C type natriuretic peptide (CNP) functions as a paracrine/autocrine vasoprotectant. CNP mRNA is up-regulated in human vascular smooth muscle cells (SMC) by PDGF-BB via a protein kinase C (PKC)-dependent pathways, and by general PKC activation with phorbol myristate acetate (PMA). In this report we examine the calcium dependence and isotype specificity of these PKC/CNP pathways. The PKC-δ-specific inhibitor rottlerin blocked the increase in CNP mRNA and immunoreactive CNP following treatment of aortic SMC (AoSMC) with PDGF-BB. A 300-400-fold PMA-induced elevation of CNP transcript levels in AoSMC and a ~40-fold increase in human aortic endothelial cells (HAEC) were reduced by PKC-α- and PKC-δ-, but not PKC-β-specific inhibitors. siRNA silencing of PKC-δ reduced PDGF-, but not PMA-stimulated CNP transcript in SMC. Inhibition of intracellular Ca(2+) mobilization abolished a PMA-stimulated increase in CNP transcript in both SMC and HAEC. The results of this study show that PDGF increases CNP in SMC via a protein kinase C-δ-dependent pathway. In contrast, PMA increases CNP expression using PKC-α- and PKC-δ-pathways in both SMC and HAEC. A 8-10-fold greater PMA-induced increase in CNP transcript in SMC than in HAEC suggests that smooth muscle cells could be selectively targeted for CNP up-regulation by PKC-α- and PKC-δ-activators.

OBJECTIVE

Periodontal ligament cells are regarded to have the capacity to differentiate into cementoblasts or osteoblasts, and are capable of forming a mineralized nodule in vitro. However, the precise mechanisms are unclear. Here we evaluated the possible involvement of growth factor receptors, such as the platelet-derived growth factor receptor (PDGFR), insulin-like growth factor-I receptor (IGF-IR), and epidermal growth factor receptor (EGFR) on periodontal ligament cells and their ligands during periodontal ligament cells differentiation in vitro.

METHODS

Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, characterized by von Kossa staining. Expressions of receptors and their ligands were analyzed by flow cytometry/reverse transcription-polymerase chain reaction.

RESULTS

During the differentiation, PDGFR-alpha was held at a lower level compared with the control. PDGFR-beta, however, was maintained at a slightly higher level that was reversed to the control level when mineralized nodules formed. In contrast, IGF-IR and EGFR were not substantially different from the control. The mineralized nodule formation was strongly inhibited by a PDGFR kinase blocker (AG1295 and AG1296), partially inhibited by an IGF-IR kinase blocker (I-Ome-AG538 and AG1024), and not inhibited by an EGFR kinase blocker (AG99). PDGF-A, PDGF-C, PDGF-D, IGF-I, and IGF-II, but not PDGF-B, were expressed on the control as well as dexamethasone/ascorbic acid-treated periodontal ligament cells during mineralized nodule formation; however, the pattern of their expressions was quite different.

CONCLUSIONS

These findings suggest that a pathway of PDGFs/PDGFR and IGFs/IGF-IR on periodontal ligament cells are involved during mineralized nodule formation, and that PDGFs and IGFs expressed by periodontal ligament cells may contribute to the formation.

Urinary trypsin inhibitor (UTI), an inhibitor of urokinase plasminogen activator relevant to proteolytic processing from the inactive into the active form of platelet-derived growth factor-D (PDGF-D) to activate PDGF-betabeta receptor (PDGF-betabetaR), inhibited fetal bovine serum-stimulated migration of human malignant mesothelioma, with the extent varying among the cell types. The more effective inhibition was found in NCIH-2052 and -2452 cells, with the higher expression of PDGF-betabetaR. The results of the present study suggest that UTI suppresses malignant mesothelioma cell migration by neutralizing active dimer of PDGF-D (PDGF-DD)/PDGF-betabetaR-mediated signal transduction.

OBJECTIVE

Biological agents like human serum or autologous platelets have recently been employed as adjuvants for macular hole surgery. However, the role of these agents on the retinal cellular level remains unclear. In the present study, we investigated the effect of human platelets, serum and PDGF on RPE migration and proliferation in cell culture.

METHODS

Human RPE were cultured in DMEM + 2% FCS and experiments performed at passages 2-4. Human platelet concentrate (PC) and serum (HS) were isolated from blood of patients previewed for macular hole surgery; human PDGF-BB was from Pepro Tech. PC and HS at protein concentrations ranging from 50-1000 micrograms/ml and PDGF at 1 and 10 ng/ml were added to 5000 cells/well in the proliferation assay and to a confluent RPE monolayer on which a central mechanical "wound" (5 mm diameter) was made. Incubation times ranged from 1 h to 5 days. Cell numbers at D 5 were indirectly determined by protein measurements. In the wound model, the cells inside the wound area were counted and results compared to the control cultures that received no supplements.

RESULTS

Cell proliferation was significantly stimulated over controls by all concentrations of PC, HS and PDGF with any incubation time. Compared to PC and PDGF, HS revealed less proliferation after 1-6 h of incubation; there was no significant difference from PC with other incubation times. In the wound model, both PC and PDGF significantly increased the number of cells migrating into the denuded area after 1 h incubation with the culture medium; longer incubation times had no further effect compared to controls.

CONCLUSIONS

The present study is the first to demonstrate that human platelet concentrate induces proliferation and migration of RPE cells in vitro. However, PDGF, a growth factor which is abundantly present in platelets, was found to be at least equally effective. We assume that the majority of the mitogenic effect of platelet concentrate is due to PDGF.

Platelet-derived growth factors (PDGFs) may play an important role in the development of atherosclerosis acting as chemoattractants and mitogens for vascular smooth muscle cells and macrophages. Three dimeric forms of PDGF (AA, AB, BB) have different activities due to distinct binding properties mediated by two types of PDGF receptors (Ralpha, Rbeta). To investigate the possible contribution of molecular variants in the human PDGF-A and PDGF-Ralpha genes to coronary heart disease we screened these genes for polymorphisms by polymerase chain reaction/single-strand conformation polymorphism analysis. A total of 600 men with myocardial infarction and 717 age-matched male controls from four populations in Northern Ireland and France (the ECTIM Study) were gneotyped for newly identified polymorphisms in the genes encoding PDGF-A (C-26IN3T, H69H, C+12IN5T) and PDGF-Ralpha [-1630 I/D (+/-AACTT), A-1506G, C-1390G, G-956A, C-908A, G-793T, +69 I/D (+/-GA)] using allele-specific oligonucleotides. All PDGF-Ralpha polymorphisms, except C-908A, involving a nucleotide change in a common consensus site for GCF and SP-1 transcription factors, were in nearly complete association, generating two major haplotypes. The PDGF-A and PDGF-Ralpha polymorphisms provided a heterozygosity of 0.69 and 0.40, respectively. Genotype and allele frequencies of the PDGF-A and PDGF-Ralpha polymorphisms did not differ between patients with myocardial infarction and controls in either country. None of the polymorphisms investigated was associated with blood pressure, coronary artery stenosis, or any biochemical parameter available in the ECTIM Study.

Platelet-derived growth factor (BB dimer; PDGF-BB) stimulates a mitogenic response in A-10 vascular smooth muscle cells. In addition, PDGF-BB stimulates phospholipase D activity against phosphatidylcholine in A-10 cells. This response was observed as a rapid metabolism of phosphatidylcholine to phosphatidate and choline; a subsequent metabolism generates sustained levels of diacylglycerol. The accumulation of phosphatidylethanol, a transphosphatidylation product of phospholipase D, was obvious in PDGF-treated cells. PDGF-BB also stimulates a chemotactic response in A-10 cells. The concentrations of PDGF-BB required to stimulate mitogenesis, phospholipase D activity and chemotaxis are similar. This finding shows that PDGF induces a variety of cellular responses and suggests that these responses may share common metabolic pathways. That conception was tested by investigating the activity of the different PDGF dimers. PDGF-AA had little or no activity in A-10 cells for any of the responses measured. PDGF-AB and PDGF-BB were equally potent in stimulating mitogenic responses. However, the AB heterodimer was only half as active as PDGF-BB with respect to activation of phospholipase D and chemotactic responses. These results demonstrate that PDGF stimulates phospholipase D in vascular smooth muscle cells. In addition, the data indicate that different PDGF dimers can transduce varying signals and suggest a link between the mechanisms by which PDGF-BB activates phospholipase D and the chemotactic response.

The influence of angiotensin-converting enzyme (ACE) inhibitory octapeptide derived from tuna muscle (tuna AI) on the bovine aorta endothelial cell (BAEC) migration was investigated, as compared with captopril. BAEC migration was quantitated 6 d after release from contact inhibition by a teflon fence assay. The culture grown in the presence of tuna AI (1 and 10 microM) clearly exhibited an increase in migration, compared with the control. The media collected from tuna AI (1 and 10 microM)-stimulated BAECs significantly exhibited the interleukin (IL) -1 activity that was detected by the thymocyte costimulation assay with phytohemagglutinin. Although tuna AI was a weaker ACE inhibitor than captopril, the increasing effect of tuna AI on the migration and the IL-1 generation in BAECs was slightly greater than that of captopril. In quiescent BAECs, tuna AI (1 microM) apparently induced c-myc and platelet derived growth factor (PDGF) A-chain messenger ribonucleic acid (mRNA) expressions within 30 min, which persisted for 6 h. In contrast, captopril induced a very low expression of c-myc mRNA, and had no relation to PDGF A-chain mRNA expression. These results suggest that the increase of BAEC migration by tuna AI, unlike captopril, is likely related to the induction or activation of IL-1, and c-myc and PDGF mRNAs, in addition to the inhibition of the conversion of endogenous angiotensin I to angiotensin II.

OBJECTIVE

To investigate whether inflammation could excessively activate platelet-derived growth factor (PDGF) signaling pathway in desoxycorticosterone (DOCA) induced salt-sensitive hypertensive rats with myocardial fibrosis (MF).

METHODS

A total of 30 male SD rats underwent right nephrectomy and then bred with 1% sodium chloride and 0.1% potassium chloride for 2 weeks. These animals were randomly divided into 3 groups: CON group, DOCA group and DOCA+FAS group. Systolic blood pressure (SBP) was measured once every 2 weeks; HE staining was done to observe myocardial inflammation; immunohistochemistry was done to detect expressions of monocyte-macrophage antigen (ectodermal dysplasia 1, ED-1), PDGFRα and PDGFRβ in the myocardium; real time fluorescence quantitative PCR was employed to detect the mRNA expressions of DGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFRα and PDGFRβ.

RESULTS

The SBP in DOCA group and DOCA+FAS group increased markedly when compared with CON group (P<0.01), but there was no marked difference between DOCA group and DOCA+FAS group (P>0.05). At 14 days, in DOCA group, the myocardial inflammation was obvious, ED-1 expression increased markedly, the mRNA expressions of PDGF-A, PDGF-B, PDGF-C, PDGFRα and PDGFRβ increased to different extents, protein expressions of PDGFRα and PDGFRβ also elevated markedly (P<0.01), but the PDGF-D mRNA expression remained unchanged, when compared with CON group. After treatment with fasudil (a drug with anti-inflammatory activity), myocardial inflammation was significantly attenuated, mRNA expressions of PDGF-A, PDGF-B, PDGF-C and PDGFRα as well as PDGFRα protein expression reduced dramatically (P<0.01), but the mRNA and protein expressions of PDGFRβ remained unchanged (P>0.05) when compared with DOCA group.

CONCLUSIONS

In DOCA/salt induced hypertensive rats with MF, excessive activation of PDGF/PDGFR signaling pathway is involved in myocardial inflammation.

OBJECTIVE

TGF-β1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-β1 from virally inactivated human platelets.

METHODS

Apheresis platelet concentrates (N=12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X-45; 31°C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7.5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1 M NaCl-PBS buffer pH 7.5 (DEAE-eluate). The content in TGF-β1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions.

RESULTS

Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-β1 at a mean concentration of about 102 ng/ml, whereas EGF, b-FGF were at about 0.72 and 0.18 ng/ml, respectively. The content in TnBP and Triton X-45 was <2 ppm.

CONCLUSIONS

A fraction enriched in TGF-β1 can be prepared from virally inactivated human platelet lysates using an easily scale process. Its interest in regenerative medicine and cell therapy will be evaluated in further studies.

OBJECTIVE

Many systemic and topical therapeutic agents such as growth hormone, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF) have been used as vulnerary agents. However, the role of nitric oxide (NO) as a wound-healing stimulant has been received with mixed reviews. NO is a potent vasodilator that is thought to be an endothelium-dependent relaxing factor, and a regulator of blood pressure and regional blood flow. It affects vascular smooth muscle proliferation and inhibits platelet aggregation and leukocyte adhesion. Therefore we compared the effects of several topical substances that have similar or reverse properties.

METHODS

Using the excisional rat wound model, we evaluated the topical effects of Dermaide Aloe (D-Aloe, Dermaide Research Corp, Palos Heights, IL), nitroglycerin, Aquaphor (Beuersdorf, Inc., Norwalk, CT) alone, with D-Aloe with nitroglycerin, 2%, and L-NAME (NO inhibitor) with Aquaphor, and L-NAME with Aquaphor and D-Aloe for a 21-day period. All wounds were measured by planimetry at 1, 7, 10, 13, 16, 18, and 21 days.

RESULTS

At day 1, all wounds had an average wound size of 2.27 cm2 (SD +/- 0.372) with no significant difference in wound size among the groups. Topically applied D-Aloe appeared to promote wound healing faster than the remaining other topicals (p < .05, Student-Newman-Keuls and Dunn's Method) over the study period. However, topicals combined with D-Aloe, the vehicle Aquaphor, and L-NAME improved the wound healing process when compared with nitroglycerin alone (p < .05).

CONCLUSIONS

D-Aloe appears to have a wound-healing advancement factor that can reverse the effects of petrolatum- and nitroglycerin-based products as observed in the remaining groups when compared with nitroglycerin alone. It appears that D-Aloe's effect of preventing dermal ischemia by reversing the effects of thromboxane synthetase (TxA2) may act synergistically with NO or could be an oxygen radical scavenger.

In vitro-fertilized bovine embryos were incubated in Menezo's B2 medium (MB2) supplemented with 2 mg/mL of BSA. In Exp. 1, eight-cell stage embryos were allotted to one of the following groups: control medium (MB2), MB2 with 20 ng/mL of platelet-activating factor (PAF), 1 x 10(7) bovine blood platelets (Platelets), oviductal cells (BOEC), BOEC and 20 ng/mL of PAF (BOEC+PAF), or BOEC and 1 x 10(7) platelets (BOEC+Platelets). In Exp. 2, eight-cell embryos were allotted to one of the following groups: control medium (MB2), MB2 with 1 x 10(7) platelets (Platelets), 1 x 10(7) platelets and 10 micrograms/mL of platelet-derived growth factor antibody (Platelets+anti-PDGF), 1 x 10(7) platelets and 1 microgram/mL of indomethacin (Platelets+Indomethacin), or 1 x 10(7) platelets and 3 micrograms/mL of mianserin (Platelets+Mianserin). Embryos were incubated at 39 degrees C in 5% CO2 in groups of five until 8 d after in vitro fertilization (IVF). In Exp. 1, Platelets stimulated embryo development to the morula, blastocyst, and expanded blastocyst stages. Embryo development was greatest in the BOEC+Platelets group on d 7 and 8 after IVF. Only embryos incubated in the BOEC+Platelets treatment group reached the hatched blastocyst stage on d 8. In Exp. 2, embryos incubated in the Platelets treatment group had the greatest (P < .05) proportion develop beyond the eight-cell stage. Embryos incubated in the Platelets + anti-PDGF group had less (P < .05) development beyond the eight-cell stage and to the morula stage. These results indicate that the stimulatory effects of PDGF on bovine embryo development may be derived from both the oviductal epithelium and platelets.

OBJECTIVE

Ex vivo expansion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) is a promising approach for overcoming the developmental delay of bone marrow (BM) reconstitution after transplantation. This project investigated the effects of culture duration, serum-free media, cytokine combinations, and chemotherapy on the outcomes of expansion.

METHODS

Enriched CD34+ cells were cultured for 8 or 10 d in serum-free media (QBSF-60 or X-Vivo 10) and four combinations of cytokines consisting of recombinant human pegylated-megakaryocyte growth and development factor, stem cell factor, flt-3 ligand, G-CSF, interleukin (IL)-6, platelet-derived growth factor (PDGF), and IL-1beta.

RESULTS

Eight days of culture in QBSF-60 significantly supported efficient expansions of CD34+ cells, CD34+ CD38- cells, colony-forming units (CFU) of myeloid, erythroid, megakaryocytic, and mixed lineages to 3.76-, 14.4-, 28.3-, 24.0-, 38.1-, and 15.7-fold, respectively. Whilst PDGF or IL-6 enhanced the expansion of early, myeloid, and erythroid progenitors, IL-1beta specifically promoted the megakaryocytic lineage. Engraftment of human CD45+ cells were detectable in all non-obese diabetic/severe-combined immunodeficient mice transplanted with expanded PBSC from donor samples, being 5.80 +/- 3.34% of mouse BM cells. The expansion and engraftment capacity of CD34+ cells from subjects postchemotherapy were significantly compromised across the panel of progenitor cells.

CONCLUSIONS

Our results provided an optimized protocol for PBSC expansion, applicable to ameliorating neutropenia and thrombocytopenia in post-BM transplant patients by the prompt provision of progenitor cells. For postchemotherapy patients, expansion products might provide committed progenitors for improving short-term engraftment, but not self-renewable stem cells.

BACKGROUND

The pathogenesis of chronic pancreatitis (CP) is a complex process of interaction between tissue injury and repair, which involves microcirculatory disturbance. Amygdalin, an effective component extracted from Semen Persicae (a kind of Chinese herbal medicine), can decrease blood viscosity and improve microcirculation. In this study, we investigated the therapeutic effects of amygdalin on pancreatic fibrosis in rats with CP.

METHODS

The rat CP model was induced by injecting dibutyltin dichloride (DBTC) into the right caudal vein. Amygdalin was administrated via the penile vein at a dose of 10 mg/(kg d) from the next day, after the induction of CP, once a day for the previous 3 days, and then once every 2 days, until the end of the experiment. Body weight was observed every 7 days. Pancreatic blood flow and histopathological changes were assessed at 28 days. The activation of pancreatic stellate cells (PSCs) was estimated by the expression of α-smooth muscle actin (α-SMA). At the same time, the expression of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β-1 (TGFβ-1), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP) of pancreatic tissues were detected.

RESULTS

Treatment of CP rats with amygdalin improved body weight and pancreatic blood flow, as well as alleviated pancreatic fibrosis and acinar destruction, accompanied by the down-regulation of the expressions of α-SMA, PDGF-BB, TGFβ-1, and ET-1, and the up-regulation of the CGRP's expression.

CONCLUSIONS

Amygdalin could reduce the production of pro-fibrotic cytokines, inhibit the activation of PSCs, and attenuate pancreatic fibrosis in a rat with CP. The mechanism probably includes improving microcirculatory disturbance by regulating the production of ET-1 and CGRP.

The growth and development of the corpus luteum after rupture of the follicle is a highly regulated process characterised by a rapid vascularization of the follicle surrounding granulosa cells. Vascularization is regulated by a large number of growth factors and cytokines whereas members of the angiopoietin family and VEGF-A are reported to play a principal role. The gonadotropic hormones luteinizing hormone and choriogonadotropin are reported to be essential for corpus luteum formation. In this study we investigated by RT PCR if the growth factors PGF, PDGF-A, PDGF-B, VEGF-A, VEGF-B, VEGF-C, VEGF-D, ANG1, ANG2, ANG3 and ANG4 are expressed in granulosa cells. We show the expression of VEGF-A, VEGF-B, PDGF-A, ANG1 and ANG2 in granulosa cells. Using RT-PCR and Real-Time PCR we demonstrate that angiopoietin 2 is downregulated in human granulosa cells in vitro after choriogonadotropin treatment whereas the expression of angiopoietin 1 is not significantly altered. The expression of VEGF on the RNA- and on the protein level was determined. It was shown that in granulosa cells VEGF is upregulated after choriogonadotropin treatment on the RNA level and that increasing concentrations of choriogonadotropin from 0 to 10 U/ml leads to an increasing amount of VEGF in the cell culture supernatants. The amount of VEGF in the supernatants reaches a plateau at 0.5 U/ml and is increased only slightly and not significantly after treatment of the cells with 10 U/ml choriogonadotropin compared to 0.5 U/ml. In total these findings suggests that in granulosa cells the mRNA of various growth factors is detectable by RT-PCR and that VEGF-A and ANG2 is regulated by the gonadotropic hormone choriogonadotropin. These findings may add impact on the hypothesis of choriogonadotropin as a novel angiogenic factor.

Platelet-derived growth factor (PDGF) induces cell proliferation together with oxidative stress. The present study investigated the effects of salvianolic acid A (Sal A) and B (Sal B) on the PDGF-induced signaling cascades in hepatic stellate cells (HSCs). HSC-T6, a rat hepatic stellate cell line, was stimulated with PDGF (10 ng/mL). The inhibitory effects of Sal A and B on oxidative stress-related signaling pathways were assessed in vitro. The protein levels were measured by Western blotting. FACS analysis was applied to detect the thioredoxin (Trx) level. Sal A and B showed different inhibitory abilities on the PDGF-related pathway. Sal A inhibited 70-kDa ribosomal S6 kinase (p70(s6k)) and associated proteins. Sal B attenuated PDGF-induced c-jun-N-terminal kinase (JNK), p38, and PKC- δ phosphorylations. Both Sal A and B diminished the activation of PKD, Trx, heme-oxygenase (HO)-1, and Nrf2. Taken together, our results showed that Sal A and B attenuated PDGF-induced ROS formation in HSCs, possibly through different signaling pathways.

PGI2 generation by the vessel wall is an agonist for cyclic-AMP-dependent cholesteryl ester hydrolysis. The process of enhanced PGI2 synthesis is stimulated, in part, by G-protein-coupled receptor ligands. Cellular cholesterol enrichment has been hypothesized to alter G-protein-mediated PGI2 synthesis. In the studies reported herein, cells generated PGI2 in response to AlF4-, GTPgammaS, and ATP in a dose-dependent manner. G-protein agonists stimulated eicosanoid production principally by activating phospholipase A2, but not phospholipase C. This is in contrast to PDGF, which stimulated phospholipase A2 and PLCgamma activities. Galphai subunits mediate G-protein agonist-induced PGI2 synthesis, since ATP- and PDGF-induced PGI2 synthesis was inhibited by pertussis toxin. Although cholesterol enrichment reduced arachidonic acid- and PDGF-induced PGI2 synthesis, cholesterol enrichment enhanced PGI2 release in response to AlF4-, GTPgammaS, and ATP. The enhancement of PGI2 release in cholesterol-enriched cells was augmented by mevalonate, which inhibits the ability of cholesterol enrichment to reduce membrane-associated G-protein subunits. Since cholesterol enrichment inhibited PDGF and AlF4--induced MAP kinase activity [Pomerantz, K., Lander, H. M., Summers, B., Robishaw, J. D., Balcueva, E. A., & Hajjar, D. P. (1997) Biochemistry 36, 9523-9531] (the major mechanism by which phospholipase A2 is activated), these results suggest that cholesterol enrichment induces other alternative signaling pathways leading to phospholipase A2 activation. A PKC-dependent pathway is described herein that is involved in enhanced eicosanoid production in cholesterol-enriched cells. This conclusion is supported by two observations: (1) G-protein-linked PGI2 production is inhibited by calphostin, and (2) cholesterol enrichment augments the specific translocation of the delta-isoform of PKC from the cytosol to the plasma membrane following treatment of cells with phorbol ester. These data support the concept that, in cells possessing normal levels of cholesterol, MAP-kinase-dependent pathways mediate eicosanoid synthesis in response to G-protein activation; however, under conditions of high cellular cholesterol levels, augmented G-protein-linked eicosanoid production results from enhanced PKCdelta activity.

When cultured in the presence of fetal calf serum, aortic vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) grow faster than those from normotensive control Wistar-Kyoto (WKY) rats. In order to investigate the mechanism underlying this growth abnormality, we measured phospholipase D (PLD) activity in VSMC taken from both SHR and WKY rats. Upon stimulation with serum, platelet-derived growth factor (PDGF) and porbol 12-myristate 13-acetate (TPA), phosphatidylethanol (PEt) was produced in the presence of ethanol. The responses of the VSMC from SHR (SHR-cells) to all stimuli were significantly greater than those of the VSMC from WKY rats (WKY-cells), which suggests an enhanced PLD activity in the SHR-cells. Since PLD is regarded as an enzyme involved in signal transduction leading to cell proliferation, this PLD hyper-reactivity in the SHR-cells may account at least partially for the growth abnormality in the SHR-cells.

The aim of this work was to study the bone repair induced by bone morphogenetic protein-2 (BMP-2), rat mesenchymal stem cells (rMSCs), and platelet-derived growth factor (PDGF-BB) incorporated in a macroporous beta-tricalcium phosphate (β-TCP) system fabricated by robocasting, and to identify the most beneficial combination in a critical rat calvaria defect. BMP-2 was formulated in microspheres to provide a prolonged, local concentration, whereas PDGF-BB, which acts during the initial stage of defect repair, was incorporated in a thin layer of crosslinked alginate. Approximately 80% of PDGF-BB and 90% of BMP-2 were released into the defect during the first 2 d and 3 weeks, respectively. Histological analyses indicated a minor synergistic effect in the BMP-2-MSC groups. In contrast, significant antagonism was found with combined BMP-2 and PDGF-BB defect treatment. The high-grade repair induced by BMP-2 rules out any advantage from combining BMP-2 with PDGF-BB or MSCs, at least with this scaffold and defect model.

Obstructive nephropathy is the most common presentation of urothelial carcinoma. The role of the urine in the obstructed kidney namely "hydronephrotic urine" in urothelial carcinoma has not been extensively explored. This study aims to evaluate whether hydronephrotic urine in the obstructed kidney could promote urothelial carcinoma. The hydronephrotic urine was collected from the obstructed kidneys of Sprague-Dawley rats induced by different periods of unilateral ureteral obstruction (UUO). By the inhibition of LY294002 and PDDDK2. It also decreases the expression of p27 and p21 in both urothelial carcinoma cells and normal urothelial cells. By the protein array study, we demonstrate that many growth factors which promote tumor cell survival and metastasis are over-expressed in a time-dependent manner in the hydronephrotic urine, including beta-FGF, IFN-γ, PDGF-BB, PIGF, TGF-β, VEGF-A, VEGF-D and EGF. These results suggest that hydronephrotic urine promotes normal and malignant urothelial cells proliferation, migration and invasion, through the activation of the mTORC2-AKT and ERK signaling pathways. Further investigation using live animal models of tumor growth may be needed to clarify aspects of these statements.

BACKGROUND

Emerging evidence implicates the platelet-derived growth factor-D (PDGF-D) in many types of human solid tumors. We investigated whether PDGF-D plays an important role in endometrial cancer (EC) in relation to clinicopathologic phenotype, angiogenesis, and patient prognosis.

METHODS

We analyzed PDGF-D protein expression by Western blotting in twenty-seven human endometrial cancer tissues, and matched normal endometrial controls collected at the third Affiliated hospital of Sun Yat-sen University during 2012-2013 (n=27). Immunohistochemical staining was performed using a human PDGF-D antibody on the endometrial cancer patients collected in the same facility during January 2001 and October 2013 (n=152). Patients were followed from the time of primary surgery in 2001-2013 until death or last follow-up. We correlated the PDGF-D expression levels with clinicopathologic parameters and prognosis in human endometrial cancer patients.

RESULTS

Compared with matched normal endometrial cases, PDGF-D was up-regulated in endometrial cancer. Expression of PDGF-D protein, found in 78% of the cases, was associated with nonendometrioid histologic type (p=0.028), FIGO stage III/IV (p=0.039), >50% solid tumor growth (p=0.048), pelvic LN metastasis (p=0.035) and ER and PR negativity (p=0.04 and 0.002). PDGF-D expression was also significantly associated with expression of VEGF-A (p=0.021). In multivariate analysis, PDGF-D expression proved to be an independent prognostic factor in addition to histologic grade and FIGO stage. Patients with high expression levels of PDGF-D had a significantly poorer overall survival rate compared with patients with no expression.

CONCLUSIONS

PDGF-D expression is frequently up-regulated in endometrial cancer, and is associated with aggressive features and poor prognosis.

Pleural malignant mesothelioma is a rare but deadly tumour mainly induced by asbestos inhalation. Despite the ban of asbestos in 1990 in 52 countries, mesothelioma cases still increase worldwide. In pleural mesothelioma, p38 mitogen activated protein kinases (MAPK) have been suggested to play a major role in carcinogenesis and aggressiveness of tumours. The aim of this study was to determine the role of the different four p38 MAPK isoforms and their effect on proliferation together with the underlying signalling pathways in a rat pleural mesothelioma cell line. Rat pleural mesothelioma cells were stimulated with platelet-derived growth factor (PDGF)-BB and/or transforming growth factor beta (TGF)-β. MAPK and transcription factor expression and activation was monitored in the cytosol and nucleus by immuno-blotting. Proliferation was determined by manual cell count and siRNAs were used to control MAPK and transcription factor expression and action. Only PDGF-BB, but not TGF-β1 induced proliferation via activated Erk1/2 and p38 MAPK. The p38α and δ isoforms were expressed in the cytosol, and upon activation p38δ translocated into the nucleus, while p38α remained in the cytosol. No other p38 isoform was expressed by rat mesothelioma cells. C/EBP-α was found in both the cytosol and the nucleus, while C/EBP-β was not expressed at all. PDGF-BB induced proliferation was suppressed by down-regulation of either Erk1/2, or p38δ MAPK, or C/EBP-α. Furthermore, TGF-β inhibited PDGF-BB induced proliferation by interruption of p38 MAPK signalling. From this rat model, we conclude that in pleural mesothelioma, p38δ in C/EBP-α mediate proliferation and thus may represent new targets in mesothelioma therapy.

PtdIns (3, 4, 5) P3 is formed rapidly in NIH-3T3 cells stimulated with platelet derived growth factor (PDGF). We have determined the pathway of formation of this lipid in these cells. Cells were labeled briefly with 32PO4 and then stimulated with PDGF under conditions where the specific activity of each phosphate group determines the order of its addition. The D-5 phosphate of this lipid contained approximately 42% of the total radioactivity present in the molecule, while approximately 32% was in the D-4 position, 25% in the D-3 position, and approximately 2% in the D-1 position. This indicates that PtdIns (3, 4) P2 and not PtdIns (4, 5)P2 is the immediate precursor of PtdIns (3, 4, 5) P3, and defines the pathway of formation of these lipids to be PtdIns (3) P----PtdIns (3, 4) P2----PtdIns (3, 4, 5) P3. This pathway is the same as that in thrombin-stimulated platelets and infers that the pathways are not different in non-growing and proliferating cells.

We have studied, at the mRNA level, the influence of various defined growth conditions on the expression of TGF-alpha, PDGF-BB, EGF-R, PDGF-R alpha, and PDGF-R beta in five different glioma cell lines (D-37MG, D-54MG, D-263MG, GaMG, and U-251MG). RNA isolated from logarithmically growing, confluent monolayer cells or multicellular spheroids was analyzed. Northern blot experiments show that with a few exceptions, specific mRNA steady-state levels were considerably higher in cells grown in a three-dimensional organization relative to cells in the logarithmic growth phase.

OBJECTIVE

Cyclopentenone prostaglandins have been shown to promote osteoblast differentiation in vitro. The aim of this study was to examine in a rat model the effects of local delivery of Delta(12)-prostaglandin J(2) (Delta(12)-PGJ(2)) on new bone formation and growth factor expression in (i) cortical defects and (ii) around titanium implants.

METHODS

Standardized transcortical defects were prepared bilaterally in the femur of 28 male Wistar rats. Ten microliters of Delta(12)-PGJ(2) at 4 concentrations (10(-9), 10(-7), 10(-5) and 10(-3) mol/l) in a collagen vehicle were delivered inside a half-cylindrical titanium chamber fixed over the defect. Contralateral defects served as vehicle controls. Ten days after surgery, the amount of new bone formation in the cortical defect area was determined by histomorphometry and expression of platelet-derived growth factor (PDGF)-A and -B, insulin-like growth factor (IGF)-I/II, bone morphogenetic protein (BMP)-2 and -6 was examined by immunohistochemistry. In an additional six rats, 24 titanium implants were inserted into the femur. Five microliters of carboxymethylcellulose alone (control) or with Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) were delivered into surgically prepared beds prior to implant installation.

RESULTS

Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) significantly enhanced new bone formation (33%, P<0.05) compared with control cortical defects. Delivery of Delta(12)-PGJ(2) at 10(-3) mol/l significantly increased PDGF-A and -B and BMP-2 and -6 protein expression (P<0.05) compared with control defects. No significant difference was found in IGF-I/II expression compared with controls. Administration of Delta(12)-PGJ(2) also significantly increased endosteal new bone formation around implants compared with controls.

CONCLUSIONS

Local delivery of Delta(12)-PGJ(2) promoted new bone formation in the cortical defect area and around titanium implants. Enhanced expression of BMP-2 and -6 as well as PDGF-A and -B may be involved in Delta(12)-PGJ(2)-induced new bone formation.