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Polydatin, a natural derivative of resveratrol, has shown many anticancer properties. However, the underlying mechanisms of its anticancer properties including its effect on the epigenetic landscape are not well understood. Here, we explored the effect of polydatin on histone deacetylase 1 (HDAC1) activity. We used in silico approaches to assess the possible binding of polydatin to the active site pockets of HDAC1 and in vitro approaches to test the potential effects of the interaction on its enzymatic activity. As compared to SAHA, an approved drug, the polydatin showed stronger and stable binding to the HDAC1. The binding energy, conformational changes, formation of extra hydrogen bonding, and other interactions within and outside the active site all favour largely stable and strong polydatin binding to the enzyme. Further, the ADME and toxicity prediction values are encouraging for the evaluation of polydatin as a drug. The laboratory leg of the study substantiated that the polydatin binding was strong and stable enough to inhibit HDAC1 activity in UMS-CC-22B cells as demonstrated by an increase in H3K9 acetylation. In addition, polydatin treated cells showed attenuated proliferation. The in vitro tube formation and migration by HUVEC and UM-SCC-22B cells were inhibited by polydatin. The decreased tube formation due to HDAC1 inhibition is possibly due to up-regulation of the anti-angiogenic gene - TSP1 in UM-SCC-22B cells. As compared to SAHA, more promising results were shown both in its computational calculations and on the cell physiology features. Stronger and stable binding, more anti-proliferative and anti-angiogenic potential were observed with respect to polydatin. Further, the cell death was more pronounced with SAHA treatment. Therefore, polydatin might be a better anticancer drug and can have a potential to replace SAHA in combinational therapeutic regimen. [ABSTRACT FROM AUTHOR]

Cluster of differentiation 47 (CD47) occupies the outer membrane of human cells, where it binds to soluble and cell surface receptors on the same and other cells, sculpting their topography and resulting in a pleiotropic receptor‐multiligand interaction network. It is a focus of drug development to temper and accentuate CD47‐driven immune cell liaisons, although consideration of on‐target CD47 effects remain neglected. And yet, a late clinical trial of a CD47‐blocking antibody was discontinued, existent trials were restrained, and development of CD47‐targeting agents halted by some pharmaceutical companies. At this point, if CD47 can be exploited for clinical advantage remains to be determined. Herein an airing is made of the seemingly conflicting actions of CD47 that reflect its position as a junction connecting receptors and signalling pathways that impact numerous human cell types. Prospects of CD47 boosting and blocking are considered along with potential therapeutic implications for autoimmune diseases and cancer. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease with a certain degree of chronic inflammation and abnormal ovarian angiogenesis in reproductive women. Mesenchymal stem cells (MSCs) have potent immunomodulatory properties to regulate ovarian function, while thrombospondin 1 (TSP1) improves the abnormal formation of ovarian vessels. The present study investigated the efficacy of the combined use of adipose‐derived mesenchymal stem cells (ADSCs) and TSP1 in PCOS mice. The PCOS model is established using dehydroepiandrosterone (DHEA) by subcutaneous injection. Ovarian apoptosis is assessed using terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL). Real‐time quantitative PCR (RT‐PCR) and western blotting are used to detect the expression of inflammatory factors and the levels of angiogenesis‐related factors in ovarian tissues. Inflammatory cells count and ovarian angiogenesis are evaluated by immunofluorescence staining. This research shows that TSP1 and ADSCs treatment can significantly reduce the inflammatory state of PCOS mice, relieve the degree of ovarian cell apoptosis, optimize the ovarian histological manifestations, and restore the levels of related hormones. The proportion of CD31‐positive cells in PCOS mice returned to near‐normal levels. The synergistic use of ADSCs and TSP1 therapy can exert a more impressive effect by inhibiting the ovarian inflammatory response and regulating the balance of angiogenesis than the single application in PCOS mice. [ABSTRACT FROM AUTHOR]

Background & Aims: Recent studies have implicated platelets, particularly α-granules, in the development of non-alcoholic steatohepatitis (NASH). However, the specific mechanisms involved have yet to be determined. Notably, thrombospondin 1 (TSP1) is a major component of the platelet α-granules released during platelet activation. Hence, we aimed to determine the role of platelet-derived TSP1 in NASH. Methods: Platelet-specific Tsp1 knockout mice (TSP1Δpf4) and their wild-type littermates (TSP1F/F) were used. NASH was induced by feeding the mice with a diet enriched in fat, sucrose, fructose, and cholesterol (AMLN diet). A human liver NASH organoid model was also employed. Results: Although TSP1 deletion in platelets did not affect diet-induced steatosis, TSP1Δpf4 mice exhibited attenuated NASH and liver fibrosis, accompanied by improvements in plasma glucose and lipid homeostasis. Furthermore, TSP1Δpf4 mice showed reduced intrahepatic platelet accumulation, activation, and chemokine production, correlating with decreased immune cell infiltration into the liver. Consequently, this diminished proinflammatory signaling in the liver, thereby mitigating the progression of NAFLD. Moreover, in vitro data revealed that co-culturing TSP1-deficient platelets in a human liver NASH organoid model attenuated hepatic stellate cell activation and NASH progression. Additionally, TSP1-deficient platelets play a role in regulating brown fat endocrine function, specifically affecting Nrg4 (neuregulin 4) production. Crosstalk between brown fat and the liver may also influence the progression of NAFLD. Conclusions: These data suggest that platelet α-granule-derived TSP1 is a significant contributor to diet-induced NASH and fibrosis, potentially serving as a new therapeutic target for this severe liver disease. Impact and implications: Recent studies have implicated platelets, specifically α-granules, in the development of non-alcoholic steatohepatitis, yet the precise mechanisms remain unknown. In this study, through the utilization of a tissue-specific knockout mouse model and human 3D liver organoid, we demonstrated that platelet α-granule-derived TSP1 significantly contributes to diet-induced non-alcoholic steatohepatitis and fibrosis. This contribution is, in part, attributed to the regulation of intrahepatic immune cell infiltration and potential crosstalk between fat and the liver. These findings suggest that platelet-derived TSP1 may represent a novel therapeutic target in non-alcoholic fatty liver disease.

Abstract Background Natural killer (NK) cells play an important first-line role against tumour and viral infections and are regulated by inhibitory receptor expression. Among these inhibitory receptors, the expression, function, and mechanism of cluster of differentiation 47 (CD47) on NK cells during human immunodeficiency virus (HIV) infection remain unclear. Methods Fresh peripheral blood mononuclear cells (PBMCs) were collected from people living with HIV (PLWH) and HIV negative controls (NC) subjects. Soluble ligand expression levels of CD47 were measured using ELISA. HIV viral proteins or Toll-like receptor 7/8 (TLR7/8) agonist was used to investigate the mechanisms underlying the upregulation of CD47 expression. The effect of CD47 on NK cell activation, proliferation, and function were evaluated by flow cytometry. RNA-seq was used to identify downstream pathways for CD47 and its ligand interactions. A small molecule inhibitor was used to restore the inhibition of NK cell function by CD47 signalling. Results CD47 expression was highly upregulated on the NK cells from PLWH, which could be due to activation of the Toll-like receptor 7/8 (TLR7/8) pathway. Compared with NC subjects, PLWH subjects exhibited elevated levels of CD47 ligands, thrombospondin-1 (TSP1), and counter ligand signal regulatory protein-α (SIRPα). The TSP1–CD47 axis drives the suppression of interferon gamma (IFN-γ) production and the activation of the Janus kinase signal transducer and activator of transcription (JAK–STAT) pathway in NK cells. After treatment with a STAT3 inhibitor, the NK cells from PLWH showed significantly improved IFN-γ production. Conclusions The current data indicate that the binding of the inhibitory receptor CD47 to plasma TSP1 suppresses NK cell IFN-γ production by activating the JAK/STAT3 pathway during HIV infection. Our results suggest that CD47 and its related signalling pathways could be targets for improving NK cell function in people living with HIV.

Background: Natural killer (NK) cells play an important first-line role against tumour and viral infections and are regulated by inhibitory receptor expression. Among these inhibitory receptors, the expression, function, and mechanism of cluster of differentiation 47 (CD47) on NK cells during human immunodeficiency virus (HIV) infection remain unclear. Methods: Fresh peripheral blood mononuclear cells (PBMCs) were collected from people living with HIV (PLWH) and HIV negative controls (NC) subjects. Soluble ligand expression levels of CD47 were measured using ELISA. HIV viral proteins or Toll-like receptor 7/8 (TLR7/8) agonist was used to investigate the mechanisms underlying the upregulation of CD47 expression. The effect of CD47 on NK cell activation, proliferation, and function were evaluated by flow cytometry. RNA-seq was used to identify downstream pathways for CD47 and its ligand interactions. A small molecule inhibitor was used to restore the inhibition of NK cell function by CD47 signalling. Results: CD47 expression was highly upregulated on the NK cells from PLWH, which could be due to activation of the Toll-like receptor 7/8 (TLR7/8) pathway. Compared with NC subjects, PLWH subjects exhibited elevated levels of CD47 ligands, thrombospondin-1 (TSP1), and counter ligand signal regulatory protein-α (SIRPα). The TSP1–CD47 axis drives the suppression of interferon gamma (IFN-γ) production and the activation of the Janus kinase signal transducer and activator of transcription (JAK–STAT) pathway in NK cells. After treatment with a STAT3 inhibitor, the NK cells from PLWH showed significantly improved IFN-γ production. Conclusions: The current data indicate that the binding of the inhibitory receptor CD47 to plasma TSP1 suppresses NK cell IFN-γ production by activating the JAK/STAT3 pathway during HIV infection. Our results suggest that CD47 and its related signalling pathways could be targets for improving NK cell function in people living with HIV. [ABSTRACT FROM AUTHOR]

Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24 h in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and inhibition of the receptor for thrombospondins prevented the increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, where neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis. [ABSTRACT FROM AUTHOR]

Abstract Objective and design An accumulating body of evidence has shown that gut microbiota is involved in regulating inflammation; however, it remains undetermined if and how gut microbiota plays an important role in modulating deep venous thrombosis (DVT), which is an inflammation-involved thrombotic event. Subjects Mice under different treatments were used in this study. Methods and treatment We induced stenosis DVT in mice by partially ligating the inferior vena cava. Mice were treated with antibiotics, prebiotics, probiotics, or inflammatory reagents to modulate inflammatory states, and their effects on the levels of circulating LPS and DVT were examined. Results Antibiotic-treated mice or germ-free mice exhibited compromised DVT. Treatment of mice with either prebiotics or probiotics effectively suppressed DVT, which was accompanied with the downregulation of circulating LPS. Restoration of circulating LPS in these mice with a low dose of LPS was able to restore DVT. LPS-induced DVT was blocked by a TLR4 antagonist. By performing proteomic analysis, we identified TSP1 as one of the downstream effectors of circulating LPS in DVT. Conclusion These results suggest that gut microbiota may play a nonnegligible role in modulating DVT by leveraging the levels of LPS in circulation, thus shedding light on the development of gut microbiota-based strategies for preventing and treating DVT.

Secretory and membrane proteins are vital for cell activities, including intra- and intercellular communication. Therefore, protein quality control in the endoplasmic reticulum (ER) is an essential and crucial process for eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) targets misfolded proteins during the protein maturation process in the ER and leads to their disposal. This process maintains the ER productive function and prevents misfolded protein stress (i.e., ER stress). The ERAD-stimulating factor ER degradation-enhancing α mannosidase-like 1 protein (EDEM1) acts on misfolded proteins to accelerate ERAD, thereby maintaining the productivity of the ER. However, the detail mechanism underlying the function of EDEM1 in ERAD is not completely understood due to a lack of established physiological substrate proteins. In this study, we attempted to identify substrate proteins for EDEM1 using siRNA. The matrix component thrombospondin-1 (TSP1) and epidermal growth factor receptor (EGFR) were identified as candidate targets of EDEM1. Their protein maturation status and cellular localization were markedly affected by knockdown of EDEM1. We also showed that EDEM1 physically associates with EGFR and enhances EGFR degradation via ERAD. Our data highlight the physiological role of EDEM1 in maintaining specific target proteins and provide a potential approach to the regulation of expression of clinically important proteins. [ABSTRACT FROM AUTHOR]

Objective and design: An accumulating body of evidence has shown that gut microbiota is involved in regulating inflammation; however, it remains undetermined if and how gut microbiota plays an important role in modulating deep venous thrombosis (DVT), which is an inflammation-involved thrombotic event. Subjects: Mice under different treatments were used in this study. Methods and treatment: We induced stenosis DVT in mice by partially ligating the inferior vena cava. Mice were treated with antibiotics, prebiotics, probiotics, or inflammatory reagents to modulate inflammatory states, and their effects on the levels of circulating LPS and DVT were examined. Results: Antibiotic-treated mice or germ-free mice exhibited compromised DVT. Treatment of mice with either prebiotics or probiotics effectively suppressed DVT, which was accompanied with the downregulation of circulating LPS. Restoration of circulating LPS in these mice with a low dose of LPS was able to restore DVT. LPS-induced DVT was blocked by a TLR4 antagonist. By performing proteomic analysis, we identified TSP1 as one of the downstream effectors of circulating LPS in DVT. Conclusion: These results suggest that gut microbiota may play a nonnegligible role in modulating DVT by leveraging the levels of LPS in circulation, thus shedding light on the development of gut microbiota-based strategies for preventing and treating DVT. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) serve as a barrier with forming a monolayer lining in the surface of vascular system. Many mature cell types are post-mitotic like neurons, but ECs have the ability to grow during angiogenesis. Vascular endothelial growth factor (VEGF) stimulates growth of vascular ECs derived from arteries, veins, and lymphatics and induces angiogenesis. Senescence of ECs is regarded as a key contributor in aging-induced vascular dysfunction via evoking increase of ECs permeability, impairment of angiogenesis and vascular repair. Several genomics and proteomics studies on ECs senescence reported changes in gene and protein expression that directly correlate with vascular systemic disorder. CD47 functions as a signaling receptor for secreted matricellular protein thrombospondin-1 (TSP1) and plays an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation, and atherosclerotic response. TSP1-CD47 signaling is upregulated with age in ECs, concurrent with suppression of key self-renewal genes. Recent studies indicate that CD47 is involved in regulation of senescence, self-renewal and inflammation. In this review, we highlight the functions of CD47 in senescent ECs, including modulation of cell cycle, mediation of inflammation and metabolism by the experimental studies, which may provide CD47 as a potential therapeutic target for aging-associated vascular dysfunction. [ABSTRACT FROM AUTHOR]

Abstract Background Radiation-induced bystander effect (RIBE) can promote tumor metastasis contributing to the failure of radiotherapy for esophageal squamous cell carcinoma (ESCC). Aberrant expression of DJ-1 has been identified in ESCC; however, the relationship between DJ-1 and RIBE in ESCC remains unknown. Methods We detected DJ-1 in the serum and cell supernatants by enzyme-linked immunosorbent assay (ELISA) and evaluated tumor metastasis by phenotypic experiments in vivo and in vitro. RNA-seq, mass spectrometry, western blot (WB), immunoprecipitation (IP), and dual-luciferase reporter assays were performed to explore the underlying mechanisms. Results DJ-1 was highly expressed in the serum of patients with ESCC receiving radiotherapy and was significantly overexpressed in the medium of ESCC cells receiving irradiation. DJ-1 promoted tumor metastasis via the TGF-β1 pathway. Mechanistic studies revealed that DJ-1 bound to HSC70 to promote Smad3 phosphorylation and nuclear aggregation in a protein-interaction manner, which activated the transcription of Thrombospondin-1 (TSP1). Subsequently, the activation of TGF-β1 by TSP1 re-promoted Smad3 phosphorylation and nuclear aggregation, constituting a positive feedback loop to strengthen the metastasis of ESCC cells, which was effectively blocked by LY2109761 and LSKL. Moreover, higher levels of serum DJ-1 in patients with ESCC were related to a poorer prognosis of radiotherapy. Conclusions Irradiation can induce ESCC cells secreting DJ-1. Secreted DJ-1 enters bystander cells to initiate activation of the TGF-β1 pathway via the DJ-1/HSC70/Smad3 signaling axis. The TSP1/TGF-β1/Smad3 positive feedback pathway constitutes the core pathway that promotes ESCC metastasis. DJ-1 is a useful biomarker for predicting the efficacy of radiotherapy and a potential therapeutic target for reversing RIBE in ESCC. Graphical Abstract Schematic diagram showing the underlying mechanism that irradiation-induced secretion of DJ-1 accelerates the metastasis of bystander ESCC cells.

Background: Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). The secreted glycoprotein thrombospondin‐1 (TSP1), a ligand for receptor CD47, is widely expressed on both systemic and pulmonary vascular cells. TSP1‐CD47 signaling has been reported to be one of the pathogeneses of pulmonary hypertension (PH). Methods: After creating a nitrofen‐induced CDH rat model, fetuses were sacrificed on D17, D19 and D21 and divided into a control group and a CDH group. Quantitative real‐time polymerase chain reaction was performed to determine the pulmonary gene expression of TSP1, CD47 and Runx3 (a regulator of TSP1). An immunofluorescence study was performed to evaluate the expression and localization of TSP1, CD47 and Runx3. Results: The relative mRNA expression of pulmonary TSP1, CD47 and Runx3 on D21 was significantly increased in the CDH group (p = 0.005, p = 0.001, p = 0.046, and p = 0.002, respectively). The immunofluorescence study also confirmed the overexpression of TSP1, CD47 and Runx3 in the CDH group. Conclusion: Our results provide evidence that TSP1‐CD47 signaling is involved in the pathogenesis of PH in a nitrofen‐induced CDH model. Our data suggest that anti‐CD47 antibodies can be novel therapeutic targets for the treatment of PH in CDH. [ABSTRACT FROM AUTHOR]

Keloids are benign fibroproliferative diseases with abnormally proliferated bulges beyond the edge of the skin lesions, and they are characterized by uncontrolled fibroblast proliferation and excessive extracellular matrix deposition in the dermis. However, the definite mechanisms that increase fibroblast proliferation and collagen deposition in keloids remain unclear. Thrombospondin 1 (TSP1) has been suggested to play an important role in wound healing and fibrotic disorders, but its role in keloids is unknown. In this study, we aimed to clarify the specific role of TSP1 in keloids and explore the potential mechanism. Our results demonstrated that TSP1 was highly expressed in keloid lesions compared to normal skin. Knockdown of TSP1 in keloid fibroblasts decreased cell proliferation and collagen I deposition. Exogenous TSP1 treatment increased cell proliferation and collagen I deposition in normal fibroblasts. We further investigated the underlying mechanism and found that TSP1 promoted fibroblast proliferation and extracellular matrix deposition by upregulating the IL6/JAK2/STAT3 pathway. Moreover, we verified that TSP1 expression was positively correlated with IL6/STAT3 signalling activity in keloids. Taken together, our findings indicate that TSP1 promotes keloid development via the IL6/JAK2/STAT3 signalling pathway and blocking TSP1 may represent a potential strategy for keloid therapy. [ABSTRACT FROM AUTHOR]

Background: Radiation-induced bystander effect (RIBE) can promote tumor metastasis contributing to the failure of radiotherapy for esophageal squamous cell carcinoma (ESCC). Aberrant expression of DJ-1 has been identified in ESCC; however, the relationship between DJ-1 and RIBE in ESCC remains unknown. Methods: We detected DJ-1 in the serum and cell supernatants by enzyme-linked immunosorbent assay (ELISA) and evaluated tumor metastasis by phenotypic experiments in vivo and in vitro. RNA-seq, mass spectrometry, western blot (WB), immunoprecipitation (IP), and dual-luciferase reporter assays were performed to explore the underlying mechanisms. Results: DJ-1 was highly expressed in the serum of patients with ESCC receiving radiotherapy and was significantly overexpressed in the medium of ESCC cells receiving irradiation. DJ-1 promoted tumor metastasis via the TGF-β1 pathway. Mechanistic studies revealed that DJ-1 bound to HSC70 to promote Smad3 phosphorylation and nuclear aggregation in a protein-interaction manner, which activated the transcription of Thrombospondin-1 (TSP1). Subsequently, the activation of TGF-β1 by TSP1 re-promoted Smad3 phosphorylation and nuclear aggregation, constituting a positive feedback loop to strengthen the metastasis of ESCC cells, which was effectively blocked by LY2109761 and LSKL. Moreover, higher levels of serum DJ-1 in patients with ESCC were related to a poorer prognosis of radiotherapy. Conclusions: Irradiation can induce ESCC cells secreting DJ-1. Secreted DJ-1 enters bystander cells to initiate activation of the TGF-β1 pathway via the DJ-1/HSC70/Smad3 signaling axis. The TSP1/TGF-β1/Smad3 positive feedback pathway constitutes the core pathway that promotes ESCC metastasis. DJ-1 is a useful biomarker for predicting the efficacy of radiotherapy and a potential therapeutic target for reversing RIBE in ESCC. Schematic diagram showing the underlying mechanism that irradiation-induced secretion of DJ-1 accelerates the metastasis of bystander ESCC cells. [ABSTRACT FROM AUTHOR]

Trichoderma virens colonizes roots and develops a symbiotic relationship with plants where the fungal partner derives nutrients from plants and offers defence, in return. Tsp1, a small secreted cysteine-rich protein, was earlier found to be upregulated in co-cultivation of T. virens with maize roots. Tsp1 is well conserved in Ascomycota division of fungi, but none of its homologs have been studied yet. We have expressed and purified recombinant Tsp1, and resolved its structure to 1.25 Å resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a β barrel fold and forms dimer in structure as well as in solution form. DALI based structure analysis revealed the structure similarity with two known fungal effector proteins: Alt a1 and PevD1. Structure and evolutionary analysis suggested that Tsp1 belongs to a novel effector protein family. Tsp1 acted as an inducer of salicylic acid mediated susceptibility in plants, rendering maize plants more susceptible to a necrotrophic pathogen Cochliobolus heterostrophus , as observed using plant defence assay and RT-qPCR analysis. • High resolution crystal structure reveals that Tsp1 adopts β-barrel fold. • Structure similarity of Tsp1 was predicted with two known fungal effector proteins: Alt a1 and PevD1. • RT-qPCR analysis and plant defence assay show that Tsp1 is an inducer of salicylic acid pathway in plants. [ABSTRACT FROM AUTHOR]

[Display omitted] • Haemonchus contortus protein Hc -SPI-I8A functions as an inhibitor of blood clotting contributed by the TIL domain. • Hc -SPI-I8 is highly expressed in the parasitic stages and at the hypodermis, intestine and gonads of H. contortus. • Hc -SPI-I8 was in the same clade as the homologues, which could inhibit coagulation by interacting with clotting factors. • Hc -SPI-I8A is most likely disturbing the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain. • Hc -SPI-I8A might also interfering in the intrinsic coagulation cascade by an unknown mechanism. Haemonchus contortus , a blood-feeding nematode, inhibits blood coagulation at the site of infection to facilitate blood-sucking and digesting for successful parasitism. However, the mechanism underlying anti-coagulation at the host-parasite interface is largely unknown. In the current study, Hc-spi-i8 , which has two greatly different transcripts named Hc-spi-i8a and Hc-spi-i8b , respectively, was described. Hc -SPI-I8A was a serine protease inhibitor containing a trypsin inhibitor-like cysteine rich (TIL) domain, while Hc -SPI-I8B was not. Hc -SPI-I8A/B were primarily expressed in the hypodermis, intestines and gonads in the parasitic stages of H. contortus. Hc -SPI-I8A interacted with Ovis aries TSP1-containing protein (Oa TSP1CP), which was determined by yeast two-hybrid, co-immunoprecipitation (Co-IP), pull down and co-localization experiments. The blood clotting time contributed by the TIL domain was prolonged by Hc -SPI-I8A. Hc -SPI-I8A is most likely interfering in the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain and intrinsic coagulation cascade by an unknown mechanism. These findings depict a crucial point in the host-parasite interaction during H. contortus colonization, which should contribute to drug discovery and vaccine development in fighting against this important parasite worldwide. [ABSTRACT FROM AUTHOR]

Abstract Pathological angiogenesis contributes to cancer progression and chronic inflammatory diseases. In inflammatory bowel disease, the microvasculature expands by intussusceptive angiogenesis (IA), a poorly characterized mechanism involving increased blood flow and splitting of pre‐existing capillaries. In this report, mice lacking the protease MT1‐MMP in endothelial cells (MT1iΔEC) presented limited IA in the capillary plexus of the colon mucosa assessed by 3D imaging during 1% DSS‐induced colitis. This resulted in better tissue perfusion, preserved intestinal morphology, and milder disease activity index. Combined in vivo intravital microscopy and lentiviral rescue experiments with in vitro cell culture demonstrated that MT1‐MMP activity in endothelial cells is required for vasodilation and IA, as well as for nitric oxide production via binding of the C‐terminal fragment of MT1‐MMP substrate thrombospondin‐1 (TSP1) to CD47/αvβ3 integrin. Moreover, TSP1 levels were significantly higher in serum from IBD patients and in vivo administration of an anti‐MT1‐MMP inhibitory antibody or a nonamer peptide spanning the αvβ3 integrin binding site in TSP1 reduced IA during mouse colitis. Our results identify MT1‐MMP as a new actor in inflammatory IA and a promising therapeutic target for inflammatory bowel disease.

Background & Aims: Thrombospondin 1 (TSP1) is a multifunctional matricellular protein. We previously showed that TSP1 has an important role in obesity-associated metabolic complications, including inflammation, insulin resistance, cardiovascular, and renal disease. However, its contribution to obesity-associated non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD or NASH) remains largely unknown; thus, we aimed to determine its role. Methods: High-fat diet or AMLN (amylin liver NASH) diet-induced obese and insulin-resistant NAFLD/NASH mouse models were utilised, in addition to tissue-specific Tsp1-knockout mice, to determine the contribution of different cellular sources of obesity-induced TSP1 to NAFLD/NASH development. Results: Liver TSP1 levels were increased in experimental obese and insulin-resistant NAFLD/NASH mouse models as well as in obese patients with NASH. Moreover, TSP1 deletion in adipocytes did not protect mice from diet-induced NAFLD/NASH. However, myeloid/macrophage-specific TSP1 deletion protected mice against obesity-associated liver injury, accompanied by reduced liver inflammation and fibrosis. Importantly, this protection was independent of the levels of obesity and hepatic steatosis. Mechanistically, through an autocrine effect, macrophage-derived TSP1 suppressed Smpdl3b expression in liver, which amplified liver proinflammatory signalling (Toll-like receptor 4 signal pathway) and promoted NAFLD progression. Conclusions: Macrophage-derived TSP1 is a significant contributor to obesity-associated NAFLD/NASH development and progression and could serve as a therapeutic target for this disease. Lay summary: Obesity-associated non-alcoholic fatty liver disease is a most common chronic liver disease in the Western world and can progress to liver cirrhosis and cancer. No treatment is currently available for this disease. The present study reveals an important factor (macrophage-derived TSP1) that drives macrophage activation and non-alcoholic fatty liver disease development and progression and that could serve as a therapeutic target for non-alcoholic fatty liver disease/steatohepatitis.

Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), known to be independently associated with cardiovascular diseases. However, the effect of IH on cardiac fibrosis and molecular events involved in this process are unclear. Here, we tested IH in angiotensin II (Ang II)-induced cardiac fibrosis and signaling linked to fibroblast activation. IH triggered cardiac fibrosis and aggravated Ang II-induced cardiac dysfunction in mice. Plasma thrombospondin-1 (TSP1) content was upregulated in both IH-exposed mice and OSA patients. Moreover, both in vivo and in vitro results showed IH-induced cardiac fibroblast activation and increased TSP1 expression in cardiac fibroblasts. Mechanistically, phosphorylation of STAT3 at Tyr705 mediated the IH-induced TSP1 expression and fibroblast activation. Finally, STAT3 inhibitor S3I-201 or AAV9 carrying a periostin promoter driving the expression of shRNA targeting Stat3 significantly attenuated the synergistic effects of IH and Ang II on cardiac fibrosis in mice. This work suggests a potential therapeutic strategy for OSA-related fibrotic heart disease.

Background & Aims: Recent studies have implicated platelets, particularly α-granules, in the development of non-alcoholic steatohepatitis (NASH). However, the specific mechanisms involved have yet to be determined. Notably, thrombospondin 1 (TSP1) is a major component of the platelet α-granules released during platelet activation. Hence, we aimed to determine the role of platelet-derived TSP1 in NASH., Methods: Platelet-specific Tsp1 knockout mice ( TSP1 Δpf4 ) and their wild-type littermates ( TSP1 F/F ) were used. NASH was induced by feeding the mice with a diet enriched in fat, sucrose, fructose, and cholesterol (AMLN diet). A human liver NASH organoid model was also employed., Results: Although TSP1 deletion in platelets did not affect diet-induced steatosis, TSP1 Δpf4 mice exhibited attenuated NASH and liver fibrosis, accompanied by improvements in plasma glucose and lipid homeostasis. Furthermore, TSP1 data revealed that co-culturing TSP1-deficient platelets in a human liver NASH organoid model attenuated hepatic stellate cell activation and NASH progression. Additionally, TSP1-deficient platelets play a role in regulating brown fat endocrine function, specifically affecting Nrg4 (neuregulin 4) production. Crosstalk between brown fat and the liver may also influence the progression of NAFLD. Δpf4 mice showed reduced intrahepatic platelet accumulation, activation, and chemokine production, correlating with decreased immune cell infiltration into the liver. Consequently, this diminished proinflammatory signaling in the liver, thereby mitigating the progression of NAFLD. Moreover, in vitro data revealed that co-culturing TSP1-deficient platelets in a human liver NASH organoid model attenuated hepatic stellate cell activation and NASH progression. Additionally, TSP1-deficient platelets play a role in regulating brown fat endocrine function, specifically affecting Nrg4 (neuregulin 4) production. Crosstalk between brown fat and the liver may also influence the progression of NAFLD., Conclusions: These data suggest that platelet α-granule-derived TSP1 is a significant contributor to diet-induced NASH and fibrosis, potentially serving as a new therapeutic target for this severe liver disease., Impact and Implications: Recent studies have implicated platelets, specifically α-granules, in the development of non-alcoholic steatohepatitis, yet the precise mechanisms remain unknown. In this study, through the utilization of a tissue-specific knockout mouse model and human 3D liver organoid, we demonstrated that platelet α-granule-derived TSP1 significantly contributes to diet-induced non-alcoholic steatohepatitis and fibrosis. This contribution is, in part, attributed to the regulation of intrahepatic immune cell infiltration and potential crosstalk between fat and the liver. These findings suggest that platelet-derived TSP1 may represent a novel therapeutic target in non-alcoholic fatty liver disease., Competing Interests: The authors of this study declare that they do not have any conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2024 The Author(s).)

The immune regulatory cell dysfunction is associated with many immune diseases including food allergy (FA). This study aims to investigate the role of vasoactive intestinal peptide (VIP) in the maintenance of regulatory B cell (Br cell)'s immune suppressive functions by stabilizing thrombospondin (TSP1) expression. In this study, blood samples were collected from patients with food allergy (FA) and healthy control (HC) subjects. Br cells were isolated from the samples through flow cytometry cell sorting and analyzed by immunological approaches to determine the immune regulatory capacity. We found that the immune suppressive functions of Br cells were impaired in FA patients. The serum VIP levels were associated with the production of immune suppressive function-related mediators (interleukin-10, IL-10) of Br cells in FA patients. VIP counteracted IL-10 mRNA decay in Br cells by up regulating the TSP1 expression. TSP1 inhibited tristetraprolin (TTP) to prevent IL-10 mRNA decay in Br cells. Administration of VIP inhibited FA response through restoration of immune suppressive functions in Br cells. In conclusion, administration of VIP can alleviate FA response through up regulating expression of TSP1 to stabilize IL-10 expression in FA Br cells and recover the immune regulatory functions. The results have translational potential for the treatment of FA and other disorders associated with immune regulatory dysfunction of Br cells. [ABSTRACT FROM AUTHOR]

Premature ovarian insufficiency (POI) is clinically irreversible and seriously damages female fertility. We previously demonstrated that menstrual blood stromal cells (MenSCs)-derived exosomes (EXOs) effectively improved ovarian functions in the POI rat model. In this study, we investigated whether TSP1 is the key component in EXOs to ameliorate ovarian functions and further explored the molecular mechanism of EXOs in improving granulosa cell (GCs) activities. Our results demonstrated that knockdown TSP1 significantly debilitated the therapeutic effect of EXOs on estrous cyclicity, ovarian morphology, follicle numbers and pregnancy outcomes in 4-vinylcyclohexene diepoxide (VCD) induced POI rat model. In addition, EXOs treatment significantly promoted the activities and inhibited the apoptosis of VCD induced granulosa cells in vitro. Moreover, EXOs stimulation markedly activated the phosphorylation of SMAD3(Ser425) and AKT(Ser473), up-regulated the expressions of BCL2 and MDM2 as well as down-regulated the expressions of CASPASE3, CASPASE8, P53 and BAX. All these effects were supressed by SIS3, a inhibitor of TGF1/SMAD3. Our study revealed the key role of TSP1 in EXOs in improving POI pathology, restoring ovarian functions and GCs activities, andprovided a promising basis for EXOs in the treatment of ovarian dysfunction. [Display omitted] • MenSCs-exosomes significantly restored the ovarian activities and fertility of POI rat models. • Knockdown of TSP1 attenuated the therapeutic effect of MenSCs-exosomes in vivo and in vitro. • MenSCs-exosomes ameliorated granulosa cell apoptosis by activating SMAD3/AKT/MDM2 to inhibit the expression of P53. [ABSTRACT FROM AUTHOR]

The matricellular protein thrombospondin-1 (TSP1) is a potent inhibitor of angiogenesis. Specifically, TSP1 has been experimentally shown to inhibit signaling downstream of vascular endothelial growth factor (VEGF). The molecular mechanism of this inhibition is not entirely clear. We developed a detailed computational model of VEGF signaling to Akt–endothelial nitric oxide synthase (eNOS) to investigate the quantitative molecular mechanism of TSP1 inhibition. The model demonstrated that TSP1 acceleration of VEGFR2 degradation is sufficient to explain the inhibition of VEGFR2 and eNOS phosphorylation. However, Akt inhibition requires TSP1-induced phosphatase recruitment to VEGFR2. The model was then utilized to test various strategies for the rescue of VEGF signaling to Akt and eNOS. Inhibiting TSP1 was predicted to be not as effective as CD47 depletion in rescuing signaling to Akt. The model further predicts that combination strategy involving depletion of CD47 and inhibition of TSP1 binding to CD47 is necessary for effective recovery of signaling to eNOS. In all, computational modeling offers insight to molecular mechanisms involving TSP1 interaction with VEGF signaling and provides strategies for rescuing angiogenesis by targeting TSP1–CD47 axis.

Thrombospondin 1 (TSP1) is a multifunctional matricellular protein. Recent studies demonstrate that TSP1 is highly expressed in adipose tissue (AT) and positively associated with AT inflammation and insulin resistance (IR). In this study, the contribution of different cellular sources of TSP1 to obesity-induced metabolic complications is determined by using mice with either adipocyte or myeloid/macrophage-specific deletion of TSP1 in a diet-induced obese model. The results demonstrated that neither adipocyte nor myeloid/macrophage-specific deletion of TSP1 affected the development of long-term high-fat diet-induced obesity. Adipocyte-specific deletion of TSP1 did not protect mice from obesity-induced inflammation and IR. On the contrary, obese mice with myeloid/macrophage loss of TSP1 had reduced macrophage accumulation in AT, which was accompanied with reduced inflammation and improved glucose tolerance and insulin sensitivity compared with obese control mice. Reduced macrophage-derived-TGF-β1 signaling and adipose tissue fibrosis were also observed in long-term high-fat-fed mice with myeloid/macrophage-specific TSP1 deletion. Moreover, in vitro experiments demonstrated an autocrine effect of TSP1-mediated TGF-β activation in macrophages in obesity. Collectively this study highlights the critical contribution of myeloid/macrophage-derived TSP1 to obesity-associated chronic inflammation and IR, which may serve as a new therapeutic target for metabolic disease. [ABSTRACT FROM AUTHOR]

The matricellular protein thrombospondin-1 (TSP1) is a potent inhibitor of angiogenesis. Specifically, TSP1 has been experimentally shown to inhibit signaling downstream of vascular endothelial growth factor (VEGF). The molecular mechanism of this inhibition is not entirely clear. We developed a detailed computational model of VEGF signaling to Akt-endothelial nitric oxide synthase (eNOS) to investigate the quantitative molecular mechanism of TSP1 inhibition. The model demonstrated that TSP1 acceleration of VEGFR2 degradation is sufficient to explain the inhibition of VEGFR2 and eNOS phosphorylation. However, Akt inhibition requires TSP1-induced phosphatase recruitment to VEGFR2. The model was then utilized to test various strategies for the rescue of VEGF signaling to Akt and eNOS. Inhibiting TSP1 was predicted to be not as effective as CD47 depletion in rescuing signaling to Akt. The model further predicts that combination strategy involving depletion of CD47 and inhibition of TSP1 binding to CD47 is necessary for effective recovery of signaling to eNOS. In all, computational modeling offers insight to molecular mechanisms involving TSP1 interaction with VEGF signaling and provides strategies for rescuing angiogenesis by targeting TSP1-CD47 axis. [ABSTRACT FROM AUTHOR]

The mechanisms underlying the progression of prostate cancer (PCa) to neuroendocrine prostate cancer (NEPC), an aggressive PCa variant, are largely unclear. Two prominent NEPC phenotypes are elevated NE marker expression and heightened angiogenesis. Identifying the still elusive direct molecular links connecting angiogenesis and neuroendocrine differentiation (NED) is crucial for our understanding and targeting of NEPC. Here we found that histone deacetylase 2 (HDAC2), whose role in NEPC has not been reported, is one of the most upregulated epigenetic regulators in NEPC. HDAC2 promotes both NED and angiogenesis. G protein-coupled receptor kinase 3 (GRK3), also upregulated in NEPC, is a critical promoter for both phenotypes too. Of note, GRK3 phosphorylates HDAC2 at S394, which enhances HDAC2's epigenetic repression of potent anti-angiogenic factor Thrombospondin 1 (TSP1) and master NE-repressor RE1 Silencing Transcription Factor (REST). Intriguingly, REST suppresses angiogenesis while TSP1 suppresses NE marker expression in PCa cells, indicative of their novel functions and their synergy in cross-repressing the two phenotypes. Furthermore, the GRK3-HDAC2 pathway is activated by androgen deprivation therapy and hypoxia, both known to promote NED and angiogenesis in PCa. These results indicate that NED and angiogenesis converge on GRK3-enhanced HDAC2 suppression of REST and TSP1, which constitutes a key missing link between two prominent phenotypes of NEPC. • Established novel roles of HDAC2, REST and TSP1 in prostate cancer progression. • GRK3 phosphorylates HDAC2 which promotes neuroendocrine phenotype and angiogenesis. • GRK3-HDAC2 pathway is a key missing link between the two prominent cancer phenotypes. • The GRK3 pathway mediates cancer progression by androgen deprivation and hypoxia. [ABSTRACT FROM AUTHOR]

VEGF signaling through VEGFR2 is a central regulator of the angiogenic response. Inhibition of VEGF signaling by the stress-induced matricellular protein TSP1 plays a role in modulating the angiogenic response to VEGF in both health and disease. TSP1 binding to CD47 inhibits VEGFR2 activation. The full implications of this inhibitory interaction are unknown. We developed a detailed rule-based computational model to inquire if TSP1- CD47 signaling through VEGF had downstream effects upon ERK1/2 and calcium. Our Simulations suggest that enhanced degradation of VEGFR2 initiated by the binding of TSP1 to CD47 is sufficient to explain the inhibition of VEGFR2 phosphorylation, calcium elevation, and ERK1/2 activation downstream of VEGF. A complementary mechanism involving the recruitment of phosphatases to the VEGFR2 complex with consequent increase in the rate of receptor dephosphorylation may augment the inhibition of the VEGF signal. The model was then utilized to simulate the effect of inhibiting external TSP1 or the depletion of CD47 as potential therapeutic strategies in restoring VEGF signaling. Results suggest that depleting CD47 is a more efficient strategy in inhibiting the effects of TSP1/CD47 on VEGF signaling. Our results highlight the utility of in silico investigations in elucidating and clarifying molecular mechanisms at the intersection of TSP1 and VEGF biology and in differentiating between competing pro-angiogenic therapeutic strategies relevant to peripheral arterial disease (PAD) and wound healing. [ABSTRACT FROM AUTHOR]

Thrombospondin 1 (TSP1) is a matricellular glycoprotein that can be secreted by many cell types. Through binding to extracellular proteins and/or cell surface receptors, TSP1 modulates a variety of cellular functions. Since its discovery in 1971, TSP1 has been found to play important roles in multiple biological processes including angiogenesis, apoptosis, latent transforming growth factor-β activation, and immune regulation. Thrombospondin 1 is also involved in regulating many organ functions. However, the role of TSP1 in liver diseases has not been extensively addressed. In this review, we summarize the findings about the possible role that TSP1 plays in chronic liver diseases focusing on non-alcoholic fatty liver diseases, liver fibrosis, and hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Platelets and their granular contents influence both angiogenesis and breast cancer progression. This study was performed to assess the effect of breast cancer and its treatment on platelet biology and the response to inhibition of the platelet P2Y12 receptor. Receptor-specific platelet activation and inhibition was studied for three platelet-associated proteins important in cancer angiogenesis and progression, vascular endothelial growth factor (VEGF), thrombospondin1 (TSP1), and transforming growth factor beta 1 (TGF-β1). Twenty-four women with active breast cancer and 10 healthy controls not receiving antiplatelet therapy participated in the study.Ex vivoactivation of platelets in whole blood was accomplished using PAR1AP, PAR4AP, convulxin, and ADP. Platelet inhibition was accomplished using the P2Y12 receptor antagonist cangrelor (the in vitro equivalent of clopidogrel). VEGF, TSP1, and TGF-β1 were measured using standard ELISA. Platelet activation by ADP, PAR1, PAR4, and collagen receptors increased VEGF, TSP1, and TGF-β1 secretion in patients with breast cancer. Agonist-induced release of VEGF was greater in cancer patients as compared to healthy controls (p= 0.02 for ADP,p< 0.001 for PAR1AP, PAR4AP, and convulxin) despite a decrease in the efficiency of VEGF secretion in patients with breast cancer. These differences were not observed for TSP1 and TGF-β1 secretion. P2Y12 receptor inhibition decreased VEGF, TSP1, and TGF-β1 secretion. In patients with cancer, cangrelor inhibited TSP1 release to a greater extent than VEGF and TGF-β1 release. In patients with breast cancer, the magnitude of platelet inhibition achieved by cangrelor was greater than that achieved with healthy controls for all agonists and platelet proteins studied. While platelets are known to influence progression of breast cancer, our results show that breast cancer and its treatment influence the platelet phenotype by increasing the secretion of pro-angiogenic proteins following platelet activation, modulating the efficiency of platelet protein release as well as increasing the response to antiplatelet therapy. [ABSTRACT FROM AUTHOR]

Hantavirus infection is associated with two frequently fatal diseases in humans: Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The pathogenesis of hantavirus infection is complex and not fully understood; however, it is believed to involve virus-induced hyperinflammatory immune responses. Thrombospondin-1 (THBS1) is a large homotrimeric protein that plays a putative role in regulating blood homeostasis. Hyperresponsiveness to inflammatory stimuli has also been associated with defects in the THBS1 gene. Our data suggest that hantavirus infection of human umbilical cord vein endothelial cells (HUVEC) suppress the accumulation of THBS1 in the extracellular matrix. Additionally, this suppression is dependent on virus replication, implying a direct mechanism of action. Our data also imply that the pathogenic Andes and Hantaan strains inhibit THBS1 expression while the non-pathogenic Prospect Hill strain showed little inhibition. These observations suggest that a dysregulation of THBS1may contribute to the pathogenesis of hantavirus infection. [ABSTRACT FROM AUTHOR]

Obesity is associated with podocyte injury and the development of proteinuria. Elevated plasma free fatty acid is one of the characteristics of obesity and has been linked to podocyte dysfunction. However, the mechanisms remain unclear. In the current study, we examined the effect of saturated free fatty acid (FFA) on human podocyte apoptosis and function in vitro. The mechanism and its in vivo relevance were also determined. We found that FFA treatment induced human podocyte apoptosis and dysfunction, which was associated with increased expression of a matricellular protein-thrombospondin1 (TSP1). FFA stimulated TSP1 expression in podocytes at the transcriptional levels through activation of MAPK pathway. Addition of purified TSP1 to cell culture media induced podocyte apoptosis and dysfunction. Tis effect is though a TGF-β independent mechanism. Moreover, peptide treatment to block TSP1 binding to its receptor-CD36 attenuated FFA induced podocyte apoptosis, suggesting that TSP1/CD36 interaction mediates FFA-induced podocyte apoptosis. Importantly, using a diet-induced obese mouse model, in vivo data demonstrated that obesity-associated podocyte apoptosis and dysfunction were attenuated in TSP1 deficient mice as well as in CD36 deficient mice. Taken together, these studies provide novel evidence that the interaction of TSP1 with its receptor CD36 contributes to obesity — associated podocytopathy. [ABSTRACT FROM AUTHOR]

Glaucoma is an optic neuropathy affecting approximately 60 million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemm's canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin. Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma. [ABSTRACT FROM AUTHOR]

Abstract: The role of CCN family proteins has been proven to be of extreme importance in the process of cartilage development and endochondral ossification. The second member, CCN2, consists of 4 conserved modules that interact with a number of cofactors to display multiple functions. Although the potentially therapeutic effect of intact CCN2 on cartilage regeneration has been indicated by a number of studies, the regenerative effect of independent modules comprising CCN2 has never been evaluated before. This study aims to discover a more robust and effective CCN2 derivative to induce regeneration through assessing the effect of CCN2 independent modules on regeneration in vitro and in vivo, in comparison to the full length CCN2. In vitro evaluation using human chondrocytic cells showed a remarkable enhancing effect of several single modules on the gene expression of cartilaginous extracellular matrix components; whereas combinations of 2 or 3 modules rather diminished such effects. Interestingly, combination of all 4 modules redeemed the effect of intact CCN2 in vitro. Suspecting the re-assembly of the 4 modules, interaction among the modules was examined by surface plasmon resonance analysis. However, the results did not support the possible formation of a tetramodular complex. Next, the thrombospondin 1 type 1 repeat module (TSP1), which was found most promising in the experiments in vitro, and the combination of 4 modules were forwarded further to in vivo confirmation using 2 rat osteoarthritis (OA) models. As a result, TSP1 displayed more prominent regenerative effects than intact CCN2 on damaged cartilage. Unexpectedly, the combination of 4 modules showed limited effects in vivo. These results indicate the utility of TSP1 in the regenerative therapeutics of OA. Possible molecular mechanism that enables conditional reconstruction of CCN2 by 4 modules is discussed as well. [Copyright &y& Elsevier]

Background: A disintegrin and metallopeptidase with thrombospondin motif type 1 (ADAMTS1) is a recently discovered metalloproteinase with antiangiogenic activity. The function of ADAMTS1 in gastric cancer remains unknown. Therefore, we were interested in examining ADAMTS1 expression in human gastric cancer, as well as its possible correlation with angiogenesis. Methods: The mRNA and protein expression of ADAMTS1, thrombospondin type I (TSP1), and vascular endothelial growth factor (VEGF) was evaluated by RT-PCR and immunohistochemistry, respectively, in 56 paired tumor and normal tissue samples, and corresponding metastatic lymph nodes ( n = 42). Microvessel density (MVD) was also evaluated by immunohistochemistry. Results: ADAMTS1 mRNA and protein levels were significantly lower in primary tumors than in corresponding normal tissues, and were significantly higher in metastatic lymph nodes compared to their matched primary tumors. High ADAMTS1 mRNA and protein expression was found to be significantly associated with lymph node metastasis in primary tumors. There was a negative correlation between ADAMTS1 and VEGF mRNA and protein expression in primary gastric tumors and normal tissues. A negative correlation was also found between ADAMTS1 protein expression and MVD in primary gastric tumors. In contrast, no correlation was detected between ADAMTS1 and TSP1 mRNA and protein expression in primary gastric tumors, normal tissues, and metastatic lymph nodes. Conclusions: These findings suggest that ADAMTS1 expression is altered in primary gastric cancer and paired lymph node metastasis. In addition, ADAMTS1 has angioinhibitory effects in primary gastric cancer due to its low expression and negative correlation with VEGF and MVD. However, it appears to lose its anti-angiogenic activity in metastatic lymph nodes in gastric cancer. [ABSTRACT FROM AUTHOR]

Abstract: Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its auto-activation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7±0.8nM) and thrombin (7.9±0.04 U/mL×10–3) generation. Addition of TSP1 significantly enhanced thrombin (23.3±0.7 U/mL×10–3) but not FXa (8.5±0.6nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFβ) as shown by phosphorylation of the SMAD pathway, an event blunted by using a TGFβ receptor I inhibitor (TGFβRI). FXa- and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFβRI. In summary, assembly of the prothrombinase complex occurs on fibroblast''s surface leading to serine proteases generation, an event enhanced by TSP1 and associated with CTGF/CCN2 upregulation. These mechanisms may play an important role in human diseases associated with fibrosis. [Copyright &y& Elsevier]

Background: We explored the predictive significance of BRCA1, TXR1 and TSP1 expression in non-small-cell lung cancer (NSCLC) patients treated with docetaxel in association with cisplatin or gemcitabine.Methods: To analyse BRCA1, TXR1 and TSP1 mRNA expression from microdissected primary tumours of 131 patients with stage IIIB (wet) and IV NSCLC, RT-qPCR was used.Results: The mRNA levels of TXR1/TSP1 were inversely correlated (Spearman's test: -0.37; P=0.001). Low TXR1 mRNA levels were associated with higher response rate (RR; P=0.018), longer median progression-free survival (PFS; P=0.029) and median overall survival (mOS P=0.003), whereas high TSP1 expression was correlated with higher RR (P=0.035), longer PFS (P<0.001) and mOS (P<0.001). Higher BRCA1 mRNA expression was associated with higher RR (P=0.028) and increased PFS (P=0.021), but not mOS (P=0.4). Multivariate analysis demonstrated that low TXR1/high TSP1 expression was an independent factor for increased PFS (HR 0.49; 95% CI 0.32-0.76; P<0.001) and mOS (HR 0.37; 95% CI 0.2-0.58; P<0.001), whereas high BRCA1 expression was correlated with increased PFS (HR 0.53; 95% CI 0.37-0.78; P=0.001).Conclusions: These data indicate that TXR1/TSP1 and BRCA1 expression could be used for the prediction of taxanes' resistance in the treatment of NSCLC. [ABSTRACT FROM AUTHOR]

In a breast tumor xenograft model, the MCT-1 oncogene increases the in vivo tumorgenicity of MCF7 cells by promoting angiogenesis and inhibiting apoptosis. Increases in the tumor microvascular density are accompanied by a strong reduction in the levels of the angiogenesis inhibitor thrombospondin-1 (TSP1), but the mechanisms underlying this process are unknown. We show that TSP1 expression is controlled, at least in part, by post-transcriptional events. Using RNA interference to knock down the expression of the RNA-binding protein HuR in MCF7 cells as well as HuR overexpression, we demonstrate that HuR plays an important role in translation of the TSP1 mRNA. Furthermore, employing the RIP-Chip assay yielded 595 transcripts with significantly altered binding to HuR in the more tumorigenic breast cancer clones compared with the weakly tumorigenic clones. These mRNAs clustered in several pathways implicated in the transformed phenotype, such as the RAS pathway (involved in mitogenesis), the PI3K pathway (evasion of apoptosis) and pathways mediating angiogenesis and the cellular response to hypoxia. These findings demonstrate for the first time that global changes in HuR-bound mRNAs are implicated in the evolution to a more tumorigenic phenotype in an in vivo tumor model and underscore the role of global mRNA-protein interactions toward tumor progression.Oncogene (2008) 27, 6151–6163; doi:10.1038/onc.2008.215; published online 21 July 2008 [ABSTRACT FROM AUTHOR]

Angiogenesis is regulated by a balance of both angiogenic inducers and inhibitors. This study was designed to evaluate the effect of both maximum-tolerated doses (MTD) and low-dose metronomic chemotherapy (LDM) on serum vascular endothelial growth factor (VEGF), thrombospondin-1 (TSP1) and VEGFR1 concentrations in patients with advanced nonsmall cell lung cancer. Forty consecutive patients with advanced stage nonsmall cell lung cancer were included in this prospective study. Twenty patients received MTD chemotherapy including 75 mg/m2 of cisplatin and 75 mg/m2 of docetaxel on day 1. The LDM treatment consisted of cisplatin 25 mg/m2 and docetaxel 25 mg/m2 were given to other 20 patients on weeks 1, 2 and 3. Serum levels were prospectively measured in serum by ELISA at four times; before chemotherapy and at 1, 2 and 3 weeks following initiation of chemotherapy. The major finding in this study that MTD chemotherapy but not LDM chemotherapy resulted in significant changes in VEGFR1 and TSP1 serum levels. Due to the effect of LDM chemotherapy, we showed no statistically significant change in patients for all serum VEGF, TSP1 and VEGFR1 levels. Similarly, serum VEGF levels did not also change under MTD chemotherapy. The MTD chemotherapy induced significant and long-lasting increase of TSP1 levels and decrease of VEGFR1 levels that persisted for at least 3 weeks after the chemotherapy initiation. No significant correlations were found between serum VEGF and TSP1 levels in cancer patients treated with both LDM and MTD chemotherapy. The circulating angiogenic balance (TSP1/VEGF) is decreased in cancer patients ( P = 0.039). The continuous/metronomic chemotherapy may not achieve a more pronounced antiangiogenic effect than MTD-scheduling chemotherapy. Future studies involving a larger number of patients are needed to confirm the present findings. [ABSTRACT FROM AUTHOR]

Abstract: In the search for androgen target genes responsible for malignant growth in S115 mouse mammary tumor cells we found that thrombospondin 1 (TSP1) expression was strongly downregulated by testosterone (Te). Experiments with cycloheximide suggested that Te repression of TSP1 was dependent on de novo protein synthesis. TSP1 repression by Te was preceded by the induction of fibroblast growth factor 8 (FGF8) expression. FGF8 has previously been shown to mediate androgen effects on proliferation of S115 cells by autocrine/paracrine mechanisms. It has also been shown to increase breast cancer cell growth as tumors in nude mice and to stimulate tumor angiogenesis. We studied here the possibility that FGF8 belonged to the Te-induced de novo synthesized proteins that mediate the effect of Te on TSP1 expression in these cells. We found that addition of FGF8b to in vitro cultures or ectopic expression of FGF8b in S115 cells repressed TSP1 expression at mRNA and protein levels even in the absence of Te. FGF2, another angiogenic member of FGF family, also downregulated TSP1 mRNA level in the in vitro cultures of S115 cells. The antisense oligonucleotides for FGF8 did not, however, prevent Te-repression of TSP1 mRNA expression and a neutralizing anti-FGF8b antibody only partially opposed Te induced downregulation of TSP1. These results suggest that both androgen and FGF8 inhibit TSP1 expression independently. They also suggest that opposite to many other androgen-induced responses in S115 cells, the effect of Te on the expression TSP1 is not mediated by FGF8. [Copyright &y& Elsevier]

Human THROMBOSPONDIN-1 play versatile roles in platelet aggregation, angiogenesis, and tumorigenesis, which forms a disulfide-linked homotrimeric complex. Here we reported that the genomic lotus of TSP1 also transcribes a natural antisense transcript (NAT) with an overlapping and reverse complement region against TSP1 . The NAT of TSP1 was expressed in human testis, lung, thymus, colon, placenta, kidney and skeleton muscle, revealed by PCR amplification. It was also expressed in some tumor cell lines. The identification of NAT of TSP1 would be of significant importance to understand the functions of TSP1 , and it would also suggest the potential attempt of using RNA interference for related tumor therapy, for instance, breast cancer. [ABSTRACT FROM AUTHOR]