Publications

The extracellular matrix protein F-spondin mediates axon guidance during neuronal development. Its N-terminal domain, termed the reelin-N domain, is conserved in F-spondins, reelins, and other extracellular matrix proteins. In this study, a recombinant human reelin-N domain has been expressed, purified, and shown to bind heparin. The crystal structure of the reelin-N domain resolved to 2.0 A reveals a variant immunoglobulin-like fold and potential heparin-binding sites. Substantial conformational variations even in secondary structure are observed between the two chemically identical reelin-N domains in one crystallographic asymmetric unit. The variations may result from extensive, highly specific interactions across the interface of the two reelin-N domains. The calculated values of buried surface area and the interface's shape complementarity are consistent with the formation of a weak dimer. The homophilic asymmetric dimer can potentially offer advantages in binding to ligands such as glycosaminoglycans, which may, in turn, bridge the two reelin-N domains and stabilize the dimer.

VEGF is a potent pro-angiogenic factor whose effects are opposed by a host of anti-angiogenic proteins, including thrombospondin-1 (TSP-1). We have previously shown that VEGF has important extravascular roles in the ovary and that VEGF and TSP-1 are inversely expressed throughout the ovarian cycle. To date, however, a causal interaction between TSP-1 and VEGF has not been identified. Here, we show that TSP-1 has a direct inhibitory effect on VEGF by binding the growth factor and internalizing it via LRP-1. Mice lacking TSP-1 are subfertile and exhibited ovarian hypervascularization and altered ovarian morphology. Treatment of ovarian cells with TSP-1 decreased VEGF levels and rendered the cells more susceptible to TNFalpha-induced apoptosis. Knockdown of TSP-1, through RNA interference, resulted in overexpression of VEGF and reduced cytokine-induced apoptosis. In conclusion, we demonstrate a direct inhibitory effect of TSP-1 on VEGF in the ovary. TSP-1's regulation of VEGF appears to be an important mediator of ovarian angiogenesis and follicle development.

PURPOSE: Recombinant adeno-associated virus (rAAV)-mediated antiangiogenic gene therapy offers a powerful strategy for cancer treatment, maintaining sustained levels of antiangiogenic factors with coincident enhanced therapeutic efficacy. We aimed to develop rAAV-mediated antiangiogenic gene therapy delivering endostatin and 3TSR, the antiangiogenic domain of thrombospondin-1.

EXPERIMENTAL DESIGN: rAAV vectors were constructed to express endostatin (rAAV-endostatin) or 3TSR (rAAV-3TSR). The antiangiogenic efficacy of the vectors was characterized using a vascular endothelial growth factor (VEGF)-induced mouse ear angiogenesis model. To evaluate the antitumor effects of the vectors, immunodeficient mice were pretreated with rAAV-3TSR or rAAV-endostatin and received orthotopic implantation of cancer cells into the pancreas. To mimic clinical situations, mice bearing pancreatic tumors were treated with intratumoral injection of rAAV-3TSR or rAAV-endostatin.

RESULTS: rAAV-mediated i.m. gene delivery resulted in expression of the transgene in skeletal muscle with inhibition of VEGF-induced angiogenesis at a distant site (the ear). Local delivery of the vectors into the mouse ear also inhibited VEGF-induced ear angiogenesis. Pretreatment of mice with i.m. or intrasplenic injection of rAAV-endostatin or rAAV-3TSR significantly inhibited tumor growth. A single intratumoral injection of each vector also significantly decreased the volume of large established pancreatic tumors. Tumor microvessel density was significantly decreased in each treatment group and was well correlated with tumor volume reduction. Greater antiangiogenic and antitumor effects were achieved when rAAV-3TSR and rAAV-endostatin were combined.

CONCLUSIONS: rAAV-mediated 3TSR and endostatin gene therapy showed both localized and systemic therapeutic effects against angiogenesis and tumor growth and may provide promise for patients with pancreatic cancer.

Thrombospondin-1 is one of most important natural angiogenic inhibitors. The three thrombospondin-1 type 1 repeats (3TSR), an anti-angiogenic domain of thrombospondin-1, is a promising novel agent for anti-angiogenic treatment. In the present study, we showed 3TSR was biologically stable at least for 7 days in mini-osmotic pumps in vivo, and continuous administration of 3TSR decreased the dosage and improved the potency of therapy in an orthotopic pancreatic cancer model. By using different dosage and delivery routes, we proved that the anti-tumor efficacy of 3TSR was correlated with its anti-angiogenic efficacy. 3TSR treatment also decreased tumor vessel patency and blood flow. The results indicate the advantage of continuous administration of angiogenic inhibitors and provide rationale for using such delivery methods for cancer treatment.

Thrombospondin-1 (TSP-1) is a multifunctional, extracellular matrix protein that has been implicated in the regulation of smooth muscle cell proliferation, migration and differentiation during vascular development and injury. Vascular injury in wildtype and TSP-1 null mice was carried out by insertion of a straight spring guidewire into the femoral artery via a muscular arterial branch. Blood flow was restored after the muscular branch was ligated. The injury completely denuded the endothelium and caused medial distension of the vessel in a manner similar to coronary artery balloon-angioplasty. After 28 days, wildtype arteries showed consistent neointima formation with smooth muscle cell hyperplasia. Injured arteries from TSP-1 null mice showed similar neointimal lesions with no significant difference in the extent of neointima formation. Unexpectedly, a high incidence of thrombus formation was observed in the TSP-1 null vessels in a region close to the entry point of the guidewire into the femoral artery. Thrombus was never observed in the injured wildtype vessels. These results provide in vivo evidence that the extent of smooth muscle cell proliferation and neointima formation following endothelial denuding injury is not affected by the absence of TSP-1. Furthermore, our results provide novel evidence for the involvement of TSP-1 in controlling thrombus growth following intra-arterial injury in areas of predicted high turbulent flow.

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.

Thrombospondins (TSPs) are a family of extracellular matrix proteins that regulate tissue genesis and remodeling. TSP-1 plays a pivotal role in the regulation of both physiological and pathological angiogenesis. The inhibitory effects of TSP-1 on angiogenesis have been established in numerous experimental models. Among other TSP members, TSP-2 has equivalent domain structure as TSP-1 and shares most functions of TSP-1. The mechanisms by which TSP-1 and -2 inhibit angiogenesis can be broadly characterized as direct effects on vascular endothelial cells and indirect effects on the various angiogenic regulators. The fact that TSP-1 and -2 are potent endogenous angiogenic inhibitors has prompted studies to explore their therapeutic applications, and detailed understanding of the mechanisms of action of TSP-1 and -2 has facilitated the design of therapeutic strategies to optimize these activities. The therapeutic effects can be achieved by up-regulation of endogenous TSPs, or by the delivery of recombinant proteins or synthetic peptides that contain sequences from the angiogenic domain of TSP-1. In this article, we review the progress in thrombospondin-based antiangiogenic therapy and discuss the perspectives on the significant challenges that remain.

OBJECTIVE: Transforming growth factor-beta (TGF-beta), the central cytokine responsible for the development of diabetic nephropathy, is usually secreted as a latent procytokine complex that has to be activated before it can bind to its receptors. Recent studies by our group demonstrated that thrombospondin-1 (TSP-1) is the major activator of latent TGF-beta in experimental glomerulonephritis in the rat, but its role in diabetic nephropathy in vivo is unknown.

RESEARCH DESIGN AND METHODS: Type 1 diabetes was induced in wild-type (n = 27) and TSP-1-deficient mice (n = 36) via streptozotocin injection, and diabetic nephropathy was investigated after 7, 9.5, and 20 weeks. Renal histology, TGF-beta activation, matrix accumulation, and inflammation were assessed by immunohistology. Expression of fibronectin and TGF-beta was evaluated using real-time PCR. Furthermore, functional parameters were examined.

RESULTS: In TSP-1-deficient compared with wild-type mice, the amount of active TGF-beta within glomeruli was significantly lower, as indicated by staining with specific antibodies against active TGF-beta or the TGF-beta signaling molecule phospho-smad2/3 or the typical TGF-beta target gene product plasminogen activator inhibitor-1. In contrast, the amount of glomerular total TGF-beta remained unchanged. The development of diabetic nephropathy was attenuated in TSP-1-deficient mice as demonstrated by a significant reduction of glomerulosclerosis, glomerular matrix accumulation, podocyte injury, renal infiltration with inflammatory cells, and renal functional parameters.

CONCLUSIONS: We conclude that TSP-1 is an important activator of TGF-beta in diabetic nephropathy in vivo. TSP-1-blocking therapies may be considered a promising future treatment option for diabetic nephropathy.

The N-terminal domain of thrombospondin-1 (TSPN-1) mediates the protein's interaction with (1) glycosaminoglycans, calreticulin, and integrins during cellular adhesion, (2) low-density lipoprotein receptor-related protein during uptake and clearance, and (3) fibrinogen during platelet aggregation. The crystal structure of TSPN-1 to 1.8 A resolution is a beta sandwich with 13 antiparallel beta strands and 1 irregular strand-like segment. Unique structural features of the N- and C-terminal regions, and the disulfide bond location, distinguish TSPN-1 from the laminin G domain and other concanavalin A-like lectins/glucanases superfamily members. The crystal structure of the complex of TSPN-1 with heparin indicates that residues R29, R42, and R77 in an extensive positively charged patch at the bottom of the domain specifically associate with the sulfate groups of heparin. The TSPN-1 structure and identified adjacent linker region provide a structural framework for the analysis of the TSPN domain of various molecules, including TSPs, NELLs, many collagens, TSPEAR, and kielin.