Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Becaplermin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources
PubMed2001

Coronary smooth muscle differentiation from proepicardial cells requires rhoA-mediated actin reorganization and p160 rho-kinase activity.

We recently reported that the first detectable expression of SMC-specific proteins during coronary smooth muscle cell (CoSMC) differentiation from isolated proepicardial cells was restricted to cells undergoing epithelial-to-mesenchymal transformation (EMT). The objectives of this study were to examine more closely the relation between actin cytoskeletal rearrangements and serum response factor (SRF)-dependent transcription, and to specifically test whether rhoA-GTPase signaling is required for CoSMC differentiation. We report here that PDGF-BB stimulates EMT and promotes SRF-dependent expression of SMC marker genes calponin, SM22alpha, and SMgamma(actin) (SMgammaA) in proepicardial cells. C3 exoenzyme or rhoGDI, inhibitors of rhoA signaling, blocked PDGF-BB-induced EMT, prevented actin reorganization into stress fibers, and inhibited CoSMC differentiation. Incubation with the selective p160 rho-kinase (p160RhoK) inhibitor Y27632 (RKI) blocked EMT, prevented the appearance of calponin and SMgammaA-positive cells, and abolished expression and nuclear localization of SRF. To test the role of RhoK signaling for CoSMC differentiation in vivo, quail proepicardial organs (PEOs) were pretreated with RKI or vehicle and then grafted into age-matched host chick embryos to produce a chimeric epicardium. The ability of grafted cells to participate in coronary vessel formation was monitored by staining with antibodies for quail cell nuclear antigen and SMC marker proteins. Proepicardial cells pretreated with RKI failed to form CoSMCs in vivo. Time course studies traced this deficiency to a failure of epicardial-derived mesenchymal cells to migrate into or survive within the myocardium. In summary, these data point to important roles for rhoA-RhoK signaling in molecular pathways controlling cytoskeletal reorganization, SRF-dependent transcription, and cell survival that are required to produce CoSMCs from proepicardial cells.

Actins↗
PubMed1994

Okadaic acid blocks PDGF-induced proliferation of AKR-2B fibroblasts at the transition from G1- to S-phase.

Okadaic acid (OA) at 100 ng/ml completely inhibited platelet-derived growth factor (PDGF)-BB-induced DNA synthesis but had no effect on early signals, i.e., PDGF receptor autophosphorylation or stimulation of inositolphosphate turnover. A detailed analysis using synchronized cells showed that OA acts at the transition from G1-phase to S-phase. These observations were confirmed by flow cytometric DNA analysis of asynchronously grown cells. Here cells were specifically arrested in the G1-phase.

Animals↗
PubMed1994

Comparison of PDGF-AA- and PDGF-BB-induced phosphoinositide formation in human and mouse fibroblasts.

In certain cells, such as human fibroblasts (AG 1523), there is a clear difference in the cell motility response induced by the different isoforms of platelet-derived growth factor (PDGF). PDGF-BB induces extensive actin reorganization and is a potent chemotactic agent, whereas PDGF-AA has a limited effect on actin reorganization and is not chemotactic. In the present study, we wanted to compare these effects on cell motility with the effects of the PDGF isoforms on phosphoinositide (PtdIns) turnover. We find that stimulation of serum-starved AG 1523 cells with PDGF-AA or PDGF-BB caused an initial increase of the phosphatidylinositol phosphate and bisphosphate (PtdInsP and PtdInsP2) pools, suggesting that activation of the phosphoinositide kinases is an initial response to PDGF stimulation. Despite a lower number of PDGF alpha-receptors than beta-receptors on these cells, the initial formation of PtdInsP and PtdInsP2 appears to be stimulated to a similar degree by the two PDGF isoforms. In contrast, PtdInsP2 hydrolysis, indirectly measured as formation of phosphatidic acid, was correlated to the number of receptors. During prolonged exposure to PDGF-BB the stimulated PtdIns turnover remained at a high level, whereas the effect of PDGF-AA appeared more transient. A marked increase in the synthesis of a component migrating as phosphatidylinositol trisphosphate (PtdInsP3) was also detected after stimulation with PDGF-BB for 5 min. With PDGF-AA minor amounts were found, indicating that activation of the PtdIns 3'-kinase occurs also via the PDGF alpha-receptor. Stimulation with PDGF-BB, but not -AA, also induced a 50% decrease in lyso-PtdIns. In murine fibroblasts (Swiss 3T3), where the two PDGF isoforms have a similar effect on cell motility, the two PDGF isoforms also similarly induced PtdIns turnover, PtdInsP3 formation, and a decrease in lyso-PtdIns. Thus, there seems to be a correlation between PDGF-induced PtdIns turnover and PDGF-induced actin reorganization. This is compatible with previous evidence suggesting the microfilament formation is directly linked to an increased turnover of polyphosphoinositides in stimulated cells.

3T3 Cells↗
PubMed1994

The differential activation of phosphatidylinositol-3 kinase and mitogen-activated protein kinases by PDGF-AA and IGF-I might explain the synergistic effect of the two growth factors on the proliferation of AKR-2B fibroblasts.

AKR-2B mouse fibroblasts express similar numbers of alpha- or beta-type PDGF receptors on their surface. Previous studies showed that PDGF-AA alone was unable to stimulate cell proliferation. Simultaneous addition together with insulin-like growth factor I (IGF-I), itself also inactive, led to a significant proliferation of the cells. In an effort to explain this synergism of the two growth factors we describe here the effects of the isoforms PDGF-AA or -BB and insulin-like growth factor I on three distinct signaling pathways, i.e., polyphosphoinositol turnover and [Ca2+]i increase, phosphatidylinositol-3 kinase, and mitogen-activated protein kinases. Whereas PDGF-BB effectively stimulated all three events, PDGF-AA failed to stimulate the phosphatidylinositol-3 kinase activity, but stimulated the mitogen-activated protein kinase to a maximum extent. In contrast IGF-I had no effect on mitogen-activated kinase but strongly stimulated phosphatidylinositol-3 kinase activity.

Animals↗
PubMed1994

Platelet-derived growth factor-BB stimulates synthesis of the integrin alpha 2-subunit in human diploid fibroblasts.

Platelet-derived growth factor (PDGF)-BB stimulates fibroblast-mediated contraction of collagen gels, as well as migration of fibroblasts through collagen-coated membranes. In the present study we examined effects of PDGF-BB stimulation on the synthesis of collagen-binding beta 1 integrins by human diploid fibroblasts (AG 1518). PDGF-BB stimulation led to an increase in the rare of integrin alpha 2-subunit synthesis. In contrast, synthesis of the integrin alpha 1- or alpha 3-subunits were not affected by PDGF-BB stimulation. Furthermore, levels of alpha 2-subunit mRNA relative to levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA increased after PDGF-BB stimulation. The latter finding is compatible with PDGF-BB stimulating transcription of the alpha 2-subunit gene. PDGF-BB stimulation did not influence the relation between levels of integrin beta 1-subunit mRNA and GAPDH mRNA. In addition, the rate of synthesis or post-translational processing of the integrin beta 1-subunit were not, or only marginally, affected by PDGF-BB stimulation. It is likely that the motility response elicited in fibroblasts by PDGF-BB involves such alterations in the synthesis of the alpha 2-subunit of the alpha 2 beta 1 collagen-binding integrin.

Becaplermin↗
PubMed1995

The effect of neomycin on PDGF-induced mitogenic response and actin organization in cultured human fibroblasts.

Actin organization and DNA synthesis were studied in cultured, serum-starved, subconfluent human fibroblasts in response to human recombinant platelet-derived growth factor (PDGF)-BB and neomycin, an inhibitor of phosphoinositide (PI) turnover. The labeling of F-actin with TRITC-phalloidin showed that PDGF (10 ng/ml) and neomycin (3.5 mM) induced, within minutes, similar changes in the actin cytoskeleton and when used together potentiated each other's effects. PDGF stimulated a twofold increase in DNA synthesis, as shown by [3H]thymidine incorporation, 48 h after the beginning of incubation. Neomycin did not induce DNA synthesis but reduced the mitogenic response to PDGF to control levels. Stimulation of DNA synthesis was correlated with an intense increase in lamellipodia formation and ruffling, whereas inhibition of the DNA synthesis was correlated with the development of elongated cell shape with few ruffles and lamellipodia, similarly to untreated control cells. Our findings support the conclusion that early changes in the actin cytoskeleton and induction of DNA synthesis are separable biological processes that are mediated by different pathways. The inhibitory effect of neomycin on PDGF-induced DNA synthesis was dose-dependent during the first 24 h of PDGF incubation, after which the cells were irreversibly and maximally stimulated to synthesize DNA. Results of experimental treatments with neomycin at various times during the incubation with PDGF indicate that the critical period during which PDGF acts to stimulate DNA synthesis is after the 1st h but before the 12th h of incubation.

Actins↗
PubMed1995

Are growth factors chemotactic agents?

We report the first direct observation of chemotaxis in slowly moving malignant cells. Two sarcoma cell lines of different metastatic potential were used. In a direct-viewing chemotaxis chamber with two concentric wells, the pooled trajectories of highly malignant rat T15 cells were strongly biased toward the outer well which contained platelet-derived and insulin-like growth factors. In individual experiments, however, the trajectories of the T15 cells showed a significant directional bias which, depending on the cell distribution, sometimes deviated by as much as 170 degrees from the gradient direction. Cells of a less malignant rat line, K2, did not respond to the gradient but a strong K2 response appeared if T15 cells were placed in the outer well along with the growth factors. We conclude that stimulated T15 cells release a chemoattractant, for both T15 and K2 cells, which overrides any chemoattractive effect of the growth factors. These results call into question whether growth factors are ever directly chemotactic in this system and demonstrate the need for direct observation in determining whether any substance is a direct chemoattractant.

Animals↗
PubMed1996

PDGF receptor-to-nucleus signaling of p91 (STAT1 alpha) transcription factor in rat smooth muscle cells.

Vascular smooth muscle cells (VSMC) are the predominant cell type in the media of a normal artery. Injury to the vessel wall leads to platelet deposition and the release of numerous factors, including PDGF, which exerts its biological effects by binding to specific surface receptors on the smooth muscle cell membrane. We demonstrate that PDGF-stimulated smooth muscle cells activate the STAT (signal transducers and activators of transcription) family of proteins in addition to other signaling pathways (e.g., RAS-RAF-MAP Kinase). We show that the transcription factor p91 (STAT1 alpha) is rapidly activated by PDGF in VSMC and specifically binds to the regulatory elements SIE or GAS. We hypothesize that signal transduction by p91 plays an important role in VSMC, especially after injury with the release of growth factors such as PDGF.

Animals↗
PubMed1996

Induction of fibroblast growth factor receptor-1 mRNA and protein by platelet-derived growth factor BB.

Expression levels of growth factor receptors are subject to complex regulation, which is of consequence for their signaling capacity in physiological and pathological processes. We examined the regulation of expression levels of fibroblast growth factor receptor 1 (FGFR-1) in human fibroblasts treated with a panel of growth regulatory factors. Only platelet-derived growth factor BB (PDGF-BB) treatment had a significant effect and induced FGFR-1 mRNA levels fourfold, with a peak around 8 h of stimulation. The increase in mRNA levels was followed by an increased synthesis of FGFR-1 protein, which responded to basic FGF (bFGF) stimulation with induction of kinase activity and biological signaling. Thus, murine brain endothelial cells displayed an augmented induction of plasminogen activator activity in response to bFGF, following treatment with PDGF-BB. These data suggest that PDGF-BB could support FGFR-1-mediated biological responses in processes such as angiogenesis.

Animals↗
PubMed1996

The synergistic effect of PDGF-AA and IGF-1 on VSMC proliferation might be explained by the differential activation of their intracellular signaling pathways.

As previous studies showed, PDGF-AA exerts a poor mitogenic effect on vascular smooth muscle cells. Simultaneous addition of insulin-like growth factor 1 (IGF-1), itself also poorly mitogenic, led to a significant increase in [3H]thymidine incorporation into the cell DNA as well as a strong increase in cell number. To explain the synergistic effect of PDGF-AA and IGF-1 on VSMC proliferation, we describe the effects of the two growth factors on distinct intracellular signals: on the activation of the signal proteins mitogen-activated protein kinase (MAPK) isoforms p42 and p44 and on the protein kinase C (PKC) isoforms alpha, delta, and epsilon, and on the induction of the transcription factor c-fos. PDGF-AA strongly activated the MAPK isoforms and PKC delta as well as the induction of c-fos. In contrast, IGF-1 exerted no effect on the signals induced by PDGF-AA, but strongly activated PKC epsilon isoform. Comparing this signal pattern to the one of the mitogenically potent PDGF isoform PDGF-BB, we found that PDGF-BB activated all of the signal proteins investigated.

Animals↗
PubMed1996

Platelet-derived growth factor is an autocrine stimulator for the growth and survival of human esophageal carcinoma cell lines.

Platelet-derived growth factors (PDGF) are important mitogens for mesenchyme-derived cells. Neither PDGF nor PDGF receptors (PDGFR) are expressed in epithelial cells under normal physiological conditions. However, we have found that PDGF-BB induces c-jun expression and promotes the growth of the human esophageal carcinoma cell line CE48T/VGH. Scatchard analysis revealed the presence of 6 x 10(5) binding sites for PDGF-BB per cell, with a Kd of 9.7 nM. Furthermore, our data indicate that CE48T/VGH expresses beta type PDGFR (PDGFRbeta) with in vitro auto-kinase activity. We have also found that CE48T/VGH expresses the mRNA of the PDGF-A and PDGF-B chains and secretes PDGF molecules. Addition of anti-PDGF neutralizing antibody significantly decreased cell numbers of CE48T/VGH under serum-free conditions. The detached cells underwent apoptosis characterized by micronucleation. These results suggest that expression of the PDGF autocrine system may not only provide the growth advantage but also prevent the apoptosis for CE48T/VGH.

Apoptosis↗
PubMed1997

Selective inhibition of platelet-derived growth factor (PDGF) receptor autophosphorylation and PDGF-mediated cellular events by a quinoline derivative.

This report describes the biological effects of our original compound, Ki6783 ((3,4-dimethoxy)-4-phenoxy-6,7-dimethoxyquinoline), a potent and selective inhibitor of platelet-derived growth factor (PDGF) receptor autophosphorylation. This compound strongly inhibited autophosphorylation of the PDGF beta-receptor in cultured rat glomerular mesangial cells (MC) bearing this receptor (IC50 0.1 microM), although it did not inhibit autophosphorylation of other growth factor receptors even at 100 microM. In a cell-free kinase experiment, it showed selective inhibition of PDGF beta-receptor tyrosine kinase. A kinetic study of the compound to this tyrosine kinase revealed a competitive mode of action to ATP. [3H]Thymidine incorporation and cell proliferation of MC were inhibited by Ki6783 in a dose-dependent manner after Ki6783 and PDGF-BB were added to the culture medium. Furthermore, this compound normalized the fibrotic cell shape of v-sis-transformed NIH3T3 cells, which grow in an autocrine manner via the PDGF receptor. These effects could be explained by the inhibition of intracellular signal transduction triggered by PDGF receptor autophosphorylation, in which activation of mitogen-activated protein kinase occurs. These results suggest that Ki6783 is one of the more potent and selective inhibitors of PDGF receptor autophosphorylation and that it may be useful in ameliorating cell abnormalities due to excess action of PDGF and its receptor systems in several diseases.

3T3 Cells↗
PubMed1997

Involvement of phosphatidylinositide 3'-kinase and Rac in platelet-derived growth factor-induced actin reorganization and chemotaxis.

Previous work has suggested a role for phosphatidylinositide 3'-kinase (PI3-kinase) in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. In support of this notion, we show in this report that the PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF beta-receptor expressing porcine aortic endothelial (PAE/PDGFR-beta) cells. Treatment with wortmannin resulted in a dose-dependent decrease in chemotaxis with an IC50 value of about 15-20 nM. Higher concentrations of wortmannin also reduced basal random migration of transfected cells in the absence of PDGF. We also investigated the role of Rac in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-beta cells led to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-beta cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurred in the absence of PDGF stimulation and was independent of PI3-kinase activity. On the other hand, PAE/PDGFR-beta cells transfected with inducible N17Rac (a dominant negative Rac mutant) failed to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway.

Actins↗
PubMed1997

1-(Carboxymethylthio)tetradecane attenuates PDGF- and TPA-induced c-fos mRNA expression and increases the formation of phosphatidylethanolamine with a shift from less to more polar molecular species in C3H/10T1/2 cells.

1-(Carboxymethylthio)tetradecane caused C3H/10T1/2Cl8 and C3H/10T1/2Cl16 to incorporate 10 times more [32P]Pi into diacylphosphatidylethanolamine than control. This 3-thia fatty acid caused a shift in incorporation of 32P-radioactivity into phosphatidylethanolamine species from species with long to species with short HPLC elution times. The increase in 32P-labeling was parallelled by a change in the apparent mass of phosphatidylethanolamine to a higher proportion of molecular species with short elution times than with long elution times. 1-(Carboxymethylthio)tetradecane caused loss of molecular species containing stearoyl groups. These results indicate that culturing the cells with 1-(carboxymethylthio)tetradecane causes looser packing and an increase in fluidity of the diacylphosphatidylethanolamine molecules in the membranes. 12-O-tetradecanoyl phorbol-13-acetate (TPA), platelet-derived growth factor (PDGF)-BB, or PDGF-AA stimulation of 1-(carboxymethylthio)tetradecane-treated cells resulted in decreased maximal levels of c-fos mRNA expression, indicating attenuation of signal transduction. Compared to cells not treated, the levels of both PDGF-alpha and PDGF-beta receptors were lower while GTPase-activating protein and phospholipase C-gamma levels were not altered in C3H/10T1/2Cl8 and C3H/10T1/2Cl16 cells cultured in the presence of 1-(carboxymethylthio)tetradecane. Our data demonstrate that 1-(carboxymethylthio)tetradecane-mediated changes in phospholipid structure and composition may affect PDGF- and TPA-mediated c-fos gene regulation in fibroblasts.

Animals↗
PubMed1998

Mitogen-activated protein kinase activation mediates PDGF-directed migration of RPE cells.

Growth factor-directed migration is a critical component of the wound healing response although little is known about the signaling pathways involved. We examined the effect of inhibiting the mitogen-activated protein kinase (MAPK) pathway on platelet-derived growth factor (PDGF) and fibronectin-induced cell migration of human retinal pigment epithelial (RPE) cells. Using transwell cell-culture chambers, the effect of PDGF-BB (10-50 ng/ml) and fibronectin on components of migration was measured with or without the MAPK pathway inhibitor PD98059 (10-30 microM) MAPK activation of serum-starved cells by PDGF-BB was demonstrated by an immunoprecipitation/kinase assay and by immunohistochemistry using antibody specific for phosphorylated MAPK. PDGF-BB (10 ng/ml) stimulated MAPK activity in RPE (10 min) and its nuclear localization (1 h). PD98059 inhibited the activation of MAPK by PDGF-BB or serum. PDGF-BB stimulated RPE chemokinesis, chemotaxis, and haptotaxis; chemokinesis was additively increased and chemotaxis synergistically increased by the presence of a fibronectin substratum. PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis but inhibited chemokinesis only at higher PDGF-BB (50 ng/ml) concentrations in the presence of fibronectin substratum. These results demonstrate that MAPK is critically involved in multiple components of RPE migration in vitro and suggest the potential of targeting MAPK to inhibit RPE migration in vivo.

Becaplermin↗
PubMed1998

Multiple intracellular pathways interfere with the activation of a CPP32-like protease induced by serum deprivation of AKR-2B cells.

As previously described, confluent AKR-2B fibroblasts rapidly disintegrate upon removal of serum. Platelet-derived growth factor isoforms AB or BB (PDGF-AB, -BB) added immediately after serum deprivation caused complete survival of the cells without initiating proliferation (Simm et al., 1994, J. Cell. Physiol. 160, 295). Here the role of cAMP as a protective agent was investigated by using forskolin or 8-Br-cAMP. Both reagents afforded high cellular protection. The phorbolester TPA, an activator of protein kinase C isoforms, also exerted a high protection against cell death (ED50 = 7 nM). Unexpectedly colchicine (ED50 = 1.5 microM) an inhibitor of tubulin polymerization also protected cells from death. The protective effects of PDGF-BB and TPA were dependent on protein synthesis as indicated by their complete suppression by cycloheximide (CHx). Surprisingly, forskolin and 8-Br-cAMP remained effective even in the presence of CHx. Detailed studies of several signalling pathways were performed. These investigations showed no interference between PDGF-BB and cAMP-dependent pathways at the early stage of signal transduction. As previously described, the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone (YVAD-cmk) protected cells from death (Simm et al., 1997, J. Cell Sci. 110, 819-828). As shown here, a substantial protection was also achieved by the addition of two other caspase inhibitors: asp-glu-val-asp-aldehyde (DEVD-cho; ED50 = 100 microM) and benzoylcarbonyl-asp-glu-val-asp-chloromethylketone (Z-DEVD-cmk; ED50 = 100 microM). The activity of caspases was studied using either tyr-val-ala-asp-aminomethylcoumarine (YVAD-amc) or asp-glu-val-asp-aminomethylcoumarine (DEVD-amc) as substrates. There was no activation of a YVADase, whereas as pronounced increase in DEVDase activity was found with a maximum 3 h after serum removal. Cross competition experiments in vitro showed that the latter activity is inhibited also by low concentrations of YVAD-cmk (300-600 nM), suggesting that both inhibitors inactivated the same target protease. Remarkably all tested protective reagents lead to an inhibition of the DEVDase activity in intact cells. Since these reagents act via distinct intracellular pathways, the existence of a regulatory element upstream of the DEVDase is proposed which integrates signals from a variety of pathways.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Tyrosine kinase cell signaling pathways of rat mesangial cells in 3-dimensional cultures: response to fetal bovine serum and platelet-derived growth factor-BB.

Cells grown in 3-dimensional collagen gels adopt a nonproliferative, contractile phenotype which is more characteristic of cells in vivo than cells grown in 2-dimensional culture. The floating collagen gel contraction assay is a well-defined system used to study cell-extracellular matrix interactions grown in 3-dimensional culture. Although the cell biology of this system is well defined, the cell signaling associated with gel contraction has not been well characterized. In this study we demonstrate that fetal bovine (FBS) and platelet-derived growth factor (PDGF)-induced mesangial cell-collagen gel contraction is associated with increased tyrosine phosphorylation of a number of proteins including focal adhesion kinase (FAK) and the 42-kDa isoform of MAPK (ERK2). FBS-induced gel contraction is not affected by the presence of the MEK inhibitor PD098059. Low concentrations of PDGF-BB (10 ng/ml) induce gel contraction; however, at higher PDGF-BB concentrations (80 ng/ml) gel contraction is not observed. PDGF-BB-induced gel contraction as well as tyrosine phosphorylation of FAK are inhibited in the presence of the PI-3 kinase inhibitor wortmanin. Minimal autophosphorylation of the PDGF-beta receptor is observed under 3-dimensional culture conditions following PDGF-BB stimulation; however, when mesangial cells grown in 2-dimensional culture are exposed to PDGF-BB, the PDGF-beta receptor was prominently phosphorylated. We conclude that induction of collagen gel contraction by FBS and PDGF-BB is associated with tyrosine kinase phosphorylation and that these responses differ substantially from what occurs in 2-dimensional cultures in the presence of the same agonists.

Androstadienes↗
PubMed1998

Platelet-derived growth factor-BB, insulin-like growth factor-I, and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration.

Vascular smooth muscle cell (VSMC) migration is an important process in the development of vascular occlusive disease. To investigate mitogen regulation of VSMC migration, a cell-layer-scrape assay was used to measure migration 20 h after stimulation of VSMC with platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor I (IGF-I), or phorbol 12-myristate 13-acetate (PMA). The contributions of cell proliferation were eliminated by treatment of VSMC with hydroxyurea, which suppressed DNA synthesis.PDGF-BB stimulated VSMC migration 2.5-fold, while PMA and IGF-I stimulated migration 1.7- and 1.5-fold, respectively. The importance of protein kinase C (PKC), ERK, and phosphoinositide-3' kinase (PI3 kinase) in mitogen-stimulated migration was investigated, using specific inhibitors of these signaling molecules. PDGF-BB-stimulated migration was inhibited by the general PKC inhibitor RO 31-8220 (40%), the MEK inhibitor PD98059 (31%), and the PI3 kinase inhibitor wortmannin (22%) but not by PMA-induced downregulation of conventional and novel PKC isoforms. IGF-I-stimulated migration was inhibited by RO 31-8220 (34%) and wortmannin (37%) but was much less affected by PD98059 (19%) or PKC downregulation (10%). PMA-stimulated migration was inhibited by RO 31-8220 (53%), PD98059 (50%), wortmannin (45%), and PKC downregulation (47%). Western analysis confirmed that ERK was strongly activated by PDGF-BB and PMA but not by IGF-I. To examine potential in vivo negative regulators of VSMC migration, we analyzed the ability of heparin, an analogue of heparan sulfate, and TGFbeta to attenuate mitogen-stimulated migration. Heparin but not TGFbeta inhibited VSMC migration stimulated by all three mitogens. Delayed-addition experiments showed that RO 31-8220 retained substantial inhibitory activity even if added 3 h after PMA or IGF-I stimulation and 5 h after PDGF-BB addition, suggesting that sustained PKC activation is important for migration. The MEK inhibitor retained some effectiveness for 5 h after PDGF-BB stimulation but only 1 h after PMA addition. Western analysis showed ERK activation was transient after PMA treatment but sustained for 6 h after PDGF-BB treatment. Heparin strongly inhibited migration even if added 5-7 h after mitogen stimulation, suggesting that heparin may inhibit both short- and long-term signals necessary for migration. The present studies indicate that PMA and IGF-I activate a limited number of second messengers resulting in moderate stimulation of migration; in contrast PDGF-BB stimulates multiple signaling pathways resulting in strong stimulation of migration and lessened sensitivity to inhibitory signals.

Animals↗