OBJECTIVES: To investigate the role of apelin in regulating proliferation, migration and angiogenesis of bladder cancer cells and the possible regulatory mechanism. METHODS: GEO database was used to screen the differentially expressed genes in bladder cancer tissues and cells. Bladder cancer and paired adjacent tissues were collected from 60 patients for analysis of apelin expressions in relation to clinicopathological parameters. In cultured bladder cancer J82 cells and human umbilical vein endothelial cells (HUVECs), the effects of transfection with an apelin-overexpressing plasmid or specific siRNAs targeting apelin, fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) on proliferation and migration of J82 cells and tube formation in HUVECs were examined using plate cloning assay, Transwell assay, and angiogenesis assay; the changes in FGF2 expression and FGFR1 phosphorylation were detected using Western blotting. RESULTS: The expression level of apelin was significantly higher in bladder cancer tissues than adjacent tissues, and bladder cancer cell lines (T24 and J82) also expressed higher mRNA and protein levels of apelin than SV-HUC-1 cells. Apelin expression level in bladder cancer tissues was correlated with tumor invasion, distant metastasis and advanced TNM stages. Apelin knockdown significantly suppressed proliferation and migration of J82 cells and decreased the total angiogenic length of HUVECs. In contrast, apelin overexpression significantly promoted proliferation and migration and enhanced FGFR1 phosphorylation in J82 cells, and increased the total angiogenesis length in HUVECs, but this effects were effectively mitigated by transfection of the cells with FGF2 siRNA or FGFR1 siRNA. CONCLUSIONS High expression of apelin promotes J82 cell proliferation and migration and HUVEC angiogenesis by promoting activation of the FGF2/FGFR1 pathway.
Humans ; Urinary Bladder Neoplasms/blood supply* ; Receptor, Fibroblast Growth Factor, Type 1/metabolism* ; Cell Proliferation ; Cell Movement ; Fibroblast Growth Factor 2/metabolism* ; Neovascularization, Pathologic ; Human Umbilical Vein Endothelial Cells ; Cell Line, Tumor ; Signal Transduction ; Apelin ; Intercellular Signaling Peptides and Proteins/genetics* ; Female ; Male ; Angiogenesis

This study aimed to explore the molecular mechanism of Juanbi Qianggu Formula(JBQGF), an empirical formula formulated by the prestigious doctor in traditional Chinese medicine, in the treatment of rheumatoid arthritis based on network pharmacology and cell function experiments. The main active components and targets of JBQGF were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM), and the core targets underwent functional enrichment analysis and signaling pathway analysis. Cytoscape 3.6.0 was used to construct a visualized "active component-target-signaling pathway" network of JBQGF. After screening, nine potential pathways of JBQGF were obtained, mainly including G protein-coupled receptor signaling pathway and tyrosine kinase receptor signaling pathway. As previously indicated, the fibroblast growth factor receptor 1(FGFR1) signaling pathway was highly activated in active fibroblast-like synoviocytes(FLS) in rheumatoid arthritis, and cell and animal experiments demonstrated that inhibition of the FGFR1 signaling pathway could significantly reduce joint inflammation and joint destruction in collagen-induced arthritis(CIA) rats. In terms of the tyrosine kinase receptor signal transduction pathway, the analysis of its target genes revealed that FGFR1 might be a potential target of JBQGF for rheumatoid arthritis treatment. The biological effect of JBQGF by inhibiting FGFR1 phosphorylation was preliminarily verified by Western blot, Transwell invasion assay, and pannus erosion assay, thereby inhibiting matrix metalloproteinase 2(MMP2) and receptor activator of nuclear factor-κB ligand(RANKL) and suppressing the invasion of fibroblasts in rheumatoid arthritis and erosive effect of pannus bone. This study provides ideas for searching potential targets of rheumatoid arthritis treatment and TCM drugs through network pharmacology.
Rats ; Animals ; Synoviocytes ; Matrix Metalloproteinase 2/metabolism* ; Network Pharmacology ; Receptor, Fibroblast Growth Factor, Type 1/therapeutic use* ; Arthritis, Rheumatoid/genetics* ; Signal Transduction ; Fibroblasts ; Drugs, Chinese Herbal/therapeutic use*

PURPOSE: To examine the usefulness of various receptor tyrosine kinase expressions as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. MATERIALS AND METHODS: We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, immunohistochemical staining was done for human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 3 (FGFR3). RESULTS: There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, significantly shorter median time to recurrence (TTR) (12.9 months vs. 49.0 months; p=0.008) and overall survival (OS) (22.3 months vs. 52.7 months; p=0.006) was found in patients with FGFR1 overexpression. By contrast, there was no difference in TTR or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% confidence interval: 1.27-3.90, p=0.006) in multivariate analysis. CONCLUSION: Our result showed that FGFR1 expression, but not FGFR3, is an adverse prognostic factor in muscle invasive UC patients after radical cystectomy. FGFR1 might be feasible for prognosis prediction and a potential therapeutic target after thorough validation in muscle invasive UC.
Adult ; Aged ; Aged, 80 and over ; Carcinoma/*metabolism/*mortality/surgery ; Cystectomy ; Female ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Muscles/pathology ; Neoplasm Invasiveness ; Prognosis ; Proportional Hazards Models ; Receptor, ErbB-2/metabolism ; Receptor, Fibroblast Growth Factor, Type 1/*metabolism ; Receptor, Fibroblast Growth Factor, Type 3/metabolism ; Retrospective Studies ; Survival Rate ; Urinary Bladder Neoplasms/*metabolism/*mortality/surgery ; Urothelium/pathology

OBJECTIVE: In order to investigate the interaction between the cytokines and keratinocyte and determine the role of cytokines in hyperproliferative of chronic otitis media with cholesteatoma, we observe the expression of matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and its receptor (KGFR) in middle ear cholesteatoma. METHOD: We examined the expression of MMP9, VEGF, KGF, KGFR and Ki-67 by immunohistochemistry in 50 specimens from chronic otitis media with cholesteatoma and 15 specimens from the normal skin of external auditory meatus. Ki-67 as an evaluation of cholesteatoma proliferation markers were used to detect the keratinocyte proliferative activity. RESULT: (1) The expression of VEGF and MMP9 in cholesteatoma specimens was higher than normal skin, and the difference was statistically significant (t = 4.914, P < 0.01; t = 3.284, P < 0.01). (2) The expression of KGF and KGFR in middle ear tissues was higher than normal skin, and the difference was statistically significant (t = 4.814, P < 0.01; t = 3.104, P < 0.01); The expression of KGF and KGFR increased, and the expression of Ki-67 also correspondly increased in the cholesteatoma. (3) In the tissue MMP9 and VEGF were positive. Mean optical density increased as well. KGF expression also increased accordingly. CONCLUSION MMP9, VEGF, KGF and KGFR proteins played an important role in hyperproliferation of cholesteatoma tissues. VEGF, MMP9 and KGF had a synergistic effect in hyperproliferation of cholesteatoma tissues.
Cholesteatoma, Middle Ear ; pathology ; Cytokines ; metabolism ; Ear Canal ; metabolism ; Ear, Middle ; metabolism ; Fibroblast Growth Factor 7 ; metabolism ; Humans ; Immunohistochemistry ; Keratinocytes ; cytology ; Ki-67 Antigen ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Otitis Media ; pathology ; Receptor, Fibroblast Growth Factor, Type 2 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo observe the expression levels of hippocampal vascular endothelial growth factor (VEGF), fms-like tyrosine kinase-1 (flt-1), basic fibroblast growth factor (bFGF), and basic fibroblast growth factor receptor (bFGF-r) in vascular dementia (VD) rats, thus studying the angiogenesis mechanism of moxibustion in VD.

METHODSSixty male elderly Wistar rats were selected. The VD rat model was prepared by bilateral carotid artery occlusion and reperfusion of sodium nitroprusside injection. The model rats were divided into 3 groups by the random digit table, i. e., the moxibustion group, the Western medicine group, and the model group. A sham-operation control group was also set up. In the moxibustion group rats was acupunctured at Baihui (GV20), Shenting (GV14), and Dazhui (GV24). Aniracetam was given to rats in the Western medicine group by gastrogavage for 2 therapeutic courses, 15 days as one course. The learning and memory results were observed by the neuroethological score in combination of step-down avoidance test before treatment and by the end of the 2nd course respectively. The expression levels of hippocampal VEGF, flt-1, bFGF, and bFGF-r of all rats were detected using immunohistochemical assay.

RESULTSAfter 2 courses of treatment, statistical difference existed in the latent period, the error times, and the neuroethological score in the moxibustion group and the Western medicine group when compared with the model group (P < 0.01, P < 0.05). Statistical difference existed in the latent period and the neuroethological score between the moxibustion group and the Western medicine group (P < 0.05), which indicated that moxibustion and Western medicine showed significant effects in improving the latent period, decreasing the error times and the neuroethological score. Better results were obtained in the moxibustion group than in the Western medicine group (P < 0.01, P < 0.05). Statistical difference of the average grey level (AGL) of hippocampal VEGF, flt-1, and bFGF existed in the moxibustion group and the Western medicine group when compared with the model group. Statistical difference of the bFGF-r expression existed only between the moxibustion group and the model group. Statistical difference of the VEGF and flt-1 expressions existed between the moxibustion group and the Western medicine group (P < 0.05).

CONCLUSIONSMoxibustion showed confirmative effects in improving the behavioral score and memory performance in VD rats. Its mechanisms might lie in that moxibustion regulated and controlled the expression levels of hippocampal VEGF, flt-1, bFGF, and bFGF-r in VD rats. Particularly it up-regulated the expression levels of key factors VEGF and flt-1, promoted the angiogenesis in the vital parts, and ultimately stimulated the repairing mechanisms of cerebral nerve injury.

Animals ; Dementia, Vascular ; metabolism ; therapy ; Fibroblast Growth Factor 2 ; metabolism ; Hippocampus ; metabolism ; Male ; Moxibustion ; Rats ; Rats, Wistar ; Receptor, Fibroblast Growth Factor, Type 2 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism

OBJECTIVETo observe the therapeutic effects of Busui Shengxue Granule (BSSXG) on chronic aplastic anemia (CAA) patients and its effects on bone marrow derived stroma cells (BMDSCs) correlated cytokines.

METHODSOne hundred and twenty-four patients with CAA were randomly assigned to two groups according to the random digit table. Patients in the test group (61 cases) were treated with BSSXG, while those in the control group (63 cases) were treated with Zaizao Shengxue Tablet (ZST). The therapeutic course was 6 months for all. Besides, 10 healthy subjects were recruited as the normal control group. Changes of the symptom integral, therapeutic efficacy judgment, and changes of peripheral hemogram of patients were observed. The mRNA expression of b-fibroblast growth factors (bFGF) and b-fibroblast growth factors receptor (bFGFR) were detected by reverse transcription PCR.

RESULTSThe total effective rate of the test group was 75.0% (45/61), higher than that of the control group (58.7%, 37/63). Its symptom integral and peripheral hemogram were obviously improved, better than those of the control group (P < 0.05, P < 0.01). The mRNA expressions of bFGF and bFGFR of the test group were obviously lower than those of the normal control group (P < 0.05, P < 0.01). They were somewhat improved after treatment in the two groups, with better results obtained in the test group.

CONCLUSIONSBSSXG showed better clinical effects. It could improve the symptom integral and peripheral hemogram of CAA patients, improve the clinical efficacy, and regulate the expression levels of bFGF and bFGFR. It improved the hematopoietic microenvironment and promoted the hematopoiesis of the bone marrow through regulating the proliferation and oriental differentiation of stroma cells, and promoting the bone marrow angiogenesis.

Adolescent ; Adult ; Anemia, Aplastic ; drug therapy ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; Child ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Fibroblast Growth Factor 2 ; metabolism ; Humans ; Male ; Middle Aged ; Phytotherapy ; Receptor, Fibroblast Growth Factor, Type 2 ; metabolism ; Stromal Cells ; drug effects ; metabolism ; Young Adult

To study the functions of human Fibroblast growth factor receptor 2IIIc (FGFR2IIIc) gene in cancer cells, breast cancer cells MDA-MB-231 were infected by recombinant adenoviruses containing FGFR2IIIc and its S252W mutant, respectively. FGFR2IIIc gene was amplified from an existing plasmid and its S252W mutant was obtained by overlapping extension PCR. These two genes were separately cloned into the adenoviral shuttle plasmid pAdTrack-CMV, confivmed by DNA sequencing linearized, and co-transformed into Escherichia coli BJ-5183 with the adenoviral vector pAdEasy-1. The resulting recombinant expression vectors Ad-FGFR2IIIc and Ad-FGFR2IIIcS252W were linearized and transfected into HEK293A cells to get adenoviral particles. GFP was used to verify the gene expression. The recombinant adenoviral particles were harvested, titrated, and then infected MDA-MB-231 cells. The expression of FGFR2IIIc and its S252W mutant were examined by RT-PCR and Western blotting, and the effect of these recombinant adenoviruses on MDA-MB-231 cell proliferation was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry. The results showed the recombinant adenoviral particles could infect MDA-MB-231 cells and express the target proteins. MTT showed that both FGFR2IIIc and its S252W mutant inhibited MDA-MB-231 cell proliferation, but the mutant was more effective. Flow cytometry showed that both FGFR2IIIc and its S252W mutant arrested MDA-MB-231 cell cycle at G0/G1 phase, resulting in low cell proliferation.
Adenoviridae ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Genetic Vectors ; genetics ; Humans ; Mutant Proteins ; biosynthesis ; genetics ; Receptor, Fibroblast Growth Factor, Type 2 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.
Adenoviridae/*genetics ; Animals ; Chondrocytes/drug effects/*metabolism ; Collagen Type II/genetics/metabolism ; Fibroblast Growth Factor 2/*genetics ; Gene Therapy/methods ; Genetic Vectors/administration & dosage/*genetics ; Humans ; Insulin-Like Growth Factor I/*genetics/metabolism ; Interleukin 1 Receptor Antagonist Protein/*genetics/metabolism ; Interleukin-1/genetics/metabolism ; Matrix Metalloproteinase 13/genetics/metabolism ; Matrix Metalloproteinase 3/genetics/metabolism ; Osteoarthritis/*therapy ; Rabbits ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Transfection

BACKGROUNDAfter myocardial infarction, specific growth factors promote cardiac angiogenisis, leading to a therapeutic effect. Although this effect is mediated by specific receptors in the endothelium of the cardiac microvasculature, few studies have investigated dynamic changes in their expression. We explored this phenomenon in a murine model.

METHODSWe observed the mRNA expression of receptors by specific angiogenesis gene microarray at day 3 and day 7 after infarction. The vascular endothelial growth factor (VEGF) receptor Flk-1 was observed at the protein level at day 3 and day 7 by immunohistochemistry. The dynamic expression of fibroblast growth factor receptor-1 (FGFR-1) mRNA in the border zone and the noninfarcted zone at day 3, day 7, day 14, and day 42 was investigated by real-time PCR. Statistical significance was analyzed with SPSS 10.0 software using one-way analysis of variance (ANOVA).

RESULTSThree days after infarction, 9 receptors in the border zone and 7 receptors in the noninfarcted zone were down-regulated. Two receptors in the infarct edge and 5 receptors in the distant myocardium were up-regulated. However, at day 7, 11 receptors in the border zone were up-regulated, and only one was down-regulated. In the border zone, Flk-1 levels decreased at day 3 but increased significantly at day 7. Real-time PCR showed that FGFR-1 mRNA decreased markedly in the border zone at day 3 but increased afterward for at least 6 weeks. In the early stage (3 days) after infarction, the expression of receptors had decreased to some extent. However, at day 7, receptor expression was active and had moved from the distant noninfarcted zone to the border zone as a part of the acute repair process.

CONCLUSIONSelecting the proper growth factors to target receptors with protective activity, and determining appropriate therapeutic timing may be important to the success of therapeutic angiogenesis.

Animals ; Endothelium, Vascular ; metabolism ; Male ; Microcirculation ; Myocardial Infarction ; metabolism ; Myocardium ; metabolism ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Fibroblast Growth Factor, Type 1 ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; analysis

Angiogenesis is considered to be an integral process to the growth and spread of solid tumors. Anti-angiogenesis therapy recently has been found to be one of the most promising anti-cancer therapeutic strategies. In this study, we provide several lines of evidences showing that KR-31831, a new benzopyran derivative, has anti-angiogenic activities. KR-31831 inhibited the proliferation, migration, invasion and tube formation of bovine aortic endothelial cells (BAECs), and suppressed the release of matrix metalloproteinase-2 (MMP-2) of BAECs. KR-31831 also inhibited in vivo angiogenesis in mouse Matrigel plug assay. Furthermore, the mRNA expressions of basic fibroblast growth factor (bFGF), fibroblast growth factor receptor-2 (FGFR-2), and vascular endothelial growth factor receptor-2 (VEGFR-2) were decreased by KR-31831. Taken together, these results suggest that KR-31831 acts as a novel angiogenesis inhibitor and might be useful for treating hypervascularized cancers.
Vascular Endothelial Growth Factor Receptor-2/metabolism ; Receptor, Fibroblast Growth Factor, Type 2/metabolism ; Neovascularization, Physiologic/drug effects ; Neovascularization, Pathologic/*drug therapy ; Models, Biological ; Mice, Inbred C57BL ; Mice ; Matrix Metalloproteinase 2/metabolism ; Male ; Ischemia/drug therapy ; Imidazoles/*pharmacology/therapeutic use ; Fibroblast Growth Factor 2/metabolism ; Endothelial Cells/drug effects ; Cells, Cultured ; Cell Movement/drug effects ; Cattle ; Benzopyrans/*pharmacology/therapeutic use ; Animals ; Angiogenesis Inhibitors/*pharmacology/therapeutic use