Publications

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.

To identify overlapping and non-overlapping functions for TSP-1 and alphavbeta6, we crossed TSP-1-null and beta6-null mice and compared the phenotype of the double-null mice with those of wild-type and single-null mice. The double-null mice exhibited focal acute and organizing pneumonia that was more severe than the wild-type and single-null mice as well as a significantly higher incidence of inflammation in tissues other than the lung. The TSP-1-null and beta6-null mice exhibited a five to eight-fold increase in granulocyte recruitment to the lung three days after exposure to lipopolysaccharide. They also had abnormalities that were infrequently observed in the wild-type and single-null mice, including heart degeneration (8.35% in wild-type and 28.1% in double-null mice), hyperplasia of the glandular of the stomach (2.8% in wild-type and 21.1% in double-null mice) and endometrial hyperplasia (0% in wild-type and 38.5% in double-null females). Furthermore, the beta6-null and double-null mice displayed a significant elevation in benign and malignant cancers. Stomach papillomas, squamous cell carcinomas of the ear and stomach, and adenocarcinomas of the lungs, vagina/cervix and colon were observed with the highest frequency. These data demonstrate that TSP-1 and alphavbeta6 are involved in regulation of the immune system and epithelial homeostasis. They also indicate that alphavbeta6 functions as a tumor suppressor gene and that activation of TGFbeta by TSP-1 and alphavbeta6 contributes to normal tissue architecture and function.

BACKGROUND: Matricellular proteins are extracellular matrix proteins that do not contribute directly to tissue integrity but are capable of modulating cell function. We hypothesized that the matricellular protein thrombospondin (TSP)-1, a potent inhibitor of angiogenesis and activator of transforming growth factor (TGF-beta), is induced in healing myocardial infarcts and plays a role in suppressing the postinfarction inflammatory response, inhibiting local angiogenesis, and limiting expansion of granulation tissue into the noninfarcted area.

METHODS AND RESULTS: We used a canine and a murine model of reperfused infarction. TSP-1 mRNA was induced in canine infarcts after 1 hour of ischemia and 3 to 7 days of reperfusion. TSP-1 protein showed a strikingly selective localization in the extracellular matrix, microvascular endothelium, and a subset of mononuclear cells of the infarct border zone after 5 to 28 days of reperfusion. Isolated canine venous endothelial cells showed low-level constitutive expression of TSP-1 mRNA, which was markedly induced by TGF-beta, and basic fibroblast growth factor. Murine infarcts also had marked TSP-1 deposition in the border zone. Infarcted TSP-1-/- mice exhibited sustained upregulation of the chemokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and interferon-gamma-inducible protein-10/CXCL10 and the cytokines interleukin-1beta, interleukin-6, and TGF-beta, suggesting an enhanced and prolonged postinfarction inflammatory response. In addition, TSP-1-/- mice had markedly increased macrophage and myofibroblast density in infarcts and in remodeling noninfarcted myocardial areas neighboring the myocardial scar, suggesting expansion of granulation tissue formation into the noninfarcted territory. TSP-1-/- animals had more extensive postinfarction remodeling than wild-type mice, although infarct size was similar in both groups.

CONCLUSIONS: The infarct border zone may be capable of modulating the healing process through its unique extracellular matrix content. The selective endogenous expression of TSP-1 in the infarct border zone may serve as a "barrier," limiting expansion of granulation tissue and protecting the noninfarcted myocardium from fibrotic remodeling.

By means of its antiangiogenic activity, thrombospondin-1 (TSP-1) exerts indirect antitumoral action on solid tumors. Here, we investigated potential antitumor action in an in vitro cell model for promyelocytic leukemia (NB4-LR1), resistant to retinoid maturation. Purified soluble TSP-1 added to cultures induced a strong dose-dependent growth inhibition and a slowly developing maturation-independent cell death. Recombinant fragments of TSP-1 allowed mapping of these activities to its type 3 repeat/C-terminal domain, features that are distinct from those of TSP-1 action on solid tumors, previously ascribed to the type 1 repeat domain. Cell death in leukemia was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization followed by membrane permeabilization. Mitochondria membrane depolarization was inherent to TSP-1 action but did not produce release of death-promoting proteins (eg, noncaspase apoptosis regulators, apoptosis-induced factor [AIF], endonuclease G, or Omi/HtrA2 or the caspase regulators, cytochrome c or second mitochondrial activator of caspase/direct inhibitor of apoptosis protein-binding protein with low isoelectric point [Smac/DIABLO]). Although detected, reactive oxygen species (ROS) production was likely not involved in the death process. Finally, receptor agonist RFYVVM and RGD peptides indicated that TSP-1 death effects are mediated by membrane receptors CD47 and alphavbeta3. These results demonstrated a new domain-specific antitumoral activity of TSP-1 on a leukemia cell line, which extends TSP-1 therapeutic potential outside the area of vascularized solid tumors.

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.

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix of the musculoskeletal system. Although COMP/TSP5 abnormalities are associated with several pathological conditions, its normal function remains unclear. This study was undertaken to delineate the function(s) of COMP/TSP5 in cartilage, especially regarding its interaction with chondrocytes. We show that COMP/TSP5 can support chondrocyte attachment and that the RGD sequence in COMP/TSP5 and the integrin receptors alpha5beta1 and alphaVbeta3 on the chondrocytes are involved in mediating this attachment. The interactions of COMP/TSP5 with the integrins are dependent on COMP/TSP5 conformation. Chondrocyte attachment to COMP/TSP5 in the calcium-replete conformation was inhibited by function-blocking integrin alpha5 and beta1 antibodies, suggesting the involvement of the alpha5beta1 integrin. Under this condition, a function-blocking antibody against alphaVbeta3 did not have any effect on cell attachment. On the other hand, chondrocyte attachment to reduced COMP/TSP5 was instead sensitive to alphaVbeta3 function-blocking antibodies, suggesting that COMP/TSP5 mediates attachment through chondrocyte alphaVbeta3 integrin under this condition. Cell attachment to reduced COMP/TSP5 was not inhibited by beta1 antibodies. These data indicate that COMP/TSP5 in different conformations can utilize different integrin receptors. These results are the first to demonstrate that COMP/TSP5 can mediate chondrocyte attachment through interactions with integrins. Through these interactions, COMP/TSP5 may be able to regulate cellular activities and respond to environment in the surrounding cartilage matrix.

BACKGROUND: Thrombospondin-1 (TSP1), a multifunctional, extracellular matrix protein, regulates cellular attachment, proliferation, migration, and differentiation in vitro, and is expressed de novo in many inflammatory diseases, including glomerulonephritis (GN).

METHODS: We investigated the role of TSP1 in the autologous phase of an accelerated model of anti-glomerular basement membrane (GBM) GN in mice deficient in TSP1. The model, induced by the injection of rabbit anti-mouse GBM antibody, is characterized by the development of proteinuria and glomerular damage over a 21-day observation period in wild-type mice.

RESULTS: Mice deficient in TSP1 developed significantly less proteinuria than their wild-type controls 21 days after induction of disease (5,793 +/- 5,456 vs. 24,293 +/- 15,336 microg albumin/mg creatinine; p = 0.002). Serum creatinine levels were significantly higher in the wild-type mice than in the TSP1 deficient animals (29.03 +/- 2.34 vs. 16.39 +/- 2.87 micromol/l; p = 0.005). Other disease indices as crescent formation, fibrin deposition and macrophage influx, were also diminished in the TSP1 knockout animals. The numbers of interstitial CD4+ and CD8+ T cells were generally less in TSP1-deficient mice and reached statistical significance in CD4+ (p = 0.01) and CD8+ T cells (p = 0.02). The difference in outcome of the disease was not due to the difference in deposition/production of heterologous and autologous antibodies in the two groups of animals.

CONCLUSION: This study suggests a proinflammatory role of TSP1 in an experimental model of GN.

The amino-terminal domain of the extracellular matrix (ECM) protein thrombospondin-1 (TSP-1) mediates binding to cell surface heparan sulfate proteoglycans (HSPG) as well as binding to the endocytic receptor, low density lipoprotein-related protein (LRP-1). We previously found that recombinant TSP-1 containing the amino-terminal residues 1-214, retained both of these interactions (Mikhailenko et al. [1997]: J Biol Chem 272:6784-6791). Here, we examined the activity of a recombinant protein containing amino-terminal residues 1-90 of TSP-1 and found that this domain did not retain high-affinity heparin-binding. The loss of heparin-binding correlated with decreased binding to the fibroblast cell surface. However, both ligand blotting and solid phase binding studies indicate that this truncated fragment of TSP-1 retained high-affinity binding to LRP-1. Consistent with this, it also retained the ability to block the uptake and degradation of (125)I-TSP-1. However, TSP-1(1-90) itself was poorly endocytosed and this truncated amino-terminal domain was considerably more effective than the full-length heparin-binding domain (HBD) of TSP-1 in blocking the catabolism of endogenously expressed TSP-1. These results indicate that TSP-1 binding to LRP-1 does not require prior or concomitant interaction with cell surface HSPG but suggest subsequent endocytosis requires high-affinity heparin-binding.

PURPOSE: Thrombospondin (TSP)-1 and -2 are important antiangiogenic factors thought to be involved in maintaining corneal avascularity (angiogenic privilege). This study was undertaken to investigate whether deficiencies of these factors altered developmental and inflammation-induced angiogenesis in the cornea and developmental angiogenesis of the iris of mice.

METHODS: Expression of TSP-1 and -2 mRNA and protein was assayed in cornea and iris stroma by RT-PCR and Western blot. Corneas and irides of TSP-1(-/-), TSP-2(-/-), and TSP-1,2(-/-) mice aged 2, 3, and 6 months, and wild-type control mice, were analyzed for spontaneous angiogenesis biomicroscopically, histologically, and with CD31 immunohistochemistry. The mouse model of suture-induced, inflammatory corneal neovascularization was used to evaluate the lack of TSP-1,2 and both TSPs on induced-corneal angiogenesis. Seven days after intrastromal placement of three 11-0 sutures, vascularized areas were analyzed morphometrically on CD31-stained corneal flatmounts.

RESULTS: Corneas and irises from normal mouse eyes constitutively expressed TSP-1 and -2 mRNAs and proteins. Corneas of TSP-1(-/-), -2(-/-), and -1,2(-/-) mice displayed no evidence of spontaneous developmental-postnatal angiogenesis, although irises of these mice contained significantly increased iris vessel density compared with wild-type animals (P < 0.01). One week after suturing, corneas of all TSP(-/-) mice had significantly greater corneal angiogenesis than those of control mice (P < 0.05). TSP-1(-/-) had a significantly greater effect on induced corneal neovascularization than did TSP-2(-/-), with the opposite being the case in developmental iris angiogenesis (P < 0.01).

CONCLUSIONS: Corneal avascularity during development is redundantly regulated, shown by the fact that lack of the antiangiogenic factors TSP-1 and/or -2 resulted in no spontaneous corneal angiogenesis. By contrast, TSP-1, more than TSP-2, helps to suppress inflammation-induced corneal angiogenesis postnatally, implying that angiogenic privilege in the cornea is actively maintained.

Low-dose cyclophosphamide (LDC) induces selective apoptosis of endothelial cells within the vascular bed of tumors. Here, we investigated a hypothesis that the effect of LDC is mediated by the pro-apoptotic action of endogenous inhibitors of angiogenesis. Tumors treated with LDC demonstrate similar expression of matrix metalloproteinases and also basement membrane-derived angiogenesis inhibitors when compared with wild-type tumors, whereas the expression of thrombospondin-1 (TSP-1) is significantly elevated in LDC-treated tumors. We used mice with an absence of type XVIII collagen (endostatin) or type IV collagen alpha3 chain (tumstatin) or TSP-1 to assess the contribution of these endogenous inhibitors of angiogenesis on LDC-mediated tumor suppression. Lack of TSP-1 in the host in addition to tumor cells leads to diminished capacity of LDC to suppress tumor growth, whereas the absence of endostatin and tumstatin did not alter the effect of LDC. LDC treatment predominantly induces selective expression of TSP-1 in tumor cells and peri-vascular cells and facilitates apoptosis of proliferating endothelial cells, with minimal direct effect on tumor cells and peri-vascular cells. These studies indicate that TSP-1 contributes to tumor growth suppression induced by LDC and suggest that tumors that express high basal level of TSP-1 may be more susceptible to tumor suppression by such a regimen. This study also makes a strong case for TSP-1 expression levels as a potential predictive marker for the successful use of LDC in cancer patients.