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The present study was aimed at characterizing the responses of human vascular smooth muscle to all three dimeric isomers of platelet-derived growth factor (PDGF-AA, -AB and -BB) in terms of mitogenesis, contraction and intracellular calcium concentration. The potential of interaction between PDGF and endothelin-1 (ET-1) was also investigated. All three PDGF isoforms (0.1-20 ng mL-1) stimulated DNA synthesis in cultured human coronary artery and saphenous vein vascular smooth muscle cells (VSMC), measured by [3H]thymidine incorporation. PDGF-AB and -BB elicited comparable large increases in DNA synthesis of maximum 595 +/- 149% (P = 0.001, n = 9) and 576 +/- 17% (P < 0.001, n = 5), respectively, whereas PDGF-AA was only weakly mitogenic (61 +/- 16% increase; P < 0.05, n = 3). At a threshold concentration, PDGF acted in synergy with ET-1 to enhance DNA synthesis (816 +/- 337% increase; P < 0.05, n = 7). In contrast to mitogenesis, none of the three PDGF isomers had any effect on contraction of human saphenous veins in vitro, nor did they affect the contractile response to ET-1, 5-HT or the thromboxane mimetic U46619. The effects of the three PDGF isomers on intracellular calcium ([Ca2+]i) rises in cultured human VSMC were heterogeneous, with PDGF-BB inducing the largest increase in [Ca2+]i (442 +/- 53 nmol L-1) vs. PDGF-AB (290 +/- 28 nmol L-1), whilst PDGF-AA had no effect. Both the responses to PDGF-AB and-BB relied upon intracellular calcium release, whilst only PDGF-AB showed additional dependence on influx of extracellular calcium. In summary, PDGF is strongly mitogenic and comitogenic with ET-1, despite not being a vasoconstrictor, for human VSMC. Also, human VSMC showed heterogeneous responses to the three PDGF isoforms. These results implicate PDGF, and in particular the PDGF receptor-beta, as important role players in the development of vascular smooth muscle-mediated intimal thickening in humans.
Rat mesenteric and epigastric small arteries were cultured to investigate influences of mitogens on contractility, proliferation and protein synthesis. Wistar rat arteries were cultured in serum-free Dulbecco's Modified Eagle Medium, first, for 24 h to equilibrate and then for a further 24-48 h either in the absence or presence of test substances: angiotensin II (AII), 1 microM; AII, 1 microM + platelet derived growth factor BB-chain (PDGF-BB), 1 ng mL-1; PDGF-BB, 1 ng mL-1; PDGF-BB, 30 ng mL-1. No mechanical stress was applied. Viability was assessed by myography, protein synthesis by 6-h incorporation of 35S-methionine and proliferation by both 48-h 3H-thymidine-incorporation and immunohistochemical analysis using the thymidine analogue 5-bromo-2'-deoxyuridine. After 3 days in culture, the contractile responses of arteries to phenylephrine, serotonin, AII and PDGF-BB were preserved. Stimulation with PDGF-BB (30 ng mL-1) increased protein synthesis 1.5- (mesenteric) and 1. 9-fold (epigastric). Similarly, stimulation with PDGF-BB (30 ng mL-1) increased 3H-thymidine incorporation of unstimulated arteries 3.4- (mesenteric) and 2.8-fold (epigastric). The other treatments affected neither protein synthesis nor proliferation. Immunohistochemical analysis showed that the proliferation was occurring primarily in the adventitia and that the levels of apoptosis were unaltered by culture. The effects of AII and PDGF-BB on remodelling did not correlate with their contractile effects: epigastric arteries responded strongly to AII and PDGF-BB, while mesenteric arteries responded weakly. The results suggest that organ culture conditions which preserve contractile function may not be sufficient to preserve trophic mechanisms.
BACKGROUND: Thrombin plays a pivotal role in the pathogenesis of arterial thrombosis and exerts a wide spectrum of effects on the cells of vessel walls. METHODS: In this paper we focus on the direct role of thrombin as a mitogen for smooth muscle cells (SMCs) derived from different vessels from the vascular tree (coronary artery, aorta, carotid artery and pulmonary artery) of different species (human and pig). All cell populations examined responded mitogenically to alpha-thrombin, however the extent of this response was dependent on both vascular origin and the species from which vessels were derived. RESULTS: Thrombin (1-100 nmol L-1) induced DNA synthesis ranging from 1.5- to 4-fold baseline depending on cell type. Porcine coronary SMCs showed the highest response to thrombin (100 nmol L-1) in terms of protein and DNA synthesis (4.0 +/- 0.2-fold) and cell division (53.4 +/- 8.8%) among the tested cells. In these cells recombinant (r)-hirudin, a specific thrombin inhibitor, exhibited maximal effectiveness to block the mitogenic effect of thrombin. Human SMC response ([3H]-thymidine incorporation) to either sera or thrombin was lower than that of porcine cells. In contrast, c-fos mRNA levels induced by thrombin in human SMCs were higher than those induced in porcine cells. In human cells, thrombin elicited an overexpression of c-fos and a lower rate of [3H]-thymidine incorporation than in porcine cells. Insulin-like growth factor I but not insulin showed additive mitogenic effects with thrombin in human coronary SMCs. The response of these cells to thrombin from different sources was a function of thrombin specific activity. CONCLUSION: These results suggest that the cell system chosen to check thrombin mitogenicity not only determines thrombin quantitative effects but also may affect the effectiveness of an inhibitor to block its biological activity.
BACKGROUND: Hrs (hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate) is an early endosomal protein that is rapidly tyrosine-phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in the endocytosis of growth factor/receptor complexes through early endosomes. In this study, we searched for Hrs-interacting molecules which may regulate the function of Hrs, using a yeast two-hybrid system. RESULTS: We isolated a cDNA clone encoding a novel Src homology 3 (SH3)-containing protein, and named it 'Hrs binding protein' (Hbp). Hbp was co-immunoprecipitated with Hrs, and its intracellular localization was similar to that of Hrs. The association between Hbp and Hrs was mediated through the coiled coil motifs in Hbp and Hrs. Deletion mutants of Hbp lacking either the SH3 domain or the Hrs binding domain showed dominantly negative effects on the intracellular degradation of a growth factor and its receptor, but not on the internalization of growth factor/receptor complexes. CONCLUSIONS: Hbp is thought to be closely associated with Hrs on early endosomes. Hbp, together with Hrs may play a regulatory role in the vesicular transport of growth factor/receptor complexes through early endosomes, for their degradation.
Human dental pulp fibroblast strains were established from explants of dental pulps using identical culture techniques. To determine proliferative activity, a 3H-thymidine uptake and a crystal violet dye-binding assay were performed at passage numbers seven and eight. Assays were performed in the presence of either 0% fetal calf serum (FCS), 0.2% FCS or 10% FCS. Considerable variation in the overall proliferative activity of the different pulp cell strains (when averaged over all other variables) was noted. All dental pulp cell strains demonstrated significantly different proliferative activity from each other. In addition, the level of proliferative response and 3H-thymidine incorporation decreased as the passage number of the cells increased. This was in accordance with the findings of Tardieu-Moreau et al. (1992). It is proposed that the differences in proliferative activity are most likely attributable to inherent variability within the established pulp cell strains. Platelet derived growth factor-BB (PDGF-BB) and insulin-like growth factor-1 (IGF-1) were added to the human pulp cells both separately and in combination. All of the pulp cells exhibited increased proliferative activity in the presence of the growth factors with the combination of PDGF-BB/IGF-1 having the greatest mitogenic effect. There was also significant variability in the level of response of all pulp cell strains to the different growth factors. This study identified significant variability in the responsiveness to the growth factors between the pulp cell strains when the results of the 3H-thymidine and dye binding assays were compared. These findings reinforce the thesis that different assay procedures may also influence the findings of biological investigations involving the human dental pulp. The results of this study confirm that when comparing the findings of different in vitro studies involving human pulp cells, variations in experimental data can be strongly influenced by the pulp cell strain used and the culture technique employed. Indeed, studies of human pulp cell proliferation using pulp cells which are not of the same transfer number may not be relevant.
Several studies have indicated that growth factors, such as platelet derived growth factor (PDGF), may be important in atherogenesis. These factors are released from platelets, or expressed by cells of the arterial wall. In order to study their role in atherogenesis more directly, rabbits were immunized with PDGF-BB, platelet cytosolic protein, or human serum albumin (HSA), until high titres of antibody were attained. Atherosclerotic lesions were subsequently induced by feeding the animals with a 2% cholesterol enriched diet. At the end of approximately 3 months, the extent of aortic lesion development was assessed by image analysis of en face preparations of aortae stained with Oil Red-O, and histological segments of aortae taken at the level of the first intercostal artery branch point. The endogenous antibodies were characterized with respect to their cross-reactivity, and ability to neutralize PDGF and platelet cytosol-induced cell proliferation and migration in vitro. The endogenous, anti-PDGF-BB antibody was isoform specific, and neutralized the mitogenic and chemotactic properties of PDGF-BB and rabbit platelet cytosolic protein in vitro. The anti-platelet cytosol antibody partially inhibited the chemotactic and mitogenic properties of rabbit platelet cytosolic protein. Compared to non-immune rabbits (n = 5), animals immunized with HSA (n = 4) had a significantly larger area of aortic lesion involvement (P < 0.01), whereas aortic lesions in rabbits immunized with PDGF-BB (n = 5), or platelet cytosolic protein (n = 7) were significantly smaller than either non-immune animals, or animals immunized with HSA (P < 0.05). The same pattern was observed for other measures of aortic lesion involvement including aortic intima:media ratio at the level of the first intercostal artery. These data suggest that PDGF-BB, and possibly other platelet-associated growth factors, are involved in cholesterol-induced atherosclerosis.
Bronchial subepithelial fibrosis is an histological characteristic of asthma. Cytokines and other mediators, such as PDGF-BB, TGF-beta1 and ET-1 found in the asthmatic submucosa can potentially activate a repair process that leads to fibroblast proliferation and collagen synthesis. The mechanisms of modulation of the repair process leading to extracellular matrix deposition are still to be documented. In this study, we assessed the in vitro proliferation and collagen synthesis of bronchial fibroblasts isolated from normal and asthmatic subjects in response to ET-1, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-beta1 alone or in combination, in the presence or absence of dexamethasone. The combination of ET-1 with one of the other two growth factors, or the triple combination, significantly increased DNA synthesis and collagen production of bronchial fibroblasts isolated from both normal and asthmatic subjects, but the same growth factors used separately had no significant effect on the same parameters. These results suggest that the simultaneous presence of ET-1, PDGF-BB and TGF-beta1 in both normal and asthmatic subjects is necessary to activate bronchial fibroblast proliferation and collagen synthesis. As these mediators are present in the submucosa of the asthmatic bronchi, they could be responsible, at least in part, for the accumulation of collagen in the mucosa.
Here we describe properties of the P2-purinoceptor, which is involved in the regulation of the key enzyme of estrogen biosynthesis, aromatase cytochrome P-450, in stromal cells from human adipose tissue. Aromatase activity induced by cortisol and platelet-derived growth factor BB (PDGF-BB) is further increased by addition of ATP, ADP, AMP and, albeit with reduced potency, adenosine, GTP and adenosine(5')tetraphospho(5') adenosine. Stable P1-purinoceptor agonists are inactive, whereas P2X-purinoceptor agonists mimic the effects of purine(s) (nucleotides). Prior incubation of cells with a P2-purinoceptor antagonist, but not P1-purinoceptor antagonists, blocks augmentation of aromatase activity by all ligands. Nucleotides, but not adenosine, retain their ability to augment aromatase activity in the presence of adenosine deaminase, indicating that they do not act via their metabolite adenosine. These results lead to the conclusion, that at least one member of the P2-subclass of purinoceptors exists in adipose tissue and is involved in modulation of aromatase activity in vitro. The pharmacological profile of the P2-site differs from those reported for cloned P2-purinoceptors, but is similar to that of the P2X-subclass. Therefore, a combined response of different members of the P2X-purinoceptor subclass or of members of different P2-purinoceptor subclasses cannot be discounted. Purinoceptor activation triggers cytoplasmic calcium transients, which, in contrast to aromatase induction, are independent from the presence of cortisol and PDGF-BB. Therefore the involved P2-purinoceptor(s) seem(s) to be constitutively expressed in human adipose tissue stromal cells. P2-purinoceptor(s) might provide a direct link between sympathetic nerve activity and estrogen biosynthesis in human adipose tissue. Furthermore it (or they) may contribute to the regulation of lipolysis.
A surface plasmon resonance assay was used to analyze the binding of platelet-derived growth factor (PDGF)-AA and PDGF-BB to various proteins of the extracellular matrix. This identified several collagen types; laminin-1, nidogen, perlecan and BM-40 as potential ligands for PDGF with Kd values in the range 2-3200 nM. Perlecan and BM-40 were used to examine the domain specificity and other parameters of the interactions. Recombinant human and mouse BM-40 were shown to bind both PDGFs in a similar manner with a Kd of about 5-10 nM. Studies with deletion mutants of human BM-40 demonstrated binding to its C-terminal extracellular calcium-binding (EC) module, yet the interaction did not require calcium. This distinguishes this from the binding of the EC module to various collagen types, which is strictly calcium dependent. Furthermore, deletion of helix alphaC or two point mutations in helix alphaA of the EC module either enhanced or abolished binding to collagen IV. Since these mutations had no effects on binding to PDGF, it demonstrated the presence of two different binding epitopes. Binding of PDGF-BB to the perlecan core protein could be mapped to its domain III-2 (Kd = 8 nM) with lower affinities shown for domains I, IV-1 and V (Kd = 34-64 nM). Other perlecan domains (II, III-1, III-3, IV-2) were inactive. PDGF-AA was also shown to bind domain III-2 but not III-1. Neither nidogen, BM-40 or perlecan domain III-2 interfered with the binding of PDGF to its alpha and beta receptors, however, suggesting that these interactions may be mainly used for storage of PDGF in the extracellular matrix.
OBJECTIVE: The physiologic mechanisms involving growth factors, including PDGF-BB, EGF, and TGF-beta 1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. PROCEDURES: Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF-BB, EGF, TGF-beta 1, keratocytes (1 x 10(5)/well) and epithelial cells (2 x 10(5)/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF-BB between 0 and 75 ng/mL; and TGF-beta 1 between 0 and 10 ng/mL. Cell proliferation was measured using 3H-thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05). RESULTS: Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF-BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF-beta 1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL. CONCLUSIONS: Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF-BB and EGF stimulate, while TGF-beta 1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.
Conditioned medium derived from a rat central nervous system neuronal cell line B104 (B104 CM) was shown previously to contain uncharacterized potent mitogen(s) for oligodendrocyte/type-2 astrocyte (O-2A) progenitor cells. In this study, we demonstrated that B104 cells produce and secrete platelet-derived growth factor (PDGF)-AA homodimer, but not PDGF-B chain. B104 cells did not express other known potent mitogens for O-2A progenitor cells, including fibroblast growth factor-2 and neurotrophin-3. Unexpectedly, B104 cells also expressed transcripts of transforming growth factor-beta1 (TGF-beta1) and -beta2 (TGF-beta2), which are known to regulate O-2A progenitor cell differentiation and proliferation, and secreted exclusively the 25-kDa active forms of TGF-beta1 and TGF-beta2. Neutralization of B104 CM with anti-PDGF-AA antibody decreased proliferation of O-2A progenitor cells, whereas neutralization with anti-TGF-beta antibodies had no effect. The combination of PDGF and TGF-beta on proliferation was not equivalent to the effect of B104 CM, indicating the possibility of an unidentified growth factor. B104 CM maintained a high expression of PDGF-alpha receptor in oligodendrocytes. The observation that both a stimulatory factor (PDGF-AA) and a regulatory factor (TGF-beta) for O-2A progenitor cell proliferation and differentiation are produced from a single neuronal cell line emphasizes the potential critical interaction between neurons and O-2A progenitor cells in myelination and possibly in remyelination.
The activation of phospholipase D (PLD) is a common response to mitogenic stimuli in various cell types. As PLD-mediated signaling is known to be disrupted in the presence of ethanol, we tested whether PLD is involved in the ethanol-induced inhibition of cell proliferation in rat cortical primary astrocytes. Readdition of fetal calf serum (FCS) to serum-deprived astroglial cultures caused a rapid, threefold increase of PLD activity and a strong mitogenic response; both effects were dependent on tyrosine kinases but not on protein kinase C. Ethanol (0.1-2%) suppressed the FCS-induced, PLD-mediated formation of phosphatidic acid (PA) as well as astroglial cell proliferation in a concentration-dependent manner. Moreover, exogenous bacterial PLD increased astroglial proliferation in an ethanol-sensitive manner, whereas exogenous PA or lysophosphatidic acid was less effective. Formation of PA and astroglial proliferation were strongly inhibited by 1-butanol (0.1-1%), a substrate of PLD, but were unaffected by t-butanol, a non-substrate; 2-butanol had intermediate effects. Platelet-derived growth factor and endothelin-1 mimicked the mitogenic effect of FCS; their effects were also inhibited by the butanols in the potency order 1-butanol > 2-butanol > tert-butanol. Our results, in particular, the differential effects of 1-, 2-, and tert-butanol with respect to PA formation and astroglial proliferation, strongly suggest that the antiproliferative effects of ethanol in glial cells are due to the disruption of the PLD signaling pathway. This mechanism may also contribute to the inhibition of astroglial growth and brain development observed in alcoholic embryopathy.
The early phase of the biphasic ventilatory response to hypoxia in mammals is critically dependent on NMDA glutamate receptor activation within the nucleus of the solitary tract. However, the mechanisms underlying the subsequent development of the typical ventilatory roll-off are unclear and could underlie important roles in the functional and molecular adaptation to oxygen deprivation. Because the growth factor platelet-derived growth factor (PDGF)-BB can modulate the open channel probability of NMDA receptors by activating PDGF-beta receptors, its contribution to hypoxic ventilatory roll-off was examined. Administration of PDGF-BB, but not PDGF-AA, in the nucleus of the solitary tract was associated with significant attenuations of the early hypoxic ventilatory response in conscious rats. Furthermore, marked reductions in the magnitude of hypoxic ventilatory roll-off occurred in mice heterozygous for a mutation in the PDGF-beta receptor. Administration of a PDGF-beta receptor antagonist to wild-type littermates elicited similar declines in hypoxic ventilatory roll-off. The relative abundance of PDGF-beta receptors was confirmed in the nucleus of the solitary tract and other nuclei implicated in the hypoxic ventilatory response. In nucleus of the solitary tract lysates, PDGF-beta receptor tyrosine phosphorylation was temporally correlated with hypoxic ventilatory roll-off formation. Increased PDGF-B chain mRNA expression was induced by hypoxia in the nucleus of the solitary tract, and PDGF-B chain immunoreactivity colocalized with approximately 40% of nucleus of the solitary tract neurons, demonstrating hypoxia-induced c-Fos enhancements. Thus, PDGF-BB release and PDGF-beta receptor activation in the nucleus of the solitary tract are critical components of hypoxic ventilatory roll-off and may have important functional implications in processes underlying survival and acclimatization to hypoxic environments.
Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood-brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent.