1.Advances on hypoxia inducible factor-1.
Chinese Medical Journal 2013;126(18):3567-3571
4.Efficient generation of recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1.
Peng LIU ; Yi WANG ; Ren-chi YANG ; Jie GU ; Ying-lin CAI ; Zhong-chao HAN
Acta Academiae Medicinae Sinicae 2003;25(3):312-315
OBJECTIVETo construct recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1 (TSP1f).
METHODSTSP1f cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was subcloned into a shuttle vector pShuttle-CMV. After sequence confirmation, the resultant plasmid was linearized by the restriction endonuclease Pme I and cotransformed with the supercoiled adenoviral vector pAdEasy-1 into Escherichia coli strain BJ5183. Recombinants were selected by Kanamycin resistance and screened by restriction endonuclease digestion. Then, the recombinant adenoviral construct was cleaved with Pac I and transfected into the packaging cell line 293. The adenoviral vector ADV-TSP1f was propagated in 293 cells and purified by cesium chloride (CsCl) density centrifugation. PCR and Western blot analysis were performed to confirm TSP1f expression.
RESULTSOf 43 Kanamycin-resistant colonies obtained from cotransformation, all of the 10 smallest ones were the correct recombinants. TSP-1f was expressed efficiently by ADV-TSP1f. The virus stock titer after CsCl banding was 1.0 x 10(11) pfu/mL.
CONCLUSIONSGenerating recombinant adenoviruses using AdEasy System results in highly efficient viral production and significantly decrease the time required to construct usable viruses. ADV-TSP1f can be further used in in vivo gene therapy studies.
Adenoviridae ; genetics ; metabolism ; Angiogenesis Inhibitors ; biosynthesis ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Neoplasms ; blood supply ; Neovascularization, Pathologic ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Thrombospondin 1 ; biosynthesis ; genetics
5.Down-regulated expression of UNC5b related to hepatocellular carcinoma angiogenesis.
Hua ZHANG ; Fan WU ; Yi-ming TAO ; Lian-yue YANG
Chinese Journal of Surgery 2009;47(20):1569-1573
OBJECTIVETo investigate the relationship between UNC5b gene expression and angiogenesis of hepatocellular carcinoma (HCC).
METHODSIn situ hybridization was performed to detect the expression of UNC5b mRNA in HCC samples, paracarcinomatous liver tissues samples and normal liver samples. The relationship between UNC5b mRNA expression and the HCC clinicopathological features were also analyzed. Human umbilical artery endothelial cells were isolated and stimulated with HCC tissues homogenate, vascular endothelial growth factor and basic fibroblast growth factor. Then RT-PCR was employed to detect the expression of UNC5b mRNA in normal HUAEC as well as activated HUAEC.
RESULTSIn situ hybridization results showed that UNC5b mRNA expression was detected majorly in endothelial cells of all normal liver tissues, and partial PCLTs but was weak or even undetectable in endothelial cells of the corresponding HCC tissues. The expression levels of UNC5b gene in PCLTs were significantly correlated with capsular formation of HCC. Furthermore, RT-PCR results showed that the expression levels of UNC5b mRNA in activated HUAEC were significantly higher than those in normal HUAEC.
CONCLUSIONSDown-regulation of UNC5b gene expression is related to angiogenesis of HCC, which may be associated with the progression of HCC.
Carcinoma, Hepatocellular ; blood supply ; genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; blood supply ; genetics ; Neovascularization, Pathologic ; genetics ; RNA, Messenger ; genetics ; Receptors, Cell Surface ; genetics
6.Establishment of transgenic mice with visualized neovascularization.
Zhen-lin LI ; Yi GAO ; Jun-shuang JIA ; Song-qin CHEN ; Hai-hong WANG ; Jing AN
Journal of Southern Medical University 2011;31(10):1748-1752
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
7.Novel Molecular Mechanisms in the Development of Non-Alcoholic Steatohepatitis.
Davide POVERO ; Ariel E FELDSTEIN
Diabetes & Metabolism Journal 2016;40(1):1-11
Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in adults and children worldwide. NAFLD has become a severe health issue and it can progress towards a more severe form of the disease, the non-alcoholic steatohepatitis (NASH). A combination of environmental factors, host genetics, and gut microbiota leads to excessive accumulation of lipids in the liver (steatosis), which may result in lipotoxicity and trigger hepatocyte cell death, liver inflammation, fibrosis, and pathological angiogenesis. NASH can further progress towards liver cirrhosis and cancer. Over the last few years, cell-derived extracellular vesicles (EVs) have been identified as effective cell-to-cell messengers that transfer several bioactive molecules in target cells, modulating the pathogenesis and progression of NASH. In this review, we focused on recently highlighted aspects of molecular pathogenesis of NASH, mediated by EVs via their bioactive components. The studies included in this review summarize the state of art regarding the role of EVs during the progression of NASH and bring novel insight about the potential use of EVs for diagnosis and therapeutic strategies for patients with this disease.
Adult
;
Cell Death
;
Child
;
Diagnosis
;
Fatty Liver*
;
Fibrosis
;
Genetics
;
Hepatocytes
;
Humans
;
Inflammation
;
Liver
;
Liver Cirrhosis
;
Liver Diseases
;
Microbiota
;
Neovascularization, Pathologic
8.Hypoxia and angiogenesis: regulation of hypoxia-inducible factors via novel binding factors.
Li CHEN ; Alexander ENDLER ; Futoshi SHIBASAKI
Experimental & Molecular Medicine 2009;41(12):849-857
The mechanisms that regulate angiogenesis in hypoxia or hypoxic microenvironment are modulated by several pro- and antiangiogenic factors. Hypoxia-inducible factors (HIFs) have been established as the basic and major inducers of angiogenesis, but understanding the role of interacting proteins is becoming increasingly important to elucidate the angiogenic processes of a hypoxic response. In particular, with regard to wound healing and the novel therapies for vascular disorders such as ischemic brain and heart attack, it is essential to gain insights in the formation and regulation of HIF transcriptional machineries related to angiogenesis. Further, identification of alternative ways of inhibiting tumor growth by disrupting the growth-triggering mechanisms of increasing vascular supply via angiogenesis depends on the knowledge of how tumor cells develop their own vasculature. Here, we review our findings on the interactions of basic HIFs, HIF-1alpha and HIF-2alpha, with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively. The present results and discussion revealed new regulatory interactions of HIF-related mechanisms.
Animals
;
Anoxia/genetics/*metabolism
;
Gene Expression Regulation
;
Histone Deacetylases/genetics/metabolism
;
Humans
;
Hypoxia-Inducible Factor 1/genetics/*metabolism
;
Neovascularization, Pathologic/genetics/*metabolism
9.Expression of Vascular Endothelial Growth Factor and p53 in Pancreatic Carcinomas.
Young Eun JOO ; Young Hae SOHN ; Wan Sik LEE ; Chang Hwan PARK ; Sung Kyu CHOI ; Jong Sun REW ; Chang Soo PARK ; Sei Jong KIM
The Korean Journal of Internal Medicine 2002;17(3):153-159
BACKGROUND: Angiogenesis has been shown to be a critical aspect of tumor growth and progression. Vascular endothelial growth factor (VEGF) has potent angiogenic activity and has been identified in a wide variety of malignancies, including pancreatic carcinoma. The tumor-suppressor gene p53 has been thought to regulate VEGF in angiogenesis. The aim of the current study was conducted to investigate the association between p53 mutation and VEGF expression and the prognostic value of these factors in pancreatic carcinoma. METHODS: Formalin-fixed, paraffin-embedded tissue specimens were obtained from 30 patients who underwent surgery for pancreatic carcinoma. We used an immunohistochemical technique to localize VEGF and p53 in pancreatic carcinoma tissues. RESULTS: Positive expression of VEGF was detected in 17 out of 30 (56.7%) tumors. Positive expression of VEGF correlated with the depth of tumor invasion (p=0.002). There was a trend towards an association between positive expression of VEGF and distant metastasis, although these associations were not statistically significant (p=0.070). p53 mutations were identified in 18 out of 30 (60.0%) tumors. However, no significant correlation was found between p53 expression and various clinicopathological parameters. The correlation between p53 mutation and VEGF expression was statistically significant (p=0.004). CONCLUSION: VEGF, a key factor for the induction of tumor-associated angiogenesis, may be involved in tumor characteristics, including tumor invasion and metastasis. And p53 mutation may be implicated in the regulation of angiogenesis through a VEGF up-regulation.
Adenocarcinoma/*genetics/pathology
;
Adult
;
Aged
;
Aged, 80 and over
;
Endothelial Growth Factors/*genetics
;
Female
;
*Genes, p53
;
Human
;
Male
;
Middle Age
;
Mutation
;
Neovascularization, Pathologic/genetics
;
Pancreatic Neoplasms/*genetics/pathology
;
Prognosis
10.Small interference RNA targeting nuclear factor-kappaB inhibits endometriotic angiogenesis in chick embryo chorioallantocic membrane.
Mu-biao LIU ; Yuan-li HE ; Jie ZHONG
Journal of Southern Medical University 2009;29(4):757-759
OBJECTIVETo investigate the effect of small interference RNA (siRNA) targeting nuclear factor-kappaB (NF-kappaB) on endometriosis.
METHODThe eutopic endometrium of women with endometriosis were transplanted into the nonvascular region of 8-day-old chicken embryo chorioallantocic membrane (CAM), and the effects of NF-kappaB p65 siRNA on the vascularization and endometriotic lesion formation were tested with proper controls.
RESULTSTransplantation of the endometrium onto the CAM resulted in a strong angiogenic response in the chicken tissue. The angiogenesis was significantly reduced and endometriotic lesion formation significantly suppressed with siRNA targeting NF-kappaB in comparison with the control group.
CONCLUSIONSThe NF-kappaB pathway is involved in the development of endometriotic lesions in vitro, and NF-kappaB gene silencing reduces endometriotic angiogenesis and promotes cell apoptosis in the endometriotic lesions, suggesting that NF-kappaB might be a good target for endometriosis treatment.
Animals ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; metabolism ; Endometriosis ; genetics ; physiopathology ; Female ; Humans ; NF-kappa B ; deficiency ; genetics ; Neovascularization, Pathologic ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics