OBJECTIVETo screen for the inhibitor of vascular endothelial growth factor (VEGF) 165 from random peptide library.

METHODSPositive phage clones were rescued after two rounds of panning and competitive elution. Its affinity activity to KDR was monitored through ELISA, immunohistochemical method, Chicken CAM assay and MTT.

RESULTSFive specific binding positive target molecule phage clones were obtained which were able to bind to cells whose surface had high KDR, among which, clone 3 and 13 could effectively block the vascularization of the chorioallantoic membrane of chick embryo, but they were not inhibitive on the proliferation of high KDR expression cells.

CONCLUSIONThe peptides, being the inhibitors of VEGF, may be useful in the treatment of cancers.

Animals ; Binding Sites ; Endothelial Growth Factors ; antagonists & inhibitors ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Lymphokines ; antagonists & inhibitors ; metabolism ; Peptide Library ; Peptides ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

After ischemic stroke, there is neovascularization around the infarcted area, which is called penumbra. Angiogenesis and arteriogenesis are responsible for the new vessel formation. Until recently, vasculogenesis has been proved to involve mechanisms in postischemic neovascularization, which was thought to be restricted to embryonic development. New blood vessels' formation is a complex pathologic process after ischemic stroke, in which many factors are properly involved. There are factors stimulating neovascularization, such as vascular endothelial growth factor, platelet-derived growth factor, basic fibroblast growth factor and angiopoietin; there are also factors inhibiting neovascularization, such as thrombospondin. Functional recovery was found after stroke, which may contribute to angiogensis in the periinfarct tissue. Thus, therapeutic angiogenesis has been initially studied in animal models, but there is still a long way to go for therapeutic angiogenesis to be used in the treatment of stroke patient.
Angiogenesis Inducing Agents ; pharmacology ; Angiopoietin-1 ; metabolism ; Brain ; blood supply ; Brain Infarction ; metabolism ; physiopathology ; Humans ; Neovascularization, Physiologic ; Platelet-Derived Growth Factor ; metabolism ; Vascular Endothelial Growth Factors ; metabolism

To explore the feasibility of mesenchymal stem cells (MSCs) acting as seed cells in tissue engineering, we isolated human bone marrow MSCs and differentiated them into vascular endothelial-like cells (ELCs) in vitro. Bone marrow mononuclear cells (BMSCs) were isolated by the method of percoll density centrifugation, and seeded in Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum. MSCs were purified through multiple adherent cultures, and differentiated into ELCs induced by endothelial cell growth medium-2 (EBM-2) medium containing vascular endothelial growth factor (VEGF), human fibroblast growth factor (hFGF), insulin like growth factors 1 (IGF-1), and human epidermal growth factor (hEGF). The relative biologic characteristics of ELCs including cell morphology and phenotype were studied by inverted microscope and flow cytometry. The induced cells were identified by immunofluorescence with CD31 and Von Willebrand factor (vWF). The results showed that the morphology of MSCs was long-spindle and vortex-like growth. After induction of differentiation, the cells were round, and similar to vascular endothelial cells (ECs). Flow cytometric analysis revealed that ELCs expressed ECs specific surface markers of CD31 and vascular endothelial cadherin (VE-cadherin), but not CD133. Immunofluorescence results also confirmed that ELCs expressed CD31 and vWF. The results suggested that ELCs possed similar cell biological characteristics with ECs. In one word, human MSCs derived from bone marrow have the potential to differentiate into ECs in vitro, and show clinical feasibility acting as ideal donor cells of vascular tissue engineering.
Antigens, CD ; metabolism ; Bone Marrow Cells ; Cadherins ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Culture Media ; chemistry ; Endothelial Cells ; cytology ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factors ; pharmacology ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; pharmacology ; von Willebrand Factor ; metabolism

To investigate the effect of arsenic trioxide (As(2)O(3)) on vascular endothelial growth factor (VEGF) expression in different courses of chronic myeloid leukemia (CML), VEGF level was measured with ELISA in the cultural supernatants of bone marrow mononuclear cells from CML patients. The results showed that supernatants of cultured bone marrow cells from 35 CML patients (20 chronic, 8 accelerated and 7 blast crisis phases) contained significantly higher VEGF levels (649.16 +/- 382.20 pg/ml, 560.27 +/- 409.14 pg/ml and 587.18 +/- 415.28 pg/ml, respectively) than that in 15 normal control samples (152.16 +/- 150.09 pg/ml; P < 0.01), but no significant differences were found in VEGF levels among different phases of CML. The bone marrow cells treated with As(2)O(3) (5 x 10(-6)mol/L) for 72 hours resulted in significant reduction of VEGF levels (down to 396.66 +/- 257.47 pg/ml, 363.42 +/- 239.85 pg/ml and 407.47 +/- 219.38 pg/ml, respectively) (P < 0.05). In conclusion, abnormal high expression of VEGF plays a role in the pathogenetic course of CML and it is probably an additional anticancer mechanism for As(2)O(3) to inhibit VEGF expression of leukemic cells.
Adolescent ; Adult ; Aged ; Arsenicals ; pharmacology ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; Child ; Culture Media, Conditioned ; chemistry ; Down-Regulation ; drug effects ; Endothelial Growth Factors ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; pathology ; Lymphokines ; metabolism ; Male ; Middle Aged ; Oxides ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVESTo investigate the inhibitory effect of macrophage migration inhibitory factor (MIF) antibody on the proliferation of HepG-2 cells and its mechanism.

METHODSHepG-2 cells were stimulated by different concentrations of MIF antibody (50, 100, 200 and 400 microg/L). The cell survival rates were evaluated by MTT assay. The cell cycles were assessed by flow cytometry (FCM) analysis. Cyclin D1 protein expression was examined by immunohistochemical methods. Vascular endothelial growth factor (VEGF) protein expression was examined by Western blot. ELISA was applied to detect the influence of MIF antibody on the production of IL-6 of HepG-2 cells.

RESULTSHepG-2 cells were inhibited by MIF antibody in a dose and time dependent manner. FCM analysis showed the cell cycles of HepG-2 cells were blocked at G0/G1 phase. With concentrations of MIF antibody of 0, 50, 100, 200, 400 microg/L, the percentages of cells in G0/G1 phase at 48 h were 61.34%+/-1.08%, 65.08%+/-2.71%, 71.19%+/-1.19%, 78.39%+/-1.00%, 83.92%+/-0.51%. With concentrations of MIF antibody of 50, 100, 200, 400 microg/L, the expressions of cyclin D1 protein were 26.06%+/-0.47%, 22.34%+/-0.75%, 18.06%+/-1.16%, 14.03%+/-0.59%, significantly lower than those of the control group (29.51%+/-1.28%). When HepG-2 cells were treated with different concentrations of MIF antibodies of 50, 100, 200, 400 microg/L the expressions of VEGF protein were 21.22%+/-0.68%, 19.64%+/-0.54%, 18.04%+/-0.42%, 16.59%+/-0.66%, significantly lower than those of the control group (23.23%+/-0.51%). With MIF antibody concentrations of 0, 50, 100, 200, 400 microg/L, the exudation amount of IL-6 were correspondingly lower [(210.67+/-9.31) pg/ml, (181.67+/-10.05) pg/ml, (160.50+/-6.60) pg/ml, (143.67+/-10.56) pg/ml, (118.01+/-7.48) pg/m].

CONCLUSIONThe proliferation of HepG-2 cells was inhibited after treatment with MIF antibody. The inhibiting effect is caused by blocking cell cycle progression at G0/G1 phase, decreasing cyclin D1 protein expression, decreasing VEGF protein expression and decreasing the exudation amount of IL-6.

Antibodies ; pharmacology ; Cell Cycle ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Hep G2 Cells ; Humans ; Interleukin-6 ; metabolism ; Macrophage Migration-Inhibitory Factors ; immunology ; Vascular Endothelial Growth Factor A ; metabolism

Ischemic stroke is a major public health problem worldwide. Although the circadian clock is involved in the process of ischemic stroke, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model, by measuring the infarct volume, neurological tests, and angiogenesis-related protein. We further report that Bmal1 plays an irreplaceable role in angiogenesis. Overexpression of Bmal1 promoted tube-forming, migration, and wound healing, and upregulated the vascular endothelial growth factor (VEGF) and Notch pathway protein levels. This promoting effect was reversed by the Notch pathway inhibitor DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.
Rats ; Animals ; Vascular Endothelial Growth Factor A/pharmacology* ; Brain Ischemia/metabolism* ; Ischemic Stroke ; Signal Transduction ; ARNTL Transcription Factors/pharmacology* ; Neovascularization, Physiologic/physiology*

OBJECTIVETo explore the response of nasal mucosa epithelial cells to hypoxia in terms of formation of nasal polyps (NP).

METHODSEpithelial cells of NP and inferior turbinate (IT) were cultured serum-free under normal oxygen and hypoxic circumstances with stimulation of IL-1 beta and TNF alpha. The vascular endothelial growth factor (VEGF) mRNA and VEGF protein levels of the cultured cells were detected using in situ hybridization and ELISA, respectively.

RESULTSThe expression of VEGF mRNA was significantly higher in epithelial cells of NP than in IT exposed to pro-inflammatory cytokines or hypoxia (P < 0.01). VEGF levels were higher in NP epithelial cells than those of IT (P < 0.01) under hypoxia.

CONCLUSIONVEGF-induced by hypoxia is very important for the early stages of forming polyps.

Cell Hypoxia ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Interleukin-1 ; pharmacology ; Lymphokines ; genetics ; Nasal Mucosa ; metabolism ; Nasal Polyps ; etiology ; metabolism ; RNA, Messenger ; analysis ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo establish an in vitro homogeneous time-resolved fluorescence immunoassay method for high throughput screening of protein tyrosine kinase (PTK) inhibitors.

METHODSSpecific fluorescence signals at 670 and 612 nm were measured by multifunctional microplate reader when the fluorescence was emitted through a resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of Sunitinib, a standard PTK inhibitor, on vascular endothelia growth factor receptor 2 (VEGFR-2) kinase activity was investigated.

RESULTSA homogeneous time-resolved fluorescence immunoassay was established for high throughput screening of PTK inhibitor. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/microl, 100 micromol/L and 1 micromol/L, respectively. Sunitinib inhibited VEGFR-2 kinase activity with an IC50 value of 86.7 nmol/L, which was close to the values tested using other methods.

CONCLUSIONThe homogeneous time-resolved fluorescence immunoassay we established can be easily used for high throughput screening of PTK inhibitors.

Fluoroimmunoassay ; methods ; High-Throughput Screening Assays ; methods ; Indoles ; pharmacology ; Peptides ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Pyrroles ; pharmacology ; Time Factors ; Vascular Endothelial Growth Factor Receptor-2 ; antagonists & inhibitors ; metabolism

OBJECTIVETo investigate the effect of losartan (an angiotensin II type I receptor antagonist) on endothelial cells exposed to high glucose in vitro and related mechanism.

METHODSVascular endothelial cells of human umbilical vein were cultured in media with high glucose levels. The activities of SOD and CAT, the level of MDA were measured by spectrophotometry in the conditioned media of endothelial cells, the VEGF mRNA expression was performed using semi-quantitative reverse transcription PCR (RT-PCR) in the cell lysates, and the protein expression of VEGF was examined by enzyme-linked immunosorbent assay (ELISA) in the supernatants of cultured cells.

RESULTWhen endothelial cells were cultured in high glucose, the activities of SOD and CAT were significantly decreased, but the level of MDA was markedly increased. However, the high glucose-induced effects were inhibited by losartan. The application of high glucose upregulated the mRNA and protein expression of VEGF in endothelial cells, which was also attenuated by losartan.

CONCLUSIONHigh glucose disrupts the oxidative equilibrium and increases the expression of VEGF in endothelial cells, which can be inhibited by losartan.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Glucose ; pharmacology ; Humans ; Losartan ; pharmacology ; Peroxidase ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Superoxide Dismutase ; metabolism ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

PURPOSE: Wound represents a major health challenge as they consume a large amount of healthcare resources to improve patient's quality of life. Many scientific studies have been conducted in search of ideal biomaterials with wound-healing activity for clinical use and collagen has been proven to be a suitable candidate biomaterial. This study intended to investigate the wound healing activity of collagen peptides derived from jellyfish following oral administration. METHODS: In this study, collagen was extracted from the jellyfish--Rhopilema esculentum using 1% pepsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fourier transform infrared (FTIR) were used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. Wound scratch assay (in vitro) was done to determine migration potential of human umbilical vein endothelial cells (HUVEC) covering the artificial wound created on the cell monolayer following treatment with collagen peptides. In vivo studies were conducted to determine the effects of collagen peptides on wound healing by examining wound contraction, re-epithelialization, tissue regeneration and collagen deposition on the wounded skin of mice. Confidence level (p < 0.05) was considered significant using GraphPad Prism software. RESULTS: The yield of collagen was 4.31%. The SDS-PAGE and FTIR showed that extracted collagen from jellyfish was type I. Enzymatic hydrolysis of this collagen using collagenase II produced collagen peptides (CP) and hydrolysis with alkaline proteinase/papain resulted into collagen peptides (CP). Tricine SDS-PAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25 kDa. Wound scratch assay showed that there were significant effects on the scratch closure on cells treated with collagen peptides at a concentration of 6.25 μg/mL for 48 h as compared to the vehicle treated cells. Overall treatment with collagen peptide on mice with full thickness excised wounds had a positive result in wound contraction as compared with the control. Histological assessment of peptides treated mice models showed remarkable sign of re-epithelialization, tissue regeneration and increased collagen deposition. Immunohistochemistry of the skin sections showed a significant increase in β-fibroblast growth factor (β-FGF) and the transforming growth factor-β (TGF-β) expression on collagen peptides treated group. CONCLUSION Collagen peptides derived from the jellyfish-Rhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future.
Administration, Oral ; Animals ; Collagen ; administration & dosage ; isolation & purification ; metabolism ; pharmacology ; Fibroblast Growth Factors ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Male ; Regeneration ; Scyphozoa ; chemistry ; Skin ; metabolism ; Skin Physiological Phenomena ; Stimulation, Chemical ; Transforming Growth Factor beta1 ; metabolism ; Wound Healing ; drug effects