BACKGROUNDThe mechanism of retinal neovascularization is not understood completely. Many growth factors are involved in the process of retinal neovascularization, such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF), which are the representatives of angiogenic and antiangiogenic molecules respectively. Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization. The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).

METHODSIn vitro, cultured EOMA cells were transfected with VEGF-siRNA (psi-HI(TM)/EGFP/VEGF siRNA) and Lipofectamine(TM) 2000 for 24, 48, and 72 hours, respectively. Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting. In vivo, OIR model mice were established, the mice (C57BL/6J) received an intra-vitreal injection of 1 µl of mixture of psi-HI(TM)/EGFP/VEGF siRNA and Lipofectamine 2000. Expressions of retinal VEGF and PEDF protein were measured by Western blotting, retinal neovascularization was observed by fluorescein angiography, and quantified.

RESULTSIn vitro psi-HI(TM)/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression. In vivo, with decreased VEGF and VEGF-PEDF ratio, significant attenuation of neovascular tufts, avascular regions, tortuous, and dilated blood vessels were observed in the interfered animals.

CONCLUSIONSVEGF plays an important role in OIR, and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo, accompanied by the downregulation of VEGF-PEDF ratio, and simultaneous attenuation of retinal neovascularization was also observed. These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression, which represents a possible therapeutic strategy.

Animals ; Eye Proteins ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factors ; analysis ; RNA, Small Interfering ; genetics ; Retinal Neovascularization ; therapy ; Serpins ; analysis ; Vascular Endothelial Growth Factor A ; analysis ; genetics ; physiology

Formation of the periodontium begins following onset of tooth-root formation in a coordinated manner after birth. Dental follicle progenitor cells are thought to form the cementum, alveolar bone and Sharpey's fibers of the periodontal ligament (PDL). However, little is known about the regulatory morphogens that control differentiation and function of these progenitor cells, as well as the progenitor cells involved in crown and root formation. We investigated the role of bone morphogenetic protein-2 (Bmp2) in these processes by the conditional removal of the Bmp2 gene using the Sp7-Cre-EGFP mouse model. Sp7-Cre-EGFP first becomes active at E18 in the first molar, with robust Cre activity at postnatal day 0 (P0), followed by Cre activity in the second molar, which occurs after P0. There is robust Cre activity in the periodontium and third molars by 2 weeks of age. When the Bmp2 gene is removed from Sp7(+) (Osterix(+)) cells, major defects are noted in root, cellular cementum and periodontium formation. First, there are major cell autonomous defects in root-odontoblast terminal differentiation. Second, there are major alterations in formation of the PDLs and cellular cementum, correlated with decreased nuclear factor IC (Nfic), periostin and α-SMA(+) cells. Third, there is a failure to produce vascular endothelial growth factor A (VEGF-A) in the periodontium and the pulp leading to decreased formation of the microvascular and associated candidate stem cells in the Bmp2-cKO(Sp7-Cre-EGFP). Fourth, ameloblast function and enamel formation are indirectly altered in the Bmp2-cKO(Sp7-Cre-EGFP). These data demonstrate that the Bmp2 gene has complex roles in postnatal tooth development and periodontium formation.
Actins ; analysis ; Activating Transcription Factor 2 ; genetics ; Age Factors ; Ameloblasts ; pathology ; Amelogenesis ; genetics ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; Cell Adhesion Molecules ; analysis ; Cell Differentiation ; genetics ; Cementogenesis ; genetics ; Dental Cementum ; pathology ; Dental Pulp ; blood supply ; Fluorescent Dyes ; Green Fluorescent Proteins ; Male ; Mice ; Mice, Knockout ; Microvessels ; pathology ; Molar ; growth & development ; Molar, Third ; growth & development ; NFI Transcription Factors ; analysis ; Odontoblasts ; pathology ; Odontogenesis ; genetics ; Periodontal Ligament ; growth & development ; Sp7 Transcription Factor ; Stem Cells ; physiology ; Tooth Root ; growth & development ; Transcription Factors ; genetics ; Vascular Endothelial Growth Factor A ; analysis ; Zinc Fingers ; genetics

Previous studies suggested that polymorphisms of proinflammatory cytokine genes are important host genetic factors in Helicobacter pylori infection. The present study evaluated whether IL-8-251 polymorphism affected H. pylori eradication rate and to investigate the effect of H. pylori eradication on angiogenesis and the inflammatory process according to the IL-8-251 polymorphism. A total of 250 H. pylori-positive patients treated by endoscopic resection of the gastric neoplasm were classified into 3 groups (134 H. pylori-eradicated group, 19 H. pylori-eradication failure group, and 97 H. pylori-infected group). H. pylori status, histology, and angiogenic factor levels were evaluated at baseline, 6 months, and 18 months. H. pylori eradication rate was 92.9% in AA genotype, 85.7% in AT genotype and 88.4% in TT genotype (P value = 0.731). Elevated IL-8 and matrix metalloproteinase-9 concentrations in H. pylori-infected gastric mucosa were reversible by successful eradication of H. pylori, independent of the IL-8-251 polymorphism. It is suggested that elevated IL-8 and MMP-9 concentrations in H. pylori-infected gastric mucosa are altered significantly after successful eradication and these conditions continue for 18 months. However, IL-8-251 polymorphism does not affect H. pylori eradication rate and the sequential changes of related angiogenic factors after H. pylori eradication in Koreans.
Aged ; Alleles ; Angiopoietin-1/analysis ; Anti-Bacterial Agents/therapeutic use ; Anti-Inflammatory Agents, Non-Steroidal/therapeutic use ; Asian Continental Ancestry Group/*genetics ; Female ; Gastric Mucosa/metabolism/pathology ; Genotype ; Helicobacter Infections/*drug therapy ; *Helicobacter pylori ; Humans ; Interleukin-8/analysis/*genetics ; Male ; Matrix Metalloproteinase 9/analysis ; Middle Aged ; *Polymorphism, Single Nucleotide ; Proton Pump Inhibitors/therapeutic use ; Republic of Korea ; Retrospective Studies ; Stomach Neoplasms/pathology/surgery ; Time Factors ; Vascular Endothelial Growth Factor A/analysis

BACKGROUNDFibroblast growth factor 21 (FGF21) is a new member of FGF super family that is an important endogenous regulator for systemic glucose and lipid metabolism. This study aimed to explore whether FGF21 reduces atherosclerotic injury and prevents endothelial dysfunction as an independent protection factor.

METHODSThe present study was designed to investigate the changes of FGF21 levels induced by oxidized-low density lipoprotein (ox-LDL), and the changes of apoptosis affected by regulating FGF21 expression. The FGF21 mRNA levels of cultured cardiac microvascular endothelial cells (CMECs) were determined by real time-PCR and the protein concentration in culture media was detected by enzyme-linked immunosorbent assay. We analyzed the different expression levels of untreated controls and CMECs incubated with ox-LDL, and the changes of CMECs apoptosis initiated by the enhancement or suppression of FGF21 levels.

RESULTSThe secretion levels of FGF21 mRNA and protein were significantly upregulated in CMECs incubated with ox-LDL. Furthermore, FGF21 levels increased by 200 µmol/L bezafibrate could reduce CMECs apoptosis, and inhibit FGF21 expression by shRNA induced apoptosis (P < 0.05).

CONCLUSIONSFGF21 may be a signal of injured target tissue, and may play physiological roles in improving the endothelial function at an early stage of atherosclerosis and in stopping the development of coronary heart disease.

Animals ; Apoptosis ; Bezafibrate ; pharmacology ; Cells, Cultured ; Coronary Artery Disease ; prevention & control ; Endothelial Cells ; physiology ; Fibroblast Growth Factors ; analysis ; antagonists & inhibitors ; genetics ; physiology ; Lipoproteins, LDL ; toxicity ; Male ; PPAR alpha ; physiology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar

OBJECTIVETo observe the effects of the three-period treatment theory of bone fracture in TCM (Traditional Chinese Medicine) on VEGF and VEGF mRNA expression in the issues of outer periosteum, endosteum and bone marrow of rabbits, and to explore the rationality of phasing method in TCM in treating fracture.

METHODS3 mm bone defection were made at lower one third part of both radius in 140 male healthy rabbits. The rabbits were randomly divided into four groups, including three-period treatment group (TTG), one-period treatment group(OTG), positive medicine treatment group(PTG) and model control group (MCG). The rabbits in TG were treated with three-period treatment, rabbits in OTG were treated with one-period treatment, rabbits in PTG were fed by Guzhe-Cuoshangsan (a Chinese patent medicine which was used to treat bone fracture), rabbits in model control group were given no prescription or drug but distilled water as same dose as that of other groups. At day 3, 6, 9, 14, 28, 42 and 56, five rabbits from every group were randomly selected and were killed by aeroembolism. The left radiuses were taken out as the research object. Immunohistochemistry stain and in situ hybridization stain were performed to examinate the VEGF and VEGF mRNA expression in the outer periosteum, endosteum and bone marrow.

RESULTSThe VEGF and VEGF mRNA expression of all TCM treatment groups were enhanced in the outer periosteum, endosteum and bone marrow at different time points in fracture healing. The VEGF and VEGF mRNA expression in the three tissues of TTG had the tendency of higher than that of the other groups at the most time points after operation.

CONCLUSIONTreating fracture in stages has more predominant effect on the expression of VEGF and VEGF mRNA in the outer periosteum, endosteum and bone marrow than that of treating fracture with single prescription or drug.

Animals ; Bone Marrow ; metabolism ; Fractures, Bone ; metabolism ; therapy ; Male ; Medicine, Chinese Traditional ; Periosteum ; metabolism ; RNA, Messenger ; analysis ; Rabbits ; Vascular Endothelial Growth Factors ; analysis ; genetics

BACKGROUND/AIMS: Human hepatocellular carcinoma (HCC) is a hypervascular tumor, and vascular endothelial growth factor (VEGF) plays a key role in the regulation of tumor-associated angiogenesis. In this study, we analyzed the significance of the expression of VEGF family members on the prognosis and clinicopathologic progress of HCC. METHODS: Surgically resected specimens of HCC and noncancerous liver tissue were obtained from 323 patients with HCC, and VEGF mRNA was examined by quantitative reverse transcriptase-polymerase chain reactions (RT-PCRs). Patients who were seropositive for hepatitis B surface antigen were selected for the analysis (n=208). The VEGF(tumor)/GAPDH (glyceraldehyde-3-phosphate dehydrogenase)(tumor)/VEGF(nontumor)/GAPDH(nontumor) ratio was calculated using a quantitative RT-PCR assay, and the relationships between the expressions of VEGF family members and clinicopathologic parameters were analyzed to evaluate their significance in the prognosis of HCC. RESULTS: The disease-free survival was significantly worse in the high-VEGF-A group than in the low-VEGF-A group (P=0.035), whereas VEGF-A expression was not significantly related to overall survival (P=0.172). The factors significantly related to poor prognosis in univariate analysis were tumor size, portal vein invasion, microvascular thrombi, intrahepatic metastasis, tumor capsule invasion, liver capsule invasion, preoperative serum albumin level, and VEGF-A ratio. Multivariate analysis showed that a poor prognosis in HCC patients was significantly related to portal vein invasion (hazard ratio=3.381, P<0.001), intrahepatic metastasis (hazard ratio=2.379, P<0.001), tumor size (hazard ratio=1.834, P=0.003), and preoperative serum albumin level (hazard ratio=2.050, P=0.006). CONCLUSIONS: Our study showed that the expression of VEGF-A is positively correlated with the recurrence rate of HCC after curative resection. Therefore, a high expression of VEGF-A might be predictive of HCC recurrence after curative resection.
Adult ; Aged ; Base Sequence ; Carcinoma, Hepatocellular/*diagnosis/*surgery/virology ; Female ; Hepatitis B/complications ; Hepatitis B Surface Antigens/blood ; Humans ; Liver Neoplasms/*diagnosis/*surgery/virology ; Male ; Middle Aged ; Molecular Sequence Data ; Multivariate Analysis ; Neoplasm Invasiveness ; Neoplasm Staging ; Predictive Value of Tests ; Prognosis ; RNA, Messenger/analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Survival Analysis ; Vascular Endothelial Growth Factors/genetics/*metabolism

OBJECTIVETo obtain some effective objective markers used to predict the early liver metastasis of colorectal tumor, the relationship of liver metastasis of colorectal tumor with associate detection three markers such as CK20mRNA, CD44v6 and VEGF was studied.

METHODSThe expression of CK20mRNA in patrol venous blood from 30 colorectal cancer patients was detected by fluorescent quantitative RT-PCR, and the results of CD44v6 and VEGF in colorectal cancer tissue were determined by means of immunohistochemistry, and then compared with those in control groups.

RESULTSThe rate of positive expression of CK20mRNA in colorectal cancer patients' patrol venous blood was obviously superior to the level of benign pathological changes controls (P < 0.01), and significantly higher than that of normal controls (P < 0.01). The rate of positive expression of CD44v6 and VEGF in colorectal tumor tissue was distinctly superior to the level of benign pathological controls, and remarkable higher than that of normal controls (P < 0.01). The positive expression of liver metastasis was also clearly higher than that of no liver metastasis (P < 0.05). The rate of positive expression of CK20mRNA in patrol venous blood was evidently correlated to the expression of CD44v6 and VEGF in tumor tissue (r(1) = 0.933, r(2) = 0.906, P < 0.05). The results of associate detection of CK20mRNA, CD44v6 and VEGF were closely related to the incidence of liver metastasis.

CONCLUSIONSIf combined detecting these markers of CK20mRNA, CD44v6 and VEGF to forecast liver metastasis of colorectal tumor, the sensitivity and specialty of prediction will be improved, there were highly clinical values in predicting in early diagnosis liver metastasis of colorectal tumor.

Adult ; Aged ; Biomarkers, Tumor ; analysis ; Colorectal Neoplasms ; metabolism ; pathology ; Early Diagnosis ; Humans ; Hyaluronan Receptors ; analysis ; Immunohistochemistry ; Keratin-20 ; blood ; genetics ; Liver Neoplasms ; diagnosis ; secondary ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity ; Vascular Endothelial Growth Factors ; analysis

OBJECTIVETo explore the possibility of using an endovascular microcoil as a gene delivery system.

METHODSAnti-adenoviral monoclonal antibodies were covalently attached to the collagen-coated surface of platinum microcoil. These antibodies were used to tether adenovirus encoding green fluorescent protein (Ad-GFP). Cell culture studies with rat arterial smooth muscle cells (A10) assessed transduction on or near the coil. Platinum coils coated with Ad-GFP were implanted into the ligated common carotid artery (CCA) of adult rats in a model of arterial stasis and pressurization. After 7 days, CCA segments were harvested, and coils were removed for histopathology and GFP expression studies, while organs were evaluated by polymerase chain reaction to assess viral biodistribution.

RESULTSIn cell culture studies, GFP-positive smooth muscle cells were detected only on the platinum coil surface. After 7 days, GFP was detected on the harvested platinum coil and in the organizing thrombus within the CCA according to fluorescence microscopy and immunohistochemistry. Morphometric analyses revealed that (13.3 +/- 2.0)% of cells within the organized thrombus were transduced with Ad-GFP via the gene delivery system. Ad-GFP was not detectable by polymerase chain reaction in lung, liver, or kidney.

CONCLUSIONSGene delivery endovascular microcoils represents an interventional device-based gene therapy system that can serve as a suitable platform for either single or multiple gene therapy vectors.

Adenoviridae ; genetics ; immunology ; Aneurysm ; surgery ; Animals ; Antibodies, Viral ; chemistry ; metabolism ; Biological Availability ; Carotid Artery, Common ; drug effects ; metabolism ; surgery ; Cells, Cultured ; Drug Delivery Systems ; instrumentation ; Embolization, Therapeutic ; methods ; Endothelial Growth Factors ; therapeutic use ; Genetic Therapy ; instrumentation ; methods ; Genetic Vectors ; administration & dosage ; chemistry ; Green Fluorescent Proteins ; analysis ; Muscle, Smooth, Vascular ; cytology ; Platinum ; chemistry ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; therapeutic use ; Transduction, Genetic ; instrumentation ; methods

BACKGROUNDHypoxia-inducible factor-1alpha (HIF-1alpha) is one of the pivotal mediators in the response of lungs to decreased oxygen availability, and increasingly has been implicated in the pathogenesis of pulmonary hypertension. Vascular endothelial growth factor (VEGF), a downstream target gene of HIF-1alpha, plays an important role in the pathogenesis of hypoxic pulmonary hypertension and hypoxic pulmonary artery remodelling. In this study, we investigated the dynamic expression of HIF-1alpha and VEGF in pulmonary artery of rats with hypoxia-induced pulmonary hypertension.

METHODSForty male Wistar rats were exposed to hypoxia for 0, 3, 7, 14 or 21 days. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were estimated. Lungs were inflated and fixed for in situ hybridisation and immunohistochemistry.

RESULTSmPAP values were significantly higher than the control values after 7days of hypoxia [(18.4 +/- 0.4) mmHg, P < 0.05]. RVHI developed significantly after 14 days of hypoxia. Expression of HIF-1alpha protein increased in pulmonary arterial tunica intima of all hypoxic rats. In pulmonary arterial tunica media, HIF-1alpha protein was markedly increased by day 3 (0.20 +/- 0.02, P < 0.05), reached the peak by day 7, then declined after day 14 of hypoxia. HIF-1alpha mRNA increased significantly after day 14 of hypoxia (0.20 +/- 0.02, P < 0.05). VEGF protein began to increase markedly after day 7 of hypoxia, reaching its peak around day 14 of hypoxia (0.15 +/- 0.02, P < 0.05). VEGF mRNA began to increase after day 7 of hypoxia, then remained more or less stable from day 7 onwards. VEGF mRNA is located mainly in tunica intima and tunica media, whereas VEGF protein is located predominantly in tunica intima. Linear analysis showed that HIF-1alpha mRNA, VEGF and mPAP were correlated with hypoxic pulmonary artery remodelling. HIF-1alpha mRNA was positively correlated with VEGF mRNA and protein (P < 0.01).

CONCLUSIONHIF-1alpha and VEGF are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.

Animals ; Blood Pressure ; Chronic Disease ; Hypertension, Pulmonary ; etiology ; Hypertrophy, Right Ventricular ; etiology ; Hypoxia ; complications ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; Male ; Pulmonary Artery ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Transcription Factors ; genetics ; physiology ; Vascular Endothelial Growth Factor A ; genetics ; physiology

In order to detect the hematopoietic growth factor gene expressed in human umbilical vein endothelial cells using gene chip, human umbilical vein endothelial cells (ECV304) were cultured in vitro and divided into VEGF group and control group in same medium. 50 ng/ml hVEGF165 was added in the VEGF group. After culture for 24 hours all cells were collected for total RNA extraction. Then, cDNAs were marked with Cy3 and Cy5 for control group and VEGF group, respectively, and hybridized with gene chip. Hybridization signals were collected and analyzed following scanning by laser co-focal microscopy. The results showed that a large number of hematopoietic growth factor and receptor genes (Epo/R, GM-CSF/R, G-CSF/R, LIF, IL-3, TPO, Flt-3, SCF) were expressed in both groups, while many other growth factors (VEGF, IGF2, PDGFA, PDGFB, TGFbeta1) and receptors (neuropilin-1, neuropilin-2, TGFbeta-R1)were expressed. The differentially expressed genes amounted to 24. It is concluded that many hematopoietic growth factors and receptors expressed by hUVECs could be analyzed in a short period by using gene chip, which provides a powerful method for further studies on characteristics of vascular endothelial cells.
Cells, Cultured ; Endothelial Cells ; metabolism ; Gene Expression Profiling ; Hematopoietic Cell Growth Factors ; genetics ; Humans ; Oligonucleotide Array Sequence Analysis ; Umbilical Veins ; metabolism