Stromal thrombospondin 1 suppresses angiogenesis in oral submucous fibrosis.

A decline in mucosal vascularity is a histological hallmark of oral submucous fibrosis (OSF), a premalignant disease that is largely induced by betel quid chewing. However, the lack of available models has challenged studies of angiogenesis in OSF. Here, we found that the expression of thrombospondin 1 (THBS1), an endogenous angiostatic protein, was elevated in the stroma of tissues with OSF. Using a fibroblast-attached organoid (FAO) model, the overexpression of THBS1 in OSF was stably recapitulated in vitro. In the FAO model, treatment with arecoline, a major pathogenic component in areca nuts, enhanced the secretion of transforming growth factor (TGF)-ß1 by epithelial cells, which then promoted the expression of THBS1 in fibroblasts. Furthermore, human umbilical vein endothelial cells (HUVECs) were incorporated into the FAO to mimic the vascularized component. Overexpression of THBS1 in fibroblasts drastically suppressed the sprouting ability of endothelial cells in vascularized FAOs (vFAOs). Consistently, treatment with arecoline reduced the expression of CD31 in vFAOs, and this effect was attenuated when the endothelial cells were preincubated with neutralizing antibody of CD36, a receptor of THBS1. Finally, in an arecoline-induced rat OSF model, THBS1 inhibition alleviated collagen deposition and the decline in vascularity in vivo. Overall, we exploited an assembled organoid model to study OSF pathogenesis and provide a rationale for targeting THBS1.

Depleted uranium induces thyroid damage through activation of ER stress via the thrombospondin 1-PERK pathway.

Depleted uranium (DU) can cause damage to the body, but its effects on the thyroid are unclear. The purpose of this study was to investigate the DU-induced thyroid damage and its potential mechanism in order to find new targets for detoxification after DU poisoning. A model of acute exposure to DU was constructed in rats. It was observed that DU accumulated in the thyroid, induced thyroid structure disorder and cell apoptosis, and decreased the serum T4 and FT4 levels. Gene screening showed that thrombospondin 1 (TSP-1) was a sensitive gene of DU, and the expression of TSP-1 decreased with the increase of DU exposure dose and time. TSP-1 knockout mice exposed to DU had more severe thyroid damage and lower serum FT4 and T4 levels than wild-type mice. Inhibiting the expression of TSP-1 in FRTL-5 cells aggravated DU-induced apoptosis, while exogenous TSP-1 protein alleviated the decreased viability in FRTL-5 cells caused by DU. It was suggested that DU may caused thyroid damage by down-regulating TSP-1. It was also found that DU increased the expressions of PERK, CHOP, and Caspase-3, and 4-Phenylbutyric (4-PBA) alleviated the DU-induced FRTL-5 cell viability decline and the decrease levels of rat serum FT4 and T4 caused by DU. After DU exposure, the PERK expression was further up-regulated in TSP-1 knockout mice, and the increased expression of PERK was alleviated in TSP-1 over-expressed cells, as well as the increased expression of CHOP and Caspase-3. Further verification showed that inhibition of PERK expression could reduce the DU-induced increased expression of CHOP and Caspase-3. These findings shed light on the mechanism that DU may activate ER stress via the TSP 1-PERK pathway, thereby leading to thyroid damage, and suggest that TSP-1 may be a potential therapeutic target for DU-induced thyroid damage.

Artemisin and human endometrial-derived stem cells improve cognitive function and synaptic plasticity in a rat model of Alzheimer disease and diabetes.

Alzheimer disease (AD) is a common form of dementia associated with loss of memory and disruption of synaptic plasticity. There is a strong correlation between the pathophysiological features of AD and diabetes, including induction of oxidative stress, inflammation, and abnormality in blood vessels. Considering the brain's limited capacity to repair damage and the potential of stem cell-derived neural cells in the repair of neurodegenerative disease, we investigated the effects of artemisinin and TSP­1­human endometrial-derived-derived stem cells (TSP­1­hEDSCs) on the cognitive function and synaptic plasticity in AD-diabetes rats. The authors previously showed that artemisinin and TSP­1­hEDSCs suppressed oxidative stress and inflammation in AD-diabetes rats. Thrombospondins-1 (TSPs-1) is a glycoprotein that inhibits angiogenesis. AD and diabetes were induced using streptozotocin. Synaptic plasticity and learning and memory function were studied using the Morris water maze and electrophysiological test, respectively. Streptozotocin increased traveled swimming distance and escape latency in the morris water maze test, decreased the percent time spent in the target quadrant, inhibited the long-term potentiation (LTP), and increased the blood glucose levels. Simultaneous or separate administration of artemisinin and TSP­1­hEDSCs decreased the blood levels of glucose and improved cognitive tasks and synaptic plasticity by considerably reducing traveled swimming distance and escape latency, increasing the percent time spent in the target quadrant, and retrieval of the LTP; therefore, they could be utilized as an adjunct treatment for AD treatment. These results may be due to a decrease in oxidative stress and inflammation.

Baicalin inhibited both the Furin/TGFß1/Smad3/TSP-1 pathway in endothelial cells and the AKT/Ca2+/ROS pathway in platelets to ameliorate inflammatory coagulopathy.

Inflammatory coagulopathy is resulted from endothelial dysfunction and platelet hyperactivation in inflammatory diseases. In this study, the effects of baicalin, an active component of the traditional Chinese medicine Huangqin, on inflammatory coagulopathy were observed both in vivo and in vitro. In LPS-induced rats, baicalin ameliorated coagulation indexes, inhibited platelet hyperactivation and decreased the expression of thrombospondin-1 (TSP-1) in vessels. In cultured endothelial cells, baicalin decreased the expression of TSP-1 and collagen as well as the TNF-α-induced increase in the levels of TSP-1 and ICAM-1. Baicalin could significantly decrease the platelet adhesion on endothelial cells treated with TNF-α. Baicalin also could inhibit the increase of ROS level and the activation of the NLRP3/Caspase-1/GSDMD pathway in TNF-α-induced endothelial cells. Furin was found to be the direct target of baicalin in HUVECs. Knockdown of Furin using siRNA could ameliorate the effects of baicalin on the activation of TGFß1/Smad3 pathway, TSP-1 expression and the adhesion of platelets on TNF-α-treated endothelial cells. At the same time, baicalin inhibited platelet aggregation induced by collagen or combination of collagen and TSP-1 peptide. Collagen-induced Ca2+ mobilization, ROS level increase, AKT1 phosphorylation, platelet degranulation and TSP-1 release could be all inhibited by baicalin. In all, baicalin ameliorated endothelial dysfunction by inhibiting Furin/TGFß1/Smad3/TSP-1 pathway and also ameliorated platelet activation by inhibiting AKT-related pathway. Both the inhibiting effects of baicalin on endothelial dysfunction and platelet activation might contribute to its ameliorating effects on inflammatory coagulopathy.

Thrombospondin-1 mimic peptide PKHB1 induced endoplasmic reticulum stress-mediated but CD47-independent apoptosis in non-small cell lung cancer.

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, Ca2+ 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.

Circulating THBS1: A Risk Factor for Nonalcoholic Fatty Liver Disease in Obese Children.

INTRODUCTION: Thrombospondin 1 (THBS1) is a highly expressed adipokine in adults with obesity. In the present study, we aimed to investigate the clinical significance of THBS1in children with obesity and nonalcoholic fatty liver disease (NAFLD) and determine the effect of metformin on THBS1 expression in dietary-induced obese (DIO) mice. METHODS: A cross-sectional study was conducted among 78 obese children and 35 nonobese children. Anthropometric parameters, clinical data, and circulating THBS1 levels were measured. The expression of THBS1 was detected in the serum and liver tissue from diet-induced obese mice (C57BL/6) with or without metformin treatment. RESULTS: Higher THBS1 levels were observed in children with NAFLD and higher SDS-BMI. Individuals in the higher THBS1 quartile had a higher prevalence of hypo-high-density lipoprotein cholesterol (HDL-C). Logistic regression analysis showed a significant correlation between THBS1 and NAFLD, as well as between hip circumference and leptin levels. Receiver-operating characteristic (ROC) analysis revealed that THBS1 was a more sensitive predictor of NAFLD than leptin. Additionally, metformin ameliorated hepatic steatosis and decreased hepatic THBS1 expression in high-fat diet (HFD)-fed mice. CONCLUSIONS: Circulating THBS1 level may be a risk factor for NAFLD in obese children. Our findings provided a novel approach of metformin administration for the prevention and treatment of NAFLD. This study also confirmed that metformin decreased the expression of hepatic THBS in DIO mice.

VR-10 polypeptide interacts with CD36 to induce cell apoptosis and autophagy in choroid-retinal endothelial cells: Identification of VR-10 as putative novel therapeutic agent for choroid neovascularization (CNV) treatment.

Choroid neovascularization (CNV) is important adverse pathological changes that contributes to the aggravation of hypoxic-ischemic eye diseases, and our preliminary work evidences that the thrombospondin-1 (TSP-1) synthetic polypeptide VR-10 may be the candidate therapeutic agent for the treatment of CNV, but its detailed effects and molecular mechanisms are not fully delineated. In this study, the CNV models in BN rats were established by using the laser photocoagulation method, which were further subjected to VR-10 peptide treatment. The RNA-seq and bioinformatics analysis suggested that VR-10 peptide significantly altered the expression patterns of genes in the rat ocular tissues, and the changed genes were especially enriched in the CD36-associated signal pathways. Next, by performing the Real-Time qPCR and Western Blot analysis, we expectedly found that VR-10 upregulated the anti-angiogenesis biomarker (PEDF) and downregulated pro-angiogenesis biomarkers (VEGF, HIF-1 and IL-17) in rat tissues. In addition, we evidenced that VR-10 downregulated CDK2, CDK4, CDK6, Cyclin D1 and Cyclin D2 to induce cell cycle arrest, upregulated cleaved Caspase-3, Bax and downregulated Bcl-2 to promote cell apoptosis, and increased LC3B-II/I ratio and facilitate p62 degradation to promote cell autophagy in RF/6A cells, which were all reversed by knocking down CD36. Moreover, VR-10 upregulated PEDF, and decreased the expression levels of VEGF, HIF-1 and IL-17 to block angiogenesis of RF/6A cells in a CD36-dependent manner. Taken together, VR-10 peptide interacts with its receptor CD36 to regulate the biological functions of RF/6A cells, and these data suggest that VR-10 peptide may be the putative therapeutic drug for the treatment of CNV in clinic.

Mechanobiological responses of astrocytes in optic nerve head due to biaxial stretch.

BACKGROUND: Elevated intraocular pressure (IOP) is the main risk factor for glaucoma, which might cause the activation of astrocytes in optic nerve head. To determine the effect of mechanical stretch on the astrocytes, we investigated the changes in cell phenotype, proteins of interest and signaling pathways under biaxial stretch. METHOD: The cultured astrocytes in rat optic nerve head were stretched biaxially by 10 and 17% for 24 h, respectively. Then, we detected the morphology, proliferation and apoptosis of the stretched cells, and performed proteomics analysis. Protein expression was analyzed by Isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. Proteins of interest and signaling pathways were screened using Gene Ontology enrichment analysis and pathway enrichment analysis, and the results were verified by western blot and the gene-chip data from Gene Expression Omnibus (GEO) database. RESULT: The results showed that rearrangement of the actin cytoskeleton in response to stimulation by mechanical stress and proliferation rate of astrocytes decreased under 10 and 17% stretch condition, while there was no significant difference on the apoptosis rate of astrocytes in both groups. In the iTRAQ quantitative experiment, there were 141 differential proteins in the 10% stretch group and 140 differential proteins in the 17% stretch group. These proteins include low-density lipoprotein receptor-related protein (LRP6), caspase recruitment domain family, member 10 (CARD10), thrombospondin 1 (THBS1) and tetraspanin (CD81). The western blot results of LRP6, THBS1 and CD81 were consistent with that of iTRAQ experiment. ANTXR2 and CARD10 were both differentially expressed in the mass spectrometry results and GEO database. We also screened out the signaling pathways associated with astrocyte activation, including Wnt/ß-catenin pathway, NF-κB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, ECM-receptor interaction, and transforming growth factor-ß (TGF-ß) signaling pathway. CONCLUSION: Mechanical stimulation can induce changes in cell phenotype, some proteins and signaling pathways, which might be associated with astrocyte activation. These proteins and signaling pathways may help us have a better understanding on the activation of astrocytes and the role astrocyte activation played in glaucomatous optic neuropathy.

[Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3].

OBJECTIVE: To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. METHODS: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. RESULTS: The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 µg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 µg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 µg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 µg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 µg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while ß-TG could not. CD36 antibody FA6-152 (25 µg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. CONCLUSION: TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.

Auranofin attenuates hepatic steatosis and fibrosis in nonalcoholic fatty liver disease via NRF2 and NF- κB signaling pathways.

BACKGROUND/AIMS: We aim to evaluate the effects of auranofin, a known antioxidant, on hepatic steatosis, inflammation, and fibrosis, contributing to non-alcoholic steatohepatitis (NASH) development in vivo and in vitro. METHODS: Transcriptome analysis of LX-2 cells was that expression patterns of genes changed by auranofin, and their related pathways were estimated. We used the gene set enrichment analysis (GSEA) program to determine the pathway involved in overall genetic change. In vitro, LX-2 and HepG2 cells were treated with transforming growth factor (TGF)-ß1 and palmitic acid (PA), respectively, and the antifibrotic and antiadipogenic effect function of auranofin was evaluated. RESULTS: Transcriptome analysis revealed that auranofin decreased the expression of 15 genes, including thrombospondin 1, endothelin 1 (ET-1), fibronectin 1, and LOX. The molecular functions of these genes are involved in collagen binding. GSEA of the overall gene expression pattern revealed that many genes increased in the reactive oxygen species pathway and decreased in the inflammatory response. Auranofin decreased nuclear factor kappa B (NF-κB) and IκBα in TGF-ß1-induced LX-2 cells, thereby reducing ET-1 and fibrosis. Furthermore, increased pNRF2 in PA-induced HepG2 cells led to increased antioxidant marker expression and decreased lipid accumulation. In the bile duct ligation model mice, auranofin reduced the fibrosis area and increased the survival rate. Auranofin reduced liver fibrosis and lipid accumulation in NASH model mice fed on a Western diet. CONCLUSION: Auranofin inhibits lipogenesis and fibrosis formation and is a proposed candidate for NASH treatment.

A biocompatible nanoparticle-based approach to inhibiting renal ischemia reperfusion injury in mice by blocking thrombospondin-1 activity.

Thrombospondin-1 (TSP-1) is a key mediator of renal ischemia-reperfusion injury (IRI), a major cause of kidney dysfunction under various disease conditions and a risk factor of renal allograft rejection. In this study, we developed a nanotechnology-based therapy targeting TSP-1 to prevent renal IRI. A biocompatible nanoparticle (NP) capable of specific binding to TSP-1 was prepared by conjugating NPs with TSP-1-binding (LSKL) peptides. LSKL/NPs not only effectively adsorbed recombinant TSP-1 proteins in vitro, but also efficiently neutralized TSP-1 in mice undergoing renal IRI. IRI-induced elevation of TSP-1 in the kidney was significantly inhibited by post-IR treatment with LSKL/NPs, but not free LSKL or NPs. Furthermore, TSP-1 proteins adsorbed on LSKL/NPs were functionally inactive and unable to induce apoptosis in renal tubular epithelial cells. Importantly, LSKL/NPs induced strong protection against renal IRI, as shown by markedly diminished serum creatinine levels and improved histological lesions of the kidney. Thus, LSKL/NPs provide a useful means of depleting and inactivating TSP-1 and a potential therapy for renal IRI.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

OBJECTIVE: The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). METHODS: HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. RESULTS: HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. CONCLUSION: Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

Sphingomyelin nanosystems decorated with TSP-1 derived peptide targeting senescent cells.

Senescent cells accumulation can contribute to the development of several age-related diseases, including cancer. Targeting and eliminating senescence cells, would allow the development of new therapeutic approaches for the treatment of different diseases. The 4N1Ks peptide, a 10 amino acid peptide derived from TSP1 protein, combines both features by targeting the CD47 receptor present in the surface of senescent cells and demonstrating senolytic activity, thereby representing a new strategy to take into account. Nonetheless, peptide drugs are known for their biopharmaceutical issues, such as low short half-life and tendency to aggregate, which reduces their bioavailability and limits their therapeutic potential. In order to overcome this problem, herein we propose the use of biodegradable and biocompatible sphingomyelin nanosystems (SNs), decorated with this peptide for the targeting of senescent cells. In order to efficiently associate the 4N1Ks peptide to the nanosystems while exposing it on their surface for an effective targeting of senescent cells, the 4N1Ks peptide was chemically conjugated to a PEGylated hydrophobic chain. The resulting SNs-4N1Ks (SNs-Ks), were extensively characterized for their physicochemical properties, by dynamic light scattering, multiple-angle dynamic light scattering, nanoparticle tracking analysis and atomic force microscopy. The SNs-Ks demonstrated suitable features in terms of size (∼100 nm), association efficiency (87.2 ± 6.9%) and stability in different biorelevant media. Cell toxicity experiments in MCF7 cancer cells indicated an improved cytotoxic effect of SNs-Ks, decreasing cancer cells capacity to form colonies, with respect to free peptide, and an improved hemocompatibility. Lastly, senescence escape preliminary experiments demonstrated the improvement of SNs-Ks senolytic activity of in chemotherapy-induced senescence model of breast cancer cells. Therefore, these results demonstrate for the first time the potential of the combination of SNs with 4N1Ks peptide for the development of innovative senolytic therapies to battle cancer.

Thrombospondin-1 Plays a Major Pathogenic Role in Experimental and Human Bronchopulmonary Dysplasia.

Rationale: Extremely preterm infants develop bronchopulmonary dysplasia (BPD), a chronic lung injury that lacks effective treatment. TSP-1 (thrombospondin-1) is an antiangiogenic protein that activates TGF-ß1 (transforming growth factor-ß1), a cytokine strongly linked to both experimental and human BPD. Objectives:1) To examine effects of inhibiting TSP-1-mediated TGF-ß1 activation (LSKL [leucine-serine-lysine-leucine]) in neonatal rats with bleomycin-induced lung injury; 2) to examine effects of a TSP-1 mimic (ABT-510) on lung morphology; and 3) to determine whether TSP-1 and related signaling peptides are increased in lungs of human preterm infants at risk for BPD. Methods: From Postnatal Days 1 to 14, rat pups received daily intraperitoneal bleomycin (1 mg/kg) or vehicle and were treated with daily subcutaneous LSKL (20 mg/kg) or vehicle alone. Separate animals were treated with vehicle or ABT-510 (30 mg/kg/d). Paraffin-embedded lung tissues from 47 autopsies (controls; death <28 d, n = 30 and BPD at risk; death ⩾28 d, n = 17) performed on infants born <29 completed weeks' gestation were semiquantified for injury markers (collagen, macrophages, and 3-nitrotyrosine), TSP-1, and TGF-ß1. Measurements and Main Results: Bleomycin or ABT-510 increased lung TGF-ß1 activity and macrophage influx, caused pulmonary hypertension, and led to alveolar and microvascular hypoplasia. Treatment with LSKL partially prevented abnormal lung morphology secondary to bleomycin. Lungs from human infants at risk for BPD had increased contents of TSP-1 and TGF-ß1 when compared with controls. TGF-ß1 content correlated with markers of lung injury. Conclusions: TSP-1 inhibits alveologenesis in neonatal rats, in part via the upregulated activity of TGF-ß1. Observations in human lungs suggest a similar pathogenic role for TSP-1 in infants at risk for BPD.

Addition of an Fc-IgG induces receptor clustering and increases the in vitro efficacy and in vivo anti-tumor properties of the thrombospondin-1 type I repeats (3TSR) in a mouse model of advanced stage ovarian cancer.

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.

Potential of thrombospondin-1 in treatment of polycystic ovary syndrome rat model: a preliminary study.

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.

Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

Effects of artemisinin and TSP­1­human endometrial­derived stem cells on a streptozocin­induced model of Alzheimer's disease and diabetes in Wistar rats.

Alzheimer's disease (AD) is an age­associated dementia disorder characterized by Aß plaques and neurofibrillary tangles. There is a strong link between cerebrovascular angiopathy, oxidative stress, inflammation, and glucose metabolism abnormalities with the development of AD. In this study, we investigated the therapeutic influences of artemisinin and TSP­1­human endometrial­derived stem cells (TSP­1­hEDSCs) on the streptozocin­induced model of AD and diabetes in rats. Hippocampal and intraperitoneal injections of streptozocin were used to induce AD and diabetes in male Wistar rats, followed by intranasal administration of a single dose of TSP­1­hEDSCs and intraperitoneal administration of artemisinin for 4 weeks. Hematoxylin together with eosin staining was performed for demonstrating Aß plaque formation and for analyzing the influence of treatments on the pyramidal cells in the hippocampus. Biochemical analysis was used to assay the serum levels of glucose, MDA, ROS, and TAC. The expression of TNF­α was measured using real­time PCR. Streptozocin induced AD and diabetes via Aß plaque formation and increasing blood glucose levels. It also increased the levels of ROS, MDA, and TNF­α and decreased the levels of TAC. Simultaneous or separate administration of artemisinin and TSP­1­hEDSCs ameliorated this influence by considerably reducing Aß plaque formation in the hippocampus, reducing glucose, MDA, ROS, and TNF­α levels, and increasing TAC levels. It appears that artemisinin and TSP­1­hEDSCs improve the adverse features of AD in a rat model of AD and diabetes. Therefore, artemisinin and TSP­1­hEDSCs could be utilized as an adjunct treatment, as well as a protective agent, in AD patients.

Age-Dependent Control of Collagen-Dependent Platelet Responses by Thrombospondin-1-Comparative Analysis of Platelets from Neonates, Children, Adolescents, and Adults.

Platelet function is developmentally regulated. Healthy neonates do not spontaneously bleed, but their platelets are hypo-reactive to several agonists. The mechanisms underlying immature platelet function in neonates are incompletely understood. This critical issue remains challenging for the establishment of age-specific reference ranges. In this study, we evaluated platelet reactivity of five pediatric age categories, ranging from healthy full-term neonates up to adolescents (11-18 years) in comparison to healthy adults (>18 years) by flow cytometry. We confirmed that platelet hypo-reactivity detected by fibrinogen binding, P-selectin, and CD63 surface expression was most pronounced in neonates compared to other pediatric age groups. However, maturation of platelet responsiveness varied with age, agonist, and activation marker. In contrast to TRAP and ADP, collagen-induced platelet activation was nearly absent in neonates. Granule secretion markedly remained impaired at least up to 10 years of age compared to adults. We show for the first time that neonatal platelets are deficient in thrombospondin-1, and exogenous platelet-derived thrombospondin-1 allows platelet responsiveness to collagen. Platelets from all pediatric age groups normally responded to the C-terminal thrombospondin-1 peptide RFYVVMWK. Thus, thrombospondin-1 deficiency of neonatal platelets might contribute to the relatively impaired response to collagen, and platelet-derived thrombospondin-1 may control distinct collagen-induced platelet responses.

Thrombospondin-1 promotes hemostasis through modulation of cAMP signaling in blood platelets.

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1-/- platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.