PDGF-B mRNA Expression Research Articles

Conditioned Medium from Human Uterine Cervical Stem Cells Regulates Oxidative Stress and Angiogenesis of Retinal Pigment Epithelial Cells

Introduction: Retinal homeostasis is essential to avoid retinal pigment epithelium (RPE) damage resulting in photoreceptor death and blindness. Mesenchymal stem cells-based cell therapy could contribute to the maintenance of the retinal homeostasis. We have explored the effect of human uterine cervical stem cells (hUCESCs)-conditioned medium (hUCESC-CM) on RPE cells under oxidative stress condition. Methods: ARPE-19 cells were treated with hydrogen peroxide (H2O2) in the presence or absence of hUCESC-CM. qRT-PCR and Western blot were used to evaluate the expression of oxidative stress-related (HO-1, GCLC, and HSPB1) and vasculogenesis-related (VEGFA, PDGFA, and PDGFB) factors. Also, we assessed in vitro effects of hUCESC-CM on endothelial-cell (HUVEC) tube formation. Results: mRNA expression of HO-1, GCLC, HSPB1, VEGFA, PDGFA, and PDGFB were significantly increased in ARPE-19 cells treated with H2O2 + hUCESC-CM compared to cells treated with H2O2 only. Regarding the tube formation assay, HUVEC treated with supernatant from ARPE-19 cells treated with H2O2 + hUCESC-CM showed a significant increase in average vessel length, number of capillary-like junctions, and average of vessels area compared with HUVEC treated with supernatant from ARPE-19 cells treated with H2O2 only. Conclusion: Our results show potential therapeutic effects of hUCESC-CM on RPE, such as protection from damage by oxidative stress, stimulation of detoxifying genes, and a better vascularization.

Sustained Surface ICAM-1 Expression and Transient PDGF-B Production by Phorbol Myristate Acetate-Activated THP-1 Cells Harboring Blau Syndrome-Associated NOD2 Mutations.

Objectives: Blau syndrome is a distinct class of autoinflammatory syndrome presenting with early-onset systemic granulomatosis. Blau syndrome-causing NOD2 mutations located in the central nucleotide-oligomerization domain induce ligand-independent basal NF-κB activation in an in vitro reporter assay. However, the precise role of this signaling on granuloma formation has not yet been clarified. Methods: Blau syndrome-causing NOD2 mutations were introduced into human monocytic THP-1 cells, and their morphological and molecular changes from parental cells were analyzed. Identified molecules with altered expression were examined in the patient’s lesional skin by immunostaining. Results: Although the production of proinflammatory cytokines was not altered without stimulation, mutant NOD2-expressing THP-1 cells attached persistently to the culture plate after stimulation with phorbol myristate acetate. Sustained surface ICAM-1 expression was observed in association with this phenomenon, but neither persistent ICAM-1 mRNA expression nor impaired ADAM17 mRNA expression was revealed. However, the transient induction of PDGF-B mRNA expression was specifically observed in stimulated THP-1 derivatives. In the granulomatous skin lesion of a Blau syndrome patient, ICAM-1 and PDGF-B were positively immunostained in NOD2-expressing giant cells. Conclusions: Sustained surface ICAM-1 expression and transient PDGF-B production by newly differentiating macrophages harboring mutant NOD2 might play a role in granuloma formation in Blau syndrome.

2000-P: Intracellular Metabolism-Dependent PDGF-B Induction in Inflammatory Macrophages Contributes to Adipose Tissue Expansion with Obesity

White adipose tissue (WAT) expands with angiogenesis. We have demonstrated that adipose-tissue-macrophages (ATMs) highly express platelet-derived growth factor (PDGF)-B in obesity, and exposure of PDGF-B stimulates detachment of pericytes (PCs) from vessels. Since vessel-attached PCs inhibit endothelial cell growth, the detachment restarts angiogenesis. In the present study, we aimed to clarify the significance of ATMs on adipose angiogenesis. Intraperitoneal injection of liposome-encapsulated clodronate (Clod) for 6 weeks achieved depletion of macrophages and decreased expression of Pdgfb mRNA in WAT of HFD-fed mice. Clod-treated mice showed smaller WAT with vessels well covered with PCs compared to control mice. We next investigated underlying mechanism of Pdgfb induction. LPS but not IL-4 stimulated Pdgfb expression in peritoneal macrophages. In RAW 264.7 macrophages, Pdgfb expression induced by high glucose plus LPS stimulation was completely blocked by pretreatment with 2-deoxyglucose (2-DG), a hexokinase inhibitor or heptelidic acid, a GAPDH inhibitor, whereas it was not affected by treatment with 6-aminonicotinamide, an inhibitor of glucose-6-phosphate dehydrogenase acting as the rate-limiting enzyme of the pentose phosphate pathway. In addition, high glucose plus LPS induced phosphorylations of S6 kinase, ERK, and JNK but not p38 MAP kinase. These phosphorylations were significantly attenuated by treatment with 2DG. Importantly, enhanced expression of Pdgfb by high glucose plus LPS was effectively suppressed by inhibitor or knockdown of Erk but not by rapamycin. The expression was also attenuated by inhibition of NFκb pathway. In conclusion, hyperglycemia and chronic inflammation reprogram metabolic pathways toward glycolysis, and subsequent activation of NFκB plays a pivotal role in the production of PDGF-B in ATMs during obesity development. Disclosure T. Wada: None. Y. Onogi: None. A. Okekawa: None. T. Matsuzawa: None. E. Watanabe: None. G. Komatsu: None. H. Tsuneki: None. T. Sasaoka: None. Funding Japan Society for the Promotion of Science (JP18K08469)

Unraveling reno-protective effects of SGLT2 inhibition in human proximal tubular cells.

Large clinical trials demonstrated that SGLT2 inhibitors (SGLT2i) slow the progression of kidney function decline in type 2 diabetes. Because the underlying molecular mechanisms are largely unknown, we studied the effects of SGLT2i on gene expression in two human proximal tubular (PT) cell lines under normoglycemic conditions, utilizing two SGLT2i, namely empagliflocin and canagliflocin. Genome-wide expression analysis did not reveal substantial differences between these two SGLT2i. Microarray hybridization analysis identified 94 genes that were both upregulated by TGF-β1 and downregulated by either of the two SGLT2i in HK-2 and RPTEC/TERT1 (renal proximal tubular epithelial cells/telomerase reverse transcriptase 1) cells. Extracellular matrix organization showed the highest significance in pathway enrichment analysis. Differential gene expression of three annotated genes of interest within this pathway was verified on mRNA level in both cell lines. Whereas TGF-β1 induced mRNA expression of thrombospondin 1 (THBS1; 4.3-fold), tenascin C (TNC; 8-fold), and platelet-derived growth factor subunit B (PDGF-B; 4.2-fold), SGLT2i downregulated basal mRNA expression of THBS1 (0.2-fold), TNC (0.5 fold), and PDGF-B (0.6-fold). Administration of SGLT2i in the presence of TGF-β1 resulted in a significant inhibition of TGF-β1-induced THBS1 and TNC mRNA expression and TGF-β1-induced THBS1, TNC, and PDGF-BB protein expression. We conclude that SGLT2i block basal and TGF-β1-induced expression of key mediators of renal fibrosis and kidney disease progression in two independent human PT cell lines.

ZNF750 inhibited the malignant progression of oral squamous cell carcinoma by regulating tumor vascular microenvironment.

Squamous cell carcinoma is often associated with the deletion or mutation of zinc finger protein 750 (ZNF750), its deletion or mutation is associated with squamous epithelial malignant biological characteristics. The present study is to explore the mechanism of ZNF750 to suppress the tumor malignant process by regulation tumor microenvironment. To evaluate the changes of tumor microenvironment in oral squamous cells carcinoma cell line CAL-27 cell, the expression of angiogenin, vascular endothelial growth factor (VEGF), prolyl hydroxylase 2 (PHD2), G protein signal regulated protein 5 (RGS5), integrin A5 (ITGA5), integrin B1 (ITGB1) and CD44 were detected by Western-blot. The changes of platelet derived growth factor (PDGFB) and tumor vascular marker CD105 (Endoglin) mRNA were estimated by qPCR. The effect of over-expressed ZNF750 on cell viability and lateral migration capacity was investigated by CCK-8 and cell scratch assay in three oral squamous cells carcinoma. ZNF750 could effectively inhibit the protein or mRNA expression of angiogenin, VEGF, RGS5 and CD105, repressed the cell adhesion molecules ITGA5, ITGB1 and CD44, but up-regulate the protein or mRNA expression of PHD2 and PDGFB. The cell viability and lateral migration ability of three oral squamous cells carcinoma were reduced by over-expression of ZNF750. ZNF750 could modulate the tumor vascular microenvironment to inhibit the oral squamous cells carcinoma malignant progression.

Dabigatran inhibits intravitreal thrombin activity.

Proliferative vitreoretinopathy (PVR) is a vitreoretinal disorder in which retinal pigment epithelial (RPE) cell activation contributes to both formation of fibrotic retinal membranes and inflammation. Vitreous of patients with PVR contains increased thrombin activity which induces profibrotic and proinflammatory programs in RPE cells. Inhibition of intravitreal thrombin activity may thus represent a therapeutic option for PVR. In this study, we examined the capacity of the clinically available directthrombin inhibitor dabigatran to inhibit thrombin activity in vitreous fluids. ARPE-19 cells were cultured with the following: (i) thrombin, (ii) vitreous without thrombin activity and (iii) vitreous with elevated thrombinactivity (PVR samples and thrombin spiked vitreous) either in the presence or absence of dabigatran (range: 10-5 to 10-7 M). Subsequently, CCL2, CXCL8, GMCSF, IL6 and PDGFB mRNA expression levels were determined by RQ-PCR and protein levels of 27 cytokines, chemokines and growth factors were detected in culture supernatants using a multiplex approach. In addition, the capacity of vitreous fluids obtained from patientsafter oral dabigatran intake was tested in an invitro thrombin activity assay. Thrombin and vitreous fluids containing thrombin activity induced CCL2, CXCL8, GM-CSF, IL-6 and PDGF-BB expression by ARPE-19 cells, which was inhibited by dabigatran. In addition, dabigatran that reached the vitreous after repeated oral intake did inhibit thrombin activity in the invitro activity assay. Proliferative vitreoretinopathy (PVR) is associated with increased intravitreal thrombin activity that activates profibrotic and proinflammatory pathways in RPE cells. Our findings provide evidence that this activation pathway can potentially be inhibited by dabigatran.

Comprehensive study of angiogenic factors in women with endometriosis compared to women without endometriosis

ObjectiveEndometriosis is a benign gynaecological disease, affecting women during their reproductive years. Angiogenesis represents a crucial step in the pathogenesis of endometriosis, because endometriotic lesions require neovascularization. In this study several angiogenesis-related genes have been studied in the context of endometriosis. Some of the analyzed angiogenic factors as well as their interactions were studied the first time regarding a possible association with endometriosis. Study designThis case-control study consisted of 205 biopsies of 114 patients comprising 61 endometriosis patients and 53 control patients. Among them in 29 cases paired samples were obtained. VEGFA, VEGFR2, HIF1A, HGF, NRP1, PDGFB, FGF18, TNFα, TGFB2, EPHB4, EPO and ANG mRNA expression was analyzed by qRT-PCR in ectopic tissue samples, in eutopic endometrium of women with and without endometriosis, and in unaffected peritoneum of women with and without endometriosis. ResultsVEGFR2, HIF1A, HGF, PDGFB, NRP1 and EPHB4 are overexpressed in ectopic lesions compared to eutopic tissues. VEGFR2, HGF, PDGFB, NRP1, and EPHB4 showed highest mRNA levels in peritoneal implants, in contrast HIF1A showed the highest expression in ovarian endometriomas. Correlation analyses of angiogenic factors in ectopic lesions revealed the strongest associations between VEGFR2, PDGFB, and EPHB4. We further showed a significant upregulation of VEGFR2, HIF1A and EPHB4 in eutopic endometrium of women with endometriosis compared to that of controls and a trend towards upregulation of HGF. Additionally, a significant downregulation for HIF1A, HGF and EPHB4 was observed in unaffected peritoneal tissues of women with endometriosis compared to controls. ConclusionWe identified new genes (EPHB4 and NRP1) that may contribute to angiogenesis in endometriosis beside known factors (VEGFA, VEGFR2, HIF1A, HGF, and PDGFB). Correlation studies revealed the putative importance of EBHB4 in association with endometriosis. Our analyses support preliminary reports that angiogenic factors seem to be differently expressed in peritoneal implants, ovarian endometriomas and deep infiltrating endometriosis. Our observation that angiogenic factors are differently expressed in the unaffected peritoneum of women with endometriosis compared to women without endometriosis underlines the importance of the peritoneum in the establishment of endometriosis.

Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid Arthritis.

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.

Towards next generation maggot debridement therapy: transgenic Lucilia sericata larvae that produce and secrete a human growth factor.

BackgroundDiabetes and its concurrent complications impact a significant proportion of the population of the US and create a large financial burden on the American health care system. FDA-approved maggot debridement therapy (MDT), the application of sterile laboratory-reared Lucilia sericata (green bottle fly) larvae to wounds, is a cost-effective and successful treatment for diabetic foot ulcers and other medical conditions. Human platelet derived growth factor-BB (PDGF-BB) is a secreted dimeric peptide growth factor that binds the PDGF receptor. PDGF-BB stimulates cell proliferation and survival, promotes wound healing, and has been investigated as a possible topical treatment for non-healing wounds. Genetic engineering has allowed for expression and secretion of human growth factors and other proteins in transgenic insects. Here, we present a novel concept in MDT technology that combines the established benefits of MDT with the power of genetic engineering to promote healing. The focus of this study is to create and characterize strains of transgenic L. sericata that express and secrete PDGF-BB at detectable levels in adult hemolymph, whole larval lysate, and maggot excretions/ secretions (ES), with potential for clinical utility in wound healing.ResultsWe have engineered and confirmed transgene insertion in several strains of L. sericata that express human PDGF-BB. Using a heat-inducible promoter to control the pdgf-b gene, pdgf-b mRNA was detected via semi-quantitative PCR upon heat shock. PDGF-BB protein was also detectable in larval lysates and adult hemolymph but not larval ES. An alternative, tetracycline-repressible pdgf-b system mediated expression of pdgf-b mRNA when maggots were raised on diet that lacked tetracycline. Further, PDGF-BB protein was readily detected in whole larval lysate as well as larval ES.ConclusionsHere we show robust, inducible expression and production of human PDGF-BB protein from two conditional expression systems in transgenic L. sericata larvae. The tetracycline-repressible system appears to be the most promising as PDGF-BB protein was detectable in larval ES following induction. Our system could potentially be used to deliver a variety of growth factors and anti-microbial peptides to the wound environment with the aim of enhancing wound healing, thereby improving patient outcome in a cost-effective manner.

Deletion of Hyaluronan Synthase 3 Inhibits Neointimal Hyperplasia in Mice.

Hyaluronan (HA) is a polymeric glucosaminoglycan that forms a provisional extracellular matrix in diseased vessels. HA is synthesized by 3 different HA synthases (HAS1, HAS2, and HAS3). Aim of this study was to unravel the role of the HAS3 isoenzyme during experimental neointimal hyperplasia. Neointimal hyperplasia was induced in Has3-deficient mice by ligation of the carotid artery. HA in the media of Has3-deficient mice was decreased 28 days after ligation, and neointimal hyperplasia was strongly inhibited. However, medial and luminal areas were unaffected. Cell density, proliferation, and apoptosis were not altered, suggesting a proportional decrease of both, the number of cells and extracellular matrix. In addition, endothelial function as determined by acetylcholine-induced relaxation of aortic rings, immunoblotting of endothelial nitric oxide synthase, and arterial blood pressure were not affected. Furthermore, the oxidative stress response was not affected as determined in total protein extracts from aortae. Transcriptome analysis comparing control versus ligated carotid arteries hinted toward a mitigated differential regulation of various signaling pathways in Has3-deficient mice in response to ligation that were related to vascular smooth muscle cell (VSMC) migration, including focal adhesions, integrins, mitogen-activated protein kinase, and phosphatidylinositol signaling system. Lentiviral overexpression of HAS3 in VSMC supported the migratory phenotype of VSMC in response to platelet-derived growth factor BB in vitro. Accordingly, knockdown of HAS3 reduced the migratory response to platelet-derived growth factor BB and in addition decreased the expression of PDGF-B mRNA. HAS3-mediated HA synthesis after vessel injury supports seminal signaling pathways in activation of VSMC, increases platelet-derived growth factor BB-mediated migration, and in turn enhances neointimal hyperplasia in vivo.

AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

Lung diseases are aggravated by exposure to diesel exhaust particles (DEPs) found in air pollution. Macrophages are thought to play a crucial role in lung immune response to these pollutants, even if the mechanisms involved remain incompletely characterized. In the present study, we demonstrated that classically and alternative human macrophages (MΦ) exhibited increased secretion of PDGF-B in response to DEP extract (DEPe). This occurred via aryl hydrocarbon receptor (AhR)-activation because DEPe-induced PDGF-B overexpression was abrogated after AhR expression knock-down by RNA interference, in both M1 and M2 polarizing MΦ. In addition, TCDD and benzo(a)pyrene, two potent AhR ligands, also significantly increased mRNA expression of PDGF-B in M1 MΦ, whereas some weak ligands of AhR did not. We next evaluated the impact of conditioned media (CM) from MΦ culture exposed to DEPe or of recombinant PDGF-B onto lung fibroblast proliferation. The tyrosine kinase inhibitor, AG-1295, prevents phosphorylations of PDGF-Rβ, AKT and ERK1/2 and the proliferation of MRC-5 fibroblasts induced by recombinant PDGF-B and by CM from M1 polarizing MΦ, strongly suggesting that the PDGF-BB secreted by DEPe-exposed MΦ is sufficient to activate the PDGF-Rβ pathway of human lung fibroblasts. In conclusion, we demonstrated that human MΦ, whatever their polarization status, secrete PDGF-B in response to DEPe and that PDGF-B is a target gene of AhR. Therefore, induction of PDGF-B by DEP may participate in the deleterious effects towards human health triggered by such environmental urban contaminants.

Yin-Chen-Hao-Tang alleviates biliary obstructive cirrhosis in rats by inhibiting biliary epithelial cell proliferation and activation.

Background:Yin-Chen-Hao-Tang (YCHT) consists of three aqueous extracts from Artemisia capillaris, Gardenia sp., and prepared Rheum rhabarbarum (rhubarb) (3:2:1). YCHT is characterized by its anti-inflammatory properties in liver regulation and relief of jaundice. We aimed to study the effects and mechanisms of action of YCHT on biliary obstructive cirrhosis.Materials and Methods:Secondary biliary fibrosis was induced in rats by bile duct ligation (BDL) and scission. One week after BDL, rats were randomly divided into a saline-treated BDL or YCHT-treated BDL group for 4 weeks. Liver function and hepatic hydroxyproline (Hyp) content were assessed. Types I and IV collagen (Col-IV), laminin, fibronectin, alpha smooth muscle actin (α-SMA), and proliferating cell nuclear antigen protein and messenger ribonucleic acid (mRNA) expression were assessed with immunohistochemistry and real-time polymerase chain reaction.Results:In the YCHT-treated BDL group, serum total bilirubin, total bile acids, aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transferase were lower than those in the sham-operated BDL group. The proliferation of bile ducts in hepatic tissues and the Hyp content and Col deposition were also significantly lower than those in control rats. In addition, α-SMA and Col-IV staining was less obvious, and mRNA expression of Procol-α1 (IV), platelet derived growth factor subunit B (PDGF)-B, connective tissue growth factor, and transforming growth factor-beta in proliferative biliary epithelial cells (BECs) in the YCHT-treated BDL group was significantly lower than those in controls.Conclusions:YCHT effectively reduces the formation of biliary obstructive cirrhosis mainly via inhibition of BEC proliferation by down-regulation of PDGF-B mRNA expression, inhibition of BEC profibrogenic paracrines, and the epithelial-mesenchymal transition pathological process.

Differential gene expression and immunolocalization of platelet-derived growth factors and their receptors in caprine ovaries

This study evaluated the messenger RNA (mRNA) expression and immunolocalization of all members of the platelet-derived growth factor (PDGF) family in caprine ovaries by quantitative PCR and immunohistochemistry, respectively. Detectable levels of PDGF-A mRNA were not observed in primordial follicles. Higher levels of PDGF-B mRNA were observed in primary follicles than in primordial follicles (P < 0.05). PDGF-D mRNA levels were higher in secondary follicles than in the other preantral follicle categories (P < 0.05). PDGF-B mRNA expression was higher than PDGF-C mRNA expression in primary follicles (P < 0.05). In antral follicles, PDGF-A mRNA expression was higher in cumulus-oocyte complexes (COCs) from small antral follicles than in those from large antral follicles and their respective granulosa/theca (GT) cells (P < 0.05). Furthermore, in COCs from small and large antral follicles, PDGF-A mRNA expression was higher than that of the other PDGF isoforms (P < 0.05). The mRNA levels of PDGF-B and PDGF-D and PDGFR-α and PDGFR-β were higher in GT cells from large antral follicles than in GT cells from small antral follicles and in their respective COCs (P < 0.05). In COCs and GT cells from small antral follicles, the mRNA levels of PDGFR-α were higher than those of PDGFR-β (P < 0.05). All proteins were observed in the cytoplasm of oocytes from all follicular categories. In granulosa cells, all PDGFs and PDGFR-β were detected from starting at the secondary stage, and in theca cells, all proteins, except PDGF-C, were detected starting at the antral stage. In conclusion, PDGF and its receptors are differentially expressed in the oocytes and ovarian cells according to the stage of follicular development, suggesting their role in the regulation of folliculogenesis in goats.

Abstract 1509: Modulation of stromal reaction by PDGF-R tyrosine kinase inhibitor in an orthotopic mice model of gastric carcinoma

Abstract Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. Many human tumors secrete platelet-derived growth factor (PDGF) ligands, and PDGF receptors (PDGF-Rs) are expressed by various stromal cell populations, including carcinoma-associated fibroblasts (CAFs). We reported recently that, in a mice model of colon cancer, bone marrow-derived mesenchymal stem cells (MSCs) traveled to tumor stroma, where they differentiated into CAF-like cells. In the present study, we examined PDGF-B and PDGF-Rβ expression in relation to clinicopathologic features of human gastric carcinomas. We also examined the effects of PDGF-R tyrosine kinase inhibitors (imatinib, nilotinib) on tumor stroma in an orthotopic nude mouse model of human gastric carcinoma. Five gastric carcinoma cell lines and 38 surgical specimens of gastric carcinoma were used. Under culture conditions, gastric cancer cell lines expressed PDGF-B at various levels but not PDGFR-β. When cells from these lines were implanted orthotopically into the gastric wall of nude mice, cells with high PDGF-B expression resulted in tumors with abundant stroma. In contrast, cells with low PDGF-B expression resulted in medullary tumors with little stromal reaction. In surgical specimens, we found PDGF-B expression to be associated with stromal reaction. PDGF-B and PDGF-Rβ mRNA expression was significantly higher in patients with lymph node metastasis than in those without and also significantly higher in diffuse-type carcinoma than in intestinal-type carcinoma. In surgical specimens and orthotopic tumors, tumor cells expressed PDGF-B, but PDGF-Rβ was expressed predominantly by stromal cells, including CAFs, pericytes, and lymphatic endothelial cells. Treatment with PDGF-R tyrosine kinase inhibitors did not inhibit tumor growth but did significantly decrease the stromal reaction, lymphatic invasion, lymphatic vessel area, and pericyte coverage of tumor microvessels. In addition, tumor tropism of MSCs was also inhibited by treatment with PDGF-R inhibitors in vivo and in vitro. The PDGF/PDGF-R signaling pathway appears to regulate cancer-stromal cell interaction, and target molecule-based inhibition of the stromal reaction may hold promise as an effective anti-tumor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1509. doi:1538-7445.AM2012-1509

Expression of platelet-derived growth factor-B mRNA during vaginal vs. dermal incisional wound healing in the rabbit

The outcome of pelvic reconstructive surgery is largely dependent on the vaginal wound healing process, but this process has not yet been fully elucidated. Platelet-derived growth factor (PDGF) is an important mediator of the wound healing process in cutaneous tissue. We sought to compare PDGF-B mRNA expression in vaginal versus cutaneous incisional wound healing in a rabbit model. Bilateral 6 mm full-thickness circular segments were excised from the vagina and abdominal skin in 36 New Zealand-White female rabbits. Animals were euthanized sequentially before, on the day of and 4, 7, 10, 14, 21, 28 and 35 days after wounding. Their wounds were evaluated for surface area and PDGF-B mRNA expression using real time PCR. In both tissues PDGF-B mRNA expression increased constantly after wounding, reaching peak levels on day 10, and declined immediately thereafter, reaching minimal values on day 21. In both tissues, the expression of PDGF-B mRNA significantly correlated with the wound closure rate. PDGF-B mRNA expression significantly correlates with incisional vaginal and cutaneous wound closure, suggesting that this factor plays an important role in the wound healing process of both tissues.

Lipopolysaccharide enhances transforming growth factor β1‐induced platelet‐derived growth factor‐B expression in bile duct epithelial cells

Platelet-derived growth factor (PDGF)-B is a potent profibrogenic mediator expressed by bile duct epithelial cells (BDECs) that contributes to liver fibrosis after bile duct ligation. However, the mechanism of PDGF-B induction in BDECs during cholestasis is not known. Transforming growth factor β (TGFβ) and lipopolysaccharide (LPS) also contribute to the profibrogenic response after bile duct ligation. We tested the hypothesis that LPS and TGFβ1 synergistically induce PDGF-B expression in BDECs. Transformed human BDECs (MMNK-1 cells) and primary rat BDECs were stimulated with LPS and/or TGFβ1, and signaling pathways through which LPS potentiates TGFβ1-induced PDGF-B mRNA expression were investigated. Stimulation of MMNK-1 cells with LPS alone did not significantly induce PDGF-B mRNA expression. However, LPS co-treatment enhanced TGFβ1 induction of PDGF-B mRNA in MMNK-1 cells and also in primary rat BDECs. Importantly, co-treatment of MMNK-1 cells with LPS and TGFβ1 also significantly increased PDGF-BB protein expression. Interestingly, LPS did not affect TGFβ1 activation of a SMAD-dependent reporter construct. Rather, stimulation of MMNK-1 cells with LPS, but not TGFβ1, increased JNK1/2 phosphorylation. Expression of dominant negative JNK2, but not dominant negative JNK1, inhibited the LPS potentiation of TGFβ1-induced PDGF-B mRNA expression in MMNK-1 cells. In addition, LPS treatment caused IκBα degradation and activation of a nuclear factor κB (NFκB)-dependent reporter construct. Expression of an IκBα super repressor inhibited activation of NFκB and attenuated LPS potentiation of TGFβ1-induced PDGF-B mRNA. The results indicate that LPS activation of NFκB and JNK2 enhances TGFβ1-induced PDGF-B expression in BDECs.

SDF-1α Induces PDGF-B Expression and the Differentiation of Bone Marrow Cells into Pericytes

Platelet-derived growth factor B (PDGF-B) and its receptor, PDGFR-β, play a critical role in pericyte maturation; however, the mechanisms by which PDGF-B is upregulated in the tumor microenvironment remain unclear. We previously showed that upregulating stromal-derived factor, SDF-1α, in VEGF(165)-inhibited Ewing's sarcoma tumors (TC/siVEGF(7-1)) induced PDGF-B mRNA expression, increased infiltration and differentiation of bone marrow cells (BMC) into pericytes and, rescued tumor growth. The purpose of this study was to investigate the mechanism by which SDF-1α increased PDGF-B expression and the role of this pathway in BM-derived pericyte differentiation. We showed that SDF-1α induced expression of PDGF-B mRNA and protein both in vitro and in vivo. In contrast, inhibiting SDF-1α downregulated PDGF-B. We cloned the 2-kb pdgf-b promoter fragment and showed that SDF-1α activates PDGF-B via a transcriptional mechanism. Chromatin immunoprecipitation showed that the ELK-1 transcription factor binds to the pdgf-b promoter in response to SDF-1α. We confirmed the correlation between the SDF-1α/PDGF-B pathway and the differentiation of PDGFR-β+ BMCs into mature pericytes using an in vitro assay. These findings show that SDF-1α regulates PDGF-B expression and that this regulation plays a critical role in the differentiation of PDGFR-β+ BMCs into mature pericytes.

Activation of Peroxisome Proliferator-Activated Receptor α in Megakaryocytes Reduces Platelet-Derived Growth Factor-BB in Platelets

Platelet-derived growth factor (PDGF)-BB plays a crucial role in atherosclerosis and vascular remodeling by promoting the migration and proliferation of vascular smooth muscle cells. The objective of this study was to clarify the pleiotropic effect of peroxisome proliferator-activated receptor α (PPARα) activators on PDGF-BB expression in megakaryocytes and platelets. The expression of PPARα in a human erythroleukemia (HEL) cells was clearly detected by reverse transcriptase-PCR and immunofluorescence microscopy. The expression level of PPARα in HEL cells was unchanged regardless of differentiation into megakaryocytic cells by treatment with phorbol 12-myristate 13 acetate (TPA). The TPA-induced expression of PDGF-B mRNA and PDGF-BB protein levels in culture media was significantly decreased by treatment with PPARα activators, Wy14643 and fenofibric acid, in a dose-dependent manner. PDGF-BB expression induced by inflammatory cytokines, including interleukin-1β or interleukin-6, was also significantly suppressed by treatment with PPARα activators. Immunohistochemistry of human bone marrow showed the expression of PPARα in both the nucleus and cytoplasm of megakaryocytes. In addition, PDGF-BB levels in platelets were significantly decreased from 1,800±870 to 1,470±840 pg/10(5) platelets (mean±SD, p<0.05) by treatment with 300 mg fenofibrate once daily for 4 weeks in 13 patients with dyslipidemia. Activation of PPARα in megakaryocytes reduces PDGF-BB expression in platelets. PPARα activators may exert vasculo-protective action through suppression of PDGF-BB production in a megakaryocyte/platelet pathway.

Expression of platelet‐derived growth factor (PDGF)‐B and PDGF‐receptor β is associated with lymphatic metastasis in human gastric carcinoma

Recent study of murine fibrosarcoma has revealed that platelet-derived growth factor (PDGF) plays a direct role in promoting lymphangiogenesis and metastatic spread to lymph nodes. Thus, we investigated the relation between PDGF and PDGF receptor (PDGF-R) expression and lymphatic metastasis in human gastric carcinoma. We examined PDGF-B and PDGF-Rβ expression in four human gastric carcinoma cell lines (TMK-1, MKN-1, MKN-45, and KKLS) and in 38 surgical specimens of gastric carcinoma. PDGF-B and PDGF-Rβ expression was examined by immunofluorescence in surgical specimens and in human gastric carcinoma cells (TMK-1) implanted orthotopically in nude mice. Groups of mice (n = 10, each) received saline (control) or PDGF-R tyrosine kinase inhibitor imatinib. PDGF-B and PDGF-Rβ mRNA expression was significantly higher in patients with lymph node metastasis than in those without and was also significantly higher in diffuse-type carcinoma than in intestinal-type carcinoma. In surgical specimens, tumor cells expressed PDGF-B, but PDGF-Rβ was expressed predominantly by stromal cells. Under culture conditions, expression of PDGF-B mRNA was found in all of the gastric cell lines, albeit at different levels. In orthotopic TMK-1 tumors, cancer cells expressed PDGF-B but not PDGF-Rβ. PDGF-Rβ was expressed by stromal cells, including lymphatic endothelial cells. Four weeks of treatment with imatinib significantly decreased the area of lymphatic vessels. Our data indicate that secretion of PDGF-B by gastric carcinoma cells and expression of PDGF-Rβ by tumor-associated stromal cells are associated with lymphatic metastasis. Blockade of PDGF-R signaling pathways may inhibit lymph node metastasis of gastric carcinoma.

Retinoic acid modulates IL-4 receptor alpha chain expression and signaling pathways in intestinal epithelial cells and macrophages (51.3)

Abstract We have shown that all-trans retinoic acid (ATRA) increased mRNA expression of several IL-4-induced chemokines in explanted porcine alveolar macrophages. We now describe that ATRA modulated IL-4-induced signaling pathways in primary macrophages, and several macrophage and epithelial cell lines. ATRA increased epithelial cell mRNA and protein for the IL-4 receptor alpha chain (IL4RA), and IL-4 induced phosphorylation of STAT6 and expression of CLCA1 mRNA in vitro. In primary macrophages and several macrophage cell lines, ATRA-induced IL4RA mRNA and enhanced IL-4-induced expression of DHSR3, MRC1, PLAT, PTPN1, TGM2, and WARS mRNA and facilitated IL-4-inhibited expression of TLR2 and CYBB mRNA. However, ATRA blocked IL-4-induced expression of DHSR9, MAF, SEPP1 and STAT5A mRNA and counteracted IL-4 inhibited expression of BCL2L1, CCL2, NCF4, and PDGFB mRNA. These activities were largely shared by the RAR-a selective retinoid, Am580. ATRA variably affected the expression of a large number of other IL-4-induced mRNAs. These data indicated that ATRA and related retinoids can modulate Th2-related responses through mechanisms that utilize IL-4 and IL-4 receptor signaling pathways.