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PubMed1998

Regulation of kappa-opioid receptor mRNA level by cyclic AMP and growth factors in cultured rat glial cells.

The mRNA of the kappa-opioid receptor (KOR) has been found recently in cultured astrocytes and in microglia. By using RT-PCR and Southern hybridization, we confirmed these observations and, in addition, we observed that KOR mRNA was expressed in oligodendrocytes and in the precursors of astrocytes and oligodendrocytes. KOR mRNA level was the highest in the immature astrocytes and decreased with their maturation. Very few data are available on the regulation of KOR level by extracellular signals. Therefore, we examined the effect of three growth factors known to be present in the adult brain, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB) and leukemia inhibitory factor (LIF) and of two cyclic AMP (cAMP) generating systems, the cAMP analog, 8-(4-chlorophenylthio)-cAMP and forskolin, on this level. It was found that in astrocytes, KOR mRNA level decreased dramatically under the effect of cAMP and less under the effect of bFGF while it did not change significantly after LIF treatment. In oligodendrocytes, it also decreased with cAMP, but increased under the effect of bFGF and PDGF-BB. In microglia, a decrease was observed with cAMP and lipopolysaccharides (LPS), the most used activators of macrophages. These results shed new evidence on the expression of opioid receptor mRNA in the glial cells of the rat CNS. The regulation of KOR mRNA level under the effect of extracellular signals suggests that opioids take part in dynamic processes in glial cells, possibly related to glial-neuron communication.

Animals↗
PubMed1999

Mechanisms through which PDGF alters intracellular calcium levels in U-1242 MG human glioma cells.

PDGF-BB induces a rapid, sustained increase in intracellular calcium levels in U-1242 MG cells. We used several calcium channel blockers to identify the types of channels involved. L channel blockers (verapamil, nimodipine, nicardipine, nitrendipine and taicatoxin) had no effect on PDGF-BB induced alterations in intracellular calcium. Blockers of P, Q and N channels (omega-agatoxin-IVA, omega-conotoxin MVIIC and omega-conotoxin GVIA) also had no effect. This indicates that these channels play an insignificant role in supplying the Ca2+ necessary for PDGF stimulated events in U-1242 MG cells. However, a T channel blocker (NDGA) and the non-specific (NS) calcium channel blockers (FFA and SK&F 9365) abolished PDGF-induced increases in intracellular calcium. This indicates that PDGF causes calcium influx through both non-specific cationic channels and T channels. To study the participation of intracellular calcium stores in this process, we used thapsigargin, caffeine and ryanodine, all of which cause depletion of intracellular calcium stores. The PDGF effect was abolished using both thapsigargin and caffeine but not ryanodine. Collectively, these data indicate that in these human glioma cells PDGF-BB induces release of intracellular calcium from caffeine- and thapsigargin-sensitive calcium stores which in turn lead to further calcium influx through both NS and T channels.

Becaplermin↗
PubMed2000

A novel approach to studying the migratory morphology of embryonic mesenchymal cells.

A polarized morphology, defined by extension of an anterior pseudopod, is essential for the amoeboid migration of embryonic mesenchymal cells. Leukocytes adopt a similar morphology immediately following suspension in simple buffers containing chemotactic factors. Polarization in suspension therefore provides a rapid and sensitive screening assay for putative regulators of leukocyte migration. The aim of the present study was to investigate whether this assay might also be used to study the motile behaviour of embryonic mesenchymal cells. Primary cultures of mesenchymal cells were established from explants of stage 28 chick embryo corneal-limbal stroma. Serum-starved, subconfluent cultures were harvested using ethylene-diamine tetra-acetic acid and resuspended in Hanks' solution for up to 15 min at 37 degrees C. A variety of cell shapes, including spherical cells, blebbed cells, and cells with either non-polarized or polarized pseudopodia were observed. The proportions of cells with pseudopodia increased significantly over time. Treatment of cells with the chemotactic mitogen platelet derived growth factor-BB (PDGF-BB, homodimer isoform) suppressed blebbing and increased both pseudopod formation and polarization. Optimal polarization occurred in concentrations of PDGF-BB that are similar to those required for optimal chemotaxis (10 ng x mL(-1)). The polarization observed in the absence of PDGF-BB suggests that the migration of cells examined in this study might be controlled at least in part by some intrinsic mechanism. In addition, the strong polarization response to PDGF-BB confirms the role for this growth factor during corneal development. Observations of mesenchymal cell morphology in suspension, therefore provide novel data regarding the motile behaviour of embryonic cells.

Animals↗
PubMed1996

Contraction of collagen lattices by cells from Dupuytren's nodules.

The aim of this study was to see if nodular cells in Dupuytren's disease differed from dermal cells in their contractile capacity and motility. Ten surgical specimens from patients with Dupuytren's disease and contracture of the finger of more than 45 degrees were harvested and the nodular cells were explanted and cultured. Dermal fibroblasts from the forearm were used as control cells. Both types of cell had the same growth pattern. The morphology on confocal laser scanning microscopy was also similar in both types of cell. Dermal control cells caused significantly more contraction of collagen lattices compared with fibroblasts from nodules of Dupuytren's contracture. The F-actin content was equal in both groups. Platelet derived growth factor, PDGF-BB (but not PDGF-AA), increased the chemotactic activity of both cell types, but there were no differences between them. The results indicate that at a late state of the disease cells from Dupuytren's nodules lose their contractile capacity and regain a phenotype resembling that of dermal fibroblasts.

Animals↗
PubMed2000

Vein interposition cuffs decrease the intimal hyperplastic response of polytetrafluoroethylene bypass grafts.

PURPOSE: The modification of the distal anastomosis of polytetrafluoroethylene (PTFE) bypass grafts with vein interposition cuffs (VCs) has been reported to increase graft patency. However, the mechanisms that are responsible for this improved patency are unclear. Because intimal hyperplasia (IH) is a primary cause of prosthetic graft failure, we hypothesized that VCs affect the distal anastomosis by decreasing the IH response of the outflow artery. METHODS: Twenty-three female domestic Yorkshire pigs (mean weight, 35 kg) underwent 42 femoral PTFE bypass grafting procedures. The PTFE bypass grafts were separated into the following three groups according to distal anastomotic configuration: end-to-side anastomoses (ES), VCs, and cuffs constructed with PTFE (PCs). Four femoral arteries from two pigs served as healthy controls. At sacrifice, the grafts were perfusion fixed, and the distal anastomoses harvested at 1 and 4 weeks. The specimens were hemisected and serially sectioned to identify the heel, toe, and mid-anastomotic regions. The sections were cut into 5-microm segments and analyzed for intima and media thickness and area, intima/media area ratio, and the distribution of IH in the vein cuff. The roles of transforming growth factor-beta1 and platelet-derived growth factor-BB in IH development were assessed with immunohistochemistry. RESULTS: IH development was significantly lower at all areas of the anastomosis, with VCs compared with ES and PCs at 4 weeks (P </=.001). IH decreased in VCs from 1 to 4 weeks in all areas of the anastomosis (P </=.001). PCs showed pronounced IH at the mid-anastomosis as compared with VCs and ES (P </=.001). IH was most pronounced at the toe with ES and PCs (P </=.001). Qualitatively, VCs altered the site of IH development, sparing the recipient artery with preferential thickening of the vein cuff and formation of a pseudointima at the vein-PTFE interface. Immunohistochemistry results showed positive staining for transforming growth factor-beta1, platelet-derived growth factor-BB, and smooth muscle alpha-actin in the hyperplastic intima. CONCLUSION: PTFE bypass grafts with VCs had less IH develop than did grafts with ES and PC anastomoses. IH regression in VCs at 4 weeks suggests compensatory vessel wall remodeling mediated by the presence of the VC. Furthermore, VCs caused a redistribution of hyperplasia to the vein-PTFE interface, delaying IH-induced outflow obstruction in the recipient artery. The marked increase in IH with PCs, despite a similar geometric configuration to VCs, suggests that the biologic properties of autogenous tissue dissipate IH development. Similarly, the flow patterns in PCs and VCs should be identical, which suggests a less important role of hemodynamic forces in VC-mediated protection.

Actins↗
PubMed1998

Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase.

PURPOSE: Mitogen-activated protein kinase (MAPK) is a ubiquitous signaling protein that has been associated with cellular proliferation; however, its role in cellular migration has not been established. In this study, we investigate the role of MAPK in platelet-derived growth factor (PDGF)-induced migration and proliferation of human vascular smooth muscle cells (SMCs). METHODS: SMC migration was measured using a microchemotaxis assay (4 hours), and proliferation was assessed using 3H-thymidine uptake and cell counts. PD098059 was used as a specific noncompetitive inhibitor of MAPK activation. RESULTS: Coincubation of SMCs with PD098059 resulted in significant inhibition of PDGF-BB (5 ng/ml)-induced SMC chemotaxis and proliferation. The IC50 for both processes was approximately 10 mumol/L with complete inhibition at 50 mumol/L. Stimulation of SMCs with PDGF produced an early peak in MAPK activity followed by a plateau of activity that persisted for 24 hours. We hypothesized that variations in the temporal activation of MAPK might explain the action of this enzyme on these two disparate cellular events. By adding PD098059 at intervals after stimulation of SMCs with PDGF, we demonstrated an association between MAPK activity within the first 15 minutes and SMC migration, whereas MAPK activity between 1 and 4 hours was associated with SMC proliferation. CONCLUSIONS: MAPK activity is essential for both SMC migration and proliferation, and distinct phases of enzyme activation are required to stimulate these two discrete cellular events. Inhibition of this signaling protein may prove to be a useful method for preventing intimal hyperplasia.

Becaplermin↗
PubMed1999

Interferon gamma up-regulates a novel protein in vascular smooth muscle cells.

PURPOSE: By means of the technique of messenger RNA (mRNA) differential display, we previously isolated a partial DNA clone found to be down-regulated at the polytetrafluoroethylene (PTFE) hyperplastic arterial anastomosis compared with the normal artery. The partial DNA gene sequence was found to be homologous with interferon gamma up-regulated protein (IGUP) first found in human psoriatic keratinocytes. We cloned the entire IGUP gene from human vascular smooth muscle cells (VSMCs) to determine its regulation by gamma interferon (gamma-IFN) and other cytokines in cultured human VSMCs. METHODS: By means of polymerase chain reaction, the IGUP gene was amplified from a QUICK-Clone complementary DNA human aorta kit using 5' and 3' oligonucleotide primers to the known IGUP sequence. Immunohistocytochemistry studies compared normal artery and distal anastomotic IH. Human VSMCs were stimulated with 1000 U/mL of gamma-IFN, 5 ng/mL of platelet-derived growth factor BB (PDGF-BB), 3. 2 ng/mL basic fibroblast growth factor, 3.3 ng/mL transforming growth factor beta(TGF-beta), 10 ng/mL of vascular endothelial growth factor, and 10% fetal bovine serum (FBS) for zero, 24, 48 and 72 hours. Western blot analysis of lysates of the stimulated VSMCs was performed to determine up-regulation of IGUP. RESULTS: DNA sequencing confirmed the cloning of the entire coding region of the IGUP gene with 100% homology to the known IGUP DNA sequence. There was strong expression of IGUP in quiescent VSMCs and marked reduction of expression of IGUP in proliferating smooth muscle cells. gamma-IFN was the only cytokine, of the cytokines evaluated, to up-regulate production of IGUP in VSMCs. CONCLUSION: IGUP is a novel protein in VSMCs found to be down-regulated in areas of anastomotic IH, as compared with a normal artery. We have now shown IGUP to be up-regulated only by gamma-IFN in human VSMCs. IGUP may, therefore, be the intermediary for the known gamma-IFN inhibition of human VSMC proliferation.

Base Sequence↗
PubMed1998

Increased plasma levels of platelet-derived growth factor (PDGF-BB + PDGF-AB) in patients with never-treated mild essential hypertension.

Platelet-derived growth factor (PDGF) could play a role in both vascular hypertrophy and atherosclerotic disease associated with hypertension. To assess whether plasma PDGF level is increased in mild essential hypertension, we measured plasma PDGF concentration in 25 never-treated patients with uncomplicated mild essential hypertension and in 22 normotensive healthy subjects. To evaluate the contribution of platelets to plasma PDGF in the two groups, we also measured plasma beta-thromboglobulin (BTG). Measurement of PDGF was carried out through an enzyme-linked immunoadsorbent assay, which detects two PDGF dimers, namely PDGF-BB and PDGF-AB. Both plasma PDGF and BTG were higher in the hypertensive than in the normotensive subjects. The ratio of PDGF to BTG was similar in the two groups. Plasma PDGF was weakly correlated with plasma BTG in the normotensive subjects, whereas this relationship was lost in the hypertensive patients. Our results suggest that the increase in plasma PDGF (PDGF-AB + PDGF-BB) in never-treated essential hypertension is mainly due to platelet activation. The increased circulating level of PDGF could play a role in the vascular structural changes associated with hypertension.

Adult↗
PubMed2000

Increased potassium concentration inhibits stimulation of vascular smooth muscle proliferation by PDGF-BB and bFGF.

The effects of changes in extracellular potassium concentration on the rate of vascular smooth muscle cell proliferation stimulated by cytokines and serum were analyzed in vitro. To analyze the DNA synthesis response, cells from swine coronary artery were grown in DMEM medium containing 3, 4, 5, or 6 mmol/L potassium together with 20 ng/mL platelet-derived growth factor BB (PDGF-BB), 25 ng/mL basic fibroblast growth factor (bFGF), or 5% fetal bovine serum (FBS), with [methyl 3H] thymidine added, for 24 h. Proliferation responses were analyzed in cells grown in medium with potassium concentrations of 3, 4, 5, or 6 mmol/L, together with either 20 ng/mL PDGF-BB, 25 ng/mL bFGF, or 5% FBS, for 7 days, then harvested and counted. Highly significant inverse relationships were observed between potassium concentration and 3H-thymidine incorporation stimulated by each of the three agonists (P < .01 for each, ANOVA), and between potassium concentration and proliferation (all P < .01, ANOVA). Elevation of potassium concentration within the physiologic range inhibits vascular smooth muscle cell DNA synthesis and proliferation.

Animals↗
PubMed2000

The permissive effect of p21(Waf1/Cip1) on DNA synthesis is dependent on cell type: effect is absent in p53-inactive cells.

The cyclin-dependent kinase inhibitors (CKI) interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but we and others have recently shown that these molecules can exert permissive effects on cell cycle transit as well. The p53 protein induces transcription of the p21(Waf1/Cip1) gene, but whether p53 has any effect on the stimulatory versus inhibitory state of p21(Waf1/Cip1) toward cell growth is not known. The focus of the current study was to examine the effect of p21(Waf1/Cip1) inhibition on growth in cells which possess an inactive p53 protein. We found that there was significant and specific inhibition of p21(Waf1/Cip1) protein transcription in human squamous carcinoma A431 cells after transfection of an antisense p21(Waf1/Cip1) oligodeoxynucleotide, yet there was no significant growth inhibition in these cells after stimulation with 10% serum or with PDGF-BB, in contrast to what was observed in vascular smooth muscle (VSM) cells. Furthermore, there was no attenuation of either cyclinD/cdk4 association or of Rb hyperphosphorylation after antisense p21(Waf1/Cip1) oligodeoxynucleotide transfection, suggesting that an alternate pathway exists to allow association and phosphorylation of these cell cycle components in the absence (or with lower levels) of p21(Waf1/Cip1). Thus, the permissive effect of p21(Waf1/Cip1) toward growth is dependent on cell type, and active p53 is likely required for this effect.

Animals↗
PubMed2001

p73 is a growth-regulated protein in vascular smooth muscle cells and is present at high levels in human atherosclerotic plaque.

p73 is a newly described homologue of the tumour suppressor p53 that was cloned serendipitously and subsequently shown to possess considerable homology in the most evolutionarily conserved p53 domains. Yet despite the fact that p53 and p73 have extensive structural similarities, their functions are proving to be quite different. We now show that p73 is a growth-regulated protein in the vasculature, being markedly increased in cultured vascular smooth muscle (VSM) cells stimulated with 10% serum, with no significant change in p73 mRNA levels. Stability of p73 is increased after serum stimulation and, probably contributing to this increase in p73 stability, the c-Abl oncogene protein displays a higher molecular weight species and is probably phosphorylated and activated in serum-stimulated VSM cells. The serum-mediated induction of p73 is not altered when the cells are incubated with inhibitors of the MAP/ERK pathway or tyrosine kinases, and is not stimulated by PDGF-BB, demonstrating that the mechanism of the increase in p73 does not involve this classical receptor tyrosine kinase growth factor signalling cascade. p73 is markedly increased in plaque tissue taken from atherosclerotic human carotid arteries, but not in comparable intimal scrapings from normal human arteries. Our data indicate that the tumour suppressor homologue p73 probably plays a role in VSM cell cycle progression, being mediated by a specific, as yet unidentified, serum component, and identifies a new function for this protein as being important in the pathogenesis of human atherosclerosis as well as other vascular diseases.

Arteriosclerosis↗
PubMed2001

Regulation of Gadd45a mRNA expression in vascular smooth muscle under growth and stress conditions.

In order to identify differentially expressed genes under growth conditions, quiescent vascular smooth muscle cells (VSMCs) were stimulated with foetal calf serum (FCS) or platelet-derived growth factor-BB (PDGF-BB) for different time periods. Analysing the gene expression by the differential display (DD) method, we identified the cDNA of the growth arrest and DNA damage inducible gene 45a (Gadd45a, also known as gadd45 and gadd45a). Treatment with FCS or PDGF-BB led to a transient down-regulating of Gadd45a expression during the G0/G1 phase and maximal expression when cells had completed division. We found that expression of p53 and BRCA1 mRNA precedes Gadd45a mRNA expression with a maximal induction in the S phase. As in smooth muscle cells, a similar pattern of the Gadd45a mRNA expression was observed in knockout Gadd45a(-/-) cultured mouse embryonic fibroblasts (MEFs). However, no differences between Gadd45a(+/+) and Gadd45a(-/-) cell lines were observed regarding their kinetics of cell division. These experiments suggest a function of Gadd45a when cells exit the cell cycle rather than when regulating the entry into the S phase.

Animals↗
PubMed1997

Effect of growth factors on dermal fibroblast contraction in normal skin and hypertrophic scar.

We have examined the effects of four 'exogenous' growth factors, i.e. PDGF-BB (5 ng/ml), TGF-beta1 (5 ng/ml), bFGF (10 ng/ml) and EGF (10 ng/ml) on the contraction of floating collagen type I lattices populated by human normal skin (NS) and hypertrophic scar (HS) fibroblasts (FPCL). Only TGF-beta1 enhanced the contractility of both NS and HS fibroblasts in the collagen lattice (P < 0.01). Other growth factors (PDGF-BB, bFGF and EGF) did not affect FPCLs contraction at 72 h (P > 0.05). The onset effect of TGF-beta1 on NS-FPCL contraction was relative early at 24 h after FPCL casting as compared to a 72 h delay on HS-FPCL contraction. Besides, PDGF-BB was found to be able to enhance HS-FPCL contraction (P < 0.05) but not on NS-FPCL contraction on day 4. On the other hand, three enzyme-linked immunosorbent assays (ELISA) were performed to demonstrate quantitatively the 'endogenous' growth factors that fibroblasts secreted into the culture medium 48 h after FPCL casting. No appreciable difference was found between 10 NS and 11 HS samples tested for PDGF-AB immunoassay (11.48 +/- 5.5 pg/ml versus 12.20 +/- 5.34 pg/ml). The same result existed in 7 NS and 13 HS samples for TGF-beta2 immunoassay (15.15 +/- 6.2 pg/ml versus 11.84 +/- 7.46 pg/ml). In bFGF immunoassay study, relative variable data was noted in both 7 NS (18.18 +/- 13.18 pg/ml) and 12 HS samples (20.41 +/- 22.36 pg/ml). In conclusion, we suppose that TGF-beta role in wound healing may be due to the secondary exogenous influences. The endogenous ability of TGF-beta2 secretion (quantity) in HS fibroblasts are the same as NS fibroblasts but with delayed timing responses (quality) to exogenous TGF-beta1 effect in the collagen lattice. Further studies with timing-regulated selective specific monoclonal antibodies against the growth factor receptors may provide the therapeutic applications on HS during wound healing.

Becaplermin↗
PubMed1997

Modulation of phospholipase D stimulation in c-src transfected mesangial cells.

Stimulation of rat mesangial cells with platelet-derived growth factor BB (PDGF-BB) enhanced phospholipase D (PLD) activity in a concentration dependent manner. Mesangial cells overexpressing the tyrosine kinase pp60c-src (c-Src) were used to determine the effect of this non transforming protooncogene on PLD activation. Overexpression of c-Src interfered with PDGF-BB-mediated activation of PLD. This modulation was dependent on the tyrosine kinase activity of c-Src, since overexpression of tyrosine kinase-negative mutants of c-Src did not affect PLD activation. No effect of c-Src overexpression was observed, when PLD was activated by ATP or guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The results indicate that the tyrosine kinase c-Src specifically interfered with PDGF-mediated but not with ATP- or GTP gamma S-mediated PLD activation.

Adenosine Triphosphate↗
PubMed2002

New technologies in vitro for analysis of cell movement on or within collagen gels.

The movement of cells through extracellular matrix (ECM) is a critical component of many normal and pathological processes in vivo. Consequently, efforts to characterize motility-associated interactions between cells and ECM have led to the development of methods to observe and quantify (assay) the movement of cells under simplified conditions in vitro. In this report, we describe a novel method (the bullseye assay) and apparatus for the concentration of cells into small, precisely sized and shaped circular disks (bullseyes) that serve as starting points for migration of cells within ECM. The same apparatus is used to form the bullseyes and position them at the center of flat disks (windows) of gelled collagen that are supported at the edges by rings of nylon mesh. Complete assemblies, each consisting of a bullseye, collagen window and nylon mesh ring, are transferred to tissue culture wells for assay of cell migration either within or on top of the collagen window. Studies of the migratory responses of three different cell types to specific cytokines demonstrated that the bullseye assay was sensitive, rapid to set up, and easy to use. In conjunction with the bullseye assay, we developed a novel annular grayscale method for quantification of cell migration from digital images. The method is easily mastered, is derived from a measurement program in the public domain, is not subjective and is more discriminative than other techniques of measurement.

Animals↗
PubMed1997

Cytokine regulation of proteoglycan production in fibroblasts: separate and synergistic effects.

We have studied the effects of cytokines, separately or in combination, on the production of proteoglycans in confluent cultures of fibroblasts. The cytokines used were the transforming growth factor-beta (TGF-beta), the platelet derived growth factor-AA (PDGF-AA), the platelet derived growth factor-BB (PDGF-BB) and the epidermal growth factor (EGF). Hyaluronan production increased in cells treated with TGF-beta, PDGF-AA and PDGF-BB. Combining pairs of factors did not contribute further to hyaluronan production, whereas the triple combination of EGF, TGF-beta and PDGF-BB induced an additional 1.9-fold increase. Proteoglycan production was only increased by TGF-beta alone. As for hyaluronan, combining pairs of the cytokines had no further effect on metabolism, whereas the combination of EGF, TGF-beta and PDGF-BB induced a further 1.6-fold increase in production and secretion. Compared with the control, an extensive increase in proteoglycan production was generated by the combination of EGF, TGF-beta and PDGF-BB, 7-fold for biglycan, approximately 5-fold for versican and hyaluronan and 2.4-4-fold for heparan sulfate proteoglycan and decorin. Compared with TGF-beta alone, this combination increased, in falling order, the production of heparan sulfate proteoglycan, hyaluronan, biglycan, decorin and versican. The mRNA levels for the various proteoglycans did not completely agree with the changes in production, suggesting that changes not only in synthesis but also in rate of degradation generate these variations. The data indicate that cytokines cooperate to produce a proper and physiological response, one needed by the organism during physiological and pathophysiological remodeling.

Anticoagulants↗
PubMed1998

Type I collagen synthesis in cultured human fibroblasts: regulation by cell spreading, platelet-derived growth factor and interactions with collagen fibers.

Type I collagen protein and pro-alpha 1(I) collagen mRNA levels were investigated in human dermal fibroblasts cultured on substrates which induced distinct morphologies. Induction of type I collagen protein synthesis required cell spreading in monolayer cultures; mere attachment to dishes coated with 2-hydroxyethyl methacrylate (poly(HEMA)) did not suffice. Spread cells or round cells cultured on poly(HEMA) differed in collagen type I production, but pro-alpha 1(I) collagen mRNA levels were similar. Recombinant human platelet-derived growth factor (PDGF)-BB could replace cell spreading as a stimulus for collagen synthesis in cells cultured on poly(HEMA). At later time points, pro-alpha 1(I) collagen mRNA levels were down-regulated, although relatively less than type I collagen synthesis. Type I collagen synthesis by fibroblasts cultured in three-dimensional collagen gels was strongly down-regulated at both the protein and RNA levels. In addition to its capacity to stimulate collagen synthesis, PDGF-BB induced elongation and the formation of long processes by fibroblasts cultured in collagen gels. The stimulatory effect by cell spreading and PDGF-BB on collagen synthesis was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. However, inhibition of PI3K only inhibited induction of collagen synthesis by actively spreading cells or by PDGF-BB and did not induce a down-regulation of collagen synthesis in cells which had already spread. These data demonstrate that type I collagen protein synthesis is partly independent of pro-alpha 1(I) collagen mRNA levels but highly regulated by cell shape, although this could be decoupled by PDGF-BB. Both cell shape- and PDGF-BB-induced stimulation of collagen type I synthesis depends on a signalling pathway involving PI3K. Furthermore, levels of pro-alpha 1(I) collagen mRNA in fibroblasts are partly cell shape independent but are down-regulated by fibroblast interactions with native collagen fibers.

Adult↗
PubMed1998

Arachidonic acid pretreatment enhances smooth muscle cell migration via increased Ca2+ influx.

It is well known that vascular smooth muscle cell (SMC) migration is an initial step in atheromatous plaque formation. In the present study, we investigated the effects of arachidonic acid (AA, 20:4 n-6) on bovine carotid artery SMC migration using the modified Boyden chamber technique. SMCs pretreated with 2.5 microg/ml of AA for 2 days, showed an enhanced migration response to fetal bovine serum. AA pretreatment (0.5-5.0 microg/ml) increased fetal bovine serum-induced SMC migration dose-dependently, and maximum stimulation was observed at a concentration of 2.5 microg/ml. However, AA pretreatment did not enhance fetal bovine serum-induced endothelial cell migration. Using lipid analysis, we found that AA was substantially incorporated into cellular phospholipids. When SMC migration was induced by platelet derived growth factor (PDGF)-BB, instead of serum, the stimulative effect of AA pretreatment was retained. SMCs pretreated with AA showed greater mobilization of intracellular Ca2+ in response to PDGF-BB than SMCs without AA pretreatment (controls). Nifedipine, a Ca2+ channel blocker, and glycoletherdiamine-tetraacetic acid (EGTA) had no effect on PDGF-induced migration of controls but both drugs reduced the enhanced PDGE-induced migration of AA-pretreated SMCs to the control level. Baicalein, an inhibitor of 12-lipoxygenase, reduced PDGF-BB-induced migration of both control and AA pretreated SMCs, however the AA-pretreated cells still showed enhanced migration compared to control cells. These findings suggest that AA accelerates SMC migration in the thickening of the intima during atheroma formation, via stimulation of extracellular Ca2+ influx.

Animals↗