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Thrombospondin-1 (TSP-1) is a large matricellular glycoprotein known to be overexpressed within tumor stroma in several cancer types. While mainly considered as an endogenous angiogenesis inhibitor, TSP-1 exhibits multifaceted functionalities in a tumor context depending both on TSP-1 concentration as well as differential receptor expression by cancer cells and on tumor-associated stromal cells. Besides, the complex modular structure of TSP-1 along with the wide variety of its soluble ligands and membrane receptors considerably increases the complexity of therapeutically targeting interactions involving TSP-1 ligation of cell-surface receptors. Despite the pleiotropic nature of TSP-1, many different antireceptor strategies have been developed giving promising results in preclinical models. However, transition to clinical trials often led to nuanced outcomes mainly due to frequent severe adverse effects. In this review, we will first expose the intricate and even sometimes opposite effects of TSP-1-related signaling on tumor progression by paying particular attention to modulation of angiogenesis and tumor immunity. Then, we will provide an overview of current developments and prospects by focusing particularly on the cell-surface molecules CD47 and CD36 that function as TSP-1 receptors; including antibody-based approaches, therapeutic gene modulation and the use of peptidomimetics. Finally, we will discuss original approaches specifically targeting TSP-1 domains, as well as innovative combination strategies with a view to producing an overall anticancer response.

Endoglin (ENG) is a TGF-β superfamily coreceptor essential for vascular endothelium integrity. ENG mutations lead to a vascular dysplasia associated with frequent hemorrhages in multiple organs, whereas ENG null mouse embryos die at midgestation with impaired heart development and leaky vasculature. ENG interacts with several proteins involved in cell adhesion, and we postulated that it regulates vascular permeability. The current study assessed the permeability of ENG homozygous null (Eng(-/-)), heterozygous (Eng(+/-)), and normal (Eng(+/+)) mouse embryonic endothelial cell (EC) lines. Permeability, measured by passage of fluorescent dextran through EC monolayers, was increased 2.9- and 1.7-fold for Eng(-/-) and Eng(+/-) ECs, respectively, compared to control ECs and was not increased by TGF-β1 or VEGF. Prolonged starvation increased Eng(-/-) EC permeability by 3.7-fold with no effect on control ECs; neutrophils transmigrated faster through Eng(-/-) than Eng(+/+) monolayers. Using a pull-down assay, we demonstrate that Ras homolog gene family (Rho) A is constitutively active in Eng(-/-) and Eng(+/-) ECs. We show that the endothelial barrier destabilizing factor thrombospondin-1 and its receptor-like protein tyrosine phosphatase are increased, whereas stabilizing factors VEGF receptor 2, vascular endothelial-cadherin, p21-activated kinase, and Ras-related C3 botulinum toxin substrate 2 are decreased in Eng(-/-) cells. Our findings indicate that ENG deficiency leads to EC hyperpermeability through constitutive activation of RhoA and destabilization of endothelial barrier function., (© FASEB.)

The multi-modular glycoprotein thrombospondin-1 (TSP-1) is considered as a key actor within the tumor microenvironment. Besides, TSP-1 binding to CD47 is widely reported to regulate cardiovascular function as it promotes vasoconstriction and angiogenesis limitation. Therefore, many studies focused on targeting TSP-1:CD47 interaction, aiming for up-regulation of physiological angiogenesis to enhance post-ischemia recovery or to facilitate engraftment. Thus, we sought to identify an innovative selective antagonist for TSP-1:CD47 interaction. Protein-protein docking and molecular dynamics simulations were conducted to design a novel CD47-derived peptide, called TAX2. TAX2 binds TSP-1 to prevent TSP-1:CD47 interaction, as revealed by ELISA and co-immunoprecipitation experiments. Unexpectedly, TAX2 inhibits in vitro and ex vivo angiogenesis features in a TSP-1-dependent manner. Consistently, our data highlighted that TAX2 promotes TSP-1 binding to CD36-containing complexes, leading to disruption of VEGFR2 activation and downstream NO signaling. Such unpredicted results prompted us to investigate TAX2 potential in tumor pathology. A multimodal imaging approach was conducted combining histopathological staining, MVD, MRI analysis and μCT monitoring for tumor angiography longitudinal follow-up and 3D quantification. TAX2 in vivo administrations highly disturb syngeneic melanoma tumor vascularization inducing extensive tumor necrosis and strongly inhibit growth rate and vascularization of human pancreatic carcinoma xenografts in nude mice.

Objective: Previously, we have identified bone morphogenetic protein endothelial cell precursor-derived regulator (BMPER) to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project, we now investigate how BMPER acts in concert with key molecules of angiogenesis to promote blood vessel formation., Approach and Results: To assess the effect of BMPER on angiogenesis-related signaling pathways, we performed an angiogenesis antibody array with BMPER-stimulated endothelial cells. We detected increased basic fibroblast growth factor (bFGF/FGF-2) expression after BMPER stimulation and decreased expression of thrombospondin-1. Additionally, FGF receptor-1 expression, phosphorylation, FGF signaling pathway activity, and cell survival were increased. Consistently, silencing of BMPER by small interfering RNA decreased bFGF and FGF receptor-1 expression and increased thrombospondin-1 expression and cell apoptosis. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased endothelial cell angiogenic activity in migration, Matrigel, and spheroid assays. To block FGF signaling, an anti-bFGF antibody was used, which effectively inhibited the proangiogenic BMPER effect. Accordingly, BMPER-silenced endothelial cells under bFGF stimulation showed decreased angiogenic activity compared with bFGF control. We confirmed these findings in vivo by subcutaneous Matrigel injections with and without bFGF in C57BL/6_Bmper(+/-) mice. Aortic ring assays of C57BL/6_Bmper(+/-) mice confirmed a specific effect for bFGF but not for vascular endothelial growth factor., Conclusions: Taken together, the proangiogenic BMPER effect in endothelial cells is mediated by inhibition of antiangiogenic thrombospondin-1 and enhanced expression and activation of the FGF signaling pathway that is crucial in the promotion of angiogenesis., (© 2014 American Heart Association, Inc.)

Background: Idiopathic pulmonary fibrosis (IPF) is associated with a marked pulmonary vascular remodeling. The aim of this study was to investigate a potential imbalance between angiogenic and angiostatic factors in this disease., Methods and Results: Sixty-four subjects with IPF and 10 healthy control subjects (60-70 years old) were prospectively included in this multicenter study. Plasma levels of vascular endothelial growth factor A (VEGF-A), thrombospondin-1 (TSP-1) and stem cell factor (SCF) were determined by Elisa. Comparisons between IPF and controls were made using the Mann-Whitney U test. We also analyzed these soluble mediators in relation with IPF severity (DLCO<40% or>40%) predicted or total lung capacity (TLC) and forced vital capacity (FVC) (both<55% or>55% predicted) using the same test. VEGF-A plasma levels were increased in IPF vs. controls (P=0.0008) as well as those of TSP-1 (P=0.008), irrespective of the severity of the disease as reflected by DLCO, TLC or FVC values. In contrast, SCF levels were similar in IPF and controls., Conclusions: Factors modulating angiogenic responses are dysregulated in patients with IPF with increases in VEGF-A and TSP-1. The serial assessment of VEGF-A and TSP-1 during the follow-up and the search for potential relationships with the outcome of the disease might give us hints to the clinical implication of these results., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Background: Although inflammation plays an important role in the development of benign prostatic hyperplasia (BPH), little is known about the exact mechanism underlying this pathogenesis. Here, we investigated the relationship between the inflammatory reaction and BPH., Methods: cDNA microarray analysis was used to identify changes in inflammation-related gene expression in a recently established rat model that mimics human BPH. To investigate the genes identified in the analysis, quantitative (q)RT-PCR, Western blotting, immunostaining, and a cell proliferation assay were conducted using BPH model tissues, human prostate tissues, and normal human prostate cultured cells., Results: Of the 31,100 genes identified in the cDNA analysis, seven inflammatory-response-related genes were expressed at a >2-fold higher level in rat BPH tissues than in normal rat prostate tissues. The levels of the most commonly expressed pro-inflammatory cytokine, IL-18, significantly increased in rat BPH tissues. In humans, IL-18 was localized in the epithelial and stromal components, while its receptor was strongly localized in smooth muscle cells. Furthermore, in human prostate smooth muscle cell line (PrSMC), IL-18 effected dose-dependent increases in the phosphorylated Akt and thrombospondin-1 (TSP-1) levels. TSP-1 promoted proliferation of the human prostate stromal cells (PrSC)., Conclusions: IL-18 may act directly in BPH pathogenesis by inducing TSP-1 production in prostatic smooth muscle cells via Akt phosphorylation., (© 2014 Wiley Periodicals, Inc.)

Purpose: To investigate the impact of radioembolization with yttrium-90 resin microspheres on the regulation of angiogenesis through observation of serial changes in a spectrum of angiogenic markers and other cytokines after therapy., Materials and Methods: This prospective pilot study enrolled 22 patients with liver-dominant disease deriving from biopsy-proven hepatocellular carcinoma (HCC) (n = 7) or metastatic colorectal carcinoma (mCRC) (n = 15). Circulating angiogenic markers were measured from serum samples drawn at baseline and at time points after therapy ranging from 6 hours to 120 days. Using multiplex enzyme-linked immunosorbent assay, several classic angiogenesis factors (vascular endothelial growth factor [VEGF], angiopoietin-2 [Ang-2], basic fibroblast growth factor [bFGF], platelet-derived growth factor subunit BB [PDGF-BB], thrombospondin-1 [Tsp-1]) and nonclassic factors (follistatin, leptin, interleukin [IL]-8) were evaluated., Results: Increases in cytokine levels ≥ 50% over baseline were observed in more than half of all patients studied for many cytokines, including classic angiogenic factors such as VEGF, Ang-2, and Tsp-1 as well as nonclassic factors IL-8 and follistatin (range, 36%-82% for all cytokines). Baseline cytokine levels in patients with overall survival (OS) < 6 months differed significantly from patients with longer survival for Ang-2 (P = .033) and IL-8 (P = .041). Patients with OS ≤ 6 months exhibited transient increases in VEGF and PDGF-BB after therapy compared with patients with OS > 6 months., Conclusions: Radioembolization is associated with early transient increases in many angiogenic cytokines. In this small sample size, some of these changes were associated with worse OS. This research has important implications for future studies of radioembolization with antiangiogenic therapy performed during and after the procedure., (© 2014 SIR Published by SIR All rights reserved.)

The biochemical mechanisms that regulate the process of cancer metastasis are still poorly understood. Because kinases, and the signaling pathways they comprise, play key roles in regulation of many cellular processes, we used an unbiased RNAi in vitro screen and a focused cDNA in vivo screen against human kinases to identify those with previously undocumented roles in metastasis. We discovered that G-protein-coupled receptor kinase 3 (GRK3; or β-adrenergic receptor kinase 2) was not only necessary for survival and proliferation of metastatic cells, but also sufficient to promote primary prostate tumor growth and metastasis upon exogenous expression in poorly metastatic cells in mouse xenograft models. Mechanistically, we found that GRK3 stimulated angiogenesis, at least in part through down-regulation of thrombospondin-1 and plasminogen activator inhibitor type 2. Furthermore, GRK3 was found to be overexpressed in human prostate cancers, especially in metastatic tumors. Taken together, these data suggest that GRK3 plays an important role in prostate cancer progression and metastasis., Competing Interests: The authors declare no conflict of interest.

Synaptic dysfunction has recently gained attention for its involvement in mood disorders. Electroconvulsive therapy (ECT) possibly plays a role in synaptic repair. However, the underlying mechanisms remain uncertain. Thrombospondin-1 (TSP-1), a member of the TSP family, is reported to be secreted by astrocytes and to regulate synaptogenesis. We investigated the effects of electroconvulsive seizure (ECS) on the expression of TSPs in the adult rat hippocampus. Single and repeated ECS significantly increased TSP-1 mRNA expression after 2h and returned to sham levels at 24h. Conversely, the TSP-2 and -4 mRNA levels did not change. Only repeated ECS induced TSP-1 proteins. ECS also induced glial fibrillary acidic protein (GFAP) expression. The GFAP expression occurred later than the TSP-1 mRNA expression following single ECS; however, it occurred earlier and was more persistent following repeated ECS. ECS had no effect on the α2δ-1 or neuroligin-1 expressions, both of which are TSP-1 receptors. Furthermore, chronic treatment with antidepressants did not induce the expression of TSP-1 or GFAP. These findings suggest that repeated ECS, but not chronic treatment with antidepressants, induces TSP-1 expression partially via the activation of astrocytes. Therefore, TSP-1 is possibly involved in the synaptogenic effects of ECS., (© 2013.)

Background and Rationale: Anaplastic thyroid cancer (ATC) is characterized by pleomorphic cells, has a poor prognosis, is highly devastating disease, and is not curable. No reliable biomarkers of metastatic potential, helpful for early diagnosis of ATC and therapeutic response have been found yet. Thrombospondin-1 (TSP-1) plays a fundamental role in cancer progression by regulating cell stromal cross-talk in the tumor microenvironment., Goals: Our goal was to understand whether TSP-1 could affect protein levels of its integrin receptors (e.g., ITGα3, α6, and β1) and cell morphology in BRAF(V600E)-ATC cells in vitro and in vivo., Experimental Design: Anaplastic thyroid cancer-derived cell cultures and western blotting were used to assess integrin protein expression upon TSP-1 silencing. Immunohistochemistry was performed on orthotopic primary human ATC and metastatic ATC in lung tissue to compare TSP-1 and integrin protein expression levels., Results: TSP-1 knock-down down-regulates ITGα3, α6, and β1 in BRAF(V600E)-human ATC cells. BRAF(V600E)-ATC cells with TSP-1 knock-down were rounded compared to control cells, which displayed a spread morphology. TSP-1 knock-down also reduced TSP-1, ITGα3, α6, and β1 protein expression levels in vivo in the ATC microenvironment, which is enriched in stromal and inflammatory cells., Conclusion: TSP-1 silencing causes changes in ITG levels and ATC cell morphology. The assessment of TSP-1 and ITG levels might contribute to earlier metastatic potential of BRAF(V600E)-positive aggressive thyroid cancers, and allow improved patient selection for clinical trials.

Hypoxia and adenosine are known to upregulate angiogenesis; however, the role of peroxisome proliferator-activated receptor alpha (PPARα) in angiogenesis is controversial. Using transgenic Tg(fli-1:EGFP) zebrafish embryos, interactions of PPARα and adenosine receptors in angiogenesis were evaluated under hypoxic conditions. Epifluorescent microscopy was used to assess angiogenesis by counting the number of intersegmental (ISV) and dorsal longitudinal anastomotic vessel (DLAV) at 28 h post-fertilization (hpf). Hypoxia (6h) stimulated angiogenesis as the number of ISV and DLAV increased by 18-fold (p<0.01) and 100 ± 8% (p<0.001), respectively, at 28 hpf. Under normoxic and hypoxic conditions, WY-14643 (10 μM), a PPARα activator, stimulated angiogenesis at 28 hpf, while MK-886 (0.5 μM), an antagonist of PPARα, attenuated these effects. Compared to normoxic condition, adenosine receptor activation with NECA (10 μM) promoted angiogenesis more effectively under hypoxic conditions. Involvement of A2B receptor was implied in hypoxia-induced angiogenesis as MRS-1706 (10nM), a selective A2B antagonist attenuated NECA (10 μM)-induced angiogenesis. NECA- or WY-14643-induced angiogenesis was also inhibited by miconazole (0.1 μM), an inhibitor of epoxygenase dependent production of eicosatrienoic acid (EET) epoxide. Thus, we conclude that: activation of PPARα promoted angiogenesis just as activation of A2B receptors through an epoxide dependent mechanism., (© 2013.)

Thrombospondin-1 (TSP-1) expression in human adipose positively correlates with body mass index and may contribute to adipose dysfunction by activating transforming growth factor-β and/or inhibiting angiogenesis. Our objective was to determine how TSP-1 is regulated in adipocytes and polarized macrophages using a coculture system and to determine whether fatty acids, including the ω-3 fatty acid docosahexaenoic acid (DHA), regulate TSP-1 expression. Coculture of M1, M2a or M2c macrophages with adipocytes induced TSP-1 gene expression in adipocytes (from 2.4- to 4.2-fold, P<.05), and adipocyte coculture induced TSP-1 gene expression in M1 and M2c macrophages (M1: 8.6-fold, M2c: 26-fold; P<.05). TSP-1 protein levels in the shared media of adipocytes and M2c cells were also strongly induced by coculture (>10-fold, P<.05). DHA treatment during the coculture of adipocytes and M2c macrophages potently inhibited the M2c macrophage TSP-1 mRNA level (97% inhibition, P<.05). Adipocyte coculture induced interleukin (IL)-10 expression in M2c macrophages (10.1-fold, P<.05), and this increase in IL-10 mRNA expression was almost completely blocked with DHA treatment (96% inhibition, P<.05); thus, IL-10 expression closely paralleled TSP-1 expression. Since IL-10 has been shown to regulate TSP-1 in other cell types, we reduced IL-10 expression with siRNA in the M2c cells and found that this caused TSP-1 to be reduced in response to adipocyte coculture by 60% (P<.05), suggesting that IL-10 regulates TSP-1 expression in M2c macrophages. These results suggest that supplementation with dietary ω-3 fatty acids could potentially be beneficial to adipose tissue in obesity by reducing TSP-1 and fibrosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The cytokine transforming growth factor-β (TGF-β) plays a pivotal role in a diverse range of cellular responses, including cell proliferation, apoptosis, differentiation, migration, adhesion, angiogenesis, stimulation of extracellular matrix (ECM) synthesis, and downregulation of ECM degradation. TGF-β and its receptors are ubiquitously expressed by most cell types and tissues in vivo. In intact adult tissues and organs, TGF-β is secreted in a biologically inactive (latent) form associated in a non-covalent complex with the ECM. In response to injury, local latent TGF-β complexes are converted into active TGF-β according to a tissue- and injury type-specific activation mechanism. Such a well and tightly orchestrated regulation in TGF-β activity enables an immediate, highly localized response to type-specific tissue injury. In the pathological process of liver fibrosis, TGF-β plays as a master profibrogenic cytokine in promoting activation and myofibroblastic differentiation of hepatic stellate cells, a central event in liver fibrogenesis. Continuous and/or persistent TGF-β signaling induces sustained production of ECM components and of tissue inhibitor of metalloproteinase synthesis. Therefore, the regulation of locally activated TGF-β levels is increasingly recognized as a therapeutic target for liver fibrogenesis. This review summarizes our present knowledge of the activation mechanisms and bioavailability of latent TGF-β in biological and pathological processes in the liver.