low as 0.1 ng/mL. However, certain bands (notably at approximately 75 to 85 kD and 100 to 120 kD) become more intense at intermediate concentrations (1 to 10 ng/mL) but then markedly decline at the concentrations of PDGF-BB that become inhibitory with respect to chemotaxis but remain maximally stimulatory with regard to DNA synthesis. Control of tyrosine phosphorylation and dephosphorylation represents a possible route of further investigation into negative chemotactic signals evoked by PDGF-Rβ.
Western blot of whole cell lysates indicating the level of protein tyrosine phosphorylation in response to increasing concentrations of platelet-derived growth factor (PDGF)-BB in human saphenous vein smooth muscle cells. Details are given in “Methods.” The uppermost arrow indicates a band of 180 kD, which becomes more intense as the concentration of PDGF-BB rises (most likely the PDGF β-receptor). The lower two arrows indicate bands that are intense in the range in which the cells respond chemotactically but diminish in the region in which the chemotactic signal becomes negative (but where the DNA synthesis signal remains maximally positive).
Discussion
This study reports the complexity and heterogeneity of control of chemotaxis and DNA synthesis in human saphenous vein SMC by PDGF isoforms and extends the previously proposed models. The main focus is on the regulation of migration by PDGF-BB. This isoform produced a biphasic response with migration occurring only in the 1 to 10 ng/mL range. A peak in the migratory response has been observed in a number of cell types including neutrophils,18 19 monocytes,20 21 and fibroblasts22 23 and in some SMC types but not others.14 24 The downturn in human saphenous vein SMC was complete (the response returned to unstimulated levels or below) and particularly sharp. Fig 2⇑ confirms the chemotactic nature of the response and is useful in addressing a few technical issues that may be raised in interpreting the results from a double well chamber. Fig 2⇑ (I-IV) shows that a gradient was required to induce migration in response to PDGF-BB. Thus it could be argued that one reason for the observed downturn in migration at high concentrations of BB is that partial dissipation of the gradient over the time course of the experiments led to a high background level of BB in both chambers. This would saturate the PDGF receptors, thereby effectively masking the presence of the gradient and leading to a loss of polarity.
However, subsequent data show that the downturn is not simply a passive response to increasing concentration, that is, the downturn is not an artifact of the experimental protocol. Fig 3⇑, 4⇑, and 7⇑ show that PDGF-BB and PDGF-AB at high concentrations were able to negate the migration induced in response to other stimulants, which indicates that a net negative signal was generated under these conditions. Such a result cannot be accounted for by a simple receptor saturation model such as that described above because that model contains no active antagonistic component. A model has been proposed in which PDGF β-receptors positively regulate and α-receptors negatively regulate SMC chemotaxis. Therefore it was determined whether activation of α-receptors could explain the downturn in the BB concentration-response curve. Figs 5⇑ and 6⇑ together demonstrate that activation of α-receptors had a small inhibitory effect that could be overcome by their specific blockade using a neutralizing antibody. However the effect of α-receptors could not explain the complete abolition of chemotaxis by 100 ng/mL BB alone. Furthermore, blockade of α-receptors with 100 ng/mL BB produced no recovery whatsoever, indicating that the negative signal originating from the β-receptors was sufficient to completely account for the abolition of chemotaxis. In further support of this argument it was observed that the AB isoform, which could not itself induce significant migration, completely inhibited BB-induced chemotaxis. The difference between application of AA and AB is the activation of β-receptors by the AB isoform. Thus it appears that the biphasic chemotactic response in these cells is predominantly under the control of PDGF-Rβ alone as opposed to a model in which PDGF-Rα and PDGF-Rβ act in an antagonistic manner to produce the overall observed effect. These findings also imply that ββ-dimer activation is necessary for significant chemotaxis to occur, but the strong inhibitory effect can arise from either ββ or αβ combinations.
The degree of tyrosine phosphorylation is often used as a marker for the level of activity of many signaling molecules, including the PDGF receptors. It has recently been observed in Swiss 3T3 cells that stimulation with high levels of PDGF results in a reduction in the level of tyrosine phosphorylation of certain molecules, including focal adhesion kinase (FAK, Reference 2525 ), a protein thought to be intimately involved in the promotion of cell migration. High concentrations of PDGF also produced a downturn in the migratory response.24 However, in rabbit aortic SMC that did not exhibit a downturn, the level of FAK tyrosine phosphorylation was sustained at high PDGF concentrations.24 The cells used in this study displayed peak PDGF-BB–induced tyrosine phosphorylation of several bands at concentrations of growth factor that parallel those that produce peak chemotactic responses and reduced tyrosine phosphorylation at high concentrations of BB (Fig 9⇑). Most prominent among these were bands at ≈80 to 90 kD and 100 to 120 kD. Other bands (including a band at ≈180 kD, presumably the PDGF β-receptor) displayed consistently elevated levels of tyrosine phosphorylation at high concentrations of PDGF-BB, similar to the sustained DNA synthesis response. These patterns of phosphorylation could provide clues as to which tyrosine-phosphorylated proteins are crucial in the control of SMC migration. Work is currently under way aimed at identifying these molecules and the mechanisms underlying the reduction in tyrosine phosphorylation.
The DNA synthesis profiles indicated that the AB and BB isoforms were very effective in terms of eliciting DNA synthesis, but the AA isoform was nonstimulatory. These data suggest that α-receptors play little or no role in PDGF-induced entry into the S-phase and therefore the early proliferative signals are also derived predominantly from β-receptors. The DNA synthesis concentration-response curves were saturable and required a 10-fold higher concentration of growth factor to achieve half-maximal stimulation than did the chemotactic response. The concentration range in which the downturn in chemotaxis began coincided with the range in which DNA synthesis was induced. This indicates a possible means by which these cells could utilize a gradient of PDGF to coordinate movement and subsequent proliferation, as illustrated in Fig 10⇓. Such a scheme could effectively encode a local sense of position that would be important in the complex and highly variable situations encountered in the wound healing process.
a, Superimposed plots of the typical chemotaxis and [3H]-thymidine incorporation dose-response relationships in response to platelet-derived growth factor (PDGF)-BB in human saphenous vein smooth muscle cells. Data are expressed as the percentage of the maximum response for both DNA synthesis and chemotaxis. b, Schematic drawing of a model system aligned with the chemotaxis and DNA synthesis profile described in a. A source of PDGF-BB (located at the vertical bar) gives rise to a concentration gradient of PDGF-BB (dotted line) declining with increasing distance away from the source. A smooth muscle cell (position I) is exposed to a small concentration of PDGF-BB sufficient to induce chemotaxis toward the source (depicted by the arrow). As the cell ascends the concentration gradient, the signal for chemotaxis is switched off (refer to a) but the signal to proliferate is switched on. The result is the location of proliferating cells at position II.
In summary, control of migration and DNA synthesis by PDGF in human saphenous vein SMC is dominated by β-receptors. A strong positive chemotactic signal arising from low levels of β-receptor activation becomes a net negative signal at higher levels of activation. The overall negative signal cannot be accounted for by activation of α-receptors (although they do play some role). Therefore, the β-receptors themselves exhibit a biphasic influence that is mirrored in the whole cell response. It is postulated that tyrosine phosphorylation and subsequent dephosphorylation of certain key signaling molecules is one mechanism by which this complex behavior may be regulated. Work is currently under way to elucidate the identity and functional importance of several such candidate molecules.
Acknowledgments
This work was supported by the British Heart Foundation and Pfizer International. We thank the surgeons and theater staff of St Mary’s Hospital for supplying samples of saphenous vein. We also gratefully acknowledge the expertise of K. Gallagher in the maintenance of cultured cells.
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