Platelet-derived growth factors (PDGF) are key mediators of organ fibrosis. We investigated whether PDGF-C(-/-) mice or mice treated with neutralizing PDGF-C antibodies are protected from bile duct ligation-induced liver fibrosis, and we compared the effects with those of PDGF-C deficiency or neutralization on kidney fibrosis induced by unilateral ureteral obstruction. Unexpectedly, and in contrast to kidney fibrosis, PDGF-C deficiency or antagonism did not protect from liver fibrosis or functional liver impairment. Furthermore, the hepatic infiltration of monocytes/macrophages/dendritic cells and chemokine mRNA expression (CC chemokine ligand [CCL]5, CCL2, and CC chemokine receptor 2 [CCR2]) remained unchanged. Transcript expression of PDGF ligands increased in both liver and kidney fibrosis and was not affected by neutralization of PDGF-C. In kidney fibrosis, PDGF-C deficiency or antagonism led to reduced expression and signaling of PDGF-receptor (R)-α- and PDGFR-β-chains. In contrast, in liver fibrosis there was either no difference (PDGF-C(-/-) mice) or even an upregulation of PDGFR-β and signaling (anti-PDGF-C group). Finally, in vitro studies in portal myofibroblasts pointed to a predominant role of PDGF-B and PDGF-D signaling in liver fibrosis. In conclusion, our study revealed significant differences between kidney and liver fibrosis in that PDGF-C mediates kidney fibrosis, whereas antagonism of PDGF-C in liver fibrosis appears to be counteracted by significant upregulation and increased PDGFR-β signaling. PDGF-C antagonism, therefore, may not be effective to treat liver fibrosis.

Dietary omega-3 fatty acids retard coronary atherosclerosis. Previously, we demonstrated that dietary omega-3 fatty acids reduce platelet-derived growth factor (PDGF)-A and PDGF-B mRNA levels in unstimulated, human mononuclear cells (MNCs). In a randomized, investigator-blinded intervention trial, we have now compared the effect of ingestion of 7 g/d omega-3, omega-6, or omega-9 fatty acids for 4 weeks versus no dietary intervention on PDGF-A, PDGF-B, heparin-bound epidermal growth factor (HB-EGF), monocyte chemoattractant protein-1 (MCP-1), and interleukin-10 gene expression in unstimulated MNCs and in monocytes that were adherence-activated ex vivo in a total of 28 volunteers. In unstimulated MNCs, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-10%, 31+/-13%, and 40+/-14%, respectively, after omega-3 fatty acid ingestion, as assessed by quantitative polymerase chain reaction (all P<0.05). In monocytes that were adherence-activated ex vivo for 4 and 20 hours, mRNA steady-state levels of PDGF-A, PDGF-B, and MCP-1 were reduced by 25+/-13%, 20+/-15%, and 30+/-8%, respectively (all P<0.05). Interleukin-10 and HB-EGF mRNA steady-state levels were not influenced by omega-3 fatty acid ingestion. Expression of all respective mRNAs in control volunteers or in those ingesting omega-6 or omega-9 fatty acids were not altered. We conclude that human gene expression for PDGF-A, PDGF-B, and MCP-1, factors thought relevant to atherosclerosis, is constitutive, is constant, and can be reduced only by dietary omega-3 fatty acids in unstimulated and adherence-activated monocytes.

We have examined the effects of either brain-derived neurotrophic factor (BDNF), the BB-isoform of platelet-derived growth factor (PDGF-BB), or a combination of these growth factors on the survival and the morphological development of embryonic striatal neurons grown under serum-free culture conditions. Striatal neurons were identified using immunocytochemistry for "dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein with a molecular weight of 32 kilodalton" (DARPP-32). BDNF and PDGF-BB promoted the survival of DARPP-32-positive neurons, with the magnitude of their effects being comparable. A combination of these growth factors exerted no significant additive effects on cell survival. BDNF stimulated morphological differentiation of DARPP-32-containing neurons by increasing the length of neurites, the number of branching points on the neurites, and the soma area. By contrast, PDGF-BB increased the neurite length and the cell body area, but not the number of branching points. BDNF also protected striatal neurons from excitotoxicity induced by N-methyl-D-aspartate, whereas PDGF-BB had no effect under the same treatment conditions as those for BDNF. Thus, BDNF is trophic for striatal DARPP-32-containing neurons in vitro by enhancing the survival, morphological differentiation and resistance to excitotoxicity, and its mechanisms of action are probably different from those of PDGF-BB.

BACKGROUND

To elucidate the roles of vascular D(1)-like receptors in atherosclerosis, the effects of the specific D(1)-like agonists on platelet-derived growth factor (PDGF)-BB-mediated oxidative stress in vascular smooth muscle cells (VSMCs) were studied.

RESULTS

Immunohistochemical studies demonstrated the coexistence of D(1A) and D(1B) dopamine receptors in VSMCs. Western blotting revealed a band of approximately 70 kDa for D(1A) and D(1B) dopamine receptors. VSMCs stimulated by PDGF-BB exhibited increased oxidative stress directly measured by flow cytometry. These effects were prevented by dopamine, SKF 38393, or YM 435, and this prevention was reversed by Sch 23390. These effects were blocked by a specific protein kinase A (PKA) inhibitor, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H 89). The PDGF-BB-mediated increase in oxidative stress of VSMCs was significantly suppressed by the indirect phospholipase D (PLD) inhibitor suramin or the specific protein kinase C (PKC) inhibitor calphostin C. Both antisense but neither sense nor scrambled oligonucleotides to D(1A) and D(1B) receptors inhibited dopamine-induced suppression of increase in oxidative stress of VSMCs induced by PDGF-BB.

CONCLUSIONS

These findings suggest that vascular D(1)-like receptors (D(1A) and D(1B) receptors) inhibit any increase in oxidative stress of VSMCs, possibly through activation of PKA and suppression of PLD and PKC.

The aim was to determine the association of tear fluid cytokine levels and post-PRK corneal haze evaluated by in vivo confocal microscopy. In addition, the possible association between subbasal neural regeneration and haze formation, or epithelial regeneration were investigated. Twenty eyes of 20 patients (16 women and four men, age 30.7 +/- 7.5 years, range 21-48 years) underwent a myopic PRK. The spherical equivalent (SE) of the intended correction was -4.7 +/- 1.5 D (range -2.75 to -9.00 D). ELISA-methods were used to assess tear fluid concentrations of TGF-beta1, PDGF-BB and TNF-alpha pre-operatively, and post-operatively on day 2 and at 3 months. Tear fluid flow in the collection capillary was recorded, and rates of cytokine release (= tear fluid flow-corrected concentrations) were calculated. In vivo confocal microscopy was performed at 3 months to evaluate the corneal morphology and to determine numerical haze estimate. There was wide interindividual variation between pre-operative and post-operative concentrations and rates of release of TGF-beta1, PDGF-BB and TNF-alpha. Subepithelial haze was observed in all corneas and the mean haze estimate was 506 +/- 401 U (100-1410 U). However, no association was found between tear fluid cytokine levels and post-PRK haze. Regenerating subbasal nerve plexus was found in 18 out of 20 corneas; in two corneas it was absent or could not be visualized due to subepithelial haze. The density of the subbasal nerve fiber bundles had a positive correlation with the epithelial thickness (Pearson correlation, r = 0.56, P = 0.011), but not with the haze estimate or the thickness of the haze area. At 3 months post-PRK, haze could be observed in all patients. The results suggest that tear fluid cytokine analysis, as measured, may not be suitable for screening the potential candidates for haze formation. We did not find any correlation between haze and regeneration of subbasal nerve plexus, but we demonstrated that the regeneration of subbasal nerve plexus might have significant influence on regulation of epithelial healing.

A number of growth factors have been implicated in the control of the proliferation of breast cancer cells and some have been reported to mediate the proliferative effects of oestradiol. MCF-7 cells were treated with growth factors in the presence and absence of oestradiol. Oestradiol increased the response of cells to the proliferative effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha) and basic fibroblast growth factor (bFGF). Platelet derived growth factor (PDGF) and cathepsin D had no effect in the presence or absence of oestradiol while TGF-beta slightly reduced the stimulation by oestradiol. In the absence of oestradiol, there was little effect of combinations of growth factors although the effects of bFGF and IGF-I were additive. In the presence of oestradiol, the effects of bFGF and TGF-alpha were additive whereas bFGF acted as an IGF-I antagonist. Overall, bFGF had the greatest effect on cell proliferation although this was less marked than the previously described effect of the IGFs and insulin. The effects of oestradiol on the sensitivity of cells to the proliferative effects of bFGF did not appear to result from regulation of bFGF receptor expression.

The platelet-derived growth factor (PDGF) family of ligands (composed of A-, B-, C-, and D-chains), potent mitogens, and chemoattractants for cells of mesenchymal origin has been implicated in numerous vascular pathologies involving smooth muscle cell (SMC) hyperplasia. Understanding the molecular mechanisms mediating PDGF transcription would provide new insights into strategies to control PDGF-dependent pathophysiologic processes. We demonstrated previously that PDGF-A expression is under the positive regulatory influence of Sp1, Sp3, and Egr-1 and is negatively controlled by GCF2, NF-1(X), and WT-1. In this article, we demonstrate that Ets-1 induces PDGF-A expression in primary rat aortic SMCs at the level of transcription and mRNA expression. Electrophoretic mobility shift, supershift, and mutational analyses revealed a functional role for the (-555)TTCC(-552) motif in the PDGF-A promoter that binds endogenous Ets-1. Chromatin immunoprecipitation analysis showed the interaction of endogenous and exogenous Ets-1 or glutathione S-transferase-tagged Ets-1, bearing only the DNA-binding domain with the authentic PDGF-A promoter. Conversely, dominant-negative mutant of Ets-1 blocked the promoter interaction of endogenous Ets-1. Overexpression of Ets-1 but not the mutant form of Ets-1 activates the PDGF-A promoter cooperatively with Sp1. Sp1, which interacts with Ets-1, failed to induce PDGF-A promoter-dependent expression if the promoter contained a site-specific mutation in this novel Ets-binding site. Small interfering RNA to Ets-1 and Sp1 blocked PDGF-BB- and serum-inducible PDGF-A expression. SMC growth was stimulated by Ets-1 and Sp1 separately and further increased by both factors together. Ets-1-inducible mitogenesis is blocked by antibodies neutralizing PDGF-A and involves activation of the PDGF alpha-receptor, which binds PDGF-A. These findings identify a functional cis-acting element for Ets-1 in the PDGF-A promoter and demonstrate that Sp1 and Ets-1 cooperatively activate PDGF-A transcription in vascular SMCs.

The effects of specific mitogens and substrates on the proliferative capacity and the differentiated phenotypic plasticity of neural precursor cell populations isolated from the adult rat subventricular zone (SVZ) were examined. SVZ cells were grown on uncoated tissue culture plastic, extracellular matrix, or poly-D-ornithine with either laminin or fibronectin. SVZ neural precursor cells could not be generated with platelet-derived growth factor (PDGF), granulocyte macrophage colony stimulating factor, stem cell factor, heparin-binding epidermal growth factor (HB-EGF), granulocyte colony stimulating factor, or ciliary neurotrophic factor (CNTF), but could be with EGF, fibroblast growth factor 2 (FGF2), and FGF2 plus heparin. Varying combinations of substrate and mitogen resulted in very different expansion rates and/or lineage potential. Neurons, oligodendrocytes, and astrocytes differentiated from all cultures, but EGF-generated neural precursor cells were more restricted to an astrocytic lineage and FGF2-generated neural precursor cells had a greater capacity for neuronal differentiation. In both EGF- and FGF2-generated cell populations, CNTF increased the number of differentiated astrocytes, triiodothyronine oligodendrocytes, PDGF neurons, and brain-derived neurotrophic factor neurons only from EGF cells. Electrophysiological analysis of differentiated cells showed three distinct phenotypes, glial, neuronal, and presumed precursor cells, although the neuronal properties were immature. Collectively, these data indicate that CNS neural precursor cell populations isolated with different mitogens and substrates are intrinsically different and their characteristics cannot be directly compared.

Meningiomas are histologically and clinically diverse CNS neoplasms with few available immunohistochemical markers of differentiation and progression. Therefore, we investigated a panel of potentially useful meningioma-associated biomarkers using high throughput tissue microarray immunohistochemistry (TMA-IHC) with a TMA that includes 9 hemangiopericytomas (HPCs) and 41 meningiomas spanning all grades, as well as two subsets of atypical meningiomas, stratified according to clinical behavior. Antibodies utilized were progesterone receptor (PR), epithelial membrane antigen (EMA), cathepsin D, E-cadherin, platelet derived growth factor (PDGF) receptor beta, PDGF BB ligand, survivin, epithelial growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). In most cases, frequencies of tumor positivity were similar to those previously reported using whole section IHC. EMA, E-cadherin, and PDGFR-beta staining patterns distinguished the anaplastic meningiomas from the HPCs (P < 0.001, P = 0.02, P = 0.015, respectively). As in prior studies, PR and cathepsin D expression were inversely proportional to tumor grade. However, PR and EGFR were also differentially expressed between symptomatic, surgically resected benign meningiomas and incidental meningiomas found at autopsy. We conclude that (1) TMA-IHC is an accurate and efficient way to rapidly assess biomarkers in meningeal tumors, (2) EMA, E-cadherin, and PDGFR-beta are useful in distinguishing anaplastic meningiomas from HPCs, and (3) the expression patterns for incidental meningiomas differ slightly from their surgically resected symptomatic counterparts.

The purpose of this study was to develop and test a novel culture model for studying fibroblast migration in 3-D collagen matrices. Human corneal fibroblasts were seeded within dense, randomly oriented compressed collagen matrices. A 6 mm diameter button of this cell-seeded matrix was placed in the middle of an acellular, less dense outer collagen matrix. These constructs were cultured for 1, 3, 5 or 7 days in serum-free media, 10% fetal bovine serum (FBS), or 50 ng/ml PDGF. Constructs were then fixed and labeled with AlexaFluor 546 phalloidin (for f-actin) and TOTO-3 (for nuclei). Cell-matrix interactions were assessed using a combination of fluorescent and reflected light confocal imaging. Human corneal fibroblasts in serum-free media showed minimal migration into the outer (non-compressed) matrix. In contrast, culture in serum or PDGF stimulated cell migration. Cell-induced collagen matrix reorganization in the outer matrix could be directly visualized using reflected light imaging, and was highest following culture in 10% FBS. Cellular contraction in 10% FBS often led to alignment of cells parallel to the outer edge of the inner matrix, similar to the pattern observed during corneal wound healing following incisional surgery. Overall, this 3-D model allows the effects of different culture conditions on cell migration and matrix remodeling to be assessed simultaneously. In addition, the design allows for ECM density, geometry and mechanical constraints to be varied in a controlled fashion. These initial results demonstrate differences in cell and matrix patterning during migration in response to serum and PDGF.

Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. In this study we show that the NO-triggered inhibition of neutral ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin II and ATP, resulting in a normalization of neutral ceramidase protein as well as activity. In vivo phosphorylation studies using (32)P(i)-labeled mesangial cells revealed that TPA, PDGF, angiotensin II, and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca(2+)-dependent isoforms, thus suggesting the involvement of a Ca(2+)-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-delta isoform and to a lesser extent the PKC-alpha isoform are efficient in directly phosphorylating neutral ceramidase. In summary, our data show that NO is able to induce degradation of neutral ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-delta isoenzyme, which can directly phosphorylate and thereby prevent neutral ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral ceramidase stability and subsequent ceramide accumulation.

The PDGF (platelet-derived growth factor) family members are potent mitogens for cells of mesenchymal origin and serve as important regulators of cell migration, survival, apoptosis and transformation. Tumour-derived PDGF ligands are thought to function in both autocrine and paracrine manners, activating receptors on tumour and surrounding stromal cells. PDGF-C and -D are secreted as latent dimers, unlike PDGF-A and -B. Cleavage of the CUB domain from the PDGF-C and -D dimers is required for their biological activity. At present, little is known about the proteolytic processing of PDGF-C, the rate-limiting step in the regulation of PDGF-C activity. In the present study we show that the breast carcinoma cell line MCF7, engineered to overexpress PDGF-C, produces proteases capable of cleaving PDGF-C to its active form. Increased PDGF-C expression enhances cell proliferation, anchorage-independent cell growth and tumour cell motility by autocrine signalling. In addition, MCF7-produced PDGF-C induces fibroblast cell migration in a paracrine manner. Interestingly, PDGF-C enhances tumour cell invasion in the presence of fibroblasts, suggesting a role for tumour-derived PDGF-C in tumour-stromal interactions. In the present study, we identify tPA (tissue plasminogen activator) and matriptase as major proteases for processing of PDGF-C in MCF7 cells. In in vitro studies, we also show that uPA (urokinase-type plasminogen activator) is able to process PDGF-C. Furthermore, by site-directed mutagenesis, we identify the cleavage site for these proteases in PDGF-C. Lastly, we provide evidence suggesting a two-step proteolytic processing of PDGF-C involving creation of a hemidimer, followed by GFD-D (growth factor domain dimer) generation.

OBJECTIVE

Hepatic stellate cell (HSC) plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis. Tyrosine protein kinase plays an important role in the proliferation, activation of HSC. The purpose of the study is to investigate the effects of the tyrosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC.

METHODS

Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient. Culture-activated HSC were serum-starved and incubated with 10(-9) to 10(-5) mol/L concentration of genistein for 24, 48 or 72 h. In PDGF-induced HSC proliferation, HSC were stimulated with 10 microg.L(-1) PDGF-BB for 15 min, and then treated with genistein for the same time. Cell proliferation was measured by MTT assay and based on flow cytometric analysis of cell cycle. The a-smooth muscle actin (alpha-SMA) expression in HSC was studied with confocal laser microscopy and flow cytometry. c-fos, c-jun and cyclin D(1) expression in HSC was also detected by flow cytometry.

RESULTS

Genistein inhibited basal and PDGF-induced proliferation of HSC at the concentration of 10(-8) to 10(-5)mol/L, and treatment with 10(-7) mol/L concentration of genistein for 48 h inhibited the HSC proliferation significantly (the inhibition rate was 70.3 %, P<0.05). Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with 10(-7) mol/L genistein for 48 h suppressed the expression of alpha-SMA significantly in HSC (the specific fluorescence intensity were 60.2+/-21.5 vs 35.3+/-11.6 and 12.8+/-10.4 vs 9.54+/-6.39, respectively, both P<0.05). The intensity of c-fos, c-jun and cyclin D(1) expression of HSCs treated with 10(-7)mol/L genistein for 48 h was also significantly decreased compared with the controls.

CONCLUSIONS

Genistein influences proliferation of HSC, suppresses the expression of alpha-SMA in HSC and t inhibits the intensity of c-fos, c-jun and cyclin D(1) expression of HSCs. Genistein has therapeutic potential against liver fibrosis.

PDGF-B is of central importance in mesangioproliferative diseases. PDGF-D, a new PDGF isoform, like PDGF-B, signals through the PDGF betabeta-receptor. The present study first determined that PDGF-D is mitogenic for rat mesangial cells and is not inhibited by a PDGF-B antagonist. Low levels of PDGF-D mRNA were detected in normal rat glomeruli. After induction of mesangioproliferative nephritis in rats by anti-Thy 1.1 mAb, glomerular PDGF-D mRNA and protein expression increased significantly from days 4 to 9 in comparison with nonnephritic rats. Peak expression of PDGF-D mRNA occurred 2 d later than peak PDGF-B mRNA expression. In addition, PDGF-D serum levels increased significantly in the nephritic animals on day 7. For investigating the functional role of PDGF-D, neutralizing fully human mAb were generated using the XenoMouse technology. Rats with anti-Thy 1.1-induced nephritis were treated on days 3 and 5 with different amounts of a fully human PDGF-DD-specific neutralizing mAb (CR002), equal amounts of irrelevant control mAb, or PBS by intraperitoneal injection. Specific antagonism of PDGF-D led to a dose-dependent (up to 67%) reduction of glomerular cell proliferation. As judged by double immunostaining for 5-bromo-2'-deoxyuridine and alpha-smooth muscle actin, glomerular mesangial cell proliferation was reduced by up to 57%. Reduction of glomerular cell proliferation in the rats that received CR002 was not associated with reduced glomerular expression of PDGF-B mRNA. PDGF-D antagonism also led to reduced glomerular infiltration of monocytes/macrophages (day 5) and reduced accumulation of fibronectin (day 8). In contrast, no effect was noted in normal rats that received an injection of CR002. These data show that PDGF-D is overexpressed in mesangioproliferative states and can act as an auto-, para-, or even endocrine glomerular cell mitogen, indicating that antagonism of PDGF-D may represent a novel therapeutic approach to mesangioproliferative glomerulonephritides.

Platelet-derived growth factor (PDGF)-D is a newly recognized member of the PDGF family with its role just now being understood. Our previous study shows that PDGF-D and its receptors (PDGFR-β) are significantly increased in the infarcted heart, where PDGFR-β is primarily expressed by fibroblasts, indicating the involvement of PDGF-D in the development of cardiac fibrosis. In continuing with these findings, the current study explored the molecular basis of PDGF-D on fibrogenesis. Rat cardiac fibroblasts were isolated and treated with PDGF-D (200 ng/ml medium). The potential regulation of PDGF-D on fibroblast growth, phenotype change, collagen turnover, and the transforming growth factor (TGF)-β pathway were explored. We found: 1) PDGF-D significantly elevated cardiac fibroblast proliferation, myofibroblast (myoFb) differentiation, and type I collagen secretion; 2) matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 protein levels were significantly elevated in PDGF-D-treated cells, which were coincident with increased expressions of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2; 3) PDGF-D significantly enhanced TGF-β1 synthesis, which was eliminated by TGF-β blockade with small-interfering RNA (siRNA); 4) the stimulatory role of PDGF-D on fibroblast proliferation and collagen synthesis was abolished by TGF-β blockade; and 5) TGF-β siRNA treatment significantly suppressed PDGF-D synthesis in fibroblasts. These observations indicate that PDGF-D promotes fibrogenesis through multiple mechanisms. Coelevations of TIMPs and MMPs counterbalance collagen degradation. The profibrogenic role of PDGF-D is mediated through activation of the TGF-β1 pathway. TGF-β1 exerts positive feedback on PDGF-D synthesis. These findings suggest the potential therapeutic effect of PDGFR blockade on interstitial fibrosis in the infarcted heart.

Phospholipase D (PLD) is a membrane protein with a double role: maintenance of the structural integrity of cellular or intracellular membranes and involvement in cell signaling through the product of the catalytic reaction, PA, and through protein-protein interaction with a variety of partners. Cross-talk during PLD signaling occurs with other cancer regulators (Ras, PDGF, TGF and kinases). Elevation of either PLDDD) is able to transform fibroblasts and contribute to cancer progression. Elevated total PLD activity, as well as overexpression, is present in a wide variety of cancers such as gastric, colorectal, renal, stomach, esophagus, lung and breast. PLD provides survival signals and is involved in migration, adhesion and invasion of cancer cells, and all are increased during PLD upregulation or, conversely, they are decreased during PLD loss of function. Eventhough the end results of PLD action as relates to downstream signaling mechanisms are still currently being elucidated, invasion, a pre-requisite for metastasis, is directly affected by PLD. This review will introduce the classical mammalian PLD's, PLDDD in cancer, mostly through its role in cell migration, invasion and metastasis, that has grown exponentially in the last few years.

Rho-kinase (ROCK) inhibitors prevent pulmonary hypertension (PHT) in adult rodents, but little is known about their effects on the neonatal lung. Our objective was to examine the effects of ROCK inhibition on chronic hypoxia (CH)-induced PHT and abnormal lung structure in the neonatal rat. Pups were exposed to air or CH from postnatal d 1-14 while receiving Y-27632 (5 or 10 mg x kg(-1) x d(-1)), fasudil (20 mg x kg(-1) x d(-1)), or saline intraperitoneally. Relative to air, CH-exposed pups had increased pulmonary vascular resistance, right ventricular hypertrophy, arterial medial wall thickening, and abnormal distal airway morphology characterized by septal thinning and decreased secondary septation. Treatment with 10 mg/kg Y-27632 or fasudil attenuated the structural and hemodynamic changes of PHT while having no effect on septal thinning or inhibited secondary septation. In addition, Y-27632 (10 mg/kg) and fasudil augmented CH-induced somatic growth restriction. Pulmonary arteries of CH-exposed pups had increased ROCK activity, up-regulated expression of PDGF-BB and increased smooth muscle DNA synthesis, all of which were attenuated by treatment with 10 mg/kg Y-27632. Systemically administered ROCK inhibitors prevented PHT in the CH-exposed neonatal rat but at the cost of inhibited somatic growth. Limiting effects on vascular remodeling likely resulted, in major part, from attenuated vascular PDGF-BB/beta-receptor signaling.

The nucleus tractus solitarii (nTS) provides the initial central synaptic relay to peripheral chemoreceptor afferent inputs elicited by changes in oxygen tension. Insofar, the overall cumulative evidence pointing towards the N-methyl-D-aspartate (NMDA) glutamate receptor as the critical receptor underlying the early component of the hypoxic ventilatory response (HVR) is reviewed in detail. In addition, we will present recent findings supporting a role for platelet-derived growth factor (PDGF) beta receptor activation in modulation of the late phase of HVR. This evidence underscores the proposal of a working model for intracellular signaling pathways, downstream to the NMDA glutamate and PDGF-beta receptors in nTS neurons, which may contribute to both the ventilatory characteristics of the acute hypoxic response and to subsequently occurring functional adaptations and synaptic plasticity phenomena.

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF-beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF-beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished.

BACKGROUND

Platelet derived growth factors (PDGFs) are mitogens for fibroblasts and smooth muscle cells. This growth factor family contains four members PDGF-A, PDGF-B, PDGF-C and PDGF-D. Biology of recently discovered PDGF-C and PDGF-D is not well-established. Here we studied the expression of PDGF-C and PDGF-D and their receptors PDGFR-alpha and PDGFR-beta in normal and atherosclerotic human arteries.

METHODS

Human arterial samples from amputations and autopsies were classified according to the atherosclerotic stage and the expression of PDGF-C and PDGF-D proteins and their receptors was studied by immunohistochemistry. In situ hybridization and reverse transcriptase-PCR were used to study mRNA expression.

RESULTS

Both growth factors were expressed in medial smooth muscle cells (SMCs) in normal arteries and atherosclerotic lesions. However, clear differences were found in the expression profiles in endothelium: PDGF-C was strongly expressed in endothelial cells in both normal arteries and lesions whereas PDGF-D was only weakly expressed in endothelium. PDGF-C expression was very prominent in lesion macrophages. PDGF-D was expressed throughout the artery wall in lesions. PDGFR-alpha expression was strong in endothelium and in lesion macrophage-rich areas, whereas PDGFR-beta was mostly expressed in SMCs.

CONCLUSIONS

Our results suggest that PDGF-C may play an important role in endothelium in normal and atherosclerotic arteries and in macrophages in lesions. PDGF-D was expressed in all types of lesions with the same intensity and thus differs from the expression of PDGF-C.

A number of reports suggest that the process of ageing impairs wound repair and that strategies to manipulate the age-related wound healing environment are necessary in order to stimulate repair. The process of cutaneous wound repair is controlled by growth factors in an autocrine and paracrine fashion: it is therefore surprising that the localisation of specific growth factors and their receptors has not been documented in wound healing with respect to chronological age. In this study the temporal profile of growth factor and receptor immunostaining was assessed within acute incisional wounds in an ageing mouse colony. A delay in appearance of platelet derived growth factor (PDGF) A and B isoforms, and PDGF-alpha and -beta receptors was evident with increasing animal age, paralleled by a similar finding for epidermal growth factor (EGF) and EGF receptor. Transforming growth factor (TGF)-beta 1 and 2 isoforms were increased at all time points in the wounds of younger animals, but the TGF-beta 3 isoform increased in intensity from d 7 postwounding in the old mice wounds, and basic fibroblast growth factor (bFGF) from d 14. The quantity and distribution patterns of the various growth factors and their receptors may explain the age-related differences in wound healing speed and quality, and possibly suggest new therapeutic targets for manipulating wound healing in the aged.

We have previously shown that treatment of postconfluent, quiescent rat vascular smooth muscle cells (SMC) with platelet-derived growth factor (PDGF) dramatically reduced smooth muscle (SM) alpha-actin synthesis and SM alpha-actin mRNA abundance, suggesting a role for this mitogen in the control of SMC differentiation. In the present studies, we explored the molecular mechanisms whereby PDGF decreases SM alpha-actin mRNA levels. Treatment of postconfluent SMC with both platelet PDGF and recombinant PDGF-BB resulted in a dramatic and concentration-dependent decrease in SM alpha-actin mRNA levels. We observed no differences in efficacy between platelet PDGF and PDGF-BB, indicating that the PDGF-A chain is not required for the effect. The rate of decrease in SM alpha-actin mRNA abundance in PDGF-treated SMC was greater than that observed in cells treated with the transcriptional inhibitor, actinomycin D, with or without PDGF, indicating that PDGF induced a transcriptionally dependent destabilization of the cytosolic SM alpha-actin mRNA pool. This effect appeared selective for SM alpha-actin, in that there was no evidence of a similar change in non-muscle (NM) beta-actin mRNA stability following PDGF treatment. Results of nuclear run-on analyses showed no differences in SM alpha-actin transcription between PDGF- and vehicle-treated SMC at either 4 or 24 hours following treatment, demonstrating that decreases in transcription of the SM alpha-actin gene did not contribute to PDGF-induced changes in SM alpha-actin mRNA abundance. Results of these studies support a possible role for PDGF in regulation of SMC differentiation via a post-transcriptional control mechanism.

Proliferation of vascular smooth muscle cells (SMC) contributes to formation of the complicated human atherosclerotic plaque. These lesions also contain macrophages, known to secrete SMC mitogens, and T lymphocytes. Many of the SMC in the lesions express class II major histocompatibility antigens, an indication that activated T cells secrete immune IFN-gamma locally in the plaque. We therefore studied the effect of IFN-gamma on the proliferation of cultured SMC derived from adult human blood vessels. IFN-gamma (1,000 U/ml) reduced [3H]thymidine (TdR) incorporation into DNA by SMC stimulated with the well-defined mitogens IL 1 (from 15.3 +/- 0.7 to 6.2 +/- 0.7 dpm X 10(-3)/24 h) or platelet-derived growth factor (PDGF) (from 18.5 +/- 1.0 to 7.3 +/- 0.7 dpm X 10(-3)/24 h). Kinetic and nuclear labeling studies indicated that this effect of IFN-gamma was not due to altered thymidine transport or specific radioactivity of TdR in the cell. In longer term experiments (4-16 d) IFN-gamma prevented net DNA accumulation by SMC cultures stimulated by PDGF. IFN-gamma also delayed (from 30 to 60 min) the time to peak level of c-fos RNA in IL 1-treated SMC. It is unlikely that cytotoxicity caused these effects of IFN-gamma, as the inhibition of growth was reversible and we detected no cell death in SMC cultures exposed to this cytokine. Activation of 2'-5' oligoadenylate synthetase gene expression may mediate certain antiproliferative and antiviral effects of interferons. Both IFN-gamma and type I IFNs (IFN-alpha or IFN-beta) induced 2'-5' oligoadenylate synthetase mRNA and enzyme activity in SMC cultures, but with concentration dependence and time course that may not account for all of IFN-gamma's cytostatic effect on SMC. The accumulation of SMC in human atherosclerotic lesions is a long-term process that must involve altered balance between growth stimulatory and inhibitory factors. The cytostatic effect of IFN-gamma on human SMC demonstrated here may influence this balance during human atherogenesis, because T cells present in the complicated atherosclerotic plaque likely produce this cytokine.