Animals ; Disease Models, Animal ; Female ; Fibrosis ; metabolism ; pathology ; Hydroxyproline ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Mice ; Thrombospondin 1 ; pharmacology

OBJECTIVE: To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. METHODS: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. RESULTS: The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. CONCLUSION TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.
Animals ; Apoptosis ; CD36 Antigens/metabolism* ; Caspase 3/metabolism* ; Cell Line ; Humans ; Leukemia, Megakaryoblastic, Acute ; Mice ; Thrombospondin 1/pharmacology*

OBJECTIVETo investigate the effect of propofol on the expression of thrombospondin-1 (THBS-1) mRNA and protein in purified newborn rat cortical astrocytes in vitro.

METHODSAstrocytes were isolated from newborn rat cortex and grown in culture before exposure to propofol at 3, 10, 30, 100 or 300 µmol/L for 6 h, 12, or 24 h. The mRNA level of THBS-1 was detected by RT-PCR, and the protein level of THBS-1 was detected by immunofluorescence cytochemistry and Western blotting.

RESULTSPropofol exposure caused significantly upregulated THBS-1 level in cultured astrocytes (P<0.05) to a level about 1.3 times higher than that in control cells. The mRNA and protein levels of THBS-1 in cultured rat cortical astrocytes were upregulated by exposures to 10, 30 and 100 µmol/L propofol (P<0.01). High expression of THBS-1 mRNA and protein was detected in the cells with exposures for different durations (P<0.05), especially in the 12 h group (P<0.01).

CONCLUSIONPropofol at clinically relevant concentrations can modulate the level of THBS-1 secreted by astrocytes of rat cerebral cortex in vitro.

Animals ; Astrocytes ; drug effects ; metabolism ; Cells, Cultured ; Cerebral Cortex ; cytology ; Propofol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Thrombospondin 1 ; metabolism

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

BACKGROUNDResearchers have recently demonstrated that thrombospondin-1 (TSP-1) has an important function in regulating neovascularization. Whether it inhibits or accelerates neovascularization, however, is still controversial. We found few reports about the correlation between TSP-1 and vascularization in mucoepidermoid carcinoma. In this research, the distribution and expression of TSP-1 in mucoepidermoid carcinoma were investigated. We also analyzed (1) the correlation between the expression of TSP-1 and microvessel density (MVD), as an indicator of neovascularization activity, and (2) the effect of TSP-1 on neovascularization and tumor growth in the subcutaneous xenotransplanted model of mucoepidermoid carcinoma.

METHOD(1) The sites and intensity of expression of TSP-1 and the MVD were analyzed in 45 cases of mucoepidermoid carcinoma after surgery by the method of streptavidin-peroxidase (SP) immunohistochemistry; and (2) recombinant human thrombospondin-1 (rhTSP-1) was injected twice a week for five consecutive weeks around the tumor in the subcutaneous xenotransplanted tumor model of mucoepidermoid carcinoma in nude mice. Each week, the tumor size was measured, in order to draw the growth curve of the xenotransplanted tumor model of mucoepidermoid carcinoma, and MVD was measured.

RESULTS(1) The positive expression of TSP-1 protein was 57.78% (26/45). Most positive staining for TSP-1 was found in the cytoplasm of the cancer cells, while some staining occurred in the extracellular matrix. The mean MVD in 45 cases of mucoepidermoid carcinoma was 58.17 +/- 19.77 per 100 visual fields. Tumors with a high expression of TSP-1 showed a low MVD value, and the TSP-1 immunocompetence and microvessel density showed a significant negative correlation (r(s) = -0.947, P < 0.001). (2) The xenotransplanted tumors with the injection doses of 1.25, 0.75 and 0.25 microg/ml respectively were 36.97%, 53.36% and 73.61% of the size of the control group ((451 +/- 92), (651 +/- 113), (898 +/- 86) and (1220 +/- 157) mm(3) respectively, F = 53.167, P < 0.001), and their weights were respectively 35.14%, 51.35% and 70.27% of the control group ((1.3 +/- 0.5), (1.9 +/- 0.5), (2.6 +/- 0.3), and (3.7 +/- 0.7) g respectively, F = 62.669, P < 0.001). Their MVDs were 25.00%, 45.93%, and 72.20% respectively of the control group and concentration dependent (15.43 +/- 3.45, 28.35 +/- 4.24, 44.57 +/- 3.35 and 61.73 +/- 5.43 per 100 visual fields respectively, F = 54.582, P < 0.001).

CONCLUSIONSThe TSP-1 has a higher expression in mucoepidermoid carcinoma and the expression has a significant negative correlation with neovascularization. The TSP-1 inhibits neovascularization and tumor growth, and it might be a new biological therapy for treatment of patients with mucoepidermoid carcinoma.

Adult ; Aged ; Animals ; Carcinoma, Mucoepidermoid ; blood supply ; chemistry ; pathology ; Female ; Humans ; Male ; Mice ; Middle Aged ; Neoplasm Transplantation ; Neovascularization, Pathologic ; pathology ; Recombinant Proteins ; pharmacology ; Thrombospondin 1 ; analysis ; pharmacology ; Transplantation, Heterologous

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

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

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

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

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