Sulfatase 2 (SULF2) Monoclonal Antibody 5D5 Suppresses Human Cholangiocarcinoma Xenograft Growth Through Regulation of a SULF2-Platelet-Derived Growth Factor Receptor Beta-Yes-Associated Protein Signaling Axis.

BACKGROUND AND AIMS: Existing therapeutic approaches to treat cholangiocarcinoma (CCA) have limited effectiveness, prompting further study to develop therapies for CCA. We report a mechanistic role for the heparan sulfate editing enzyme sulfatase 2 (SULF2) in CCA pathogenesis. APPROACH AND RESULTS: In silico analysis revealed elevated SULF2 expression in human CCA samples, occurring partly through gain of SULF2 copy number. We examined the effects of knockdown or overexpression of SULF2 on tumor growth, chemoresistance, and signaling pathway activity in human CCA cell lines in vitro. Up-regulation of SULF2 in CCA leads to increased platelet-derived growth factor receptor beta (PDGFRß)-Yes-associated protein (YAP) signaling activity, promoting tumor growth and chemotherapy resistance. To explore the utility of targeting SULF2 in the tumor microenvironment for CCA treatment, we tested an anti-SULF2 mouse monoclonal antibody, 5D5, in a mouse CCA xenograft model. Targeting SULF2 by monoclonal antibody 5D5 inhibited PDGFRß-YAP signaling and tumor growth in the mouse xenograft model. CONCLUSIONS: These results suggest that SULF2 monoclonal antibody 5D5 or related agents may be potentially promising therapeutic agents in CCA.

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model.

BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. METHODS: bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. RESULTS: Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. CONCLUSIONS: Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.

Activity of sunitinib in extraskeletal myxoid chondrosarcoma.

BACKGROUND: Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue sarcoma, marked by NR4A3 rearrangement. Herein we report on the activity of sunitinib in a series of 10 patients, strengthening what initially observed in two cases. PATIENTS AND METHODS: From July 2011, 10 patients with progressive metastatic translocated EMC have been consecutively treated with sunitinib 37.5mg/day, on a named-use basis. In an attempt to interpret the activity of sunitinib in EMC, genotype/phenotype correlations were carried out by fluorescence in situ hybridization (FISH) analyses. Moreover, transcriptome, immunohistochemical and biochemical analyses of a limited set of samples were performed focusing on some putative targets of sunitinib. RESULTS: Eight of 10 patients are still on therapy. Six patients had a Response Evaluation Criteria in Solid Tumours (RECIST) partial response (PR), two were stable, two progressed. Positron emission tomography (PET) was consistent in 6/6 evaluable cases. One patient underwent surgery after sunitinib, with evidence of a pathologic response. At a median follow-up of 8.5 months (range 2-28), no secondary resistance was detected. Median progression free survival (PFS) has not been reached. Interestingly, all responsive cases turned out to express the typical EWSR1-NR4A3 fusion, while refractory cases carried the alternative TAF15-NR4A3 fusion. Among putative sunitinib targets, only RET was expressed and activated in analysed samples. CONCLUSIONS: This report confirms the therapeutic activity of sunitinib in EMC. Genotype/phenotype analyses support a correlation between response and EWSR1-NR4A3 fusion. Involvement of RET deserves further investigation.

Morphine stimulates platelet-derived growth factor receptor-ß signalling in mesangial cells in vitro and transgenic sickle mouse kidney in vivo.

BACKGROUND: Pain and renal dysfunction occur in sickle cell disease. Morphine used to treat pain also co-activates platelet-derived growth factor receptor-ß (PDGFR-ß), which can adversely affect renal disease. We examined the influence of morphine in mesangial cells in vitro and in mouse kidneys in vivo. METHODS: > Mouse mesangial cells treated with 1 µM morphine in vitro or kidneys of transgenic homozygous or hemizygous sickle or control mice (n=3 for each), treated with morphine (0.75, 1.4, 2.14, 2.8, 3.6, and 4.3 mg kg(-1) day(-1) in two divided doses during the first, second, third, fourth, fifth, and sixth weeks, respectively), were used. Western blotting, bromylated deoxy uridine incorporation-based cell proliferation assay, reverse transcriptase-polymerase chain reaction, immunofluorescent microscopy, and blood/urine chemistry were used to analyse signalling, cell proliferation, opioid receptor (OP) expression, and renal function. RESULTS: Morphine stimulated phosphorylation of PDGFR-ß and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) to the same extent as induced by platelet-derived growth factor-BB (PDGF-BB) and promoted a two-fold increase in mesangial cell proliferation. The PDGFR-ß inhibitor, AG1296, OP antagonists, and silencing of µ- and κ-OP abrogated morphine-induced MAPK/ERK phosphorylation and proliferation by ~100%. Morphine treatment of transgenic mice resulted in phosphorylation of PDGFR-ß, MAPK/ERK, and signal transducer and activator of transcription 3 (Stat3) in the kidneys. Morphine inhibited micturition and blood urea nitrogen (BUN) clearance and increased BUN and urinary protein in sickle mice. CONCLUSION: Morphine stimulates mitogenic signalling leading to mesangial cell proliferation and promotes renal dysfunction in sickle mice.

Ellagic acid inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and prevents atheroma formation in streptozotocin-induced diabetic rats.

Plant-derived polyphenolic compounds have beneficial health effects. In the present study, we determined the ability of ellagic acid (EA) to prevent platelet-derived growth factor-BB (PDGF-BB)-induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs). We also determined the ability of EA to prevent atherosclerosis in streptozotocin-induced diabetic rats. Proliferation of cells was measured via Alamar Blue assay and through propidium iodide-based cell cycle analysis in flow cytometer. Reactive oxygen species (ROS) were measured via 2',7'-dichlorofluorescin diacetate and Amplex red methods. Expression of proliferation markers and activation of kinases were assessed by immunoblot analysis. Cotreatment of primary cultures of RASMCs with 25 µmol/L of EA significantly reduced PDGF-BB (20 ng/ml)-induced proliferation by blocking S-phase entry. EA effectively blocked PDGF receptor-ß (PDGFR-ß) tyrosine phosphorylation, generation of intracellular ROS and downstream activation of extracellular signal-regulated kinase 1/2. It also blocked PDGF-BB-induced expression of cyclin D1. Computational molecular docking of EA with the PDGFR-ß-PDGF-BB complex revealed two putative inhibitor binding sites which showed similar binding energies with the known PDGFR-ß inhibitor AG1295. In diabetic rats, supplementation of diet with 2% EA significantly blocked diabetes-induced medial thickness, and lipid and collagen deposition in the arch of aorta. These were assessed through haematoxylin and eosin, Oil Red O and Masson's trichome staining, respectively. EA treatment also blocked cyclin D1 expression in medial smooth muscle cells in experimental animals. Thus, EA is effective in reducing atherosclerotic process by blocking proliferation of vascular smooth muscle cells.

Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-ß receptor-mediated ERK and p38 pathways.

Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H(2)O(2)), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H(2)O(2) at 5µM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H(2)O(2)-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-ß receptor (PDGF-ßR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-ßR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-ßR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H(2)O(2) stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H(2)O(2)-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis.

B-cell depletion therapy in patients with diffuse systemic sclerosis associates with a significant decrease in PDGFR expression and activation in spindle-like cells in the skin.

INTRODUCTION: Recently, several studies assessing the clinical efficacy of rituximab (RTX) in systemic sclerosis (SSc) have reported encouraging results. We aimed at exploring whether RTX exerts its beneficial effects on fibrosis through attenuation of platelet-derived growth factor receptor (PDGFR) pathway activation. METHODS: We immunohistochemically assessed skin biopsies obtained from eight patients with SSc prior to and 6 months following RTX treatment, three control SSc patients (at the same time points) and three healthy subjects. We assessed the expression of platelet-derived growth factor, PDGFR and phosphorylated (activated) PDGFR. RESULTS: We found a strong correlation of PDGFRα and PDGFRß expression on spindle-like cells and collagen deposition in SSc biopsies (r = 0.97 and r = 0.96 for PDGFRα and PDGFRß, respectively; P < 0.0001 for both), indicating a strong link between PDGFR expression and fibrosis. Expression of PDGFRα and PDGFRß in the papillary dermis significantly decreased following RTX administration (mean ± standard error of the mean at baseline vs. 6 months, respectively: PDGFRα, 42.05 ± 5.03 vs. 26.85 ± 3.00, P = 0.004; and PDGFRß, 37.14 ± 4.94 vs. 24.01 ± 3.27, P = 0.012). Similarly, expression of phosphorylated PDGFRα and PDGFRß in the papillary dermis significantly decreased following RTX administration (P = 0.006 and P = 0.013 for phospho-PDGFRα and phospho-PDGFRß, respectively). No changes in platelet-derived growth factor tissue expression or serum levels were found following RTX treatment. CONCLUSION: RTX may favorably affect skin fibrosis through attenuation of PDGFR expression and activation, a finding that supports a disease-modifying role of RTX in SSc. Large-scale, multicenter studies are needed to further explore the efficacy of RTX in SSc.

Shattering the underpinnings of neoplastic architecture in LNCap: synergistic potential of nutraceuticals in dampening PDGFR/EGFR signaling and cellular proliferation.

OBJECTIVE: Prostate cancer is a polyfactorial molecular anomaly that is offering refractoriness against a broad range of therapeutic drugs. Growth factor receptors are actively implicated in oncogenesis. PDGFR/EGFR mediated exacerbated signaling has a central participation and is contributory in fueling the signal transductions that gear up prostate cancer progression. MATERIALS AND METHODS: In this particular study, androgen sensitive, Prostate cancer cell line (LNCaP) was used. Pretreatment of cell line with PDGF resulted in an enhanced proliferation of cells which was evaluated by MTT assay. Treatment of cell line with either alone Curcumin, EGCG, sulforaphane or in combination was evaluated. PDGFR/EGFR activation (phosphorylation) was studied using western blot. RESULTS: Results indicated that phosphorylation was gradually downregulated after treatment with individual compound. However there was a remarkable decrease in cellular proliferation after a combinatorial approach which is indicative of the fact that PDGFR phosphorylation was decreased outstandingly as evaluated by MTT assay. That also gave a prominent decline in the expression and subsequent decrease in proliferation pattern of cells. CONCLUSION: Despite the fact that little is still known regarding the mechanistic insights by which phytonutrients act as barrier to cancer, and attempts to translate the studies from benchtop to bedside are in progress. A detailed analysis of nutraceuticals will help a lot in identifying the stumbling blocks in the standardization of therapeutic interventions.

Receptor heterodimerization: a new mechanism for platelet-derived growth factor induced resistance to anti-epidermal growth factor receptor therapy for bladder cancer.

PURPOSE: Human bladder cancer cells resistant to anti-epidermal growth factor receptor therapy often co-express platelet-derived growth factor receptor-ß. We determined whether there is functional crosstalk between epidermal growth factor receptor and platelet-derived growth factor receptor-ß, and how this regulates biological functions in bladder cancer cases. MATERIALS AND METHODS: We determined heterodimerization and co-localization of epidermal growth factor receptor and platelet-derived growth factor receptor-ß by immunoprecipitation and confocal microscopy, respectively. We tested the antiproliferative effects of specific inhibitory monoclonal antibodies to each receptor by (3)H-thymidine uptake assay. We transfected the nonplatelet-derived growth factor receptor-ß expressing bladder cancer cell line UMUC5 with the platelet-derived growth factor receptor-ß gene. These cells were analyzed in vitro by (3)H-thymidine uptake and by Matrigel™ invasion assay, and in vivo for tumorigenicity, metastatic potential and orthotopic growth. In a treatment study nude mice were inoculated with orthotopic tumors and treated with the inhibitory antibodies alone and in combination. RESULTS: Immunoprecipitation revealed epidermal growth factor receptor/platelet-derived growth factor receptor-ß heterodimers in all platelet-derived growth factor receptor-ß expressing cell lines. Forced expression of platelet-derived growth factor receptor-ß in epidermal growth factor receptor sensitive UMUC5 cells (50% inhibitory concentration less than 10 nM) significantly decreased responsiveness to epidermal growth factor receptor inhibition (50% inhibitory concentration greater than 100 nM) and increased invasive potential 3-fold as well as tumorigenicity. Increased invasiveness was associated with epidermal growth factor triggered platelet-derived growth factor receptor-ß transactivation, increased mitogen activated protein kinase and glycogen synthase kinase-3ß phosphorylation, and decreased E-cadherin. Inhibition of epidermal growth factor receptor and platelet-derived growth factor receptor-ß receptors blocked cell invasion, decreased cell proliferation, reduced xenograft tumor growth and increased E-cadherin expression. CONCLUSIONS: In epidermal growth factor receptor expressing bladder cancer co-expression of platelet-derived growth factor receptor-ß has implications for tumor biology. Thus, it should be further evaluated as a strategy involving dual receptor targeting.

Target-driven exploratory study of imatinib mesylate in children with solid malignancies by the Innovative Therapies for Children with Cancer (ITCC) European Consortium.

AIM: To explore imatinib efficacy and pharmacokinetics in children and adolescents with refractory/relapsing solid tumours, expressing imatinib-sensitive receptor tyrosine kinases. METHODS: Exploratory study on imatinib in tumours expressing, at least, one of the receptors KIT or platelet-derived growth factor receptor (PDGFR). Standard radiological response evaluation, pharmacokinetics, gene mutations and positron emission tomography imaging were assessed. RESULTS: Thirty-six patients (median age: 13.7 years) with brain (12), mesenchymal/bone (14) or other solid tumours, received imatinib 340 mg/m(2)/d over a total of 255 months. Fifteen tumours expressed KIT in 30% cells, 19 expressed PDGFRA and 25 expressed PDGFRB. Twenty patients experienced grades 1-2 treatment-related toxicities. Ten patients achieved stable disease; one chordoma had metabolic response. Pharmacokinetic data showed high inter-patient variability (variation coefficient: 44% and 53% for plasma imatinib and CGP 74588 AUCs, respectively). CONCLUSIONS: Imatinib was tolerated well, but failed to show efficacy according to standard criteria in paediatric malignancies expressing KIT or PDGFR.

Combination of imatinib and vinorelbine enhances cell growth inhibition in breast cancer cells via PDGFR beta signalling.

INTRODUCTION: Imatinib mesylate is a tyrosine kinase receptor inhibitor targeted against PDGFR alpha and beta, c-kit and bcr-abl. These receptors regulate cellular processes such as proliferation, differentiation, and survival. This study was performed to evaluate the effects of imatinib on breast cancer cell lines with respect to the activity of PDGFR beta and Akt: a downstream modulator of cell growth and survival. METHODS: Expression of imatinib targets was analyzed with reverse transciptase PCR and immunoblotting assays in the breast cell lines MDA MB 231, MCF 7, ZR 75-1, and T 47-D. Changes on receptor expression and phosphorylation status under imatinib were evaluated using drug concentrations of 2 to 10 microM. The anti-proliferative and pro-apoptotic effects of imatinib alone and in combination with vinorelbine were investigated with an MTT and TUNEL assay. RESULTS: Imatinib inhibited growth and induced apoptosis of all cell lines examined. This effect was increased when combined with vinorelbine. A dose-dependent inhibitory effect on the phosphorylation of PDGFR beta and Akt was detected. CONCLUSIONS: The growth inhibitory effect of imatinib on breast cell lines may be caused by inhibiting the activity of the tyrosine kinases PDGFR beta and Akt. Imatinib is a promising novel drug for targeted therapy of breast cancer patients.

Imatinib inhibits in vitro proliferation of cells derived from a pleural solitary fibrous tumor expressing platelet-derived growth factor receptor-beta.

We examined the in vitro effects of imatinib (Novartis Pharma AG, Basel, Switzerland) as a possible inhibitor of PDGFR pathway on cells derived from a recurrence of a pleural malignant solitary fibrous tumor (SFT). Primary cell culture was characterised by immunofluorescence. SFT-derived cells were treated with imatinib at different time points. Western blotting for PDGFR-beta, phospho-PDGFR-beta or smooth muscle actin (SMA) was performed before and after 96 h of treatment with imatinib. SFT-derived cells treated with imatinib for 96 h showed a dose dependent decrease of Ki67 expression. Results were confirmed by growth curve. Western blotting showed that PDGFR-beta was highly expressed and phosphorylated in SFT-derived cells and imatinib treatment reduced PDGFR-beta phosphorylation and SMA expression. With the limit of experimental findings, our results support a possible future application of imatinib as a candidate molecule in the target therapy of malignant SFTs over-expressing wild-type PDGFR.

Impact of vessel maturation on antiangiogenic therapy in ovarian cancer.

OBJECTIVE: The purpose of this study was to examine the functional and therapeutic significance of pericytes in ovarian cancer vasculature. STUDY DESIGN: Tumor vessel morphologic condition and efficacy of endothelial and pericyte targeting were examined with the use of in vivo ovarian cancer models. The expression of platelet-derived growth factor (PDGF) ligands and receptors was examined in endothelial, pericyte-like, and ovarian cancer cells. RESULTS: Relative to normal vessels, tumor vasculature was characterized by loosely attached pericytes in reduced density. PDGF-BB was expressed predominantly by the endothelial and cancer cells, whereas PDGFRbeta was present in pericyte-like cells. PDGF-BB significantly increased the migration of and VEGF production by pericyte-like cells; PDGFRbeta blockade abrogated these effects. Dual VEGF (VEGF-Trap) and PDGF-B (PDGF-Trap) targeted therapy was more effective in inhibiting in vivo tumor growth than either agent alone. CONCLUSION: Aberrations in the tumor microenvironment contribute to endothelial cell survival. Strategies that target both endothelial cells and pericytes should be considered for clinical trials.

The effect of imatinib (Glivec) on scleroderma and normal dermal fibroblasts: a preclinical study.

BACKGROUND: Scleroderma skin overexpresses the platelet-derived growth factor receptor beta-subunit (PDGFR-beta) in dermal vessels and PDGFR-beta messenger RNA in cultured fibroblasts. Moreover, increased levels of PDGF and stimulatory autoantibodies to PDGFR have been identified in the serum of scleroderma patients. OBJECTIVE: Imatinib being an inhibitor of tyrosine kinase receptors such as PDGFR, its effect on scleroderma fibroblasts was evaluated in vitro as a preclinical therapeutic step. METHODS: The effect of imatinib on fibroblasts grown from normal or involved/uninvolved scleroderma skin was studied by Western blot and the methyltetrazolium test. The pattern of distribution of PDGFR-beta in scleroderma versus normal skin was studied by immunohistochemistry. RESULTS: In vitro, imatinib inhibited the proliferation of normal dermal and scleroderma fibroblasts at least partly via the inhibition of the phosphorylation of PDGFR. PDGFR-beta was expressed in the epidermis and adnexae in 5 lesional scleroderma biopsies and not in controls. CONCLUSION: This study suggests that imatinib can serve as therapy to limit dermal fibroblast proliferation in scleroderma.

Melittin, a major bioactive component of bee venom toxin, inhibits PDGF receptor beta-tyrosine phosphorylation and downstream intracellular signal transduction in rat aortic vascular smooth muscle cells.

Studies previously reported that melittin, a major bioactive component of bee venom, inhibits vascular smooth muscle cell (VSMC) proliferation through suppression of nuclear factor (NF)-kappaB and Akt activation and through enhancement of proapoptotic protein expression. In this study, the effects of melittin were investigated on the tyrosine phosphorylation of platelet-derived growth factor (PDGF) beta receptor (Rbeta) and its downstream intracellular signal transduction. When combined with PDGF-Rbeta inhibitor, melittin exhibited a synergic inhibitory effect on PDGF-BB-induced rat aortic VSMC proliferation. In addition, melittin inhibited PDGF-Rbeta phosphorylation in a concentration-dependent manner. Accordingly, the downstream signal transduction of PDGF-Rbeta, such as ERK1/2, Akt, and PLCgamma1 phosphorylation, was also inhibited by melittin in the same manner. These findings suggest that, in addition to suppressing NF-kappaB activation, the antiproliferative effect of melittin in VSMC may be mediated, at least in part, by the inhibition of PDGF-Rbeta tyrosine phosphorylation and its downstream intracellular signal transduction.

Effects of KTJ740, a novel antithrombotic agent, on platelet-derived growth factor-induced rat aortic smooth muscle cell proliferation and cell cycle progression.

Hyperproliferation of platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (VSMC) is a hallmark of atherosclerosis and related vascular disorders. In the previous study, we reported that KTJ740 [2-chloro-3-(4-(ethylcarboxy)-phenyl)-amino-1,4-naphthoquinone], a newly synthesized vitamin K derivative, has potent antithrombotic effects in mice and antiplatelet activity in vitro and ex vivo. In the present study, we have tested that KTJ740 could inhibit PDGF-BB-stimulated VSMC proliferation. We have examined the potential inhibitory effect of this compound on rat aortic smooth muscle cells (RASMCs). Our results show that this compound significantly inhibits PDGF-BB-stimulated RASMC number and DNA synthesis in a concentration-dependent manner. Furthermore, we have examined its effect on cell cycle progression by flow cytometry. KTJ740 treatment resulted in a significant arrest in cell cycle progression of RASMCs induced by PDGF-BB, and this effect was achieved by suppressing activation of PDGF-beta receptor (PDGF-Rbeta) tyrosine kinase pathway. These results suggest that a possibility of KTJ740 can be a potential agent to control vascular disorders and its antiproliferative mechanism may be mediated through PDGF-Rbeta tyrosine kinase-dependent signaling pathway.

Mesenchymal stem cell-organized bone marrow elements: an alternative hematopoietic progenitor resource.

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent postnatal stem cells that have been used for the treatment of bone defects and graft-versus-host diseases in clinics. In this study, we found that subcutaneously transplanted human BMMSCs are capable of organizing hematopoietic progenitors of recipient origin. These hematopoietic cells expressed multiple lineages of hematopoietic cell associated markers and were able to rescue lethally irradiated mice, with successful engraftment in the recipient, suggesting a potential bone marrow (BM) resource for stem cell therapies. Furthermore, we found that platelet-derived growth factor (PDGF) promotes the formation of BMMSC-generated BM niches through upregulation of beta-catenin, implying that the PDGF pathway contributes to the formation of ectopic BM. These results indicate that the BMMSC-organized BM niche system represents a unique hematopoietic progenitor resource possessing potential clinical value.

Morphine stimulates vascular endothelial growth factor-like signaling in mouse retinal endothelial cells.

Go/Gi coupled G-protein receptor mediated transactivation is critical in the activation of receptor tyrosine kinases (RTK). Here we show that mu opioid receptor (MOR) transactivates Flk1 and platelet-derived growth factor-beta (PDGF-beta) receptors and its agonist morphine stimulates pro-angiogenic and survival-promoting signaling in mouse retinal endothelial cells (mREC). Morphine stimulates mREC proliferation in a dose dependent fashion and promotes survival to the same extent as vascular endothelial growth factor164 (VEGF164). Morphine stimulates mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and Akt phosphorylation in a time dependent manner like VEGF in mREC. Moreover, analogous to VEGF, morphine stimulates oncogenic signal transducer and activator of transcription 3 (STAT3) signaling. Morphine as well as VEGF-induced phospho-STAT3 and phospho-Flk1 immunoprecipitated with MOR-associated proteins. In addition morphine also stimulated MOR associated PDGF-beta receptor phosphorylation. Consistent with the relationship between VEGF and MOR we found that VEGF upregulates MOR protein and RNA expression in mREC. These data suggest that MOR associates and transactivates RTKs for Flk1 and PDGF-beta, which may have a compounding effect on angiogenic signaling in endothelium. Therefore, G-Protein coupled receptors including MOR provide novel targets to develop anti-angiogenic agents.