Fibroblast-like synoviocytes (FLS) play a major role in invasive joint destruction in rheumatoid arthritis (RA). This prodestructive phenotype has been shown to involve autocrine TGF-β that triggers formation of matrix-degrading invadosomes through molecular mechanisms that are not fully elucidated. The platelet-derived growth factor (PDGF) receptor (PDGFR) family of receptor tyrosine kinases (RTK) has been shown to cooperate with TGF-β in various pathological conditions. We therefore sought to determine whether RTK activity played a role in invadosome biogenesis. We demonstrated that, among the common RTKs, PDGFR-αβ was specifically phosphorylated in FLS from RA patients. Phosphorylation of PDGFR-αβ was also elevated in RA synovial tissues. Interference with PDGFR activation or PDGF neutralization inhibited invadosome formation in RA synoviocytes, indicating the presence of an autocrine PDGFR activation loop that involved endogenous PDGF. Among the PDGF-A-D isoforms, only PDGF-B was found both significantly elevated in FLS lines from RA patients, and related to high-invadosome forming cells. Addition of TGF-β upregulated invadosome formation, PDGF-B mRNA expression, and phosphorylation of PDGFR. All of these functions were efficiently suppressed by TGF-β neutralization or interference with the Smad/TβR1or PI3K/Akt pathway. Among the class 1 PI3K family proteins known to be expressed in RA synoviocytes, PI3Kα was selectively involved in PDGF-B expression, whereas both PI3Kα and PI3Kδ participated in invadosome formation. Our findings demonstrate that PDGFR is a critical RTK required for the prodestructive phenotype of RA synovial cells. They also provide evidence for an association between autocrine TGF-β and PDGFR-mediated invadosome formation in RA synoviocytes that involves the production of PDGF-B induced by TGF-β.

Wound healing evaluation is important in forensic pathology, in which angiogenesis plays an important role. We have already shown that vascular endothelial growth factor A (VEGF) is produced in the rat skin incision wounds by neutrophils, endothelial cells, and fibroblasts. In this study, we assessed the changes in the mRNA expressions of various factors possibly involved in angiogenesis including angiopoietin (ANGPT) 1 and 2, cadherin 5 (CDH5), granulocyte-macrophage colony stimulating factor (CSF2/GM-CSF), granulocyte colony stimulating factor (CSF3/G-CSF), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand12 (CXCL12/SDF1), endothelin 1 (ET1), fibroblast growth factor 1 (FGF 1), hepatocyte growth factor (HGF), hypoxia inducible factor 1 alpha (HIF1a), leptin, matrix metallopepitidase 9 (MMP9), serpine/plasminogen activator inhibitor1 (PAI1), platelet-derived growth factor-A (PDGF-A), transforming growth factor alpha and beta 1 (TGFa and b1), tenomodulin (TNMD), and troponin I type 2 (TNNI2) in the early stage of the rat skin incision wounds by real time RT-PCR. Factors reported to be involved in lymphangiogenesis such as fibroblast growth factor 2 (FGF 2), c-fos induced growth factor (FIGF/VEGF-D), forkhead box C2 (FOXC2), and prospero homeobox 1 (PROX1) were also studied. One and 3 days after the dorsal skin incisions, wounds on male Sprague-Dawley rats showed the statistically significant increases in the mRNA expressions for CXCL2, CSF3, MMP9, PAI1, and CSF2, whereas TGFa, TNNI2, FGF1, TNMD, leptin, and CXCL12 showed the statistically significant decreases. Interestingly, lymphgangiogenic factors FOXC2, PROX1, and FGF2 also showed the statistically significant decreases. In situ hybridization and immunohistochemistry showed the mRNA and protein positivity in endothelial cells, fibroblasts, and some leukocytes at the bottom of the wound tissue for PAI1, CSF3, and MMP9, 1 day after the skin incisions. Our novel findings show the possible involvement of several factors involved in angiogenesis and lymphangiogenesis in the early stage of wound healing process, which may be useful for forensic wound evaluations.

BACKGROUND

Podocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases.

RESULTS

By microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation.

CONCLUSIONS

Schip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling via interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated.

Intrapleural instillation of tetracycline hydrochloride (TCN) is an effective means of achieving pleural fibrosis. However, its mechanism of action remains unknown. To evaluate the hypothesis that TCN stimulates pleural mesothelial cells to release growth-factor-like activity for fibroblasts we performed the following experiments. Rat visceral pleural mesothelial cells were incubated with TCN at doses ranging from 0.01 microgram/ml to 100 mg/ml. The conditioned media (CM) were collected after incubation for 2 to 48 h. CM caused fibroblasts to increase incorporation of thymidine when compared with CM that was unexposed to TCN (p less than 0.05). This growth-factor-like activity continued to be produced by mesothelial cells for 48 h after removal of TCN from the medium. There was a dose-response relationship since increasing doses of TCN to as much as 1 mg/ml caused increasing production of growth-factor-like activity without mesothelial cell injury as measured by trypan blue exclusion. The growth factor activity was a competence-type activity. It coeluted with human PDGF at a molecular weight of 31,000. It was heat-stable (100 degrees C for 10 min) and sensitive to trypsin and papain but not to heat-inactivated trypsin. Addition of cycloheximide or actinomycin D inhibited its production. TCN did not have any direct effect on fibroblasts. Bleomycin CM did not contain growth-factor-like activity for fibroblasts. These data demonstrate that TCN stimulates mesothelial cells to release a growth-factor-like activity for fibroblasts. This phenomenon may play an important role in TCN-induced pleural fibrosis.

In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.

Meningothelial hyperplasia is a poorly characterized entity, often associated with advanced age, chronic renal failure, trauma, hemorrhage, and neoplasia. In order to elucidate the nature of this lesion, 11 cases defined by the presence of nests of 10 or more cell layers thick, were compared with normal arachnoidal cap cells and meningiomas. Immunohistochemistry and FISH were performed to determine NF2 (merlin), protein 4.1B, EMA, progesterone receptor (PR), EGFR, survivin, VEGF, PDGF-BB, PDGFR-beta, E-cadherin, and cathepsin D status. All cases had at least one putative predisposing factor, including hemorrhage (7), chronic renal disease (5), old age (5), trauma (1), and an adjacent optic nerve pilocytic astrocytoma (1). There was typically a discontinuous growth pattern, with no invasion of surrounding normal tissue. No gene deletions were found, though scattered polyploid cells were seen in 2 cases. The immunoprofile was similar to normal cap cells with one exception; whereas normal cells were uniformly negative for PR, nuclear positivity was seen in 64% of hyperplasias, a frequency similar to that of benign meningiomas. Our data suggest that meningothelial hyperplasia is a reactive process that is usually distinguishable from meningioma based on clinicopathologic and genetic features. It may be preneoplastic in some, though further studies are needed to test this hypothesis.

2,3,4',5-tetrahydroxystilbene-2-0-β-D glucoside (TSG) has been recognized to suppress the proliferation of vascular smooth muscle cells (VSMCs). The aim of the present study was to determine whether TSG inhibits neointimal hyperplasia in a rat carotid arterial balloon injury model. Balloon injury was induced in the left common carotid artery of rats. TSG (30, 60, 120 mg/kg/day) was treated from 3 days prior to, until 14 days after the induction of balloon injury. The ratio of intima-to-media was significantly reduced in the TSG-treated rats at 14 days after the induction of injury, which was associated with reduced expressions of proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA) and platelet-derived growth factor-BB (PDGF-BB), as markers of VSMCs proliferation and migration. Additionally, TSG significantly inhibited PDGF-BB induced cell migration in cultured VSMCs. Furthermore, we explored the underlying mechanisms for such effects of TSG. The result showed that TSG markedly reduced balloon injury-induced AKT, extracellular signal-regulated kinase (ERK1/2) and nuclear factor kappaB (NF-κB) activation as well as mRNA expressions of c-myc, c-fos and c-jun, which is important signal pathway for VSMCs proliferation. And in both vivo and vitro model, TSG markedly regulated matrix metalloproteinase-2, 9 expressions and collagen I, III expressions, which are key factors in extracellular matrix for VSMCs migration. These results suggest that the anti-proliferative and anti-migrative effects of TSG on VSMCs could help to explain the beneficial effects of TSG on neointima hyperplasia induced by balloon injury.

To investigate the effect of naringenin eye drops in corneal neovascularization induced by alkali (1 N NaOH) burn in mice.

Corneal neovascularization in the right eye of male Swiss mice was induced by alkali. Treatment with naringenin eye drops (0.08-80 μg; 8 μL of 0.01-10 g/L solution) or vehicle (saline) started 2 days before corneal neovascularization was induced and was performed twice a day. Mice were treated up until the time animals were euthanized and cornea tissue was collected for testing, which was 2, 4, and 6 hours after alkali stimulus for cytokine and antioxidant capacity measurements, and 3 and/or 7 days after alkali stimulus for the assessment of corneal epithelial thickness and neovascularization, neutrophil, and macrophage recruitment, and vascular endothelial growth factor (Vegf), platelet-derived growth factor (Pdgf), matrix metalloproteinase-14 (Mmp14), and pigment epithelium-derived factor (Pedf) mRNA expression.

Naringenin eye drops inhibited alkali burn-induced neutrophil (myeloperoxidase activity and recruitment of Lysm-GFP+ cells) and macrophage (N-acetyl-β-D glucosaminidase activity) recruitment into the eye, decrease in epithelial thickness, and neovascularization in the cornea. Further, naringenin inhibited alkali-induced cytokine (IL-1β and IL-6) production, Vegf, Pdgf, and Mmp14 mRNA expression, and the reduction of ferric reducing antioxidant power and Azinobis-(3-Ethylbenzothiazoline 6-Sulfonic acid) radical scavenging capacity as well as increased the reduced glutathione and protein-bound sulfhydryl groups levels.

Collectively, these results indicate that naringenin eye drops are protective in alkali-induced corneal burn by inhibiting leukocyte recruitment, the proangiogenic factor expression, inflammatory cytokine production, and loss of antioxidant defenses.

Rheumatoid arthritis (RA) is an immune-mediated disease with the characteristics of progressive joint destruction, deformity, and disability. Epigenetic changes have been implicated in the development of some autoimmune disorders, resulting in an alteration of gene transcription. Here, we investigated how Jumonji C family of histone demethylases (JMJ<em>D</em>3) regulated the proliferation and activation of fibroblast-like synoviocytes (FLSs), which are involved in RA joint destruction and pathologic process. The JMJ<em>D</em>3 expression and proliferation markers in RA-FLS were higher than those in healthy-FLS and were upregulated in platelet-derived growth factor (<em>PDGF</em>)-induced FLS. Elevated JMJ<em>D</em>3 promoted the proliferation and migration of FLS. Treatment with JMJ<em>D</em>3 small interfering RNA or inhibitor glycogen synthase kinase (GSK) J4 led to decreased proliferation and migration of FLS. Interestingly, induction of proliferating cell nuclear antigen (PCNA), a major player of the cell-cycle regulation, was correlated with trimethylated lysine 27 in histone H3 loss around the gene promoters. The knockdown of JMJ<em>D</em>3 abolished PCNA expression in <em>PDGF</em>-induced FLS and further inhibited cell proliferation and migration, suggesting that JMJ<em>D</em>3/PCNA played a crucial role in aspects of FLS proliferation and migration. In vivo, the ability of GSK J4 to hinder collagen-induced arthritis (CIA) in <em>D</em>BA/1 mice was evaluated. We found that GSK J4 markedly attenuated the severity of arthritis in CIA mice. The therapeutic effects were associated with ameliorated joint swelling and reduced bone erosion and destruction. This study revealed how JMJ<em>D</em>3 integrated with epigenetic processes to regulate RA-FLS proliferation and invasion. These data suggested that JMJ<em>D</em>3 might contribute to rheumatoid synovial hyperplasia and have the potential as a novel therapeutic target for RA.-Jia, W., Wu, W., Yang, <em>D</em>., Xiao, C., Su, Z., Huang, Z., Li, Z., Qin, M., Huang, M., Liu, S., Long, F., Mao, J., Liu, X., Zhu, Y. Z. Histone demethylase JMJ<em>D</em>3 regulates fibroblast-like synoviocyte-mediated proliferation and joint destruction in rheumatoid arthritis.

Due to a lack of sufficient diagnostic tools to predict aggressive disease, there is a significant overtreatment of patients with prostate cancer. Platelet derived growth factors (PDGFs) and their receptors (PDGFRs) are key regulators of mesenchymal cells in the tumor microenvironment, and has been associated with unfavorable outcome in several other cancers. Herein, we aimed to investigate the prognostic impact of PDGFR-β and its ligands (PDGF-B and PDGF-D) in a multicenter prostatectomy cohort of 535 Norwegian patients. Using tissue microarrays and immunohistochemistry, the expression of ligands PDGF-B and PDGF-D and their corresponding receptor, PDGFR-β, was assessed in neoplastic tissue and tumor-associated stroma. PDGFR-β was expressed in benign and tumor associated stroma, but not in epithelium. High stromal expression of PDGFR-β was independently associated with clinical relapse (HR = 2.17, p = 0.010) and biochemical failure (HR = 1.58, p = 0.002). This large study highlights the prognostic importance of PDGFR-β expression, implicating its involvement in prostate cancer progression even in early stage disease. Hence, analyses of PDGFR-β may help distinguish which patients will benefit from radical treatment, and since PDGFR-β is associated with relapse and shorter survival, it mandates a focus as a therapeutic target.

The purpose of this study was to test the hypotheses that development of mature vimentin+/α-smooth muscle actin+/desmin+ (V+A+D+) myofibroblasts from corneal fibroblasts is regulated by transforming growth factor (TGF) β and platelet-derived growth factor (PDGF); and that myofibroblast development in vitro follows a similar developmental pathway as it does in vivo. Mouse corneal stromal fibroblasts (MSF) were isolated from the corneas of Swiss Webster mice and cultured in serum-free media augmented with DMEM/F12 and varying doses of TGFβ (0.1-2.0 ng/ml), with and without mouse PDGF-AA and/or PDGF-BB (2.0 ng/ml), to study the transition of the MSF to V+A+D+ myofibroblasts. The mean percentage of vimentin+, α-SMA+ and desmin+ cells was determined at each time point (2-15 days), with each growth factor concentration. MSF in vitro were noted to undergo the same developmental transition from V+A-D- to V+A+D- to V+A+D+ myofibroblasts as precursors undergo in vivo. TGFβ at a dose of 0.5 ng/ml and 1.0 ng/ml with 2.0 ng/ml PDGF-AA and 2.0 ng/ml PDGF-BB in DMEM/F12 serum-free media was optimal for the development of V+A+D+ myofibroblasts. This study defines optimal in vitro conditions to monitor the development of MSF into myofibroblasts. The combined effects of TGFβ and PDGF promote the full development of V+A+D+ myofibroblasts from MSF.

Alternative splicing results in two distinct forms of the platelet-derived growth factor (PDGF) A-chain that differ by a hydrophilic carboxyl terminus consisting of 18 amino acids (A194-211). The functional significance of this region is unclear. Previous results indicate that a radioiodinated tyrosinated synthetic peptide corresponding to A194-211 binds specifically, saturably, and with low affinity to a large population of sites on Balb/c 3T3 and several other cell lines (Khachigian, L. M., Owensby, D. A., and Chesterman, C. N. (1992) J. Biol. Chem. 267, 1660-1666). In this paper, we report that (Y)A194-211 and A194-211 can modulate the cellular proliferative response to normal human serum and several individual polypeptide growth factors as a consequence. When these peptides were coincubated separately with whole serum, PDGF, epidermal growth factor, or fibroblast growth factor, DNA synthesis was inhibited in a dose-dependent fashion and maximally by 200 microM peptide. In addition, both peptides could attenuate the stimulation of cell division by serum and PDGF. Peptides with similar charge or length failed to modify the level of proliferation. These observations were not due to cytotoxicity. Synthetic peptides such as (Y)A194-211 and A194-211 that influence cellular proliferation may be useful in modulating the cellular response to selected growth factors.

Abnormal vascular smooth muscle cell (VSMC) proliferation and migration are involved in restenosis following percutaneous transluminal angioplasty (PTCA) as well as in the development and progression of atherosclerosis. We investigated the mechanisms underlying the inhibitory effect of the sesquiterpene 3-oxo-5alphaH,8betaH-eudesma-1,4(15),7(11)-trien-8,12-olide (1) on rat VSMC proliferation and migration. VSMCs were isolated from rat aorta, and then the effect of 1 on cell proliferation and migration was examined using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and chemotaxis assays, respectively. Compound 1 had a potent inhibitory effect on fetal calf serum-induced VSMC proliferation. This effect correlated with reduced expression of cyclin D(1). In addition, 1 also inhibited platelet derived growth factor (PDGF)-induced migration of VSMCs. These results indicate that 1 is a promising candidate for additional biological evaluation to further define its potential as an inhibitory modulator of VSMC responses that contribute to restenosis following PTCA and to the development and progression of atherosclerosis.

BACKGROUND

Platelet-derived growth factor (PDGF) antagonists have demonstrated beneficial effects on neointima formation, but in studies using PDGF inhibitors and extended follow-up, the lesions reoccur. These findings implicate a need to combine targeting of PDGF with other strategies. Stimulation of reendothelialization by treatment with endothelial cell mitogens of the vascular endothelial growth factor (VEGF) family counteracts restenosis, but there are also concerns regarding the durability of the effect with this approach.

RESULTS

To explore whether a combined use of PDGF antagonist and stimulation of reendothelialization confers better results than each therapy alone, we combined systemic administration of imatinib mesylate (STI571/Gleevec, 10 mg/kg(-1) per d(-1)), a tyrosine kinase inhibitor with activity against PDGF receptors, with local intravascular adenovirus-mediated VEGF-C gene transfer (1.15x10(10) pfu) in cholesterol-fed, balloon-injured rabbits. Throughout the course of the STI571 therapy, the circulating concentrations were able to suppress PDGF receptor phosphorylation. At 3 weeks, the treatment with STI571 led to a transient decrease in intralesion macrophages and to an increase in intimal smooth muscle cell apoptosis. VEGF-C application reduced neointima formation and accelerated reendothelialization. However, none of the therapies alone reduced intimal thickening at a 6-week time point, whereas the combined treatment led to a persistent reduction (55% versus control) in lesion size at this time point.

CONCLUSIONS

Our study provides one of the first successful examples of gene therapy combined with a pharmacological treatment to modulate 2 distinct ligand-receptor signaling systems and suggests combination of local VEGF-C gene therapy with systemic inhibition of PDGF signaling as a novel principle to prevent intimal hyperplasia after vascular manipulations.

Polyoma virus (Py) causes neoplastic transformation in vitro and multiple tumors in vivo. The role played by large and middle T antigens (LT, MT) and their mechanisms of action are focused here. Py-transformed Balb-3T3 cells become independent of platelet-derived growth factor (PDGF) for growth. JE, c-fos, c-jun and c-myc are 'immediate early' genes induced in response to PDGF. To test whether these cellular genes play a role in malignant transformation by Py, we generated a number of transfectant cell lines overexpressing LT, MT or both. Characterization of these cell lines revealed that: (a) MT but not LT causes morphological transformation, ability to grow in agarose suspension; (b) cooperation between LT and MT is evident in vitro, however, high and simultaneous LT and MT expression does not warrant tumorigenic potential; (c) MT expression does not correlate with tumorigenic potential but alters the probability of eliciting tumors; (d) JE and c-myc (but not c-fos or c-jun) are constitutively expressed in MT transfectants. MT induction is followed by c-myc induction 1.5 h later. We conclude that some of the 'immediate-early' genes may play pivotal roles in Py transformation.

The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a key feature of the preneoplastic cirrhotic liver. Fibrogenic cells activate in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. We investigated the effects of quercetin on the amphiregulin/EGFR signal and on the activation of downstream pathways leading to cell growth. Rats were divided into 4 groups (8 rats/group): rats subjected to common bile duct ligation (CBDL), Sham (rats subjected to simulated CBDL), quercetin-treated sham, and quercetin-treated CBDL (CBDL-Q). Quercetin (50 mg/kg i.p. injection) was administered daily for 2 wk starting on d 14 after surgery. Overexpression of amphiregulin, EGFR, TNFα, IL-6, TGFβ, platelet-derived growth factor (PDGF), extracellular regulated kinase, protein kinase B (Akt), cycloxygenase (COX)-2, and glioma-associated oncogenes (GLI)-1 and-2 were observed in liver of CBDL rats after 4 wk of bile duct ligation. CBDL-Q rats had a significantly diminished expression of amphiregulin and EGFR compared with untreated CBDL rats. Furthermore, mRNA levels of TNFα, IL-6, TGFβ, and PDGF and the protein content of COX-2, GLI-1, and GLI-2 were significantly lower in CBDL-Q rats than in untreated CBDL rats. The findings indicate that quercetin ameliorated activation of survival pathways and downregulated the expression of genes related to inflammation and precancerous conditions. Suppression of amphiregulin/EGFR signals may contribute to this effect.

OBJECTIVE

Our earlier study suggested that platelet-derived growth factor (PDGF)- ββ receptor regulates chemotaxis of human malignant mesothelioma cells such as MSTO-211H, NCIH-2052, NCIH-2452, and NCIH-28 cells, but not non-malignant Met5A cells. The present study was designed to gain further insight into the PDGF-ββ receptor signals underlying the chemotaxis.

METHODS

PDGF-D secreted from cells, activation of Akt and ERK, and cell migration were monitored for cells with and without knocking-down PDGF-ββ receptor.

RESULTS

FBS significantly stimulated PDGF-D secretion from malignant mesothelioma cells, but not Met5A cells. PDGF-D activated Akt and ERK in both the non-malignant and malignant cells. PDGF-D significantly facilitated migration of malignant mesothelioma cells, but not Met5A cells, with the extent varying among the cell types. The facilitatory action of PDGF-D was clearly prevented by knocking-down PDGF-ββ receptor or inhibitors of PI3 kinase, PDK1, Akt, Rac1, ROCK, and MEK.

CONCLUSIONS

The results of the present study indicate that PDGF-D promotes malignant mesothelioma cell chemotaxis through PDGF-ββ receptor signaling pathways along a PI3 kinase/PDK1/Akt/Rac1/ROCK axis and relevant to ERK activation.

Actinomycin D was revealed as an inhibitor of Shc/Grb2 interaction in cell lines from our recent study. Shc and Grb2 proteins are important molecules in Ras signaling pathways leading to cellular differentiation and proliferation, which require dramatic morphological changes. It was detected by transmission electron microscopy that actinomycin D induced significant changes in cellular ultrastructures of B104-1-1 cells and confirmed that the changes were due to inhibition of Shc/Grb2 interaction by actinomycin D rather than its inhibitory effect on transcription. Because actinomycin D was dispersed mainly in cytoplasm and Shc peptide (synthetic 13 amino acid tyrosine phosphorylated polypeptide) successfully displaced actinomycin D binding to its cellular targets while the other polypeptide from PDGF receptor could not. We examined the effect of actinomycin D on growth of B104-1-1 tumor xenografted in nude mice. Tumor growth was inhibited in vivo after treatment with this inhibitor. Efficacy was correlated with a reduction in the levels of Shc/Grb2 binding in excised tumors. These results suggest that actinomycin D inhibited Shc/Grb2 interaction in B104-1-1 tumor xenografted in nude mice.

BACKGROUND

Increased platelet activation caused by an immunosuppressive therapy regimen may contribute to the high incidence of death from cardiovascular disease in renal transplant patients. Cyclosporine (INN, ciclosporin) and azathioprine are reported to activate platelets, but data are rare and controversial for tacrolimus and mycophenolate mofetil.

METHODS

This cross-sectional study assessed markers of platelet degranulation (P-selectin; CD62), the activated glycoprotein IIb/IIIa receptor (PAC1, indicating the fibrinogen binding site), platelet aggregation, and secretion of platelet-derived growth factor (PDGF(AB)) in renal transplant patients treated with 4 different therapy regimens. Immunosuppression was based on low-dose steroids (5 mg/d prednisone) in combination with a single agent: (1) cyclosporine (n = 16), (2) azathioprine (n = 18), (3) tacrolimus (n = 17), or (4) mycophenolate mofetil (n = 13). Effects were compared with those in an age-matched control group of patients with hypertension (n = 11).

RESULTS

In all renal transplant patient groups, unactivated platelets exhibited an increased expression of CD62. When stimulated with 2-micromol/L thrombin receptor-activating peptide, CD62 expression in platelets from patients treated with azathioprine (63% +/- 17%; P <.05), cyclosporine (51% +/- 23%; P <.05), and tacrolimus (50% +/- 22%; P <.05) was elevated compared with control subjects (33% +/- 19%). PAC1 expression was significantly increased in the patient groups that received azathioprine and cyclosporine. PDGF(AB) secretion was elevated in patients treated with azathioprine only (51 +/- 24 ng/10(9) platelets [versus 35 +/- 17 ng/10(9) platelets for control subjects]; P <.05). Platelet aggregation in response to collagen (0.5 microg/mL) was decreased in patients treated with tacrolimus (49% +/- 29%; P <.05) and mycophenolate mofetil (55% +/- 32%; P <.05) compared with control subjects (73% +/- 25%).

CONCLUSIONS

This is the first study to compare the effects on platelet function of different immunosuppressive regimens that are based on monotherapy. All renal transplant patients showed preactivated platelets compared with those of patients with hypertension. However, the "newer" immunosuppressive agents tacrolimus and mycophenolate mofetil seemed to have fewer unfavorable effects on platelet CD62 expression and PAC1 expression and aggregation. Whether this finding is accompanied by fewer cardiovascular events remains to be elucidated.

Our previous studies demonstrated coordinate expression of platelet-derived growth factor (PDGF) -B chain and beta-receptor in neurons at risk in the rat brain with focal ischemia. To clarify a role of the -B chain in the brain further, we examined whether PDGF-A or -B chain protects CA1 pyramidal neurons from delayed neuronal death after forebrain ischemia in rats. Pretreatment with PDGF-BB, but not -AA, at 120 ng/d for 2 days until forebrain ischemia was performed markedly ameliorated delayed neuronal death in CA1 pyramidal neurons on day 7 after ischemia. This neuroprotective effect of PDGF-BB was dose-dependent, and pretreatment with PDGF-BB at 240 ng/d showed almost complete inhibition of delayed neuronal death. In contrast, posttreatment with PDGF-BB at 120 ng/d starting 20 minutes after ischemia demonstrated no significant neuroprotective effect. The current study established marked neuroprotective actions of PDGF-BB in ischemic neuronal damage.

We examined the massive early cell death that occurs in the ventral horn of the cervical spinal cord of the chick embryo between embryonic days 4 and 5 (E4 and E5). Studies with immunohistochemical, in situ hybridization, and retrograde-tracing methods revealed that many dying cells express Islet proteins and Lim-3 mRNA (motoneuron markers) and send their axons to the somatic region of the embryo before cell death. Together, these data strongly suggest that the dying cells are somatic motoneurons. Cervical motoneurons die by apoptosis and can be rescued by treatment with cycloheximide and actinomycin D. Counts by motoneuron numbers between E3.5 and E10 revealed that, in addition to cell death between E4 and E5, motoneuron death also occur between E6 and E10 in the cervical cord. Studies with [3H]thymidine autoradiography and morphological techniques revealed that in the early cell-death phase (E4-E5), genesis of motoneurons, axonal elongation, and innervation of muscles is still ongoing. However, studies with [3H]thymidine autoradiography also revealed that the cells dying between E4 and E5 become postmitotic before E3.5. Increased size of peripheral targets, treatment with neuromuscular blockade, and treatment with partially purified muscle or brain extracts and defined neurotropic agents, such as NGF, BDNF, neurotrophin-3, CNTF, bFGF, PDGF, S100-beta, activin, cholinergic differentiation factor/leukemia inhibitory factor, bone morphogenetic protein-2, IGF-I, interleukin-6, and TGF-beta 1, were all ineffective in rescuing motoneurons dying between E4 and E5. By contrast, motoneurons that undergo programmed cell death at later stages (E6-E10) in the cervical cord are target-dependent and respond to activity blockade and trophic factors. Experimental approaches revealed that early cell death also occurs in a notochord-induced ectopic supernumerary motoneuron column in the cervical cord. Transplantation of the cervical neural tube to other segmental regions failed to alter the early death of motoneurons, whereas transplantation of other segments to the cervical region failed to induce early motoneuron death. These results suggest that the mechanisms that regulate motoneuron death in the cervical spinal cord between E4 and E5 are independent of interactions with targets. Rather, this novel type of cell death seems to be determined by signals that either are cell-autonomous or are derived from other cells within the cervical neural tube.

BACKGROUND

The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates.

RESULTS

Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV.

CONCLUSIONS

Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.