Down-regulation Of Thrombospondin-1 Expression Research Articles

Trivalent chromium inhibits TSP-1 expression, proliferation, and O-GlcNAc signaling in vascular smooth muscle cells in response to high glucose in vitro.

Trivalent chromium (Cr(3+)) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. In vitro and in vivo studies suggest that Cr(3+) supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr(3+) in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study we show that a therapeutically relevant concentration of Cr(3+) (100 nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr(3+) occurs at the level of transcription. The inhibitory effects of Cr(3+) on TSP-1 were accompanied by significant reductions in O-glycosylation of cytoplasmic and nuclear proteins. Using Western blotting and immunofluorescence studies, we demonstrate that reduced protein O-glycosylation by Cr(3+) is mediated via inhibition of glutamine: fructose 6-phosphate amidotransferase, a rate-limiting enzyme of the hexosamine pathway, and O-linked N-acetylglucosamine (O-GlcNAc) transferase, a distal enzyme in the pathway that controls intracellular protein O-glycosylation. Additionally, we found that Cr(3+) attenuates reactive oxygen species formation in glucose-stimulated HASMC, suggesting an antioxidant effect. Finally, we report an antiproliferative effect of Cr(3+) that is specific for high glucose and conditions triggering elevated protein O-glycosylation. Taken together, these findings provide the first cellular evidence for a novel role of Cr(3+) to modulate aberrant vascular smooth muscle cell function associated with hyperglycemia-induced vascular complications.

Effects of Cilostazol on Thrombospondin-1 (TSP-1) Expression Changes in the Myocardium of Diabetic Rats

Myocardial protective effects of the phosphodiesterase 3 inhibitor, cilostazol, in streptozotocin-induced diabetic rats were investigated. Four weeks after induction of diabetes, we measured thrombospondin-1 (TSP-1) expression in the left ventricular myocardium. Microstructural and ultrastructural changes were also analysed. Four weeks after the induction of diabetes there were significant differences in body weight and blood glucose between the control, diabetic and cilostazol-treated diabetic animals. TSP-1 expression was significantly increased in the myocardium of diabetic rats compared with the control group. Although significantly higher than the control group, TSP-1 expression was significantly lower in the cilostazol group compared with the diabetes group. There were obvious ultrastructural changes in the myocardium of diabetic rats, which were rarely seen in cilostazol-treated diabetic rats. In conclusion, this study provides experimental evidence that cilostazol treatment of diabetic rats effectively prevents pathological myocardial alterations, possibly via the down-regulation of TSP-1 expression.

The C-terminal CD47/IAP-binding domain of thrombospondin-1 prevents camptothecin- and doxorubicin-induced apoptosis in human thyroid carcinoma cells

Camptothecin and doxorubicin belong to a family of anticancer drugs that exert cytotoxic effects by triggering apoptosis in various cell types. However there have only been few investigations showing that matricellular proteins like thrombospondin-1 (TSP-1) could be involved in the underlying mechanism of this cytotoxicity. In this report, using Hoechst reagent staining, reactive oxygen species production and caspase-3 activity measurement, we determined that both camptothecin and doxorubicin induced apoptosis in human thyroid carcinoma cells (FTC-133). On the one hand, we demonstrated that camptothecin and doxorubicin inhibited TSP-1 expression mainly occurring at the transcriptional level. On the other hand, drug-induced apoptosis determined by western blot analysis for PARP cleavage and caspase-3 activity measurement, was significantly decreased in presence of exogenous TSP-1. In order to identify the sequence responsible for this effect, we used the CD47/IAP-binding peptide 4N1 (RFYVVMWK), derived from the C-terminal domain of TSP-1, and known to play a role in apoptosis . Thus, in presence of 4N1, camptothecin and doxorubicin-induced pro-apoptotic activity was considerably inhibited. These findings suggest that induction of apoptosis by camptothecin or doxorubicin in FTC-133 cells is greatly dependent by a down-regulation of TSP-1 expression and shed new light on a possible role for TSP-1 in drug resistance.

Molecular Mechanisms Underlying the Proangiogenic Effect of Factor XIII

Coagulation Factor XIII (FXIII) was previously shown by us to induce angiogenesis. The aim of this study was to elucidate the molecular events underlying the proangiogenic effects of activated FXIII (FXIIIa) on human umbilical vein endothelial cells (HUVECs). As shown by coimmunoprecipitation studies, FXIIIa crosslinked alpha(v)beta3 with vascular endothelial growth factor receptor 2 (VEGFR-2) and enhanced the noncovalent interaction between the 2 receptors. In addition, FXIIIa induced tyrosine phosphorylation of VEGFR-2 in both the crosslinked high-molecular-weight and the noncovalent VEGFR-2/alpha(v)beta3 complexes. These effects as well as FXIIIa-induced proliferation and migration of HUVECs were abolished by iodoacetamide treatment of FXIIIa (I-FXIII) or by PTKI, an inhibitor of VEGFR-2. FXIIIa induced upregulation of c-Jun and Egr-1 as revealed by quantitative RT-PCR. Electrophoretic mobility-shift assay experiments showed that FXIIIa treatment of HUVECs enhanced binding of Wilm's tumor-1 (WT-1) but not of early growth response (Egr)-1 to the thrombospondin-1 (TSP-1) promoter sequence, suggesting that WT-1 but not Egr-1 is involved in downregulation of TSP-1 expression. The proangiogenic effect of FXIIIa is mediated by (1) enhancement of crosslinked and noncovalent alpha(v)beta3/VEGFR-2 complex formation; (2) tyrosine phosphorylation and activation of VEGFR-2; (3) upregulation of c-Jun and Egr-1; and (4) downregulation of TSP-1 induced indirectly by c-Jun through WT-1. These processes may clarify FXIII role in vascular remodeling and tissue repair.

Thrombospondin-1, PECAM-1, and regulation of angiogenesis.

Thrombospondin-1 (TSP1) is a multidomain glycoprotein expressed by many cell types. It is a multifunctional protein with important roles in regulation of vascular cell functions. Mutation or loss of tumor suppressor genes results in down regulation of TSP1 expression during malignant transformation. Thus, suggesting that down regulation of TSP1 may contribute to development of the tumor angiogenic phenotype and perhaps tumor metastasis. TSP1 was demonstrated to be a natural inhibitor of angiogenesis. Peptides from procollagen-like domain and type 1 repeats of TSP1, like whole TSP1, inhibit the angiogenic response to a variety of angiogenic stimuli in vivo and endothelial cell (EC) migration in vitro by directly acting on ECs. The molecular mechanisms which mediate these inhibitory effects of TSP1 and its peptides are not understood. TSP1 expression is down regulated in the Polyoma middle T transformed mouse brain ECs (bEND.3). This may remove the TSP1 inhibitory effects allowing ECs to rapidly proliferate in culture and form hemangiomas in vivo. Re-expression of TSP1 in bEND.3 cells restores a normal phenotype and suppresses their ability to form hemangiomas. This is mediated by modulating expression of several genes in concert favoring a differentiated state of endothelium. TSP1 transfected bEND.3 cells down regulate expression of PECAM-1, a multifunctional endothelial cell adhesion molecule with essential roles in angiogenesis. A similar phenotype to that of TSP1 transfected cells was observed when endogenous PECAM-1 levels were down regulated by anti-sense transfection of bEND.3 cells. The anti-sense PECAM-1 transfected cells turn on expression of endogenous TSP1 and its angioinhibitory receptor, CD36. Expression of other genes with potential roles in regulation of EC phenotype were also affected in patterns very similar to those observed in TSP1 transfected bEND.3 cells. Therefore, it appears that a reciprocal relationship exists between TSP1 and PECAM-1 such that they are constituents of a "switch" that regulates in concert many components of the angiogenic and differentiated phenotype of ECs.

Clinical significance of the determination of angiogenic factors

INTRODUCTION ANGIOGENIC ACTIVITY is known to depend upon a balance between positive and negative angiogenesis regulators [ 11. The alteration of this balance by mechanisms, such as the induction of vascular endothelial growth factor (VEGF), expression of TP53 or RAS mutations, SRC overexpression, and downregulation of thrombospondin-1 expression by TP53 mutation, can switch the angiogenic phenotype and facilitate the growth of solid tumours [2-51. Currently, measurement of intratumoral microvessel density (IMD) by immunocytochemistry appears to be the most reliable method of measuring angiogenic activity [6]. However, the determination of levels of each endothelial growth regulator in tumour tissues and in serum or urine can provide more information on the angiogenic characteristics of each tumour and might be an alternative method for evaluating angiogenic activity. For example, the level of intratumoral VEGF varies greatly between tumours and can be a valuable prognostic factor [7]. The endogenous angiogenesis inhibitor, angiostatin, which can cause tumour regression in human tumour xenografts models, has been detected in serum [8]. Since the biological properties of each endothelial growth factors is different, it will be of particular importance to know the role of each factor in human tumours. Therefore, we have focused on the determination of angiogenic factors, particularly positive endothelial growth regulators including VEGF, platelet-derived endothelial cell growth factorithymidine phosphorylase (I’D-ECGFITP), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and metalloproteinases (MM&), in breast cancer patients. In this article, we review our experience and the most relevant data in the literature on the clinical significance of the determination of these endothelial growth regulators in human tumours.