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 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

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

Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767~+756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407~+756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.
Binding Sites/genetics ; Cell Line, Tumor ; DNA-Binding Proteins/*metabolism ; Down-Regulation/genetics ; Electrophoretic Mobility Shift Assay ; Genes, Reporter/genetics ; Humans ; Luciferases/analysis/genetics ; Promoter Regions (Genetics)/*genetics ; Proto-Oncogene Proteins c-jun/genetics/*metabolism ; Repressor Proteins/*metabolism ; Research Support, Non-U.S. Gov't ; Sequence Deletion/genetics ; Thrombospondin 1/*genetics/metabolism ; Transcription Factor AP-1/metabolism ; Transcription Factors/*metabolism

BACKGROUNDTrans-arachidonic acids (TAAs), newly discovered markers of nitrative stress and the major products of nitrogen dioxide (NO2(·))-mediated isomerization of arachidonic acid (AA), represent a new mechanism of NO2(·)-induced toxicity. It has been reported that TAAs were generated in oxygen-induced microvascular degeneration model and TAAs were also generated in a diabetic retinopathy (DR) model. In this study, we examined high glucose-induced nitrative stress damage and TAAs levels and explored the possible mechanisms for DR caused by reactive nitrogen species.

METHODSDiabetic rats were induced by intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. Bovine retinal capillary endothelial cells (BRECs) were selectively cultured and incubated with normal or high glucose. The serum TAAs and AA in diabetic rats were measured by the gas chromatography and mass spectrometry (GC/MS) method. The ratio of peak area of TAAs to AA with selected ion of 79 was estimated by a group t-test. Thrombospondin-1 (TSP-1) in the rat retinas and BRECs extracts were examined by Western blotting. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) protein was examined by Western blotting in BRECs incubated with high glucose.

RESULTSThe TAAs to AA ratio (TAAs/AA) was significantly increased in the serum at 8, 12 and 16 weeks after STZ injection (P < 0.05), with no noticeable change found at 2 or 4 weeks (P > 0.05). Expression of TSP-1 in the retina of diabetic rats was progressively elevated according to the duration of diabetes. TSP-1 expression was increased in BRECs incubated with high glucose at 48 hours. Moreover, high glucose also increased ERK1/2 expression, which peaked at 30 minutes and then decreased in the following 48 hours.

CONCLUSIONAn elevation of TAAs/AA is associated with high glucose-induced nitrative stress, which probably involves upregulation of TSP-1 through activating ERK1/2.

Animals ; Arachidonic Acid ; metabolism ; Blotting, Western ; Cattle ; Cells, Cultured ; Diabetes Mellitus, Experimental ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Gas Chromatography-Mass Spectrometry ; Male ; Rats ; Rats, Sprague-Dawley ; Reactive Nitrogen Species ; metabolism ; Streptozocin ; Thrombospondin 1 ; genetics ; Up-Regulation

OBJECTIVETerminal stage renal failure is the final common fate of chronic nephropathies independent of the type of initial insult. Abnormally filtered proteins have an intrinsic renal toxicity linked to their over-reabsorption by proximal tubular cells and activation of tubular-dependent pathways of interstitial inflammation and fibrosis. The functional importance of tubulointerstitial events in progressive renal disease is supported by evidence that the severity of tubular interstitial damage strongly correlates with the risk of renal failure. The present study aimed to investigate the expressive tendency of some pro-fibrosis genetic factors mRNA, including thrombospondin-1 (TSP-1), transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF) and the major component of extracellular matrix-fibronectin (FN) in renal tissues at different time points during early stage of renal lesions caused by proteinuria in bovine serum albumin (BSA) injection-induced proteinuria overload nephrotic young rats and the significance of these factors on tubulointerstitial fibrosis development.

METHODSFemale young Wistar rats aged 3-4 weeks with proteinuria overload nephrosis induced by BSA (1.0 g/d) injected intraperitoneally were used as experimental models. The 80 young rats were divided into control group (n = 40) and BSA injected group (n = 40). At different time points (weeks 1, 2, 3 and 4), the urinary protein was measured by Coomassie brilliant blue colorimetric assay; the renal tissues morphologic changes were evaluated after HE staining; the P(65)/Rel-A, TSP-1, TGF-beta1 and CTGF mRNA expression in renal tissues was determined by in situ hybridization method and the FN mRNA expression was detected by Northern blot. The experimental data were evaluated by statistics software SPSS10.0.

RESULTS(1) Three to four weeks after BSA injection, heavy proteinuria was observed in the rats of BSA group (week 3: 104.3 +/- 21.8 mg/24 h; week 4: 131.1 +/- 18.3 mg/24 h). The proteinuria deteriorated progressively afterwards. Histopathological examination revealed that inflammatory cells infiltrated into tubulointerstitial areas extensively, protein casts were seen in tubules and edema occurred in tubulointerstitial areas. (2) In situ hybridization showed that NF-kappaB (P(65)/Rel-A) mRNA expression was up-regulated progressively in nuclei of tubular epithelial cells, the semi-quantitative scores (at 1, 2, 3 and 4 weeks) were 2.33 +/- 0.20, 2.76 +/- 0.12, 2.96 +/- 0.19, and 3.76 +/- 0.18, respectively (F = 37.34, P < 0.01). (3) At 1 week after BSA injection, the TSP-1 mRNA expression appeared in glomeruli and increased, but was light in tubulointerstitial areas, its expressive peak was observed at week 2, and declined to mild after weeks 3 and 4. The semi-quantitative scores at different time points suggested that TSP-1 mRNA was expressed mainly in early stage of lesion in this model, then, this tendency turned to a flat roof smoothly. (4) TGF-beta1 and CTGF mRNA was up-regulated simultaneously in tubular epithelial cells (F = 8.80, P < 0.01 F = 19.41, P < 0.01). (5) Northern blotting showed that FN mRNA was considerably up-regulated at second week in the kidneys of rats in BSA group, 2.7-fold higher at week 4 than that at week 1 in BSA group rats, and was 3.6-fold higher than that of control group.

CONCLUSIONThe present study suggested that NF-kappaB (P(65)/Rel-A) mRNA expression and its activity was enhanced significantly by proteinuria-loading and synchronized with high expression of TSP-1, TGF-beta1, and CTGF mRNA in the kidney, at the same time, FN mRNA was up-regulated in renal tissues and an aggravating tendency in tubulointerstitial lesions was observed in nephrotic young rats with heavy proteinuria.

Animals ; Connective Tissue Growth Factor ; metabolism ; Female ; Fibronectins ; genetics ; metabolism ; Kidney ; metabolism ; physiopathology ; NF-kappa B ; genetics ; metabolism ; Nephrosis ; metabolism ; physiopathology ; Proteinuria ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Thrombospondin 1 ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVEPancreatic cancer is one of the most deadly cancers, which is characterized by its high metastatic potential. S100A4 is a major prometastatic protein involved in tumor invasion and metastasis which precise role in pancreatic cancer has not been fully investigated. We knocked down the S100A4 gene in the Bxpc-3 pancreatic cancer cell line via RNA interference to study the changes in cell behavior.

METHODSReal-time polymerase chain reaction and western blotting were used to detect mRNA and protein expression levels of S100A4, matrix metalloproteinase (MMP)-2, E-cadherin and thrombospondin (TSP)-1. Transwell chambers were used to detect the migration and invasion abilities; a cell adhesion assay was used to detect adhesion ability; colony forming efficiency was used to detect cell proliferation; flow cytometry was used to detect apoptosis.

RESULTSS100A4 mRNA expression was reduced to 17% after transfection with S100A4-siRNA, and protein expression had a similar trend. mRNA and protein expression of MMP-2 was reduced and that of E-cadherin and TSP-1 was elevated, indicating that S100A4 affects their expression. S100A4-silenced cells exhibited a marked decrease in migration and invasiveness and increased adhesion, whereas overall proliferation and apoptosis were not overtly altered.

CONCLUSIONS100A4 and its downstream factors play important roles in pancreatic cancer invasion, and silencing A100A4 can significantly contain the invasiveness of pancreatic cancer.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cadherins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Pancreatic Neoplasms ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; S100 Calcium-Binding Protein A4 ; S100 Proteins ; genetics ; metabolism ; Thrombospondin 1 ; genetics ; metabolism

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

Thrombospondin-1 (TSP-1), a multifunctional protein that is able to function as a negative regulator of solid tumor progression and angiogenesis, is normally present at a very low level but rapidly elevated in pathological tissues. To understand the cellular regulation of TSP-1 expression, the mode of it's expression in Hep3B, SK-HEP-1, and porcine aortic endothelial (PAE) cells was examined in the presence of all-trans retinoic acid (ATRA), interleukin-6 (IL-6), interferon-gamma (IFN-gamma), or phorbol 12-myristate 13-acetate (PMA). ATRA or IL-6 induced a dose-dependent increase of TSP-1 protein and mRNA levels in PAE cells, while they negatively regulated TSP-1 expression in the Hep3B and SK-HEP-1 cells. In contrast, PMA showed just the opposite effects on the TSP-1 expression in the same cells. IFN-gamma had little effect on TSP-1 level in Hep3B and PAE cells. The TSP-1 expression in SK-HEP-1 cells by these agents showed a close resemblance to that of liver cells rather than that of the endothelial cell line. Possible TSP-1 promoter-mediated responses by ATRA, IL-6, IFN-gamma, or PMA in Hep3B and PAE cells examined with luciferase activity of TSP-LUC reporter plasmid showed that levels of TSP-1 promoter activity were lower than that of the expressed TSP-1 protein and mRNA levels. Transfection of c-Jun and/or RARalpha expression vectors into Hep3B and PAE cells resulted in the enhanced TSP-1 promoter activity as well as the increments of of its protein and mRNA level. These results suggest that regulatory agents-induced TSP-1 expression may be attributed to mRNA stability and/or translational activation in concert with transcriptional activation and TSP-1 expression may be independently controlled via each signal pathway stimulated by PMA or ATRA.
Animal ; Cell Line ; Endothelium, Vascular/cytology/drug effects/metabolism ; *Gene Expression Regulation/drug effects ; Genes, Reporter ; Genes, jun ; Human ; Immunoblotting ; Interferon Type II/pharmacology ; Interleukin-6/pharmacology ; *Promoter Regions (Genetics) ; Proto-Oncogene Proteins c-jun/genetics/metabolism ; Receptors, Retinoic Acid/genetics/metabolism ; Recombinant Fusion Proteins/metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; Thrombospondin 1/*genetics/metabolism ; Transcription, Genetic ; Tretinoin/pharmacology

OBJECTIVETo investigate the effect of Qingre Quyu Granule (清热祛瘀颗粒, QRQYG) on stabilizing vulnerable plaques in apolipoprotein E (ApoE) deficient mice.

METHODSSeventy-two male ApoE deficient mice were given a high-fat diet from 6 weeks of age. At the 16th week, all the mice were randomized into 3 groups: the QRQYG group, the simvastatin group, and the control group. Sixteen weeks after administration of 0.9 g/kg QRQYG, 3 mg/kg simvastatin or 10 mg/kg sodium chloride per day to the respective groups, the animals were euthanized. The pathological morphologic changes in the vulnerable plaques were evaluated, the matrix metalloprotease-9 (MMP-9) expression was measured by immunohistofluorescence, the soluble intercellular adhesion molecule 1 (ICAM-1) was determined by ELISA, the nuclear factor kappaB (NF-κB) subunit p65 was measured by quantitative RT-PCR, and, finally, thrombospondin-1 (TSP-1) was determined by the immunohistochemical method.

RESULTSThe plaque cross-sectional area in the brachiocephalic artery (23.7%, P<0.01), the lipid core of the plaque (43.1%±3.1%), and the number of buried fibrotic caps of the plaque were significantly decreased in the QRQYG group compared to the control group (both P<0.01); furthermore, the thickness of the fibrotic cap of the plaque increased and the intra-plaque hemorrhage of the plaque decreased. The serum soluble ICAM-1 (27.1±5.1 μg/mL), the protein expression of MMP-9 and TSP-1 and the p65 mRNA expression increased in the QRQYG group in comparison with the control group (P<0.05 or P<0.01).

CONCLUSIONQRQYG could stabilize the vulnerable plaque through inhibition of the inflammatory response.

Animals ; Apolipoproteins E ; genetics ; Atherosclerosis ; pathology ; Brachiocephalic Trunk ; drug effects ; enzymology ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Knockout ; NF-kappa B ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Simvastatin ; pharmacology ; Sodium Chloride ; pharmacology ; Thrombospondin 1 ; metabolism