Publications

BACKGROUND: Orthotopic mouse models of human cancer represent an important in vivo tool for drug testing and validation. Most of the human thyroid carcinoma cell lines used in orthotopic or subcutaneous models are likely of melanoma and colon cancer. Here, we report and characterize a novel orthotopic model of human thyroid carcinoma using a unique thyroid cancer cell line.

METHODS: We used the cell line 8505c, originated from a thyroid tumor histologically characterized by anaplastic carcinoma cell features. We injected 8505c cells engineered using a green fluorescent protein-positive lentiviral vector orthotopically into the thyroid of severe combined immunodeficient mice.

RESULTS: Orthotopic implantation with the 8505c cells produced thyroid tumors after 5 weeks, showing large neck masses, with histopathologic features of a high-grade neoplasm (anaplasia, necrosis, high mitotic and proliferative indexes, p53 positivity, extrathyroidal invasion, lymph node and distant metastases) and immunoprofile of follicular thyroid cell origin with positivity for thyroid transcription factor-1 and PAX8, and for cytokeratins.

CONCLUSIONS: Here we describe a novel orthotopic thyroid carcinoma model using 8505c cells. This model can prove to be a reliable and useful tool to investigate in vivo biological mechanisms determining thyroid cancer aggressiveness, and to test novel therapeutics for the treatment of refractory or advanced thyroid cancers.

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias.

Through its interactions with proteins and proteoglycans, thrombospondin-1 (TSP-1) functions at the interface of the cell membrane and the extracellular matrix to regulate matrix structure and cellular phenotype. We have previously determined the structure of the high affinity heparin-binding domain of TSP-1, designated TSPN-1, in association with the synthetic heparin, Arixtra. To establish that the binding of TSPN-1 to Arixtra is representative of the association with naturally occurring heparins, we have determined the structures of TSPN-1 in complex with heparin oligosaccharides containing eight (dp8) and ten (dp10) subunits, by x-ray crystallography. We have found that dp8 and dp10 bind to TSPN-1 in a manner similar to Arixtra and that dp8 and dp10 induce the formation of trans and cis TSPN-1 dimers, respectively. In silico docking calculations partnered with our crystal structures support the importance of arginine residues in positions 29, 42, and 77 in binding sulfate groups of the dp8 and dp10 forms of heparin. The ability of several TSPN-1 domains to bind to glycosaminoglycans simultaneously probably increases the affinity of binding through multivalent interactions. The formation of cis and trans dimers of the TSPN-1 domain with relatively short segments of heparin further enhances the ability of TSP-1 to participate in high affinity binding to glycosaminoglycans. Dimer formation may also involve TSPN-1 domains from two separate TSP-1 molecules. This association would enable glycosaminoglycans to cluster TSP-1.

The thrombospondins (TSPs) are a family of five proteins that are involved in the tissue remodeling that is associated with embryonic development, wound healing, synaptogenesis, and neoplasia. These proteins mediate the interaction of normal and neoplastic cells with the extracellular matrix and surrounding tissue. In the tumor microenvironment, TSP-1 has been shown to suppress tumor growth by inhibiting angiogenesis and by activating transforming growth factor beta. TSP-1 inhibits angiogenesis through direct effects on endothelial cell migration and survival, and through effects on vascular endothelial cell growth factor bioavailability. In addition, TSP-1 may affect tumor cell function through interaction with cell surface receptors and regulation of extracellular proteases. Whereas the role of TSP-1 in the tumor microenvironment is the best characterized, the other TSPs may have similar functions. (Part of a Multi-author Review).

Thrombospondins are large secreted, multimodular, calcium-binding glycoproteins that have complex roles in mediating cellular processes. Determination of high-resolution structures of thrombospondins has revealed unique and interesting protein motifs. Here, we review this progress and discuss implications for function. By combining structures of modules from thrombospondins and related extracellular proteins it is now possible to prepare an overall model of the structure of thrombospondin-1 and thrombospondin-2 and discern features of other thrombospondins. (Part of a multi-author Review).

OBJECTIVE: Vascular abnormalities and expression of proangiogenic factors have been repeatedly reported in inflammatory bowel disease (IBD). Thrombospondin 1 (TSP-1) is a protein well known for its antiangiogenic and anti-inflammatory properties. Using the dextran sulfate sodium (DSS) model, the role of TSP-1 in IBD has been investigated in vivo.

METHODS: TSP-1-deficient mice (TSP-1-/-) and WT mice were treated with DSS for 7 days. Disease activity indices, myeloperoxidase activity (MPO) and histology were analyzed. Microvascular density (MVD) was quantified using immunohistochemistry (IMH) with CD31 antibody. TGF-beta(1), basic FGF, VEGF, TNF-alpha and MMPs protein levels were evaluated by IMH and enzyme-linked immunoabsorbent assay (ELISA). Mice were treated with ABT-510 (Abbott Laboratories), an antiangiogenic TSP peptide, using miniosmotic pumps for 7 days.

RESULTS: TSP-1(-/-) mice had a worse clinical outcome and exhibited severe signs of rectal bleeding compared to the WT controls. The TSP-1-/- mice showed a higher level of crypt damage and deeper lesions. The grade of inflammation and the levels of MPO activity were also significantly higher in colons of TSP-1-/- mice. TSP-1-/- mice displayed higher MVD in focal areas of the colon after only 3 days of DSS treatment. Furthermore, clinical severity of the colitis and angiogenesis was significantly diminished when mice was treated with ABT-510.

CONCLUSIONS: These findings directly link TSP-1 as a protective factor in IBD and suggest antiangiogenesis treatment, including compounds such as ABT-510 as an adjuvant therapy for IBD.

Latency Associated Peptide (LAP) binds TGF-beta1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-beta1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-beta1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-beta1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.

OBJECTIVE: Insufficient development of a new intra-islet capillary network after transplantation may be one contributing factor to the failure of islet grafts in clinical transplantation. The present study tested the hypothesis that the angiostatic factor thrombospondin-1 (TSP-1), which is normally present in islets, restricts intra-islet vascular expansion posttransplantation.

RESEARCH DESIGN AND METHODS: Pancreatic islets of TSP-1-deficient (TSP-1(-/-)) mice or wild-type islets transfected with siRNA for TSP-1 were transplanted beneath the renal capsule of syngeneic or immunocompromised recipient mice.

RESULTS: Both genetically TSP-1(-/-) islets and TSP-1 siRNA-transfected islet cells demonstrated an increased vascular density when compared with control islets 1 month after transplantation. This was also reflected in a markedly increased blood perfusion and oxygenation of the grafts. The functional importance of the improved vascular engraftment was analyzed by comparing glucose-stimulated insulin release from islet cells transfected with either TSP-1 siRNA or scramble siRNA before implantation. These experiments showed that the increased revascularization of grafts composed of TSP-1 siRNA-transfected islet cells correlated to increments in both their first and second phase of glucose-stimulated insulin secretion.

CONCLUSIONS: Our findings demonstrate that inhibition of TSP-1 in islets intended for transplantation may be a feasible strategy to improve islet graft revascularization and function.

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.