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

Thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, inhibits neovascularization and is implicated in the regression of tumor growth and metastasis. We found that the synthesis of TSP-1 in porcine aortic endothelial (PAE) cells was decreased in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) treatment in porcine aortic endothelial (PAE) cells. In this study, a responsive site on the TSP-1 promotor affected by PMA treatment in PAE was characterized. The level of TSP-1 mRNA was also decreased by PMA after 1 h and persisted that way for at least 24 h. PMA treatment and c-Jun overexpression suppressed the transcription of TSP-1 promotor-luciferase reporter gene. A deletion between -767 and -657 on the TSP-1 promotor neutralized the PMA-induced down-regulation. In addition, oligo a (-767 approximately -723) was responsive to PMA-induced repression, while oligo b (-734 approximately -689) and c (-700 approximately -656) was not. Electrophoretic mobility shift assays showed that this PMA responsive element specifically bound a nuclear protein and that the binding activity was diminished by PMA treatment in PAE cells but not in Hep 3B cells. In supershift assay, potential regulatory elements in this region, SP1 and GATA-1, were not responsive to the inhibition of TSP-1 expression by PMA. Our results suggest that the repression of TSP-1 synthesis by PMA is mediated by blocking a particular unknown nuclear protein binding to the responsive site (-767 approximately -735), which is regulated by c-Jun.
Animal ; Aorta/cytology ; Cell Line ; Down-Regulation (Physiology) ; Endothelium, Vascular/drug effects* ; Endothelium, Vascular/cytology ; Promoter Regions (Genetics)* ; Proto-Oncogene Proteins c-jun/metabolism ; Response Elements* ; Swine ; Tetradecanoylphorbol Acetate/pharmacology* ; Thrombospondin 1/genetics* ; Thrombospondin 1/biosynthesis

OBJECTIVETo explore the effects of pioglitazone on MKP-1 and TSP-1 expression in the early stages of diabetic retinopathy induced by streptozotocin (STZ) and the relevant mechanism in it.

METHODSDiabetic rats were induced by an intraperitoneal injection of STZ in SD rats. Thirty male SD rats were randomly divided into 3 groups: diabetes adding pioglitazone group (intragastric administration pioglitazone 20 mg x kg(-1) x d(-1)), diabetes adding BBS group and normal control group. The body weight and blood glucose were measured every two weeks. Eight weeks later, all rats were killed and the expression of TSP-1 and MKP-1 mRNA was quantified in retinal tissue by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) respectively.

RESULTTSP-1 and MKP-1 concentrations were significantly increased in the diabetic rats' retinal tissue compared to the control rats. Diabetes groups adding pioglitazone caused the upregulation of TSP-1 and MKP-1 expression in the retina among the three groups.

CONCLUSIONPioglitazone treatment can significantly attenuate the evolutionary in the early stages of experimental diabetic retinopathy. Further studies should address the possible involvement of TSP-1 and MKP-1 in the correlational pathophysiology between pioglitazone and diabetic retinopathy.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Diabetic Retinopathy ; drug therapy ; metabolism ; Hypoglycemic Agents ; therapeutic use ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; therapeutic use ; Thrombospondin 1 ; biosynthesis ; genetics

Total saponins of Panax notoginseng (PNS) have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rg1 on renal fibrosis in rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into 3 groups: sham-operation (n=15), UUO (n=15) and UUO with ginsenoside Rg1 treatment (n=15, 50 mg per kg body weight, intraperitoneally (i.p.) injected). The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rg1 significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition. alpha-smooth muscle actin (alpha-SMA) and E-cadherin are two markers of tubular epithelial-myofibroblast transition (TEMT). Interestingly, ginsenoside Rg1 notably decreased alpha-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA (mRNA) of transforming growth factor-beta1 (TGF-beta1), a key mediator to regulate TEMT, in the obstructed kidney increased dramatically, but was found to decrease significantly after administration of ginsenoside Rg1. Further study showed that ginsenoside Rg1 considerably decreased the levels of both active TGF-beta1 and phosphorylated Smad2 (pSmad2). Moreover, ginsenoside Rg1 substantially suppressed the expression of thrombospondin-1 (TSP-1), a cytokine which can promote the transcription of TGF-beta1 mRNA and the activation of latent TGF-beta1. These results suggest that ginsenoside Rg1 inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.
Actins ; biosynthesis ; Animals ; Cadherins ; biosynthesis ; Collagen Type I ; genetics ; metabolism ; Fibronectins ; genetics ; metabolism ; Ginsenosides ; pharmacology ; Immunohistochemistry ; Male ; Nephritis, Interstitial ; drug therapy ; genetics ; metabolism ; pathology ; Panax notoginseng ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Smad2 Protein ; biosynthesis ; Thrombospondin 1 ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; biosynthesis ; genetics ; Ureteral Obstruction ; metabolism ; pathology

OBJECTIVETo observe the effect of sodium tanshinone II (A) sulfonate (STS) on Ang II -induced atrial fibroblast collagen synthesis and TGF-beta1 activation.

METHODAtrial fibroblasts of neonatal rats were cultured to determine the content of collagen protein. The original synthesis rate determined by the [3H]-proline incorporation method was taken as the index for myocardial fibrosis. The content of active TGF-beta1 and total TGF-beta1 in cell culture supernatants were tested and cultured by ELISA. The expression of thrombospondin-1 (TSP-1) was assessed by using Western blot.

RESULTAng II could significantly increase the content of atrial fibroblast collagen and the collagen synthesis rate, the TSP-1 expression and the concentration of active TGF-beta1, without any obvious change in total TGF-beta1. After the STS treatment, all of the indexes, apart from total TGF-beta1, were obviously down-regulated.

CONCLUSIONSTS could decrease the secretion of Ang II -induced atrial fibroblast collagen and the synthesis rate. Its mechanism is related to the inhibition of TSP-1/TGF-beta1 pathway.

Angiotensin II ; pharmacology ; Animals ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Heart Atria ; cytology ; Phenanthrenes ; pharmacology ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Thrombospondin 1 ; metabolism ; Transforming Growth Factor beta1 ; metabolism

Expression of thrombospondin-1 (TSP-1), which is a known inhibitor of tumor growth and angiogenesis, is reciprocally regulated by positive regulators, such as VEGF. Additionally, trichostatin A (TSA) suppresses tumor progression by altering VEGF levels and VEGF-mediated signaling. Thus, understanding TSA-regulated TSP-1 expression and the effects of altered TSP-1 levels might provide insights into the mechanism of action of TSA in anti-tumorigenesis, and provide an approach to cancer therapy. Here, we examined the effect of TSA on TSP-1 expression, and the effects of TSA-induced TSP-1 on cell motility and angiogenesis, in HeLa and bovine aortic endothelial cells. TSA remarkably increased TSP-1 expression at the mRNA and protein levels, by controlling the TSP-1 promoter activity. Both TSA and exogenous TSP-1 reduced cell migration and capillary-like tube formation and these activities were confirmed by blocking TSP-1 with its neutralizing antibody and small-interfering RNA. Our results suggest that TSP-1 is a potent mediator of TSA-induced anti- angiogenesis.
Angiogenesis Inhibitors/*pharmacology ; Animals ; Cattle ; Cell Line ; Cell Movement/*drug effects ; Endothelial Cells/drug effects/*physiology ; Humans ; Hydroxamic Acids/*pharmacology ; Neovascularization, Pathologic/metabolism/prevention & control ; Neovascularization, Physiologic/*drug effects ; RNA, Messenger/biosynthesis ; RNA, Small Interfering/*genetics ; Thrombospondin 1/*biosynthesis/genetics/pharmacology