OBJECTIVETo investigate the feasibility of endothelialization of bioprosthesis by transfer of vascular endothelial growth factor (VEGF) gene.

METHODSBovine pericardium treated with glutaraldehyde and L-glutamic acid was positioned into the pig right atrium. pcD(2)/hVEGF(121) gene (1 mg) was transferred into the right ventricular myocardium using surgical sutures Reverse transcri ption polymerase chain reaction (RT PCR) was employed to evaluate the expression of myocardial VEGF mRNA. The determination of concentrations of VEGF protein in blood from both the right atrium and peripheral vein, and histological and ultrastructural analysis of implanted bovine pericardium were completed simultaneously.

RESULTSThe concentration of VEGF derived from the right atrium in pcD(2)/hVEGF(121) group was significantly higher than that in the pcD(2) group 10 days after VEGF gene transfer (P < 0.01). The expression of myocardial VEGF mRNA in pcD(2)/hVEGF(121) group was much higher in comparison with that in the pcD(2) group. The morphological analysis demonstrated that the coverage rate of host endothelium in the pcD(2)/hVEGF(121) group was 2.6 times as fast as that in the pcD(2) group at 16 days after VEGF(121) gene transfer (P < 0.01). Entire endothelialization occurred at 30 days after VEGF gene transfer. In addition, higher expression of myocardial VEGF mRNA was still available.

CONCLUSIONSVEGF gene transfer by surgical suture can remarkably accelerate endothelialization of bioprosthesis, which may provide a new approach for inhibiting biological valve calcification and improve biocompatibility and long-term durability of the bioprosthesis.

Animals ; Bioprosthesis ; Endothelial Growth Factors ; analysis ; genetics ; Endothelium, Vascular ; physiology ; Female ; Gene Transfer Techniques ; Heart Valve Prosthesis ; Humans ; Lymphokines ; analysis ; genetics ; Male ; RNA, Messenger ; analysis ; Swine ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

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

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart
Animals ; Chloramphenicol O-Acetyltransferase/analysis/genetics ; Comparative Study ; Cytomegalovirus/*genetics ; DNA, Viral/*administration & dosage/*genetics ; Endothelial Growth Factors/analysis/*genetics ; *Enhancer Elements (Genetics) ; Gene Expression Regulation, Viral ; Gene Fusion ; *Gene Transfer Techniques ; Genes, Viral ; Genetic Vectors ; Intercellular Signaling Peptides and Proteins/analysis/*genetics ; Lymphokines/analysis/*genetics ; Male ; Myocardium/*metabolism ; Plasmids/genetics ; *Promoter Regions (Genetics) ; Rats ; Rats, Sprague-Dawley ; Time Factors ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart
Animals ; Chloramphenicol O-Acetyltransferase/analysis/genetics ; Comparative Study ; Cytomegalovirus/*genetics ; DNA, Viral/*administration & dosage/*genetics ; Endothelial Growth Factors/analysis/*genetics ; *Enhancer Elements (Genetics) ; Gene Expression Regulation, Viral ; Gene Fusion ; *Gene Transfer Techniques ; Genes, Viral ; Genetic Vectors ; Intercellular Signaling Peptides and Proteins/analysis/*genetics ; Lymphokines/analysis/*genetics ; Male ; Myocardium/*metabolism ; Plasmids/genetics ; *Promoter Regions (Genetics) ; Rats ; Rats, Sprague-Dawley ; Time Factors ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.

METHODSThe HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.

RESULTSHFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.

CONCLUSIONHFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.

Cell Adhesion ; Cell Cycle ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; metabolism ; Epidermis ; Fetus ; G1 Phase ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Keratinocytes ; cytology ; Keratins ; analysis ; Luminescent Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Microscopy, Fluorescence ; Plasmids ; genetics ; Stem Cells ; chemistry ; cytology ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

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

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 study the apoptosis of alveolar type II cells, alterations of vascular endothelial growth factor (VEGF), VEGF receptor (Flt1) in serum and lung and expression of VEGF mRNA in lung in pulmonary edema mice induced by phosgene.

METHODSTwenty-six BALB/C mice were randomly divided into 2 groups: control group, exposed group (13 mice in each group). Mice of exposed group were intoxicated by inhalation of phosgene 11.9 mg/L for 5 minutes. Mice of control group were treated as the same way by inhalation of air. Isolation of mice alveolus type II cells 4 h after intoxication was carried out to observe their apoptosis under electron microscope. Contents of VEGF and Flt1 in lung and serum by ELISA, and expression of VEGF mRNA were determined.

RESULTSAlveolar type II cells were identified by tannic acid staining and electron microscopy. After exposed to 11.9 mg/L of phosgene for 5 minutes, the apoptotic body in alveolus type II cells was found in exposed group. The contents of VEGF in serum and lung and Flt1 in lung of exposed mice [(134.07 +/- 120.26), (477.76 +/- 98.06), (1,2818.48 +/- 2,304.15) pg/ml] were significantly lower than those of control group [(445.57 +/- 173.30), (1,026.87 +/- 474.56), (21,976.51 +/- 7,421.01) pg/ml, P < 0.05] but the content of Flt1 in serum [(2,369.56 +/- 381.70) pg/ml] was higher than that in control group [(1,898.00 +/- 453.69) pg/ml, P < 0.05]. The expression of VEGF mRNA in pulmonary edema mice was decreased.

CONCLUSIONPhosgene can induce apoptosis of alveolar type II cells, and decrease in the content of VEGF and Flt1, and expression of VEGF mRNA in lung.

Animals ; Apoptosis ; drug effects ; Cells, Cultured ; Chemical Warfare Agents ; toxicity ; Endothelial Growth Factors ; biosynthesis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Male ; Mice ; Mice, Inbred BALB C ; Phosgene ; toxicity ; Pulmonary Alveoli ; drug effects ; metabolism ; pathology ; Pulmonary Edema ; chemically induced ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Vascular Endothelial Growth Factor A ; analysis ; genetics ; physiology ; Vascular Endothelial Growth Factor Receptor-1 ; analysis ; genetics

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

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