Overexpression Of Thrombospondin-1 Research Articles

Thrombospondin‐1/HIV‐1 Tat protein interaction: modulation of the biological activity of extracellular Tat

Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.

Correlation of thrombospondin-1 and transforming growth factor-beta expression with malignancy of glioma.

The expression of thrombospondin-1 (TSP-1) and its role in gliomas have not been well examined. In the present study TSP-1 expression in a panel of malignant glioma cell lines and the expression of TSP-1 and transforming growth factor (TGF-beta) proteins in low-grade and malignant glioma tissues were investigated. Reverse transcription-polymerase chain reaction analysis showed that nine of nine malignant glioma cell lines expressed TSP-1 mRNA, and seven of nine glioma lines expressed TSP-2 mRNA. Production and secretion of TSP-1 were examined in the T98G glioblastoma cell line by western blot analysis. Total TSP-1 protein content in the supernatant was 10 times higher than that in the cell lysate. Secretion of TSP-1 was examined in these glioma cell lines by western blot analysis. All glioma lines secreted significant levels of TSP-1. Bioassay showed that all tumor lines had the capacity to activate latent TGF-beta. Localization of TSP-1, TGF-beta1, -beta2, and -beta3 was examined immunohistochemically in surgically resected glioma tissues, including 11 glioblastomas, six anaplastic astrocytomas, and eight astrocytomas. Most glioblastomas expressed high levels of both TSP-1 and TGF-beta. Anaplastic astrocytomas expressed moderate levels of TSP-1 and TGF-beta. Most malignant gliomas expressed various levels of TGF-beta1, -beta2, and -beta3. The expression of both proteins, however, was weak in low-grade gliomas. Normal brain tissues around the tumors were negatively or very weakly positively stained for TSP-1 and TGF-beta. These results indicate that most malignant glioma cells express TSP-1 in vitro and in vivo, and the expression of TSP-1 and TGF-beta in vivo correlates with the histologic malignancy of glioma. Overexpression of both TSP-1 and TGF-beta may increase the biologic malignancy of malignant gliomas, through generating the active form of TGF-beta in tumor tissues.

Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP-1 in the skin, using a keratin 14 expression cassette. TSP-1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full-thickness skin wounds was greatly delayed in TSP-1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP-1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP-1 preferentially interfered with wound healing-associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.

Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis.

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.

Overexpression of Thrombospondin-1 Decreases Angiogenesis and Inhibits the Growth of Human Cutaneous Squamous Cell Carcinomas

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in epithelial tumor development has remained controversial. We studied the in vitro growth characteristics and the in vivo tumor xenograft growth of the human squamous cell carcinoma cell lines A431 and SCC-13, stably transfected to overexpress human TSP-1. Overexpression of TSP-1 inhibited tumor growth of A431 xenotransplants, and completely abolished tumor formation by SCC-13 cells. TSP-1 overexpressing A431 tumors were characterized by extensive areas of necrosis and by decreased tumor vessel number and size. The effects of TSP-1 on tumor cell growth were indirect since tumor cell proliferation rates in vivo and in vitro, anchorage-dependent and -independent growth in vitro, and susceptibility to induction of apoptosis by serum withdrawal were unchanged in TSP-1 overexpressing tumor cells. However, TSP-1 overexpression up-regulated the TSP-1 receptor CD36, leading to enhanced adhesion of A431 cells to TSP-1. These findings establish TSP-1 as a potent inhibitor of angiogenesis and tumor growth in carcinomas of the skin.

Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization.

The development of skin carcinomas presently is believed to be correlated with mutations in the p53 tumor suppressor and ras gene as well as with the loss of chromosome 9. We now demonstrate that, in addition, loss of chromosome 15 may be a relevant genetic defect. Reintroduction of an extra copy of chromosome 15, but not chromosome 4, into the human skin carcinoma SCL-I cells, lacking one copy of each chromosome, resulted in tumor suppression after s.c. injection in mice. Transfection with thrombospondin-1 (TSP-1), mapped to 15q15, induced the same tumor suppression without affecting cell proliferation in vitro or in vivo. Halted tumors remained as small cysts encapsulated by surrounding stroma and blood vessels. These cysts were characterized by increased TSP-1 matrix deposition at the tumor/stroma border and a complete lack of tumor vascularization. Coinjection of TSP-1 antisense oligonucleotides drastically reduced TSP-1 expression and almost completely abolished matrix deposition at the tumor/stroma border. As a consequence, the tumor phenotype reverted to a well vascularized, progressively expanding, solid carcinoma indistinguishable from that induced by the untransfected SCL-I cells. Thus, these data strongly suggest TSP-1 as a potential tumor suppressor on chromosome 15. The data further propose an unexpected mechanism of TSP-1-mediated tumor suppression. Instead of interfering with angiogenesis in general, in this system TSP-1 acts as a matrix barrier at the tumor/stroma border, which, by halting tumor vascularization, prevents tumor cell invasion and, thus, tumor expansion.

Angiogenesis inhibitor SR 25989 upregulates thrombospondin-1 expression in human vascular endothelial cells and foreskin fibroblasts

The aim of this work was to evaluate the effects of SR 25989, a member of the thienopyridine family devoid of antiplatelet activity but possessing anti-angiogenic properties, on the regulation of proteins involved in matrix remodeling during wound healing or tumor progression. Human endothelial cells grown in the presence of SR 25989 showed moderate increases in the production of activators (tissue plasminogen activator and urokinase) and one inhibitor (plasminogen activator inhibitor type 1) of fibrinolysis, together with a significant rise in intracellular thrombospondin-1. SR 25989 induced a similar increase in thrombospondin-1 in human foreskin fibroblasts. This over-expression of thrombospondin-1 was correlated to a decrease in cell density. A concomitant increase in the tumor suppressor gene protein p53 was observed in endothelial cells and in fibroblasts, in which the slowing down of proliferation could be related to an accumulation of cells in the S and G2 M phases of the cell cycle. Northern blot analysis revealed a temporary rise in thrombospondin-1 transcripts, followed by a decrease along with a moderate increase in p53 transcripts. Thus the anti-angiogenic properties of SR 25989 appear to result from an upregulation of thrombospondin-1 which is possibly mediated by p53. The thienopyridine SR 25989 could therefore be a good candidate for adjuvant anti-angiogenic therapy in cancer.

Angiogenesis inhibitor SR 25989 upregulates thrombospondin‐1 expression in human vascular endothelial cells and foreskin fibroblasts

The aim of this work was to evaluate the effects of SR 25989, a member of the thienopyridine family devoid of antiplatelet activity but possessing anti-angiogenic properties, on the regulation of proteins involved in matrix remodeling during wound healing or tumor progression. Human endothelial cells grown in the presence of SR 25989 showed moderate increases in the production of activators (tissue plasminogen activator and urokinase) and one inhibitor (plasminogen activator inhibitor type 1) of fibrinolysis, together with a significant rise in intracellular thrombospondin-1. SR 25989 induced a similar increase in thrombospondin-1 in human foreskin fibroblasts. This over-expression of thrombospondin-1 was correlated to a decrease in cell density. A concomitant increase in the tumor suppressor gene protein p53 was observed in endothelial cells and in fibroblasts, in which the slowing down of proliferation could be related to an accumulation of cells in the S and G2/M phases of the cell cycle. Northern blot analysis revealed a temporary rise in thrombospondin-1 transcripts, followed by a decrease along with a moderate increase in p53 transcripts. Thus the anti-angiogenic properties of SR 25989 appear to result from an upregulation of thrombospondin-1 which is possibly mediated by p53. The thienopyridine SR 25989 could therefore be a good candidate for adjuvant anti-angiogenic therapy in cancer.

Regulation of tumor growth and metastasis by thrombospondin‐1

Thrombospondin-1 (TSP1) is an extracellular matrix glycoprotein that influences cell adhesion, motility, and growth. Based on its effects on tumor and endothelial cell behavior, this member of the thrombospondin gene family has attracted interest as a potential regulator of tumor growth and metastasis. Initial studies have confirmed that increased TSP1 expression suppresses growth or metastasis of some tumors in vivo and inhibits angiogenesis. These activities are cell type specific, however, since overexpression of TSP1 in some tumors causes increased tumor progression. One basis for these apparently conflicting observations may be the complexity of the protein. TSP1 interacts specifically with several cell-surface receptors, heparan sulfate proteoglycans, growth factors, and other matrix components. These multiple binding specificities, combined with the ability of TSP1 to activate latent transforming growth factor beta and inhibit several proteases, suggest that exposure to TSP1 may initiate several intracellular signals. The integration of these signals may allow varied responses to TSP1. Furthermore, these signals may be received by the tumor cells, endothelial cells responsible for neovascularization, stromal cells, or cells of the host immune system. TSP1 influences specific behaviors of each cell type. Relating these phenomena to the molecular interactions of TSP1 observed in vitro may lead to novel therapeutic strategies for controlling cancer progression and metastasis.-Roberts, D. D. Regulation of tumor growth and metastasis by thrombospondin-1.