Mark Duquette

Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia.

The thrombospondins are a family of extracellular calcium-binding proteins that modulate cellular phenotype. Thrombospondin-1 (TSP-1) reportedly regulates cellular attachment, proliferation, migration, and differentiation in vitro. To explore its function in vivo, we have disrupted the TSP-1 gene by homologous recombination in the mouse genome. Platelets from these mice are completely deficient in TSP-1 protein; however, thrombin-induced platelet aggregation is not diminished. TSP-1-deficient mice display a mild and variable lordotic curvature of the spine that is apparent from birth. These mice also display an increase in the number of circulating white blood cells, with monocytes and eosinophils having the largest percent increases. The brain, heart, kidney, spleen, stomach, intestines, aorta, and liver of TSP-1-deficient mice showed no major abnormalities. However, consistent with high levels of expression of TSP-1 in lung, we observe abnormalities in the lungs of mice that lack the protein. Although normal at birth, histopathological analysis of lungs from 4-wk-old TSP-1-deficient mice reveals extensive acute and organizing pneumonia, with neutrophils and macrophages. The macrophages stain for hemosiderin, indicating that diffuse alveolar hemorrhage is occurring. At later times, the number of neutrophils decreases and a striking increase in the number of hemosiderin-containing macrophages is observed associated with multiple-lineage epithelial hyperplasia and the deposition of collagen and elastin. A thickening and ruffling of the epithelium of the airways results from increasing cell proliferation in TSP-1-deficient mice. These results indicate that TSP-1 is involved in normal lung homeostasis.

Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication

Thrombospondin-1 (TSP-1) contains three type 1 repeats (TSRs), which mediate cell attachment, glycosaminoglycan binding, inhibition of angiogenesis, activation of TGFβ, and inhibition of matrix metalloproteinases. The crystal structure of the TSRs reported in this article reveals a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan and arginine residues from respective strands, capped by disulfide bonds on each end. The front face of the TSR contains a right-handed spiral, positively charged groove that might be the “recognition” face, mediating interactions with various ligands. This is the first high-resolution crystal structure of a TSR domain that provides a prototypic architecture for structural and functional exploration of the diverse members of the TSR superfamily.

Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficient mice

In vitro and in vivo data indicate that thrombospondin-1 (TSP1) inhibits tumor progression in several ways including direct effects on cellular growth and apoptosis in the stromal compartment. To evaluate the importance of TSP1 for the progression of naturally arising tumors in vivo, we have crossed TSP1-deficient mice with p53-deficient mice. In p53-null mice, the absence of TSP1 decreases survival from 160 +/- 52 days to 149 +/- 42 days. A log-rank test comparing survival curves for these two populations yields a two-sided P value of 0.0272. For mice that are heterozygous for the p53-null allele, survival is 500 +/- 103 days in the presence of TSP1 expression, and 426 +/- 125 days in its absence (P = 0.0058). Whereas TSP1 expression did not cause a measurable change in the incidence of the majority of tumor types, a statistically significant (P < or = 0.05) decrease in the incidence of osteosarcomas is observed in the absence of TSP1. To determine more directly if host TSP1 inhibits tumor growth, B16F10 melanoma and F9 testicular teratocarcinoma cells have been implanted in C57BL/6J and 129Sv TSP1-null mice, respectively. The B16F10 tumors grow approximately twice as fast in the TSP1-null background and exhibit an increase in vascular density, a decrease in the rate of tumor cell apoptosis, and an increase in the rate of tumor cell proliferation. Increased tumor growth is also observed in the absence of TSP1 on the 129Sv genetic background. These data indicate that endogenous host TSP1 functions as a modifier or landscaper gene to suppress tumor growth.

Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level

Vascular endothelial growth factor (VEGF) is a well‐established stimulator of vascular permeability and angiogenesis, whereas thrombospondin‐1 (TSP‐1) is a potent angiogenic inhibitor. In this study, we have found that the TSP‐1 receptors CD36 and ßl integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, this complex may represent a molecular switch that regulates angiogenesis and determines endothelial cell behavior. In this context, physiological levels of TSP‐1 appear to support VEGFR2 function on both the cellular and tissue level, because phosphorylation of VEGFR2 and vascular permeability in response to VEGF are decreased in TSP‐1‐null mice and isolated endothelial cells. A therapeutic agent based on the antiangiogenic domain of TSP‐1, designated 3TSR (for three TSP‐1 type 1 repeats), has significant antiangiogenic and antitumor efficacy. Systemic treatment of wild‐type mice with 3TSR significantly decreased VEGF‐induced permeability. Consistent with this result, VEGF‐stimulated phosphorylation of VEGFR2 was also significantly decreased in lung extracts from 3TSR‐treated mice. Moreover, 3TSR significantly decreased VEGF‐stimulated VEGFR2 phosphorylation in human dermal microvascular endothelial cells in culture. Taken together, the results indicate that TSP‐1 and 3TSR modulate the function of VEGFR2.—Zhang, X., Kazerounian, S., Duquette, M., Perruzzi, C., Nagy, J. A., Dvorak, H. J., Parangi, S., and Lawler, J. Thrombospondin‐1 modulates vascular endothelial growth factor activity at the receptor level. FASEB J. 23, 3368–3376 (2009). www.fasebj.org

The evolution of the thrombospondin gene family

SummaryThrombospondin-1 is an adhesive glycoprotein that is involved in cellular attachment, spreading, migration, and proliferation. To date, four genes have been identified that encode for the members of the thrombospondin gene family. These four genes are homologous to each other in the EGF-like (type 2) repeats, the calcium-binding (type 3) motifs, and the COOH-terminal. The latter has been reported to be a cell-binding domain in thrombospondin-1. Phylogenetic trees have been constructed from the multisequence alignment of thrombospondin sequences from human, mouse, chicken, and frog. Two different algorithms generate comparable results in terms of the topology and the branch lengths. The analysis indicates that an early form of the thrombospondin gene duplicated about 925 million years ago. The gene duplication that produced the thrombospondin-1 and -2 branches of the family is predicted to have occurred 583 million years ago, whereas the gene duplication that produced the thrombospondin-3 and -4 branches of the family is predicted to have occurred 644 million years ago. These results indicate that the members of the thrombospondin gene family have existed throughout the evolution of the animal kingdom and thus probably participate in functions that are common to most of its members.

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase.

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

The crystal structure of the signature domain of cartilage oligomeric matrix protein: implications for collagen, glycosaminoglycan and integrin binding

Cartilage oligomeric matrix protein (COMP), or thrombospondin‐5 (TSP‐5), is a secreted glycoprotein that is important for growth plate organization and function. Mutations in COMP cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). In this study, we determined the structure of a recombinant protein that contains the last epidermal growth factor repeat, the type 3 repeats and the C‐terminal domain (CTD) of COMP to 3.15‐Ä resolution limit by X‐ray crystallography. The CTD is a β‐sandwich that is composed of 15 antiparallel P‐strands, and the type 3 repeats are a contiguous series of calcium binding sites that associate with the CTD at multiple points. The crystal packing reveals an exposed potential metal‐ion‐ dependent adhesion site (MIDAS) on one edge of the P‐sandwich that is common to all TSPs and may serve as a binding site for collagens and other ligands. Disease‐ causing mutations in COMP disrupt calcium binding, disulfide bond formation, intramolecular interactions, or sites for potential ligand binding. The structure presented here and its unique molecular packing in the crystal identify potential interactive sites for glycosaminoglycans, integrins, and collagens, which are key to cartilage structure and function.— Tan, K.,Duquette, M., Joachimiak, A., Lawler, J. The crystal structure of the signature domain of cartilage oligomeric matrix protein: implications for collagen, glycosaminoglycan and integrin binding. FASEB J. 23, 2490–2501 (2009)

A Double Hit to Kill Tumor and Endothelial Cells by TRAIL and Antiangiogenic 3TSR

As tumor development relies on a coordination of angiogenesis and tumor growth, an efficient antitumor strategy should target both the tumor and its associated vessels. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner. Additionally, thrombospondin-1, a naturally occurring inhibitor of angiogenesis, and a recombinant protein containing functional domains of thrombospondin-1, 3TSR, have been shown to be necessary and sufficient to inhibit tumor angiogenesis. Here, we show that a combination of a TRAIL receptor 2 agonist antibody, Lexatumumab, and 3TSR results in a significantly enhanced and durable tumor inhibition. We further observed that 3TSR induces apoptosis in primary endothelial cells by up-regulating the expression of TRAIL receptors 1 and 2 in a CD36 and Jun NH(2)-terminal kinase-dependent manner leading to the activation of both intrinsic and extrinsic apoptotic machineries. The modulation of these pathways is critical for 3TSR-induced apoptosis as disrupting either via specific inhibitors reduced apoptosis. Moreover, 3TSR attenuates the Akt survival pathway. These studies indicate that 3TSR plays a critical role in regulating the proapoptotic signaling pathways that control growth and death in endothelial cells and that a combination of TRAIL and 3TSR acts as a double hit against tumor and tumor-associated vessels.

Antiangiogenic Treatment with Three Thrombospondin-1 Type 1 Repeats versus Gemcitabine in an Orthotopic Human Pancreatic Cancer Model

Purpose: In this study, we investigated the antitumor efficacy of thrombospondin-1 three type 1 repeats (3TSR), the antiangiogenic domain of thrombospondin-1, in comparison and in combination with gemcitabine, in an orthotopic pancreatic cancer model. Experimental Design: Human pancreatic cancer cells were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR, gemcitabine, 3TSR plus gemcitabine, or vehicle for 3 weeks. Subsequently, the effects of 3TSR and/or gemcitabine on tumor growth, tumor necrosis, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. Results: After 3 weeks of treatment, 3TSR reduced tumor volume by 65%, and gemcitabine by 84%. Tumor volume was not statistically different between gemcitabine group and combinatorial treatment group. Extensive necrotic areas were observed in tumors from 3TSR-treated mice, whereas tumors from gemcitabine and combinatorially treated mice were less necrotic than control tumors. 3TSR reduced tumor microvessel density and increased tumor blood vessel endothelial cell apoptosis. In contrast, gemcitabine induced apoptosis and inhibited proliferation of cancer cells. Conclusion: 3TSR, the antiangiogenic domain of thrombospondin-1, showed comparable antitumor efficacy to gemcitabine in a human pancreatic cancer orthotopic mouse model. No synergistic effect was found when the two drugs were combined and possible reasons are discussed in detail. A delicate balance between normalization and excessive regression of tumor vasculature is important when initiating alternative combinatorial regimens for treatment of patients with pancreatic cancer.

Combined therapy with thrombospondin-1 type I repeats (3TSR) and chemotherapy induces regression and significantly improves survival in a preclinical model of advanced stage epithelial ovarian cancer

Most women are diagnosed with epithelial ovarian cancer (EOC) at advanced stage, where therapies have limited effectiveness and the long‐term survival rate is low. We evaluated the effects of combined anti‐angiogenic and chemotherapy treatments on advanced stage EOC. Treatment of EOC cells with a recombinant version of the thrombospondin‐1 type I repeats (3TSR) induced more apoptotic cell death (36.5 ± 9.6%) in vitro compared to untreated controls (4.1 ± 1.4). In vivo, tumors were induced in an orthotopic, syngeneic mouse model of advanced stage EOC. Mice were treated with 3TSR (4 mg/kg per day) alone or in combination with chemotherapy drugs delivered with maximum tolerated dose or metronomic scheduling. Pretreatment with 3TSR induced tumor regression, normalized tumor vasculature, and improved uptake of chemotherapy drugs. Combination 3TSR and metronomic chemotherapy induced the greatest tumor regression (6.2‐fold reduction in size compared to PBS‐treated controls) and highest survival when treatment was initiated at advanced stage. 3TSR binding to its receptor, CD36 (cluster of differentiation 36), increased binding of CD36 and SHP‐1, which significantly inhibited phosphorylation of the VEGF receptor. In this study, we describe a novel treatment approach and mechanism of action with 3TSR and chemotherapy that induces regression of advanced stage EOC and significantly improves survival.—Russell, S., Duquette, M., Liu, J., Drapkin, R., Lawler, J., Petrik, J. Combined therapy with thrombospondin‐1 type I repeats (3TSR) and chemotherapy induces regression and significantly improves survival in a preclinical model of advanced stage epithelial ovarian cancer. FASEB J. 29, 576‐588 (2015). www.fasebj.org