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Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world. Current therapy includes monthly intraocular injections of anti-VEGF antibodies, which are ineffective in up to one third of patients. Thrombospondin-1 (TSP1) inhibits angiogenesis via CD36 binding, and its down-regulated expression is negatively associated with the onset of nAMD. Here, we describe TSP1 mimetic protein-like polymers (TSP1 PLPs). TSP1 PLPs bind CD36 with high affinity, resist degradation, show prolonged intraocular half-lives (13.1 hours), have no toxicity at relevant concentrations in vivo (40 μM), and are more efficacious in ex vivo choroidal sprouting assays compared to the peptide sequence and Eylea (aflibercept), the current standard of care anti-VEGF treatment. Furthermore, PLPs exhibit superior in vivo efficacy in a mouse model for nAMD compared to control PLPs consisting of scrambled peptide sequences, using fluorescein angiography and immunofluorescence. Since TSP-1 inhibits angiogenesis by VEGF-dependent and independent mechanisms, TSP1 PLPs are a potential therapeutic for patients with anti-VEGF treatment-resistant nAMD.

Epithelial ovarian cancer (EOC) is the most common and fatal subtype of ovarian malignancies, with no effective therapeutics available. Our previous studies have demonstrated extraordinary suppressive efficacy of enterolactone (ENL) on EOC. A chemotherapeutic agent, trabectedin (Trabe), is shown to be effective on ovarian cancer, especially when combined with other therapeutics, such as pegylated liposomal doxorubicin or oxaliplatin. Thrombospondin 1 (THBS1), a kind of matrix glycoprotein, plays important roles against cancer development through inhibiting angiogenesis but whether it is involved in the suppression of EOC by ENL or Trabe remains unknown. To test combined suppressive effects of ENL and Trabe on EOC and possible involvement of THBS1 in the anticancer activities of ENL and Trabe. The EOC cell line ES-2 was transfected with overexpressed THBS1 by lentivirus vector. We employed tube formation assay to evaluate the anti-angiogenesis activity of ENL and of its combined use with Trabe after THBS1 overexpression and established drug intervention and xenograft nude mouse cancer models to assess the in vivo effects of the hypothesized synergistic suppression between the agents and the involvement of THBS1. Mouse fecal samples were collected for 16S rDNA sequencing and microbiota analysis. We detected strong inhibitory activities of ENL and Trabe against the proliferation and migration of cancer cells and observed synergistic effects between ENL and Trabe in suppressing EOC. ENL and Trabe, given either separately or in combination, could suppress the tube formation capability of human microvascular endothelial cells, and this inhibitory effect became even stronger with THBS1 overexpression. In the ENL plus Trabe combination group, the expression of tissue inhibitor of metalloproteinases 3 and cluster of differentiation 36 was both upregulated, whereas matrix metalloproteinase 9, vascular endothelial growth factor, and cluster of differentiation 47 were all decreased. With the overexpression of THBS1, the results became even more pronounced. In animal experiments, combined use of ENL and Trabe showed superior inhibitory effects to either single agent and significantly suppressed tumor growth, and the overexpression of THBS1 further enhanced the anti-cancer activities of the drug combination group. ENL and Trabe synergistically suppress EOC and THBS1 could remarkably facilitate the synergistic anticancer effects of ENL and Trabe., (© 2023 John Wiley & Sons, Ltd.)

Objective: To explore the underlying molecular mechanism of ()., Methods: We used a tandem mass tag-based quantitative proteomic method to determine the differentially expressed proteins. Network pharmacology analysis was used to analysis the main components of and construct the compound-target network. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the analyses results., Results: The expression levels of thrombospondin-1 (TSP-1) and transforming growth factor (TGF)-β1/Smad3 signaling pathway proteins were significantly upregulated in focal segmental glomeruloscleosis (FSGS) rats. The reduced the expression levels of TSP-1 and TGF-β1 signaling pathway proteins. Network pharmacology analysis revealed that protocatechualdehyde was the main active component. Subsequent and experiments validated the results of proteomic and network pharmacology analyses., Conclusions: Our results suggested that may inhibit renal sclerosis by inhibiting TSP-1-activated TGF-β1 signaling and may have potential applications in the treatment of FSGS.

Background: Anaplastic thyroid carcinoma (ATC) is a rapidly fatal cancer with a median survival of a few months. Enhanced therapeutic options for durable management of ATC will rely on an understanding of genetics and the role of the tumor microenvironment. The prognosis for patients with ATC has not improved despite more detailed scrutiny of underlying tumor genetics. Pericytes in the microenvironment play a key evasive role for thyroid carcinoma (TC) cells. Lenvatinib improves outcomes in patients with radioiodine-refractory well-differentiated TC. In addition to the unclear role of pericytes in ATC, the effect and mechanism of lenvatinib efficacy on ATC have not been sufficiently elucidated. Design: We assessed pericyte enrichment in ATC. We determined the effect of lenvatinib on ATC cell growth cocultured with pericytes and in a xenograft mouse model from human BRAF WT/V600E -ATC-derived cells coimplanted with pericytes. Results: ATC samples were significantly enriched in pericytes compared with normal thyroid samples. BRAF WT/V600E -ATC-derived cells were resistant to lenvatinib treatment shown by a lack of suppression of MAPK and Akt pathways. Moreover, lenvatinib increased CD47 protein (thrombospondin-1 [TSP-1] receptor) levels over time vs. vehicle. TSP-1 levels were downregulated upon lenvatinib at late vs. early time points. Critically, ATC cells, when cocultured with pericytes, showed increased sensitivity to this therapy and ultimately decreased number of cells. The coimplantation in vivo of ATC cells with pericytes increased ATC growth and did not downregulate TSP-1 in the microenvironment in vivo . Conclusions and Implications: Pericytes are enriched in ATC samples. Lenvatinib showed inhibitory effects on BRAF WT/V600E -ATC cells in the presence of pericytes. The presence of pericytes could be crucial for effective lenvatinib treatment in patients with ATC. Degree of pericyte abundance may be an attractive prognostic marker in assessing pharmacotherapeutic options. Effective durable management of ATC will rely on an understanding not only of genetics but also on the role of the tumor microenvironment.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies with high morbidity and mortality. PKHB1, a serum-stable Thrombospondin-1 (TSP-1) mimic peptide, has shown some effective ability in triggering cell death against several cancers. Here, we aimed to study the potential biological function of PKHB1 and its molecular mechanism in NSCLC. Our results revealed that PKHB1 significantly suppressed NSCLC cell proliferation, cell migration, and induced apoptosis in a dose-dependent manner. Additionally, we found that PKHB1 treatment resulted in mitochondrial transmembrane potential depolarization, Ca 2+ overloading as well as the upregulation of proapoptotic proteins. Mechanistically, PKHB1 induced NSCLC cells apoptosis in a CD47-independent manner. Further study revealed that PKHB1 provoked endoplasmic reticulum (ER) stress principally through the activation of CHOP and JNK signaling, which could be alleviated in the presence of 4-PBA, an ER stress inhibitor. Furthermore, xenograft tumor models showed that PKHB1 treatment could notably inhibit NSCLC tumor growth in vivo. In conclusion, these findings suggested that PKHB1 exerted antitumor efficacy in NSCLC via triggering ER stress-mediated but CD47-independent apoptosis, potentially functioned as a promising peptide-based therapeutic agent for NSCLC., (© 2023 Wiley Periodicals LLC.)

Background: Cardiovascular (CV) morbidity, mortality and metabolic syndrome are associated with rheumatoid arthritis (RA). A recent trial has suggested increased risk of major CV events (MACE) upon the Janus kinase (JAK) inhibitor tofacitinib compared with anti-tumor necrosis factor α (TNF-α) therapy. In our study, we evaluated lipids and other metabolic markers in relation to vascular function and clinical markers in RA patients undergoing one-year tofacitinib therapy. Patients and methods: Thirty RA patients treated with either 5 mg or 10 mg bid tofacitinib were included in a 12-month follow-up study. Various lipids, paraoxonase (PON1), myeloperoxidase (MPO), thrombospondin-1 (TSP-1) and adipokine levels, such as adiponectin, leptin, resistin, adipsin and chemerin were determined. In order to assess flow-mediated vasodilation (FMD), common carotid intima-media thickness (IMT) and arterial pulse-wave velocity (PWV) ultrasonography were performed. Assessments were carried out at baseline, and 6 and 12 months after initiating treatment. Results: One-year tofacitinib therapy significantly increased TC, HDL, LDL, APOA, APOB, leptin, adipsin and TSP-1, while significantly decreasing Lp(a), chemerin, PON1 and MPO levels. TG, lipid indices (TC/HDL and LDL/HDL), adiponectin and resistin showed no significant changes. Numerous associations were found between lipids, adipokines, clinical markers and IMT, FMD and PWV (p < 0.05). Regression analysis suggested, among others, association of BMI with CRP and PWV (p < 0.05). Adipokines variably correlated with age, BMI, CRP, CCP, FMD, IMT and PWV, while MPO, PON1 and TSP-1 variably correlated with age, disease duration, BMI, RF and PWV (p < 0.05). Conclusions: JAK inhibition by tofacitinib exerts balanced effects on lipids and other metabolic markers in RA. Various correlations may exist between metabolic, clinical parameters and vascular pathophysiology during tofacitinib treatment. Complex assessment of lipids, metabolic factors together with clinical parameters and vascular pathophysiology may be utilized in clinical practice to determine and monitor the CV status of patients in relation with clinical response to JAK inhibition.

The transmembrane protein, CD47, is recognized as an important innate immune checkpoint, and CD47-targeted drugs have been in development with the aim of inhibiting the interaction between CD47 and the regulatory glycoprotein SIRPα, for antitumor immunotherapy. Further, CD47 mediates other essential functions such as cell proliferation, caspase-independent cell death (CICD), angiogenesis and other integrin-activation-dependent cell phenotypic responses when bound to thrombospondin-1 (TSP-1) or other ligands. Mounting strategies that target CD47 have been developed in pre-clinical and clinical trials, including antibodies, small molecules, siRNAs, and peptides, and some of them have shown great promise in cancer treatment. Herein, the authors endeavor to provide a retrospective of ligand-mediated CD47 regulatory mechanisms, their roles in controlling antitumor intercellular and intracellular signal transduction, and an overview of CD47-targetd drug design., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022. Published by Elsevier B.V.)

Objectives: Tumor vasculature is structurally abnormal, with anatomical deformities, reduced pericyte coverage and low tissue perfusion. As a result of this vascular dysfunction, tumors are often hypoxic, which is associated with an aggressive tumor phenotype, and reduced delivery of therapeutic compounds to the tumor. We have previously shown that a peptide containing the thrombospondin-1 type I repeats (3TSR) specifically targets tumor vessels and induces vascular normalization in a mouse model of epithelial ovarian cancer (EOC). However, due to its small size, 3TSR is rapidly cleared from circulation. We now introduce a novel construct with the 3TSR peptide fused to the C-terminus of each of the two heavy chains of the Fc region of human IgG1 (Fc3TSR). We hypothesize that Fc3TSR will have greater anti-tumor activity in vitro and in vivo compared to the native compound., Methods: Fc3TSR was evaluated in vitro using proliferation and apoptosis assays to investigate differences in efficacy compared to native 3TSR. In light of the multivalency of Fc3TSR, we also investigate whether it induces greater clustering of its functional receptor, CD36. We also compare the compounds in vivo using an orthotopic, syngeneic mouse model of advanced stage EOC. The impact of the two compounds on changes to tumor vasculature morphology was also investigated., Results: Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR. This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer., Conclusion: The development of Fc3TSR which is greater in size, stable in circulation and enhances receptor activation compared to 3TSR, facilitates its translational potential as a therapy in the treatment of metastatic advanced stage ovarian cancer., Competing Interests: Declaration of Competing Interest J.P. and J.L. are co-inventors on US patent US20140271641A1 for the treatment of ovarian cancer with 3TSR., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease in reproductive women, and the endocrine levels are also affected by diseases. The aim of this study was to determine the effect of thrombospondin-1 (TSP-1) on PCOS rat model., Methods: We established the PCOS rat model, the serum hormones including TSP-1 expression were determined and morphological characteristics were investigated to evaluate the model. These above endocrine and morphological features were investigated again to evaluate the effect of TSP-1 treatment., Results: In the PCOS model group, the serum hormones change (higher luteinizing hormone, testosterone and estrogen) and decreased TSP-1 expression levels were found compared with the control group. Besides, the morphological characteristics of PCOS were also observed in the model group. After TSP-1 treatment, the higher TSP-1, ANGPT2, PDGFB and PDGFD expression levels, the lower LH and T levels, decreased vessel density as well as VEGFA and ANGPT1 expression levels were found compared with the control group, and the ovary morphological changes were also observed in the TSP-1 experimental group., Conclusions: TSP-1 delivery system might be an alternative therapy for PCOS treatment.

Alzheimer's disease (AD) is characterized by impaired cognitive function and memory loss, which are often the result of synaptic pathology. Thrombospondin (TSP) is an astrocyte-secreted protein, well known for its function as a modulator of synaptogenesis and neurogenesis. Here, we investigated the effects of TSP-1 on AD pathogenesis. We found that the level of TSP-1 expression was decreased in AD brains. When we treated astrocytes with amyloid beta (Aβ), secreted TSP-1 was decreased in autophagy-dependent manner. In addition, treatment with Aβ induced synaptic pathology, such as decreased dendritic spine density and reduced synaptic activity. These effects were prevented by coincubation of TSP-1 with Aβ, which acts through the TSP-1 receptor alpha-2-delta-1 in neurons. Finally, intrasubicular injection with TSP-1 into AD model mouse brains mitigated the Aβ-mediated reduction of synaptic proteins and related signaling pathways. These results indicate that TSP-1 is a potential therapeutic target in AD pathogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ocular angiogenesis is one of the underlying causes of blindness and vision impairment and may occur in a spectrum of disorders, including diabetic retinopathy, neovascular age-related macular degeneration, retinal artery or vein occlusion, and retinopathy of prematurity. As such, strategies to inhibit angiogenesis by suppressing vascular endothelial growth factor activity have proven to be effective in the clinic for the treatment of eye diseases. A complementary approach would be to increase the level of naturally occurring inhibitors of angiogenesis, such as thrombospondin (TSP)-1. This article summarizes the development of TSP-1-based inhibitors of angiogenesis.

Rationale: Histological examination of abdominal aortic aneurysm (AAA) tissues demonstrates extracellular matrix destruction and infiltration of inflammatory cells. Previous work with mouse models of AAA has shown that anti-inflammatory strategies can effectively attenuate aneurysm formation. Thrombospondin-1 is a matricellular protein involved in the maintenance of vascular structure and homeostasis through the regulation of biological functions, such as cell proliferation, apoptosis, and adhesion. Expression levels of thrombospondin-1 correlate with vascular disease conditions., Objective: To use thrombospondin-1-deficient (Thbs1(-/-)) mice to test the hypothesis that thrombospondin-1 contributes to pathogenesis of AAAs., Methods and Results: Mouse experimental AAA was induced through perivascular treatment with calcium phosphate, intraluminal perfusion with porcine elastase, or systemic administration of angiotensin II. Induction of AAA increased thrombospondin-1 expression in aortas of C57BL/6 or apoE-/- mice. Compared with Thbs1(+/+) mice, Thbs1(-/-) mice developed significantly smaller aortic expansion when subjected to AAA inductions, which was associated with diminished infiltration of macrophages. Thbs1(-/-) monocytic cells had reduced adhesion and migratory capacity in vitro compared with wild-type counterparts. Adoptive transfer of Thbs1(+/+) monocytic cells or bone marrow reconstitution rescued aneurysm development in Thbs1(-/-) mice., Conclusions: Thrombospondin-1 expression plays a significant role in regulation of migration and adhesion of mononuclear cells, contributing to vascular inflammation during AAA development., (© 2015 American Heart Association, Inc.)

Background: Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides., Methods and Findings: In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease., Conclusions: Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL.

Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this cross-talk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling is lost. Work over the last several years indicates that regulation of NO is much more complex than previously believed. It is now apparent that the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and disease-related responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Angiogenesis is an important process for tumor growth and progression of various solid tumors including urological cancers. Thrombospondins (TSPs), especially TSP-1, are representative "anti"-angiogenic molecules and many studies have clarified their pathological role and clinical significance in vivo and in vitro. In fact, TSP-1 expression is associated with clinicopathological features and prognosis in many types of cancers. However, TSP-1 is a multi-functional protein and its biological activities vary according to the specific tumor environments. Consequently, there is no general agreement on its cancer-related function in urological cancers, and detailed information regarding regulative mechanisms is essential for a better understanding of its therapeutic effects and prognostic values. Various "suppressor genes" and "oncogenes" are known to be regulators and TSP-1-related factors under physiological and pathological conditions. In addition, various types of fragments derived from TSP-1 exist in a given tissue microenvironment and TSP-1 derived-peptides have specific activities. However, a detailed pathological function in human cancer tissues is not still understood. This review will focus on the pathological roles and clinical significance of TSP-1 in urological cancers, including prostate cancer, renal cell carcinoma, and urothelial cancer. In addition, special attention is paid to TSP-1-derived peptide and TSP-1-based therapy for malignancies.

Chronic wounds are a significant socio-economic problem, thus, the improvement of the effectiveness of their treatment is an important objective for public health strategies. The predominant stage of the chronic wound is the inflammatory reaction which is associated with the damage of tissues, possibly due to the excessive secretion and activation of matrix metalloproteinases (MMPs). Several reports have suggested that amnion dressing inhibits tissue destruction and accelerates wound healing. Our recent study revealed that sterilized amnion stimulates keratinocyte proliferation in vitro, while the present study focused on the clinical application of radiation-sterilized amnion in chronic venous leg ulcers and aimed to explain the possible mechanism of its in vivo action. The study involved 25 individuals suffering from venous leg ulceration with a surface area of 10-100 cm2 and a healing rate below 10% per week, as verified during a 2-week screening period. The effectiveness of the amnion dressing was estimated following 4 weeks of treatment. The wound assessment, based on a modified Bates-Jensen Questionnaire, revealed a good and satisfactory response to the treatment in 23 of the 25 patients. The measurement of MMP-2 and MMP-9 activities in wound exudates revealed a decrease in activity in response to amnion application. This effect resulted from the presence of the potent MMP inhibitors, tissue inhibitor of metalloproteinases-1 (TIMP-1), type-1 plasminogen activator inhibitor (PAI-1) and thrombospondin-1 (TSP-1) in the amnion dressings, as shown by real-time fluorescence zymography and protein microarrays. Thus, unlike modern synthetic dressing materials, radiation-sterilized amnion dressings may have a multidirectional beneficial effect on chronic wounds.

Endometriosis is a common cause of pelvic pain and infertility in women, and a common indication for hysterectomy, yet the disease remains poorly diagnosed and ineffectively treated. Because endometriotic lesions require new blood supply for survival, inhibiting angiogenesis could provide a novel therapeutic strategy. ABT-898 mimics the antiangiogenic properties of thrombospondin-1, so we hypothesized that ABT-898 will prevent neovascularization of human endometriotic lesions and that ABT-898 treatment will not affect reproductive outcomes in a mouse model. Endometriosis was induced in BALB/c-Rag2(-/-)Il2rg(-/-) mice by surgical implantation of human endometrial fragments in the peritoneal cavity. Mice received daily injections of ABT-898 for 21 days. Flow cytometry was performed to measure circulating endothelial progenitor cells in peripheral blood. Cytokines were measured in plasma samples. Half of the ABT-898-treated and control mice were euthanized to assess neovascularization of endometriotic lesions, using CD31(+) immunofluorescence. The remaining mice were mated and euthanized at gestation day 12. Endometriotic lesions increased circulating endothelial progenitor cells 13 days after engraftment, relative to baseline. Endometriotic lesions from ABT-898-treated mice exhibited reduced neovascularization, compared with controls, and lesions had fewer CD31(+) microvessels. Chronic treatment with ABT-898 did not lead to any fetal anomalies or affect litter size at gestation day 12, compared with controls. Our results suggest that ABT-898 inhibits neovascularization of human endometriotic lesions without affecting mouse fecundity., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Thrombospondin-1 (TSP-1) is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR) domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS) to induce colitis in wild-type (WT) mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1) the three type 1 repeats of the TSP-1 (3TSR), (2) the second type 1 repeat (TSR2), or (3) TSR2 with the RFK sequence (TSR2+RFK). Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE) genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease.

Background: Despite the discovery of novel inhibitors of tumor angiogenesis, protein-based antiangiogenic cancer therapy suffers some limitations that antiangiogenic gene therapy could overcome. We investigated whether intra-tumoral electrotransfer of three angiogenic plasmids could inhibit tumor growth and metastasis., Methods: Plasmids encoding recombinant disintegrin domain of ADAM-15 (RDD), thrombospondin 1 (TSP-1), and the soluble isoform of the VEGF receptor 1 (sFlt-1) were injected into B16F10 melanoma-bearing C57BL/6 mice followed by electroporation. Tumor volume was measured daily using a digital caliper. Metastasis was monitored by in vivo bioluminescence after surgical removal of the primary luciferase-encoding B16F10 tumor 5 days after intra-tumoral electrotransfer. Markers of vascularization and cell proliferation were quantified by immunohistochemistry., Results: Intra-tumoral electrotransfer of the antiangiogenic plasmids induced a significant inhibition of tumor growth, doubling of mean survival time and long-term survivors (∼40% vs 0% in control). When the tumor was removed by surgery after intra-tumoral plasmid electrotransfer, a significant decrease in tumor metastasis was observed leading to long-term tumor-free survival especially after treatment with pRDD plasmid (84% vs 0% in control). Unlike pTSP-1 and psFlt-1, pRDD significantly decreased cell proliferation in B16F10 primary tumors which express αvβ3 and α5β1 integrins. No effect of antiangiogenic plasmid electrotransfer on normal skin blood flow was detected., Conclusion: The intra-tumoral electrotransfer of the three antiangiogenic plasmids is a promising method for the treatment of melanoma. The plasmid encoding RDD seems to be particularly effective due to its direct antitumoral activity combined with angiogenesis suppression, and its marked inhibition of metastasis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In osteoarthritis, angiogenesis, which occurs in the osteochondral junction and synovium, may accelerate inflammation and contribute to the severity of the disease. We used anterior cruciate ligament-transection (ACLT) to investigate the therapeutic effect of an angiogenesis inhibitor, thrombospondin-1 (TSP-1), in a rat model of osteoarthritis. Osteoarthritis was induced in Wistar rats in the knee of one hind leg. After ACLT, AdTSP-1 (adenoviral vector encoding mouse TSP-1) was intraarticularly injected into the knee joints. Transgene expression, angiogenesis, and inflammatory responses in the knee joints were examined. They were also assessed morphologically, radiographically, and histologically for manifestations of disease. The levels of TSP-1 peaked on day 3 and were substantially maintained for at least 9 days after AdTSP-1 infection. Adenovirus-mediated gene expression was detected in the synovial membrane and chondrocytes. TSP-1 gene transfer induced transforming growth factor-β (TGF-β) production, but it reduced microvessel density, macrophage infiltration, and interleukin-1β (IL-1β) levels. Gross morphological and histopathological examinations revealed that rats treated with AdTSP-1 had less severe osteoarthritis than controls. In vivo adenovirus-mediated TSP-1 gene transfer significantly reduced microvessel density, inflammation, and suppressed the progression of osteoarthritis. This study provides potential applications of TSP-1 gene delivery for treating osteoarthritis., (Published by Wiley Periodicals, Inc. J Orthop Res 28:1300-1306, 2010.)

The endothelium was the first non-hematopoietic tissue to be identified as a physiological target for erythropoietin (EPO). EPO is involved in recruitment and mobilization of endothelial progenitors and stimulates the production of erythroid cell regulatory factors in endothelial cells. Production of these EPO-dependent factors is inhibited by IL-3 in vitro. Furthermore, EPO-dependent red cell formation in anemic mice is equally repressed by IL-3. The number of IL-3 receptors on endothelial cells increases in chronic inflammation and IL-3 may be one of the inflammatory cytokines, together with TNF-alpha, IFN-gamma or IL-6, which prevents optimal red cell formation in many patients with kidney failure receiving high doses of EPO. These patients could benefit from the administration of some of the EPO-stimulated endothelial factors, such as C21 (the C-terminal segment thrombospondin-4), thrombospondin-1 and chaperonin 10, because these proteins bypass EPO receptors and signaling pathways that are usually compromised in EPO resistance. C21 stimulates red cell formation in anemic mice, increases human hematopoietic cell proliferation in vitro and could eventually fight inflammation, because it is an osteopontin antagonist. Thrombospondin-1 prevents inflammation, stimulates erythroblast proliferation and counteracts IGFBP-3-mediated erythroid inhibition. Finally, chaperonin 10 stimulates hemoglobin synthesis and has anti-inflammatory properties through the inhibition of Toll-like receptor signaling pathways., (Copyright 2010 Elsevier Ltd. All rights reserved.)

CVX-22 is a CovX-Body, produced by covalently attaching a thrombospondin-1 (TSP-1) type 1 repeat peptide mimetic to a humanized IgG1 molecule. To dissect the antiangiogenic mechanism of CVX-22, the numbers and proliferative status of defined tumor endothelial cell (TEC) subsets from the B16 and C32 melanoma models were examined. CVX-22 treatment reduced the numbers of activated, vascular endothelial growth factor receptor 2 (VEGFR2)-positive TECs. Because the vast majority of mitotically active TECs reside in the VEGFR2 subset, a reduction in numbers of this compartment resulted in an 82% overall decrease in BrdU labeling of TEC. However, the rate of proliferation and VEGFR2 receptor density of this VEGFR2-positive subpopulation were unaffected. Instead, CVX-22 induced endothelial cell apoptosis both in vitro and in vivo, indicating that CVX-22 acts by selective deletion of activated, VEGFR2-positive TEC. The overrepresentation of activated cells in sites of tumor angiogenesis may confer a unique specificity of CVX-22 for tumor vasculature.

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).

Objective: To evaluate the effect of a thrombospondin 1 (TSP1)-derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis., Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan-polysaccharide (PG-PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1-derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT-PCR., Results: Histological data indicated that the TSP1-derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1-derived peptide treatment. Also, immunofluorescence and RT-PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1-derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1-derived peptide treated animals. Moreover, macrophage/monocyte-specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1-derived peptide treated animals., Conclusion: Both inflammation and angiogenesis were decreased after TSP1-derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues., ((c) 2007 Wiley-Liss, Inc.)

A large body of evidence now demonstrates that angiostatic therapy represents a promising way to fight cancer. This research recently resulted in the approval of the first angiostatic agent for clinical treatment of cancer. Progress has been achieved in decrypting the cellular signaling in endothelial cells induced by angiostatic agents. These agents predominantly interfere with the molecular pathways involved in migration, proliferation and endothelial cell survival. In the current review, these pathways are discussed. A thorough understanding of the mechanism of action of angiostatic agents is required to develop efficient anti-tumor therapies.

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.

Purpose: Thrombospondin-I (TSP-I) is a natural antiangiogenic protein that enhances apoptosis of activated endothelial cells. A modified nonapeptide from TSP-I, ABT-526, has been found to be active in mouse cancer models and in dogs with naturally occurring cancers. To further assist in the development of ABT-526, we report herein on its evaluation in combination with cytotoxic chemotherapy in pet dogs with relapsed non-Hodgkin's lymphoma (NHL)., Experimental Design: Ninety-four pet dogs with naturally occurring first-relapse NHL were entered into a prospective randomized placebo controlled double-blinded trial of ABT-526 plus CeeNu (Bristol-Myers Squibb, New York, NY) versus CeeNu alone. Endpoints included response rate, duration of response, time to progression, and incidence of toxicoses., Results: No significant ABT-526-specific toxicities were seen. CeeNu-associated toxicities, including neutropenia, thrombocytopenia, gastroenteritis, and elevated alanine transaminase, were similar. No significant difference in objective response rate was seen (ABT-526 + CeeNu versus placebo + CeeNu, 23/49 versus 23/37; P > 0.25). Cooperative activity between ABT-526 and CeeNu chemotherapy was evident based on a significant increase in the median response duration of dogs receiving ABT-526 plus CeeNu compared with placebo plus CeeNu (35 versus 15 days; P < 0.05). The time to progression for responding cases was also significantly greater in dogs receiving ABT-526 plus CeeNu compared with placebo plus CeeNu (41 versus 21 days; P < 0.05)., Conclusions: Results of this preclinical trial suggest that the activity of ABT-526 is sustained when combined with cytotoxic chemotherapy; furthermore, the activity seems to be associated with the maintenance of CeeNu-induced treatment responses. Further studies of TSP-I peptide antiangiogenic therapy in pet dogs and humans with NHL are warranted.

Purpose: The angiogenic phenotype of malignant cancers has been established as a target for cancer therapy. ABT-526 and ABT-510, two peptide mimetics of thrombospondin-1 (TSP-1), block angiogenesis in vitro and in vivo and slow tumor growth in mice. To guide the clinical development of these drugs, translational studies in dogs with naturally occurring cancers were initiated., Experimental Design: A prospective open-label trial using ABT-510 or ABT-526 in pet dogs with measurable malignant spontaneously arising tumors. Endpoints included safety, pharmacokinetics, antitumor activity, and preliminary assessment of changes in circulating endothelial cell populations., Results: Two-hundred and forty-two dogs were sequentially entered to this open-label trial. The elimination half-life for ABT-510 and ABT-526 was 0.7 and 0.8 h, respectively (range, 0.5-1 h). No dose-limiting toxicities were seen in any dogs (N = 242). Forty-two dogs receiving peptide had objective responses (>50% reduction in tumor size; n = 6) or significant disease stabilization. Most objective responses were seen after 60 days of exposure to the TSP-1 peptide. Antitumor activity was similar for both peptides and was seen in several histologies, including mammary carcinoma, head and neck carcinoma, soft tissue sarcoma, cutaneous T-cell lymphoma, and non-Hodgkin's lymphoma. Assessment of circulating endothelial cell populations in a small subset of dogs suggested that effective exposure to TSP-1 peptides may be associated with reductions in circulating endothelial cells., Conclusions: These results support the safety and activity of ABT-526 and ABT-510 in dogs with naturally occurring malignant cancers. Data from this preclinical trial support the development of TSP-1 mimetic peptides as anticancer agents.

Purpose: This study investigates the antiangiogenesis and antitumor efficacy of a recombinant protein composed of the three type 1 repeats (3TSR) of thrombospondin-1 in an orthotopic human pancreatic cancer model and provides useful preclinical data for pancreatic cancer treatment., Experimental Design: Human pancreatic cancer cells (AsPC-1) were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR (3 mg per kg per day) or PBS for 3 weeks. Subsequently, the effects of 3TSR on tumor growth, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. The in vitro effects of 3TSR on human pancreatic cancer cells were also studied., Results: 3TSR treatment significantly reduced angiogenesis and tumor growth of orthotopic pancreatic cancer. 3TSR-treated mice had a 69% reduction in tumor volume (316.6 +/- 79.3 versus 1,012.2 +/- 364.5 mm(3); P = 0.0001), and a significant increase in tumor necrotic area. After 3TSR treatment, both the vessel number and average microvessel size were significantly decreased, and microvessel density was decreased from 8.0% to 3.7% (P < 0.0001). The apoptotic rate of tumoral endothelial cells in 3TSR-treated tumors increased to 14.7% comparing to 4.2% in control tumors (P < 0.0001). 3TSR showed no direct effects on pancreatic cancer cell proliferation or apoptosis either in vivo or in vitro., Conclusion: 3TSR, a domain of a natural occurring angiogenesis inhibitor, showed potent therapeutic effect in pancreatic cancer by inhibiting tumor angiogenesis and may prove to be a promising agent for clinical pancreatic cancer treatment.

Antiangiogenic or metronomic chemotherapy, the frequent administration of conventional cytotoxic agents at low doses, is believed to target activated tumor endothelial cells. The mechanisms of action of such regimen remain poorly understood. In the March 2004 issue of Cancer Research, Hamano et al. demonstrated that low-dose cyclophosphamide inhibits tumor growth by upregulating the endogenous angiogenesis inhibitor thrombospondin-1 in tumor and perivascular cells. Thrombospondin-1, in turn, promotes endothelial cell apoptosis. It was also proposed that thrombospondin-1 levels might be used as a surrogate marker to monitor response to low-dose cyclophosphamide therapy in the clinic.

The TSR superfamily is a diverse family of extracellular matrix and transmembrane proteins, many of which have functions related to regulating matrix organization, cell-cell interactions and cell guidance. This review samples some of the contemporary literature regarding TSR superfamily members (e.g. F-spondin, UNC-5, ADAMTS, papilin, and TRAP) where specific functions are assigned to the TSR domains. Combining these observations with the published crystal structure of the TSRs of thrombospondin-1 may hold a key to the development of therapeutic agents for fighting parasitic infection and tumor growth.

The thrombospondins (TSPs) are a family of proteins that regulate tissue genesis and remodeling. In many tumors, down-regulation of TSPs accompanies activation of oncogenes or inactivation of tumor suppresser genes and appears to be a prerequisite for the aquisition of a pro-angiogenic phenotype. The normal suppression of angiogenesis by TSP-1 and -2 involves multiple mechanisms including direct interaction with vascular endothelial cell growth factor (VEGF), inhibition of matrix metalloproteinase 9 (MMP9) activation, inhibition of endothelial cell migration and induction of endothelial cell apoptosis. The importance of down-regulation of TSPs for tumor progression is further established by the fact that several different approaches that are designed to increase the levels of TSP-1 or -2 in tumor tissue inhibit tumor growth. These approaches include cell-based gene therapy, low dose chemotherapeutics and systemic delivery of recombinant proteins or synthetic peptides that include type 1 repeat (TSR) sequences. Initial studies indicate that these reagents, in combination with established approaches for the treatment of cancer, will offer more efficacious therapies.

Chemotherapy for the treatment of advanced or metastatic colon cancer, utilizing agents such as 5-fluorouracil (5-FU) and irinotecan (CPT-11), produce a 5-year survival of about 10%. Thus, the identification of new, effective, therapeutic regimens to treat this disease remains critically important. To this end, selected antiangiogenic agents, compounds that inhibit neovascularization, have been shown to produce a modest tumor growth-inhibitory effect with little systemic toxicity. Thus these agents are attractive candidates for use with conventional chemotherapeutic agents to treat this disease. To evaluate this approach, experiments were undertaken to assess the cytotoxic and antineoplastic activity of CPT-11 and the antiangiogenic agent thrombospondin-1 (TSP-1) in the HT-29 model of human colon cancer. These agents were chosen since CPT-11 is a camptothecin analogue efficacious in the treatment of colon cancer and TSP-1 is a human glycoprotein that possess antiangiogenic activity. As expected, in vitro studies revealed that a 5-day exposure to TSP-1 at concentrations up to 130 microg/ml was not cytotoxic alone and did not affect the cytotoxicity of CPT-11, or of its active metabolite SN38, in HT-29 cells. Similarly, in human umbilical vein endothelial cells, TSP-1 alone induced only a slight cell growth-inhibitory effect and did not significantly increase the cytotoxicity of either CPT-11 or SN38. The antineoplastic activities of TSP-1 and CPT-11 were assessed in athymic (nude) female mice bearing advanced subcutaneous xenografts of HT-29 cells. Mice received TSP-1 alone (5-40 mg/kg per day) intraperitoneally (i.p.), CPT-11 alone (100-300 mg/kg, i.p.), TSP-1 (10 mg/kg per day) plus CPT-11 (125 mg/kg), or TSP-1 (20 mg/kg per day) plus CPT-11 (150 mg/kg). TSP-1 was injected daily (Monday through Friday) for 4 weeks (20 injections in total) whereas CPT-11 was administered once weekly on days 0, 7, 14 and 21. By day 28, treatment with TSP-1 alone (5, 10 or 20 mg/kg per day) induced a dose-dependent inhibition of xenograft growth. Further, treatment with 10 or 20 mg/kg per day resulted in an average treated tumor size/control tumor size (T/C) on day 28 of 0.68 (range 0.64-0.71) or 0.58 (range 0.54-0.60), respectively. CPT-11 at all doses significantly inhibited tumor growth with an average T/C value of 0.21 (range 0.15-0.27). However, the 250 and 300 mg/kg regimens induced significant toxicity and mortality. When TSP-1 was combined with CPT-11, a significant ( P< or = 0.05) inhibition of tumor growth also was observed (T/C < or = 0.17, range 0.11-0.20). Importantly, this enhanced tumor growth inhibition was obtained without significant toxicity. The therapeutic implications of these findings are discussed.

Purpose: Intradermal D-12 human melanoma xenografts develop pulmonary micrometastases in BALB/c nu/nu mice, and these metastases are kept dormant for prolonged times, because the primary tumor secretes thrombospondin-1 (TSP-1) into the blood circulation of the host. In this study, we report on the development of macroscopic metastases after surgical resection and curative radiation treatment of the primary tumor, the mechanisms involved, and the effects of treating the host with exogenous TSP-1 after the eradication of the primary tumor., Methods and Materials: Xenografted tumors of the D-12 human melanoma were used as tumor model. Macroscopic metastases were scored by using a stereomicroscope. Micrometastases were detected by histologic examinations. Angiogenesis was studied by using an intradermal angiogenesis assay. Apoptotic endothelial cells were detected by immunohistochemistry by using an in situ apoptosis detection kit., Results: Surgical resection as well as curative radiation treatment of the primary tumor resulted in accelerated growth of dormant micrometastases. This growth could be prevented by treating the host with exogenous TSP-1 after the surgery or the irradiation. Endogenous and exogenous TSP-1 prevented metastatic growth by suppressing angiogenesis, i.e., by inducing apoptosis in activated endothelial cells adjacent to dormant micrometastases., Conclusions: TSP-1 has antiangiogenic and antimetastatic effects that should be investigated further in clinical studies. Cancer patients with TSP-1-producing primary tumors may benefit from combined local treatment and antiangiogenic/antimetastatic treatment with TSP-1.

Thrombospondin-1 (TSP-1) was one of the first endogenous inhibitors of angiogenesis to be discovered. This large multimodular protein of around 600 kDa inhibits endothelial cell proliferation, migration and morphogenic organization into capillary tubes. TSP-2 shares homology with TSP-1 in primary sequence, structural organization and angiostatic properties. TSP-1-null and TSP-2-null mice display increased tissue vascularity and enhanced sensitivity to carcinogenesis. Conversely, overexpression of TSP-1 or TSP-2 in cancer cells results in reduced tumor vascularization and tumor growth. In this review, we focus on the preclinical data obtained in experimental anti-tumorigenic assays using either TSP-1, TSP-2 or shorter peptides derived from the type 1 repeats of these molecules. In contrast with the full length thrombospondin molecules, which present a poor bioavailability and are highly susceptible to proteolytic degradation, TSP-derived angiostatic peptides appear as potent and promising therapeutic agents in anti-angiogenic therapy.

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies., (Copyright 2002 Wiley-Liss, Inc.)

Ovarian cancer claims the lives of more women in North America each year than all other gynecologic malignancies combined. Despite the high initial response rates of patients with advanced ovarian cancer to aggressive primary surgical debulking followed by combination chemotherapy, the majority of patients will ultimately develop disease recurrence. The high risk of relapse and nearly guaranteed incurability after relapse is due to genetic instability and a high mutation rate of neoplastic cells that together allow for a high risk of selection for drug resistance. Given the seemingly insurmountable obstacle that acquired drug resistance presents in a setting of minimal, often undetectable, residual tumor burden in women with ovarian cancer, antiangiogenic-targeted therapies offer an attractive strategy for enhanced long-term disease-free survival. The past decade has witnessed a substantial proliferation in our knowledge regarding tumor angiogenesis, which has spurred interest in antiangiogenesis drug development. Current clinical trials are evaluating these agents in a variety of solid tumors, including ovarian cancer. Preliminary work has provided hope that the addition of antiangiogenic therapies may be incorporated into the treatment of women afflicted with ovarian cancer and may translate into enhanced survival.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis that acts directly on endothelial cells via the CD36 surface receptor molecule to halt their migration, proliferation, and morphogenesis in vitro and to block neovascularization in vivo. Here we show that inhibitory signals elicited by TSP-1 did not alter the ability of inducers of angiogenesis to activate p42 and p44 mitogen-activated protein kinase (MAPK). Rather, TSP-1 induced a rapid and transient activation of c-Jun N-terminal kinases (JNK). JNK activation by TSP-1 required engagement of CD36, as it was blocked by antagonistic CD36 antibodies and stimulated by short anti-angiogenic peptides derived from TSP-1 that act exclusively via CD36. TSP-1 inhibition of corneal neovascularization induced by bFGF was severely impaired in mice null for JNK-1, pointing to a critical role for this stress-activated kinase in the inhibition of neovascularization by TSP-1.

We recently reported that a p53 encoding plasmid (BAP-p53) complexed to liposomes administered intravenously markedly attenuates the growth of a malignant human breast tumor. We now have found that systemically delivered liposomes complexed to a plasmid expressing an established antiangiogenic peptide of thrombospondin I (BAP-TSPf) decreased the growth of MDA-MB-435 tumors compared to controls in nude mice. Compared to BAP-p53, the BAP-TSPf group had a similar antitumor efficacy. More importantly, liposomes complexed with BAP-TSPf and BAP-p53 synergistically decreased the growth of MDA-MB-435 tumors when compared to either BAP-p53 or BAP-TSPf alone. Furthermore, we also determined that the combination therapy of p53 and TSPf inhibited endothelial cells in vitro more than either p53 or TSPf alone. There was also a significant decrease of the blood vessel density in the combination p53 and TSPf treatment group compared to the control groups. These results suggest that liposomes complexed to a tumor suppressor and antiangiogenic genes may be effective in treating metastatic tumors.

Thrombospondin-1 is a glycoprotein that is released from platelet alpha-granules in response to thrombin stimulation and that is also a transient component of extracellular matrix in developing and repairing tissues. It is a 420 kDa homotrimer, each subunit of which consists of multiple structural domains. A variety of factors regulate thrombospondin-1 expression and the protein is degraded by both extracellular and intracellular routes. Thrombospondin-1 functions as a cell adhesion molecule and also modulates cell movement, cell proliferation, neurite outgrowth and angiogenesis. The molecular mechanisms underlying these activities are beginning to be examined. Medical interest in thrombospondin-1 centres on its roles in haemostasis and its effects on angiogenesis.