Adult ; Aged ; China ; ethnology ; Female ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; genetics ; Polymorphism, Genetic ; Thrombospondin 1 ; 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 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 investigate the association of thrombospondin-1 (TSP- 1) gene A8831G (N700S) polymorphism with coronary artery disease (CAD).

METHODSThis study was conducted with a case-control design including 178 patients with CAD (55 AMI) and 158 healthy subjects. The TSP-1 N700S polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphism analysis.

RESULTSNo significant difference of the AG genotype in CAD group and control group (1.7% compared with 0.6%, P=0.375) was detected. None of the homozygotes was detected for the G allele. The prevalence of the G allele was not significantly different between CAD group and controls (0.8% compared with 0.3%, P=0.376). No significant difference of the AG genotype in AMI group and control group (3.6% compared with 0.6%, P=0.104). The prevalence of G allele was not significantly different between AMI patients and controls (1.8% compared with 0.3%, P=0.364).

CONCLUSIONThere are TSP-1 N700S polymorphisms in Chinese Zhejiang Han people, but the TSP-1 N700S variant shows a much lower prevalence compared with Western populations and may be not a potential risk for CAD and AMI.

Alleles ; Case-Control Studies ; Coronary Artery Disease ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Thrombospondin 1 ; 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

Breast cancer is the most common cancer in women worldwide. It is necessary to identify biomarkers for early detection, to make accurate prognoses, and to monitor for any recurrence of the cancer. In order to identify potential breast cancer biomarkers, we analyzed the plasma samples of women diagnosed with breast cancer and age-matched normal healthy women by mTRAQ-based stable isotope-labeling mass spectrometry. We identified and quantified 204 proteins including thrombospondin-1 (THBS1) and bromodomain and WD repeat-containing protein 3 (BRWD3) which were increased by more than 5-fold in breast cancer plasma. The plasma levels of the two proteins were evaluated by Western blot assay to confirm for their diagnostic value as serum markers. A 1.8-fold increase in BRWD3 was observed while comparing the plasma levels of breast cancer patients (n = 54) with age-matched normal healthy controls (n = 30), and the area under the receiver operating characteristic curve (AUC) was 0.917. THBS1 was detected in pooled breast cancer plasma at the ratio similar to mTRAQ ratio (> 5-fold). The AUC value for THBS1 was 0.875. The increase of THBS1 was more prominent in estrogen receptor negative and progesterone receptor negative patients than receptor-positive patients. Our results are evidence of the diagnostic value of THBS1 in detecting breast cancer. Based on our findings, we suggest a proteomic method for protein identification and quantification lead to effective biomarker discovery.
Adult ; Breast Neoplasms/*diagnosis/genetics/pathology ; Early Detection of Cancer ; Female ; Gene Expression Profiling ; Humans ; Middle Aged ; Pathology, Molecular/methods ; Predictive Value of Tests ; Prognosis ; *Proteomics ; Thrombospondin 1/*blood ; Transcription Factors/*blood ; Tumor Markers, Biological/*blood

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

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