A small proportion of many cancers are due to inherited mutations in genes, which result in a high risk to the individual of developing specific cancers. There are several classes of genes that may be involved: tumour suppressor genes, oncogenes, genes encoding proteins involved in DNA repair and cell cycle control, and genes involved in stimulating the angiogenic pathway. Alterations in susceptibility to cancer may also be due to variations in genes involved in carcinogen metabolism. This review discusses examples of some of these genes and the associated clinical conditions caused by the inheritance of mutations in such genes.
DNA Repair ; genetics ; Genes, Tumor Suppressor ; Genetic Predisposition to Disease ; Humans ; Neoplasms ; genetics ; Neovascularization, Physiologic ; genetics ; Oncogenes

OBJECTIVETo explore a new method for the therapy of avascular necrosis of the femoral head.

METHODSThe recombinant plasmid pCD-hVEGF165 was mixed with collagen and was implanted in the necrotic femoral head. The expression of vascular endothelial growth factor (VEGF) was examined by RNA dot hybridization and immunohistochemical techniques. Repair of the femoral head was observed by histological and histomorphometric analysis.

RESULTSThe expression of VEGF was detected in the femoral head transfected with the VEGF gene. The femoral head transfected with the VEGF gene showed a significant increase in angiogenesis 2 and 4 weeks after gene transfection and a significant increase in bone formation 6 and 8 weeks after gene transfection on histomorphometric analysis (P < 0.01).

CONCLUSIONSTransfection of the VEGF gene enhances bone tissue angiogenesis. Repair of osteonecrosis could be accelerated accordingly, thus providing a potential method for therapy of osteonecrosis.

Animals ; Femur Head Necrosis ; therapy ; Neovascularization, Physiologic ; Rabbits ; Random Allocation ; Transfection ; Vascular Endothelial Growth Factor A ; genetics

This study was purposed to investigate the angiogenesis-promoting activities of human mesenchymal stem cells (hMSCs) modified by hepatocyte growth factor (HGF) and the underlying mechanisms. The hMSCs were transfected by recombinant adenoviral vector carrying human HGF gene and seeded onto the chicken chorioallantoic membrane. Three days later, the number of blood vessels was counted and their angiogenic response was compared with those of hMSCs of same generation, recombinant basic fibroblast growth factor (bFGF) and alpha-MEM as control. The expression levels of bFGF, VEGF, angiopoietin-1 and angiopoietin-2 were evaluated by RT-PCR assay. The results showed that gene-modified hMSCs exhibited greatest activity to promote angiogenesis while the angiogenic response was nearly same between groups treated by hMSCs and bFGF, all of which were significantly higher than that observed in control (p < 0.01). RT-PCR analysis revealed that hMSCs constitutively expressed multiple angiogenesis-associated growth factors and their levels seemed up-regulated by HGF gene transfer. It is concluded that HGF gene-modified hMSCs show a potent angiogenesis-promoting function and may be useful in the treatment of ischemic disorders.
Animals ; Cells, Cultured ; Chick Embryo ; Chickens ; Hepatocyte Growth Factor ; genetics ; Humans ; Mesenchymal Stromal Cells ; Neovascularization, Physiologic ; genetics ; Transfection

Anemia ; metabolism ; physiopathology ; Animals ; Cell Hypoxia ; Genetic Therapy ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; metabolism ; physiology ; Neovascularization, Pathologic ; metabolism ; pathology ; Neovascularization, Physiologic ; Osteogenesis ; RNA, Messenger ; metabolism

Tumor angiogenesis plays a pivotal role in the progress of tumor. Among the various endogenous angiogenic inhibitors discovered, the human plasminogen kringle 5 (K5) has been demonstrated to be a potential inhibitor of the proliferation and migration of vascular endothelial cells in vitro. The replication-incompetent adenovirus (Ad) vector Adeno-X-CMV-K5 (Ad-K5) (where CMV is cytomegalovirus) was constructed and its antiangiogenic effect was tested on vascular endothelial cell and tumor cell. For the construction, the K5 cDNA was fused in-frame with human plasminogen signal sequence and inserted into the eukaryotic expression vector pcDNA3 to form pcDNA3K5. The recombinant plasmid was subcloned into the shuttle plasmid pShuttle under the control of the constitutive CMV immediate-early promoter. The plasmid carrying the cDNA for K5 (pShuttleKS) was then recombined with the Adeno-X viral DNA and transformed into E. coli DH5alpha. The resultant recombinant plasmid pAd-K5 was transfected into human embryonic kidney (HEK) 293 cells with liposome. The adenovirus expressing human plasminogen kringle 5 (Ad-K5) was successfully packaged and propagated in 293 cells, as detected by the cytopathic effect (CPE) on the cells, and the viral titer in the supernatant was 5 x 10(8) pfu/mL by plaque assay. Both human umbilical vein endothelial cell line ECV304 and human breast carcinoma cell line MDA-MB-231 were infected with Ad-K5 and Ad-LacZ, which was used the negative control, and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared with uninfected control and Ad-LacZ infected control, Ad-K5 infected group at 80 MOI (multiplicity of infection) significantly inhibited ECV304 proliferation; the difference between uninfected control and Ad-LacZ infected control was not significant. In contrast, there was no significant difference in the proliferation of MDA-MB-231 among all the treatments. In addition, the Ad-K5 at 100 MOI inhibited the differentiation and tube formation of ECV304 on ECMatrix gel. These results suggested that the recombinant replication-defective Adenovirus expressing human plasminogen kringle 5 inhibited the proliferation, differentiation and tube formation of ECV304 and had no effect on the proliferation of MDA-MB-231. Adenovirus mediated human plasminogen kringle 5 gene therapy may be a potential treatment of cancer through angiogenesis inhibition.
Adenoviridae ; genetics ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; genetics ; Humans ; Neovascularization, Physiologic ; genetics ; physiology ; Peptide Fragments ; genetics ; physiology ; Plasminogen ; genetics ; physiology ; Polymerase Chain Reaction

OBJECTIVETo establish transgenic mice with GFP expression in the vascular endothelium during neovascularization.

METHODSThe vector nestin-hsp68-gfp containing nestin second intron was introduced into U251 cells and the expression level of GFP was detected by fluorescence microscopy. Transgenic mice were produced by microinjection. The genome of the offspring mice was screened by PCR, and GFP expression in the vascular endothelium was detected using immunohistochemistry.

RESULTSThirteen offspring mice were obtained and 2 of them were positive for GFP in the vascular endothelium as detected by PCR. GFP was detected in the offspring mice both at the embryonic stage and after birth.

CONCLUSIONSThe transgenic mice with GFP expression in the vascular endothelium during neovascularization have been successfully established.

Animals ; Animals, Newborn ; Base Sequence ; Endothelium, Vascular ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Intermediate Filament Proteins ; biosynthesis ; genetics ; Mice ; Mice, Transgenic ; Molecular Sequence Data ; Neovascularization, Pathologic ; genetics ; Neovascularization, Physiologic ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Nestin

Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells (ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin (ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.
Cell Movement ; genetics ; Cytoskeletal Proteins ; genetics ; metabolism ; Cytoskeleton ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; physiology ; Humans ; Neovascularization, Physiologic ; genetics

OBJECTIVETo establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study.

METHODSAn endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry.

RESULTSDigital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01).

CONCLUSIONSThe endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

Animals ; Cell Line, Tumor ; Cells, Cultured ; Chorioallantoic Membrane ; blood supply ; Diagnostic Imaging ; methods ; Homeodomain Proteins ; genetics ; metabolism ; physiology ; Human Umbilical Vein Endothelial Cells ; Humans ; Neovascularization, Pathologic ; Neovascularization, Physiologic ; Software ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transcription Factors ; genetics ; metabolism ; physiology ; Transfection

CD151 is a member of the tetraspanin family that is implicated as a promoter of pathological or physiological angiogenesis. C-Met is expressed on a variety of cells including vascular endothelial cells (VECs) and up-regulated during angiogenesis. In this study, we investigated whether CD151 regulated migration, proliferation, tube formation and angiogenesis of human umbilical VECs (HUVECs) with activation of C-Met. Moreover, we studied whether CD151 could affect the angiogenic molecules such as nitric oxide (NO), vascular cell adhesion molecule-1 (VCAM-1) and vascular endothelial growth factor (VEGF). The expression of CD151 was determined by Western blotting. The cell proliferation assay was performed using the cell counting kit-8 (CCK-8) method and cell migration was assessed in microchemotaxis chambers by using fetal bovine serum (FBS) as the chemotactic stimulus. The angiogenic molecules were evaluated using ELISA. The NO level was detected using NO detection kit. The potential involvement of various signaling pathways was explored using relevant antibodies. We found that proliferation, migration and tube formation of HUVECs were promoted by CD151 with activation of C-Met, FAK and CDC42, while they were suppressed with CD151 knockdown by RNAi. Similarly, the levels of NO, VCAM-1 and VEGF in HUVECs were increased by CD151, but they were inhibited with CD151 knockdown by RNAi. These data suggested that CD151 could promote migration, proliferation, tube formation and angiogenesis of HUVECs, which was possibly related to the C-Met signaling pathways.
Base Sequence ; Human Umbilical Vein Endothelial Cells ; Humans ; Neovascularization, Physiologic ; RNA, Small Interfering ; genetics ; Receptor Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Tetraspanin 24 ; genetics ; metabolism

Vascular endothelial growth factor-D (VEGF-D) and vascular endothelial growth factor receptor-2, -3 (VEGFR-2, -3) with their corresponding signaling pathway play significant roles in the development of the embryonic vascular system and pathological lymphangiogenesis. The study was aimed to express and purify the GST-VEGF-D fusion protein, and to explore the angiogenesis effect of VEGF-D. The total RNA was extracted from human fetal lung tissue, and the mature form of VEGF-D was expanded by polymerase chain reaction (PCR), then the plasmid pGEX-5X-1/VEGF-D was reconstructed and the GST-VEGF-D fusion protein expressed in transformed E.coli BL21-DE3. The results showed that the molecular mass of this fusion protein was 38 kD and compassed more than 15% of the total bacteria proteins. The fusion protein was recognized by anti-GST and anti-VEGF-D antibodies. The soluble GST-VEGF-D fusion protein could interact with VEGFR-3/Fc and was able to stimulate the proliferation of human erythroleukemia cell line (HEL) cells. The data of chick embryo chorioallantoic membrane (CAM) experiments indicated that GST-VEGF-D could induce the CAM angiogenesis. It is concluded that the GST-VEGF-D fusion protein with biological activity was successfully expressed, and which may provide an experimental model for the investigation of the VEGF-D-induced angiogenesis and lymphangiogenesis.
Animals ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; Humans ; Neovascularization, Physiologic ; drug effects ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Vascular Endothelial Growth Factor D ; biosynthesis ; genetics ; pharmacology