This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1control group (group A, n=6); SIRT1osteoarthritis group (group B, n=6); SIRT1control group (group C, n=6); SIRT1osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1osteoarthritis group and SIRT1control group, SIRT1 protein expression was not obviously changed in the SIRT1osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.
Animals ; Cartilage ; pathology ; Chondrocytes ; metabolism ; Collagen Type II ; metabolism ; Disease Models, Animal ; Humans ; Knee Joint ; metabolism ; pathology ; Mice ; Mice, Knockout ; Oncogene Protein v-akt ; genetics ; Osteoarthritis ; genetics ; pathology ; Phosphatidylinositol 3-Kinases ; genetics ; Signal Transduction ; genetics ; Sirtuin 1 ; genetics ; Sterol Regulatory Element Binding Protein 2 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis

In order to investigate the expression of nerve growth factor (NGF) and hypoxia inducible factor-1α (HIF-1α) and its correlation with angiogenesis in non-small cell lung cancer (NSCLC), paraffin-embedded tissue blocks from 20 patients with NSCLC were examined. Twenty corresponding para-cancerous lung tissue specimens were obtained to serve as a control. The expression of NGF, HIF-1α, and vascular endothelial growth factor (VEGF) in the NSCLC tissues was detected by using immunohistochemistry. The microvascular density (MVD) was determined by CD31 staining. The results showed that the expression levels of NGF, HIF-1α and VEGF in the NSCLC tissues were remarkably higher than those in the para-cancerous lung tissues (P<0.05). There was significant difference in the MVD between the NSCLC tissues (9.19±1.43) and para-cancerous lung tissues (2.23±1.19) (P<0.05). There were positive correlations between NGF and VEGF, between HIF-1α and VEGF, and between NGF and HIF-1α in NSCLC tissues, with the spearman correlation coefficient being 0.588, 0.519 and 0.588, respectively. In NSCLC tissues, the MVD had a positive correlation with the three factors (P<0.05). Theses results suggest that NGF and HIF-1α are synergically involved in the angiogenesis of NSCLC.
Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; blood supply ; metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; Immunohistochemistry ; Lung ; blood supply ; metabolism ; pathology ; Lung Neoplasms ; blood supply ; metabolism ; Male ; Middle Aged ; Neovascularization, Pathologic ; metabolism ; Nerve Growth Factor ; biosynthesis ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

OBJECTIVETo investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation.

METHODSDPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction.

RESULTSThe relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P < 0.05). For in vitro tube formation assay, tubular structures were formed on the matrigel by differentiated DPC. A total of 47 miRNA were differentially expressed, in which 15 miRNA were up-regulated and 32 miRNAs down-regulated in differentiated DPC compared with the control. Of these, 4 miRNA were confirmed by qRT-PCR. The target genes of differential miRNA were predicted to associate with several biological functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway.

CONCLUSIONSThe differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

Antigens, CD ; Cadherins ; Cell Differentiation ; Collagen ; Computational Biology ; Dental Pulp ; metabolism ; Drug Combinations ; Fibroblast Growth Factor 2 ; Humans ; Laminin ; MicroRNAs ; Platelet Endothelial Cell Adhesion Molecule-1 ; biosynthesis ; Proteoglycans ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Vascular Endothelial Growth Factor A ; Wnt Signaling Pathway ; von Willebrand Factor

OBJECTIVETo observe the therapeutic angiogenesis effect of naotai recipe (NR) on local ischemia/reperfusion (I/R) injury of rats by observing signaling pathway of hypoxia-inducible factor-lα (HIF-1α) and vascular endothelial growth factor (VEGF).

METHODSTotally 120 Sprague-Dawley (SD) rats were randomly divided into 4 groups, namely, the normal control group (n =12), the sham-operation group (n =12), the I/R model group (n =48), and the NR group (n =48). Cerebral I/R injury models were established using thread suture method. Rats in the I/R model group and the NR group were sub-divided into 4 sub-groups according to the 1st, 3rd, 5th, and 7th I/R day (n =12). The phenomenon of neovasculization was observed by immunofluorescence staining. The protein and mRNA expression levels of HIF-la, VEGF-A, and VEGFR II receptor were detected by RT-PCR.

RESULTSThere were a large amount of labels for neovasculization in the ischemic area of the NR group. Double-immunofluorescence labeling [vWF (red) and BrdU (green)] was observed in the NR group. Compared with the model group, the HIF-1α protein expression was obviously enhanced on the 1 st day of I/R (P <0.01), and the VEGF protein expression started to enhance on the 3rd day in the NR group (P <0.01). The VEGFR protein expression level was the highest in the NR group on the 5th day of I/R (P <0.01). The protein expression of VEGF and HIF-1α started to decrease on the 7th day of I/R.

CONCLUSIONNR could strengthen angiogenesis after I/R by elevating the expression of HIF-lα and activating HIF-lα/VEGF signaling pathway.

Animals ; Brain Ischemia ; metabolism ; Cerebral Infarction ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Hypoxia-Ischemia, Brain ; metabolism ; Ischemia ; Neovascularization, Pathologic ; Rats, Sprague-Dawley ; Reperfusion Injury ; Signal Transduction ; Vascular Endothelial Growth Factor A ; biosynthesis

The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.
Animals ; Anti-HIV Agents/pharmacology ; Bone Marrow Cells/cytology ; Chemokine CXCL12/biosynthesis ; Endothelial Progenitor Cells/*cytology ; Hematopoietic Stem Cells/*cytology ; Heterocyclic Compounds/*pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Male ; Neovascularization, Physiologic ; Nitric Oxide Synthase Type III/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, CXCR4/antagonists & inhibitors ; Surgical Flaps/*blood supply/surgery ; Tissue Expansion/*methods ; Vascular Endothelial Growth Factor A/biosynthesis ; Vascular Endothelial Growth Factor Receptor-2/biosynthesis/metabolism

Cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) is an angiogenic factor for vascular angiogenesis. The aim was to investigate the effect of an intracavernosal injection of COMP-Ang1 on cavernosal angiogenesis in a diabetic rat model. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats made up the experimental group (1 yr old) and Long-Evans Tokushima Otsuka (LETO) rats made up the control group. The experimental group was divided into vehicle only, 10 microg COMP-Ang1, and 20 microg COMP-Ang1. COMP-Ang1 was injected into the corpus cavernosum of the penis. After 4 weeks, the penile tissues of the rats were obtained for immunohistochemistry and Western blot analysis. The immunoreactivity of PECAM-1 and VEGF was increased in the COMP-Ang1 group compared with the vehicle only group. Moreover, the expression of PECAM-1 and VEGF was notably augmented in the 20 microg Comp Ang-1 group. In the immunoblotting study, the expression of PECAM-1 and VEGF protein was significantly less in the OLEFT rats than in the control LETO rats. However, this expression was restored to control level after intracavernosal injection of COMP-Ang1. These results show that an intracavernosal injection of COMP-Ang1 enhances cavernous angiogenesis by structurally reinforcing the cavernosal endothelium.
Angiopoietin-1/genetics/*metabolism ; Animals ; Antigens, CD31/metabolism ; Blood Glucose/analysis ; Blotting, Western ; Body Weight ; Cartilage Oligomeric Matrix Protein/genetics/*metabolism ; Diabetes Mellitus, Experimental/*pathology ; Immunohistochemistry ; Male ; Neovascularization, Physiologic/*drug effects ; Penis/metabolism/pathology ; Rats ; Rats, Long-Evans ; Recombinant Fusion Proteins/biosynthesis/genetics/*pharmacology ; Vascular Endothelial Growth Factor A/metabolism

OBJECTIVE: To determine whether quantitative perfusion parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) correlate with immunohistochemical markers of angiogenesis in rectal cancer. MATERIALS AND METHODS: Preoperative DCE-MRI was performed in 63 patients with rectal adenocarcinoma. Transendothelial volume transfer (Ktrans) and fractional volume of the extravascular-extracellular space (Ve) were measured by Interactive Data Language software in rectal cancer. After surgery, microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression scores were determined using immunohistochemical staining of rectal cancer specimens. Perfusion parameters (Ktrans, Ve) of DCE-MRI in rectal cancer were found to be correlated with MVD and VEGF expression scores by Spearman's rank coefficient analysis. T stage and N stage (negative or positive) were correlated with perfusion parameters and MVD. RESULTS: Significant correlation was not found between any DCE-MRI perfusion parameters and MVD (rs = -0.056 and p = 0.662 for Ktrans; rs = -0.103 and p = 0.416 for Ve), or between any DCE-MRI perfusion parameters and the VEGF expression score (rs = -0.042, p = 0.741 for Ktrans ; r = 0.086, p = 0.497 for Ve) in rectal cancer. TN stage showed no significant correlation with perfusion parameters or MVD (p > 0.05 for all). CONCLUSION: DCE-MRI perfusion parameters, Ktrans and Ve, correlated poorly with MVD and VEGF expression scores in rectal cancer, suggesting that these parameters do not simply denote static histological vascular properties.
Adult ; Aged ; Aged, 80 and over ; Contrast Media/*diagnostic use ; Female ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Magnetic Resonance Imaging/*methods ; Male ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic/diagnosis/metabolism ; Rectal Neoplasms/blood supply/*diagnosis/metabolism ; Retrospective Studies ; Tumor Markers, Biological/biosynthesis ; Vascular Endothelial Growth Factor A/*biosynthesis

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.
Antibodies, Bispecific ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Humans ; Plasmids ; Protein Binding ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

OBJECTIVECelastrus orbiculatus Thunb. has been used for thousands of years in China as a remedy against cancer and inflammatory diseases. This study aims to investigate whether C. orbiculatus extract (COE) could inhibit angiogenesis, which is the pivotal step in tumor growth, invasiveness, and metastasis.

METHODSIn this study, the extract from the stem of C. orbiculatus was used. Mouse hepatic carcinoma cells (Hepa1-6) were treated with COE in different nontoxic concentrations (10, 20, 40, 80, and 160 μg/mL). The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively; the active fractions were further tested on C57BL/6 mice and human umbilical vein endothelial cells (HUVEC) for any antiangiogenic effects.

RESULTSCOE significantly inhibited proliferation and induced apoptosis in Hepa1-6 cells and inhibited VEGF expression at both mRNA and protein levels. Furthermore, this agent inhibited the formation of the capillary-like structure in primary cultured HUVEC in a dose-dependent manner. In vivo, COE significantly reduced the volume and weight of solid tumors with low adverse effects and decreased tumor angiogenesis.

CONCLUSIONSIn summary, COE could be used to treat hepatic carcinoma. The mechanisms of the antitumor activity of COE may be due to its effects against tumor angiogenesis by targeting the VEGF protein.

Administration, Oral ; Angiogenesis Inhibitors ; pharmacology ; therapeutic use ; Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; blood supply ; drug therapy ; pathology ; Celastrus ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Collagen ; metabolism ; Drug Combinations ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Laminin ; metabolism ; Liver Neoplasms ; blood supply ; drug therapy ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; drug therapy ; pathology ; Neovascularization, Physiologic ; drug effects ; Phytotherapy ; Plant Extracts ; administration & dosage ; pharmacology ; therapeutic use ; Plant Stems ; chemistry ; Proteoglycans ; metabolism ; Signal Transduction ; drug effects ; Transcriptional Activation ; drug effects ; genetics ; Tumor Burden ; drug effects ; Vascular Endothelial Growth Factor A ; biosynthesis ; metabolism

OBJECTIVETo express granzyme B-vascular endothelial growth factor (VEGF) receptor-binding peptide (GrB-VRB) fusion protein in Bifidobacteria longum (B. longum) and investigate the effects of this fusion protein on the proliferation and apoptosis of cells expressing VEGF receptor II, the kinase domain receptor (KDR).

METHODSThe recombinant expression vectors pBBADx-VRB, pBBADx-GrB and pBBADx-GrB-VRB were separately transformed into B. longum cells by electroporation. The expressed products were identified by enzyme-linked immunosorbent assay and Western blotting, and their effects on KDR-positive cells were analyzed using proliferation assay and TUNEL assay.

RESULTSThe expressed products were detected in both the supernatant and cellular fractions of B. longum cells. The recombinant GrB-VRB fusion protein reacted with such KDR-positive cells as human umbilical vein endothelial cells (HUVEC) and mouse colon cancer cell line CT-26, and caused obvious cell proliferation inhibition, cytotoxicity and cell apoptosis in these cells.

CONCLUSIONThe recombinant GrB-VRB fusion protein secreted by the engineered B. longum cells can induce KDR-positive cell death as the result of GrB-induced cell apoptosis following the cell recognition by VRB.

Animals ; Apoptosis ; Bifidobacterium ; metabolism ; Carrier Proteins ; Cell Line, Tumor ; Cell Proliferation ; Granzymes ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Mice ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism