Expression Of PDGF-D Research Articles

Expression of PDGF-D in Patients with Valvular Atrial Fibrillation

Expression of PDGF-D in Patients with Valvular Atrial Fibrillation

Abnormal Fetal Lung of Hoxa1-/- Piglets Is Rescued by Maternal Feeding with All-Trans Retinoic Acid.

Neonatal Hoxa1-/- piglets were characterized by dyspnea owing to the Hoxa1 mutation, and maternal administration with ATRA alleviated the dyspnea of neonatal Hoxa1-/- piglets. The purpose of this experiment was to explore how maternal ATRA administration rescued the abnormal fetal lungs of Hoxa1-/- piglets. Samples of the lungs were collected from neonatal Hoxa1-/- and non-Hoxa1-/- piglets delivered by sows in the control group, and from neonatal Hoxa1-/- piglets born by sows administered with ATRA at 4 mg/kg body weight on dpc 12, 13, or 14, respectively. These were used for the analysis of ELISA, histological morphology, immunofluorescence staining, immunohistochemistry staining, and quantitative real-time PCR. The results indicate that the Hoxa1 mutation had adverse impacts on the development of the alveoli and pulmonary microvessels of Hoxa1-/- piglets. Maternal administration with ATRA at 4 mg/kg body weight on dpc 14 rescued the abnormal lung development of Hoxa1-/- piglets by increasing the IFN-γ concentration (p < 0.05), airspace area (p < 0.01) and pulmonary microvessel density (p < 0.01); increasing the expression of VEGFD (p < 0.01), PDGFD (p < 0.01), KDR (p < 0.01), ID1 (p < 0.01), and NEDD4 (p < 0.01); and decreasing the septal wall thickness (p < 0.01) and the expression of SFTPC (p < 0.01) and FOXO3 (p < 0.01). Maternal administration with ATRA plays a vital role in rescuing the abnormal development of lung of Hoxa1-/- fetal piglets.

PDGFR-β As a New Therapeutic Target in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. General prognosis of T-ALL has been improving over time. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains dismal. PDGFR-β is a transmembrane glycoprotein dimer molecule that functions as a receptor tyrosine kinase. Ligand binding (PDGF-B/PDGF-D) results in receptor dimerization and subsequent activation via autophosphorylation, causing the downstream activation of various signaling pathways such as PI3K/AKT, MAPK/ERK, JAK/STAT and Notch. During normal T cell development, PDGFR-β is expressed in the double negative stage in CD34+ T-cells and drops dramatically once CD34 is lost. The expression of both ligands, PDGF-B and PDGF-D, seems to be restricted to γδ CD3+ CD1- T-cells. We looked in vivo to the effect of PDGFR-β overexpression on normal T cell development making use of CD2-iCre driven overexpression of PDGFR- β in a C57Bl6 background. Overexpression of PDGFR-β in the R26-locus did not affect normal T-cell development. In T-ALL patients, high PDGFR-β expression can be observed in the HOXA and TLX3 subgroups. These subgroups have significantly higher PDGFR-β expression compared to normal developing T-cells. As constitutively active PDGFR-β is observed in a variety of malignancies, multi-target kinase inhibitors are already used in the clinic. However, they are not specific and often result in adverse side-effects. CP-673451 is a novel, potent and selective PDGFR inhibitor. Mechanistically, CP-673451 inhibits autophosphorylation of dimeric PDGFR-β and in this way prevents phosphorylation of downstream PI3K/AKT, GSK-3α and GSK-3β. Previous studies showed growth inhibition of multiple tumor xenografts (lung and colon carcinomas, glioblastoma) mainly due to a direct antitumor effect. Previously, we demonstrated the efficacy of CP-673451 on a T-ALL (IC50=544 nM) and a B-ALL cell line (IC50=500nM) with high PDGFR-β expression, as well as on a T-ALL PDX model with high levels of PDGFR-β (IC50=1,6 nM). Both cell lines and PDX models showed phosphorylation of the PDGFR-β receptor. After PDGFR-β inhibition, a decrease of PDGFR-β could be observed as a negative feedback loop. Also, phosphorylation of the receptor itself decreased which resulted downstream in a downregulated phosphorylation levels of GSK3β (Ser9), STAT3 (Tyr705) and STAT5 (Tyr694). Combination experiments with glucocorticoids (Fig. A) and chemotherapy (Fig. B), currently used as conventional therapy in T-ALL, showed a synergistic effect when combined with PDGFR-β inhibition. In conclusion, we have shown that specific PDGFR-β inhibitors can be used as a novel and targeted therapy in PDGFR-βhigh T-ALL patients and that they can be of added value to the current chemotherapy treatment for T-ALL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cancer-Associated Fibroblasts in Cholangiocarcinoma: Current Knowledge and Possible Implications for Therapy.

Cholangiocarcinoma (CCA) is an aggressive neoplasia with an increasing incidence and mortality. It is characterized by a strong desmoplastic stroma surrounding cancer cells. Cancer-associated fibroblasts (CAFs) are the main cell type of CCA stroma and they have an important role in modulating cancer microenvironments. CAFs originate from multiple lines of cells and mainly consist of fibroblasts and alpha-smooth muscle actin (α-SMA) positive myofibroblast-like cells. The continuous cross-talking between CCA cells and desmoplastic stroma is permitted by CAF biochemical signals, which modulate a number of pathways. Stromal cell-derived factor-1 expression increases CAF recruitment to the tumor reactive stroma and influences apoptotic pathways. The Bcl-2 family protein enhances susceptibility to CAF apoptosis and PDGFRβ induces fibroblast migration and stimulates tumor lymphangiogenesis. Many factors related to CAFs may influence CCA prognosis. For instance, a better prognosis is associated with IL-33 expression and low stromal IL-6 (whose secretion is stimulated by microRNA). In contrast, a worst prognosis is given by the expression of PDGF-D, podoplanin, SDF-1, α-SMA high expression, and periostin. The maturity phenotype has a prognostic relevance too. New therapeutic strategies involving CAFs are currently under study. Promising results are obtained with anti-PlGF therapy, nintedanib (BIBF1120), navitoclax, IPI-926, resveratrol, and controlled hyperthermia.

Platelet-Derived Growth Factors Affect Clinical Features and Prognosis of Gastric Cancer.

Purpose To investigate the association of platelet-derived growth factors (PDGFs), clinicopathological features, and prognosis in gastric cancer patients. Methods Tumor specimens of 180 individuals with gastric cancer treated between 2016 and 2020 were collected. Immunohistochemical staining and Western blot (WB) were used to detect the expression of PDGF-B and PDGF-D. The relationship between the expression of PDGF-B and PDGF-D and relapse-free subsistence (RFS) time was assessed using Kaplan–Meier curves. Univariate and multivariate Cox proportional hazards regression models were used to evaluate the relationship between the expression of PDGF-B and PDGF-D and the prognosis and clinicopathological features in gastric cancer patients. Results High expression of PDGF-B and PDGF-D was detected in 108 (60%) and 137 (76%) tumor specimens, respectively. The expressions of PDGF-B and PDGF-D were independent predictive indicators in multivariate analysis when compared to tumor depth, tumor stage, lymph node metastasis, and RFS (P < 0.01). Conclusion The high expression of PDGF-B and PDGF-D in gastric cancer tissues is associated with poor prognosis and poor survival rate of the patients. The expression of PDGF-B and PDGF-D can be used as important indicators to evaluate the biological behavior and prognosis of gastric cancer.

MO066: The Role of Platelet-Derived Growth Factor in Focal Segmental Glomerulosclerosis

Abstract BACKGROUND AND AIMS Focal segmental glomerulosclerosis (FSGS) describes a primary disease of podocytes (podocytopathy) and a secondary consequence of severe or progressive glomerular damage. No specific treatment options exist for FSGS, with many patients progressing to end-stage kidney disease. While podocyte injury is considered to initiate FSGS, parietal epithelial cells (PECs) were shown to be one of the main effector cells forming FSGS. Given the involvement of platelet-derived growth factor (PDGF) in fibrotic processes, we hypothesized that PDGF signaling is involved in the formation and progression of FSGS. METHOD We analyzed the role of PDGF in FSGS development and progression using a murine Thy1.1 trangenic FSGS model, patient kidney biopsies and primary murine PECs. In a second step, we therapeutically treated mice with FSGS with a neutralizing anti-PDGF-B antibody. RESULTS The podocyte-specific Thy1.1 transgenic mice 3 weeks after anti-Thy1.1 antibody injection showed reduced kidney function due to FSGS development. In this model, compared with control mice, PECs were progressively activated (63.3-fold increase in CD44 expression), showed a significantly increased proliferation (14-fold increase in Ki67 expression), followed by glomerular extracellular matrix deposition (2.6-fold increase in collagen 4) and FSGS development. Fluorescence in situ hybridization showed that mRNA of Pdgfb and Pdgfd was elevated in podocytes and Pdgfrb was upregulated in PECs during the time course of the disease. Similarly, in human FSGS biopsies, gene expression of both ligands, PDGFB and PDGFD, was elevated in podocytes and PDGFRB was increased in PECs compared with healthy glomeruli. Stimulation of primary mouse PECs with PDGF-B and PDGF-D in vitro led to significant activation (1.37- and 1.44-fold increase in CD44 expression, respectively), proliferation (1.46- and 1.36-fold increase, respectively), migration and extracellular matrix production (1.40-fold increase in fibronectin after PDGF-D stimulation) which could be inhibited by imatinib or a neutralizing PDGF-B antibody. In mice with FSGS, both a short- and long-term treatment with PDGF-B neutralizing antibody improved kidney function and reduced FSGS, PEC proliferation and profibrotic activation. CONCLUSION PDGFR-β and its ligands PDGF-B and PDGF-D were upregulated in experimental and clinical samples with FSGS in PECs and podocytes, respectively, and the inhibition of PDGF-B reduced PEC proliferation and FSGS development. This suggests paracrine cross-talk between injured podocytes and PECs, leading to PECs activation toward a profibrotic (pro-FSGS) phenotype and FSGS development. Thus, PDGF represents a novel molecular pathway in FSGS pathogenesis and potential target for therapy.

SU16f inhibits fibrotic scar formation and facilitates axon regeneration and locomotor function recovery after spinal cord injury by blocking the PDGFR\u03b2 pathway

BackgroundExcessively deposited fibrotic scar after spinal cord injury (SCI) inhibits axon regeneration. It has been reported that platelet-derived growth factor receptor beta (PDGFRβ), as a marker of fibrotic scar-forming fibroblasts, can only be activated by platelet-derived growth factor (PDGF) B or PDGFD. However, whether the activation of the PDGFRβ pathway can mediate fibrotic scar formation after SCI remains unclear.MethodsA spinal cord compression injury mouse model was used. In situ injection of exogenous PDGFB or PDGFD in the spinal cord was used to specifically activate the PDGFRβ pathway in the uninjured spinal cord, while intrathecal injection of SU16f was used to specifically block the PDGFRβ pathway in the uninjured or injured spinal cord. Immunofluorescence staining was performed to explore the distributions and cell sources of PDGFB and PDGFD, and to evaluate astrocytic scar, fibrotic scar, inflammatory cells and axon regeneration after SCI. Basso Mouse Scale (BMS) and footprint analysis were performed to evaluate locomotor function recovery after SCI.ResultsWe found that the expression of PDGFD and PDGFB increased successively after SCI, and PDGFB was mainly secreted by astrocytes, while PDGFD was mainly secreted by macrophages/microglia and fibroblasts. In addition, in situ injection of exogenous PDGFB or PDGFD can lead to fibrosis in the uninjured spinal cord, while this profibrotic effect could be specifically blocked by the PDGFRβ inhibitor SU16f. We then treated the mice after SCI with SU16f and found the reduction of fibrotic scar, the interruption of scar boundary and the inhibition of lesion and inflammation, which promoted axon regeneration and locomotor function recovery after SCI.ConclusionsOur study demonstrates that activation of PDGFRβ pathway can directly induce fibrotic scar formation, and specific blocking of this pathway would contribute to the treatment of SCI.

Identification of HBEGF+ fibroblasts in the remission of rheumatoid arthritis by integrating single-cell RNA sequencing datasets and bulk RNA sequencing datasets

BackgroundFibroblasts are important structural cells in synovium and play key roles in maintaining the synovial homeostasis. By single-cell RNA sequencing (scRNA-seq), subpopulation of synovium-resident cells has been reported to protect intra-articular structures from chronic inflammation and promote tissue repair. However, a significant number of researchers have concentrated on the role of fibroblasts in the progress of rheumatoid arthritis (RA) while few reports had described the contribution of distinct fibroblast subsets in the RA remission. It is helpful to understand the role of fibroblast subpopulations in the RA process to provide predictive biomarkers and address RA remission mechanisms. Here, we found HBEGF+ fibroblasts that contributed to RA remission by integrating scRNA-seq datasets and bulk RNA sequencing (bulk RNA-seq) datasets.MethodThree single-cell RNA datasets of cells harvested from RA patients were processed and integrated by Seurat and Harmony R packages. After identifying cell types by classic marker genes, the integrated dataset was used to run CellChat for analysis of cell-cell communication. Specially, EGF signaling pathway was found and HBEGF+ fibroblasts were identified based on HBEGF expression. Differential expressed genes of HBEGF+ were shown in heatmap and volcano plot and used to run gene ontology (GO) enrichment analysis. Next, bulk RNA-seq datasets of synovium under different conditions (health, osteoarthritis (OA), rheumatoid arthritis, before and after classical treatment) were compared to show expression change of HBEGF and gene markers that are mainly expressed by HBEGF+ fibroblasts such as CLIC5, PDGFD, BDH2, and ENPP1. Finally, two single-cell RNA sequencing datasets of synovial cells from mice were integrated to identify Hbegf+ fibroblasts and calculate the population of Hbegf+ fibroblasts under different joint conditions (health, K/BxN serum transfer arthritis (STA), and remission of STA).ResultAfter integrating three single-cell RNA sequencing datasets, we identified 11 clusters of synovial cells, such as fibroblasts, mural cells, endothelial cells, CD4+ T cells, CD8+ T cells, natural killer cells, synovium macrophage, peripheral blood macrophages, plasma cells, B cells, and STMN1+ cells. We found fibroblasts had an extensive communication network with other clusters and interacted with synovial macrophages through EGF signaling pathway via analysis of cell-cell communication between synovial cells. HBEGF, ligand to EGF signaling pathway, was highly expressed by a subset of fibroblasts and macrophages, and EGFR, receptor to EGF signaling pathway, was highly expressed by fibroblasts and meniscus cells. Moreover, HBEGF was downregulated under RA state and it had an increase after classical treatment. We then defined fibroblasts with high expression of HBEGF as HBEGF+ fibroblasts. In addition, we also found that HBEGF+ fibroblasts highly expressed CRTAC1, ITGB8, SCARA5, THBS4, and ITGBL1, genes relative to encoding extracellular matrix proteins and engaged in positive regulation of cell migration and motility, cellular component movement, and cell growth by GO enrichment analysis. We eventually identified HBEGF+ fibroblasts specially expressed CLIC5, PDGFD, BDH2, and ENPP1, which positively correlated with the expression of HBEGF. Moreover, the expression of CLIC5, PDGFD, BDH2, and ENPP1 was downregulated under RA state and elevated by classical therapy. On the contrary, the HBEGF+ macrophages specially expressed SLAMF8, GK, L1RN, and JAK2, which negatively correlated with the expression of HBEGF. The expression was upregulated in SLAMF8, GK, L1RN, and JAK2 under the RA state, whereas it was decreased after classical treatment. In mice, the number of Hbegf+ fibroblasts was reduced in the RA synovium but increased in the RA remitting synovium.ConclusionsHBEGF+ fibroblasts play a role in the remission of rheumatoid arthritis, and HBEGF has potential to become a novel biomarker for prediction of RA progress.

The HIF1\u03b1-PDGFD-PDGFR\u03b1 axis controls glioblastoma growth at normoxia/mild-hypoxia and confers sensitivity to targeted therapy by echinomycin

BackgroundGlioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies.MethodsTarget genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence.ResultsHIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway.ConclusionsHIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.

Neuropilin-2 and Its Transcript Variants Correlate with Clinical Outcome in Bladder Cancer.

Urothelial bladder cancer ranks among the 10 most frequently diagnosed cancers worldwide. In our previous study, the transmembrane protein neuropilin-2 (NRP2) emerged as a predictive marker in patients with bladder cancer. NRP2 consists of several splice variants; the most abundant of these, NRP2a and NRP2b, are reported to have different biological functions in lung cancer progression. For other cancer types, there are no published data on the role of these transcript variants in cancer progression and the clinical outcome. Here, we correlate NRP2 and its two most abundant transcript variants, NRP2A and NRP2B, with the clinical outcome using available genomic data with subsequent validation in our own cohort of patients with muscle-invasive bladder cancer. In addition to NRP2, NRP1 and the NRP ligands PDGFC and PDGFD were studied. Only NRP2A emerged as an independent prognostic marker for shorter cancer-specific survival in muscle-invasive bladder cancer in our cohort of 102 patients who underwent radical cystectomy between 2008 and 2014 with a median follow-up time of 82 months. Additionally, we demonstrate that high messenger expression of NRP2, NRP1, PDGFC and PDGFD associates with a more aggressive disease (i.e., a high T stage, positive lymph node status and reduced survival).

Exosomes Released from CaSR-Stimulated PMNs Reduce Ischaemia/Reperfusion Injury.

Ischemia-reperfusion (I/R) injury caused by acute myocardial infarction (AMI) can initiate a strong inflammatory response. Polymorphonuclear cells (PMNs) are the most important inflammatory cells. Our previous studies found that the calcium-sensing receptor (CaSR) regulates the proinflammatory effects of PMNs. However, the role and mechanism of CaSR-regulated PMNs in I/R injury remain uncertain. A rat AMI model was developed in this study and showed that the expression of CaSR on PMNs increased in AMI; however, the levels of Bcl-xl and SOD in myocardial tissue decreased, while Bax and MDA levels increased. Then, after coculture with CaSR-stimulated PMNs, the expression of Bcl-xl in cardiomyocytes significantly increased, Bax expression and the apoptotic rate decreased, and ROS production was significantly inhibited. At the same time, the cardiomyocyte damage caused by hypoxia-reoxygenation was reduced. Furthermore, we found that exosomes derived from PMNs could be taken up by cardiomyocytes. Additionally, the exosomes secreted by CaSR-stimulated PMNs had the same effect on cardiomyocytes as CaSR-stimulated PMNs, while the increased phosphorylation level of AKT in cardiomyocytes could be revered by AKT transduction pathway inhibitors. Subsequently, we identified the exosomes derived from CaSR-stimulated PMNs by second-generation sequencing technology, and increased expression of lncRNA ENSRNOT00000039868 was noted. The data show that this lncRNA can prevent the hypoxia-reoxygenation injury by upregulating the expression of PDGFD in cardiomyocytes. In vivo, exosomes from CaSR-stimulated PMNs played a significant role against AMI and reperfusion injury in myocardial tissue. Thus, we propose that exosomes derived from CaSR-stimulated PMNs can reduce I/R injury in AMI, and this effect may be related to the AKT signaling pathway.

High expression of PDGFR-\u03b2 in prostate cancer stroma is independently associated with clinical and biochemical prostate cancer recurrence

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.

Clinical significance of platelet derived growth factor-C and -D in gastric cancer.

Platelet-derived growth factor (PDGF)-C and PDGF-D are frequently upregulated in human cancers and play important roles in tumor progression, angiogenesis and metastasis. However, the distribution, frequency and prognostic value of PDGF-C and PDGF-D expression in gastric cancer have not been clarified. The present study evaluated the association between expression of PDGF-C and PDGF-D, clinicopathological factors and outcomes, in patients with gastric cancer. Gastric adenocarcinoma tumor samples were obtained from 204 patients who underwent curative gastrectomy between 2003 and 2007. The expression of PDGF-C and PDGF-D was analyzed by immunohistochemical staining. High expression of PDGF-C and PDGF-D was detected in 114 (56%) and 151 (74%) tumors, respectively. PDGF-D expression was significantly associated with tumor depth (P=0.039), histopathology (P<0.01), tumor stage (P=0.01) and recurrence (P<0.01), whereas PDGF-C expression correlated only with histopathology (P=0.05). High PDGF-D expression was also associated with significantly shorter relapse-free survival (RFS) time (P<0.01), whilst high PDGF-C expression was associated with marginally, but not significantly, shorter RFS (P=0.10). On multivariate analysis, high PDGF-D expression was determined to be an independent prognostic factor (hazard ratio, 3.3; 95% confidence interval, 1.20–9.4; P=0.02). These findings indicate that high PDGF-D expression is strongly associated with tumor progression, recurrence, distant metastasis and poor outcomes in patients with gastric cancer. PDGF-D may therefore be an independent prognostic factor and a novel therapeutic target.

Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells.

Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice.

P-005 Clinical significance of Eph, FGFR, and PDGF expressions in advanced gastric cancers

Introduction: Various tyrosine kinases receptors and those ligands are involved in growth of malignant disease. Many studies showed the relation between HER1, HER2, VEGFRs, or cMET and gastric cancers. Inhibition of HER2 or VEGFR has been used for treatment for unresectable or metastatic gastric cancer in clinical practice. In this study, we investigated expression of EphA1, EphA2, EphA4, ephrinA1, FGFR1, FGFR 2, FGFR 4, PDGFC, and PDGFD and evaluated the relation to clinical outcomes in advanced gastric cancers resected completely, because those expressions was involved with tumor progression of gastric cancer in our previous studies.Methods: Our study group comprised 117 patients with primary advanced gastric adenocarcinoma who underwent surgical complete resection between 2003 and 2007 in the Department of Esophagogastric Surgery, Tokyo Medical and Dental University. The expression of EphA1, A2, A4, ephrinA1, FGFR1, 2, 4, PDGFC, and PDGFD was measured in the resected tumors by immunohistochemical analysis. Interpretation of immunostaining results was based on a scoring system comprised of both staining intensity and extensity.Results: Based on the 7th TNM classification, T2, T3, and T4 tumors were 26, 32, and 43%, and N0, N1, N2, and N3 tumors were 34, 17, 21, and 27%. Stage IB, II, and III tumors were17, 30, and 53%. Overexpression of EphA1, EphA2, EphA4, ephrinA1, FGFR1, FGFR 2, FGFR 4, PDGFC, and PDGFD were 74, 79, 67, 61, 41, 64, 91, 61, and 85%. Serosa invasion was significantly associated with high expression of EphA2, EphA4, and PDGFD (p = 0.01, 0.025, and 0.015) and lymph node metastasis was done with EphA2, EphA4, and FGFR4 (p = 0.01, 0.006, and 0.044). Stage III significantly related to EphA4, FGFR4, and PDGFC (p = 0.005, 0.021, and 0.01). On univariate analysis of relapse-free survival, overexpression of EphA2, EphA4, or PDGFD was significantly associated with poor survival (p = 0.015, 0.001, and 0.046). EphA4 was an independent prognostic factor (HR = 2.4, 95% CI: 1.1-5.4; p = 0.034)Conclusion: Eph, FGFR, or PDGF overexpressed in many patients with advanced gastric cancer. The expression of EphA2, EphA4, or PDGFD was significantly associated with survival of those patients. EphA4 might become a prognostic factor in those patients. Introduction: Various tyrosine kinases receptors and those ligands are involved in growth of malignant disease. Many studies showed the relation between HER1, HER2, VEGFRs, or cMET and gastric cancers. Inhibition of HER2 or VEGFR has been used for treatment for unresectable or metastatic gastric cancer in clinical practice. In this study, we investigated expression of EphA1, EphA2, EphA4, ephrinA1, FGFR1, FGFR 2, FGFR 4, PDGFC, and PDGFD and evaluated the relation to clinical outcomes in advanced gastric cancers resected completely, because those expressions was involved with tumor progression of gastric cancer in our previous studies. Methods: Our study group comprised 117 patients with primary advanced gastric adenocarcinoma who underwent surgical complete resection between 2003 and 2007 in the Department of Esophagogastric Surgery, Tokyo Medical and Dental University. The expression of EphA1, A2, A4, ephrinA1, FGFR1, 2, 4, PDGFC, and PDGFD was measured in the resected tumors by immunohistochemical analysis. Interpretation of immunostaining results was based on a scoring system comprised of both staining intensity and extensity. Results: Based on the 7th TNM classification, T2, T3, and T4 tumors were 26, 32, and 43%, and N0, N1, N2, and N3 tumors were 34, 17, 21, and 27%. Stage IB, II, and III tumors were17, 30, and 53%. Overexpression of EphA1, EphA2, EphA4, ephrinA1, FGFR1, FGFR 2, FGFR 4, PDGFC, and PDGFD were 74, 79, 67, 61, 41, 64, 91, 61, and 85%. Serosa invasion was significantly associated with high expression of EphA2, EphA4, and PDGFD (p = 0.01, 0.025, and 0.015) and lymph node metastasis was done with EphA2, EphA4, and FGFR4 (p = 0.01, 0.006, and 0.044). Stage III significantly related to EphA4, FGFR4, and PDGFC (p = 0.005, 0.021, and 0.01). On univariate analysis of relapse-free survival, overexpression of EphA2, EphA4, or PDGFD was significantly associated with poor survival (p = 0.015, 0.001, and 0.046). EphA4 was an independent prognostic factor (HR = 2.4, 95% CI: 1.1-5.4; p = 0.034) Conclusion: Eph, FGFR, or PDGF overexpressed in many patients with advanced gastric cancer. The expression of EphA2, EphA4, or PDGFD was significantly associated with survival of those patients. EphA4 might become a prognostic factor in those patients.

MP31-14 MICRORNAS AND GENES RELATED TO EPITHELIAL-MESENCHYMAL TRANSITION IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2014MP31-14 MICRORNAS AND GENES RELATED TO EPITHELIAL-MESENCHYMAL TRANSITION IN PROSTATE CANCER Betina Katz, Sabrina Reis, Nelson Dip, Nayara Viana, Denis Morais, Caio Moura, Iran Silva, Alexandre Iscaife, Miguel Srougi, and Katia RM. Leite Betina KatzBetina Katz More articles by this author , Sabrina ReisSabrina Reis More articles by this author , Nelson DipNelson Dip More articles by this author , Nayara VianaNayara Viana More articles by this author , Denis MoraisDenis Morais More articles by this author , Caio MouraCaio Moura More articles by this author , Iran SilvaIran Silva More articles by this author , Alexandre IscaifeAlexandre Iscaife More articles by this author , Miguel SrougiMiguel Srougi More articles by this author , and Katia RM. LeiteKatia RM. Leite More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.923AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) is the most common cancer in men and the second leading cause of cancer-related mortality. Although most patients present with localized disease at diagnosis, 5% already have metastatic disease. One of the main mechanisms of tumor progression is the epithelial-mesenchymal transition (EMT). In EMT, epithelial cells lose intercellular adhesion and acquire a mesenchymal phenotype with invasiveness and metastatic potential. The main event is the repression of E-cadherin by transcriptional factors, including ZEB1, ZEB2, Snail, Slug and Twist. microRNAs are also involved in EMT, and one of the most important is the miR-200 family, which induces epithelial differentiation by inhibiting ZEB. Our aim was to evaluate the expression of genes and miRNAs involved in EMT in localized PCa and to correlate with clinicopathological features and outcome after surgery. METHODS Fifty-one fresh-frozen samples from patients with localized PCa submitted to radical prostatectomy were selected. Gene expression of E-cadherin, N-cadherin, Vimentin, TGFβ, ZEB1, ZEB2, Snail, Slug, Twist and PDGF-D and miRNA expression of miRNAs 200b, 429, 141, 203, 205, 183, 373, 21, 9, 1, 495, 29b, 30a, 34a, 155 and 10b were assessed by qRT-PCR. Controls were comprised by 10 cases of benign prostatic hyperplasia. The genes and miRNAs expression were compared with Gleason score (GS), pathological stage (pT), preoperative PSA and biochemical recurrence. RESULTS The majority of cases showed overexpression of E-cadherin and Twist and underexpression of N-cadherin, Vimentin, TGFβ, ZEB1, Slug and PDGF-D. miRNAs 200b, 429, 141, 203, 205, 183, 373 and 21 were overexpressed, while miRNAs 9, 495, 29b and 1 were underexpressed. The remaining showed a variable expression pattern. There was a significant correlation between lower expression of miR 200b, 30a and 1 and non-organ-confined disease (pT3). miRNAs 200b and 30a levels were also underexpressed in cases with GS ≥ 8 comparing to GS ≤ 6 (6.94 vs 18.67, p=0.035) and in patients categorized as having high risk disease in relation to low risk (1.70 vs 6.37; p=0.03), respectively. Yet, Vimentin and Twist were significantly overexpressed in the high risk group. CONCLUSIONS Localized PCa still maintains the epithelial phenotype. However, underexpression of miRNAs 200b, 30a and 1 were significantly associated with more advanced stage and higher GS. The mesenchymal markers Vimentin and Twist were also overexpressed in patients with high risk disease. These miRNAs and genes could be used in the clinical practice as tumor markers to characterize tumor aggressiveness. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e327 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Betina Katz More articles by this author Sabrina Reis More articles by this author Nelson Dip More articles by this author Nayara Viana More articles by this author Denis Morais More articles by this author Caio Moura More articles by this author Iran Silva More articles by this author Alexandre Iscaife More articles by this author Miguel Srougi More articles by this author Katia RM. Leite More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MMP-13 regulates growth of wound granulation tissue and modulates gene expression signatures involved in inflammation, proteolysis, and cell viability.

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13 −/−) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13 −/− mice. Granulation tissue in Mmp13 −/− mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13 −/− mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13−/− mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13 −/− granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13 −/− mice compared to WT mice. Mmp13 −/− mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.

A novel function for platelet-derived growth factor D: induction of osteoclastic differentiation for intraosseous tumor growth

Although increasing evidence suggests a critical role for platelet-derived growth factor (PDGF) receptor β (β-PDGFR) signaling in prostate cancer (PCa) progression, the precise roles of β-PDGFR and PDGF isoform-specific cell signaling have not been delineated. Recently, we identified the PDGF-D isoform as a ligand for β-PDGFR in PCa and showed that PDGF-D is activated by serine protease-mediated proteolytic removal of the CUB domain in a two-step process, yielding first a hemidimer (HD) and then a growth factor domain dimer. Herein, we demonstrate that the expression of PDGF-D in human PCa LNCaP cells leads to enhanced bone tumor growth and bone responses in immunodeficient mice. Histopathological analyses of bone tumors generated by PDGF-D-expressing LNCaP cells (LNCaP-PDGF-D) revealed osteolytic and osteoblastic responses similar to those observed in human PCa bone metastases. Importantly, we discovered a novel function of PDGF-D in the regulation of osteoclast differentiation, independent of the RANKL/RANK signaling axis. Although both PDGF-B and -D were able to activate β-PDGFR, only PDGF-D was able to induce osteoclastic differentiation in vitro, and upregulate the expression and nuclear translocation of nuclear factor of activated T cells 1, a master transcription factor for osteoclastogenesis. Taken together, these results reveal a new function of PDGF-D as a regulator of osteoclastic differentiation, an activity critical for the establishment of skeletal metastatic deposit in PCa patients.

Molecular Markers of Epithelial-to-Mesenchymal Transition Are Associated with Tumor Aggressiveness in Breast Carcinoma

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a transient process occurring during developmental stages and carcinogenesis, characterized by phenotypic and molecular alterations, resulting in increased invasive and metastatic capabilities of cancer cells and drug resistance. Moreover, emerging evidence suggests that EMT is associated with increased enrichment of cancer stem-like cells in neoplastic tissues. We interrogated the molecular alterations occurring in breast cancer using proposed EMT markers such as E-cadherin, vimentin, epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF) D, and nuclear factor κB (NF-κB) to decipher their roles in the EMT and breast cancer progression. METHODS: Fifty-seven invasive ductal adenocarcinomas of the breast were assessed for the expression of E-cadherin, vimentin, EGFR, NF-κB, and PDGF-D using immunohistochemical analysis. Tumors were categorized into three groups: A (ER+, and/or PR+, HER-2/neu-), B (ER+, and/or PR+, HER-2/neu+), and C (triple-negative: ER-, PR-, and HER-2/neu-). Immunostained slides were microscopically evaluated and scored using intensity (0, 1+, 2+, and 3+) and percentage of positive cells, and data were statistically analyzed. RESULTS: Membranous E-cadherin was positive in all 57 cases (100%), whereas cytoplasmic E-cadherin was predominantly positive in groups B and C compared with group A (21%, 7%, and 0%, respectively). All group A cases were negative for vimentin and EGFR. There was statistically significant increased expression of vimentin (P < .0002), EGFR (P < .0001), and NF-κB (P < .02) in triple-negative cases when compared with groups A and B. CONCLUSIONS: Vimentin, EGFR, and NF-κB were significantly increased in triple-negative tumors, which is consistent with the aggressiveness of these tumors. These markers could be useful as markers for EMT in breast cancers and may serve as predictive markers for designing customized therapy in the future.

Expression and Significance of PDGF-D and Its Receptor in Gastric Carcinoma Tissues

Objective To investigate the correlation between mRNA expression of PDGF-D and PDGFRβ and clinicopathological features in gastric cancer.Methods The reverse transcription-polymerase chain reaction(RT-PCR)technique was used to examine mRNA expression of PDGF-D and PDGFRβ in 58 paired specimens of gastric carcinoma and tissues adjacent carcinoma.Results The positive rates of PDGF-D mRNA and PDGFRβ mRNA(98.28%,87.93%)were higher in gastric cancer tissues than(72.41%,53.45%)in tissues adjacent carcinoma respectively.While the positive rate of PDGFRβmRNA was 72.41%,which was positively correlated with PDGF-D(r=0.40,P0.05).PDGF-D expression was correlated with lymph node metastasis and relapse(P0.05).No correlation was found between PDGF-D and other clinicopathological features of gastric carcinoma(P0.05).Conclusion PDGF-D may play a crucial role in carcinogenesis,development,metastasis and relapse of gastric cancer.It can be used as prognostic markers of gastric cancer in clinical practice.