Thrombospondin-1 Levels Research Articles

Assessment of Thrombospondin-1 Level in Type II Diabetic Patients

The study is intended to asses serum levels of Thrombospondin-1, fasting blood glucose in type 2 diabetic patients, also correlation between Thrombospondin-1 in both males and females. The study was conducted on randomly selected 65 type 2 diabetic patients (34 males and 31 females) attending the diabetes mellitus center in Al-Sadder Teaching City in Al- Najaf province , Iraqi and a group of 24 apparently healthy subjects (12 Males and 12 Females) were included as a control group . The Study was carried out from August 2013 to February 2014.The patients' age was ranging from 35 to 64 years old. The results indicated a significant increase (p<0.05) in serum FBG and TSP-1 levels in diabetic patients in comparing with healthy groups. The results also revealed no significant differences (p> 0.05) in serum TSP-1 levels between males and females in both patients and healthy groups, while the results of FBG levels significantly (p<0.05) increase in females than males in both patients and healthy groups. The results have been shown significant positive correlation (P<0.05) between TSP-1 and FBG in (males and females), males, females DM patients. The present study concluded that Thrombospondin-1 were marker for detection and diagnosis of diabetic patients type 2.

Predicting value of thrombospondin-2 for coronary artery dilatation in patients with Kawasaki disease

To investigate the predictive value of thrombospondin-2 in children with coronary artery dilatation secondary to Kawasaki disease. This study was a retrospective controlled study, the patients were divided into Kawasaki disease group and control group.Patients first time hospitalized in Children's Hospital Affiliated to Capital Institute of Pediatrics during the acute phase of Kawasaki disease were enrolled in Kawasaki disease group from May 2013 to August 2014.Sixty-four Kawasaki disease patients were enrolled into the Kawasaki disease group based on inclusion and exclusion criteria, including 39 males and 25 females, with an average age of 2.3 years.According to the results of echocardiography of children with Kawasaki disease, patients were divided into coronary artery dilatation group and non-coronary artery dilatation group, there were 33 cases in the coronary dilatation (CAD) group, 26 males and 7 females, the average age was 2.0 years, and 31 cases in the non-coronary dilatation (NCAD) group, 13 males and 18 females, the average age was 2.6 years.The control group consisted of children with fever but excluding vascular inflammatory diseases and healthy children, 32 cases in fever group, 19 males and 13 females, mean age was 4.5 years; in which healthy group had 32 cases, 18 males and 14 females and the average age was 3.3 years. Enzyme linked immunosorbent assay (ELISA) method was used to measure the levels of thrombospondin-1 (TSP-1) and TSP-2 in the plasma of each group, and the related laboratory parameters, and general information was analyzed.Multiple sets of normal distribution of data were compared by analysis of unifactorial analysis of variance and the two groups were compared by two independent samples t-test.Analysis of covariance was used to remove the effect of age.The χ(2) test was used to analyze categeorical data and receiver operating characteristic (ROC) curve for evaluating the predictive value of TSP-2. Compared with the febrile group and healthy group, plasma TSP-2 and TSP-1 of Kawasaki disease group was significantly elevated ((33.6±12.5) vs. (21.4±8.8) and (16.4±2.9) μg/L, (10 949±7 241) vs. (7 174±4 807) and (3 661±2 672) μg/L, F=36.861 and 17.339, P both<0.01). The TSP-2 concentration in the group with coronary artery dilatation was significantly higher than the group without it ((36.7±13.1) vs. (30.4±11.1)μg/L, t=2.062, P=0.043). Regarding the predictive effect of TSP-2 for coronary artery dilatation, sensitivity was 54.5%, specificity was 80.6%, and the cut-off point was 33.9 μg/L.When TSP-2 was combined with albumin<35 g/L to predict coronary artery dilatation, the area under the ROC curve was 0.701, sensitivity was 60.0%, specificity was 82.4%, and the cut-off point was 33.8 μg/L. During the acute phase of Kawasaki disease, TSP-2 levels were significantly elevated.It could be used to predict the occurrence of coronary artery dilatation.The predictive value of TSP-2 was significantly improved when combined with albumin.

Decreased Serum Thrombospondin-1 Levels in Pancreatic Cancer Patients Up to 24 Months Prior to Clinical Diagnosis: Association with Diabetes Mellitus.

Identification of serum biomarkers enabling earlier diagnosis of pancreatic ductal adenocarcinoma (PDAC) could improve outcome. Serum protein profiles in patients with preclinical disease and at diagnosis were investigated. Serum from cases up to 4 years prior to PDAC diagnosis and controls (UKCTOCS,n= 174) were studied, alongside samples from patients diagnosed with PDAC, chronic pancreatitis, benign biliary disease, type 2 diabetes mellitus, and healthy subjects (n= 298). Isobaric tags for relative and absolute quantification (iTRAQ) enabled comparisons of pooled serum from a test set (n= 150). Validation was undertaken using multiple reaction monitoring (MRM) and/or Western blotting in all 472 human samples and samples from a KPC mouse model. iTRAQ identified thrombospondin-1 (TSP-1) as reduced preclinically and in diagnosed samples. MRM confirmed significant reduction in levels of TSP-1 up to 24 months prior to diagnosis. A combination of TSP-1 and CA19-9 gave an AUC of 0.86, significantly outperforming both markers alone (0.69 and 0.77, respectively;P< 0.01). TSP-1 was also decreased in PDAC patients compared with healthy controls (P< 0.05) and patients with benign biliary obstruction (P< 0.01). Low levels of TSP-1 correlated with poorer survival, preclinically (P< 0.05) and at clinical diagnosis (P< 0.02). In PDAC patients, reduced TSP-1 levels were more frequently observed in those with confirmed diabetes mellitus (P< 0.01). Significantly lower levels were also observed in PDAC patients with diabetes compared with individuals with type 2 diabetes mellitus (P= 0.01). Circulating TSP-1 levels decrease up to 24 months prior to diagnosis of PDAC and significantly enhance the diagnostic performance of CA19-9. The influence of diabetes mellitus on biomarker behavior should be considered in future studies.

Immunological and angiogenic markers during metronomic temozolomide and cyclophosphamide in canine cancer patients.

Metronomic chemotherapy stimulates the immune response via depletion of regulatory T cells (Tregs) and suppresses angiogenesis by modulating the secretion of thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF). In this study, blood was collected from 10 healthy dogs and from 30 canine cancer patients before and 2 and 4 weeks after treatment with metronomic temozolomide (6.6 mg m-2 ), cyclophosphamide (12.5 mg m-2 ) or cyclophosphamide and temozolomide. The percentage of circulating CD25+ Foxp3+ CD4+ Tregs and the plasma levels of TSP-1 and VEGF were measured. There was a significant difference in the percentage of Tregs between cancer patients and healthy dogs. A significant decrease in Tregs was noted in patients treated with metronomic cyclophosphamide and the combination. Treatment with temozolomide had no effect on the percentage of Tregs. TSP-1 and VEGF levels were, respectively, significantly lower and higher in cancer patients than in healthy dogs, but they were not influenced by any of the studied metronomic treatment regimens.

Thrombospondin-1 levels correlate with macrophage activity and disease progression in dysferlin deficient mice

Dysferlinopathy is associated with accumulation of thrombospondin (TSP)-1 and macrophages, both of which may contribute to the pathogenesis of the disease. The purpose of this study was to determine whether TSP-1 levels can predict macrophage activity and disease progression in dysferlin deficient BlaJ mice, focusing on the early disease process. In 3 month-old BlaJ mice, muscle TSP-1 levels exhibited strong positive correlations with both accumulation of F4/80hi macrophages and with their in vivo phagocytic activity in psoas muscles as measured by magnetic resonance imaging and flow cytometry. Muscle TSP-1 levels also exhibited a strong negative correlation with muscle mass and strong positive correlations with histological measurements of muscle fiber infiltration and regeneration. Over the course of disease progression from 3 to 12 months of age, muscle TSP-1 levels showed more complicated relationships with macrophage activity and an inverse relationship with muscle mass. Importantly, blood TSP-1 levels showed strong correlations with macrophage activity and muscle degeneration, particularly early in disease progression in BlaJ mice. These data indicate that TSP-1 may contribute to a destructive macrophage response in dysferlinopathy and pose the intriguing possibility that TSP-1 levels may serve as a biomarker for disease progression.

Effects of taxol resistance gene 1 expression on the chemosensitivity of SGC-7901 cells to oxaliplatin.

The present study aimed to evaluate the role of taxol resistance gene 1 (Txr1) in the development of oxaliplatin (L-OHP) resistance in gastric cancer (GC). Using SGC-7901 cells as a model, Txr1 was exogenously expressed or knocked down using small interfering RNA. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to establish whether the Txr1 gene is involved in chemoresistance, and cell proliferation was assessed using an MTS assay. To this end, the mRNA and protein levels of Txr1, thrombospondin-1 and excision repair cross-complementing 1 protein were measured using qPCR and western blotting, respectively. Txr1-knockdown significantly increased the sensitivity of the SGC-7901 cells to L-OHP, whereas Txr1 overexpression promoted the resistance of the SGC-7901 cells to L-OHP. Exogenous Txr1 expression in the SGC-7901 cells induced L-OHP resistance, and the siRNA knockdown of Txr1 sensitized the human GC cells to L-OHP. Txr1 is, therefore, likely to play a role in L-OHP resistance, acting via TSP1, and should be investigated as a potential therapeutic target in the treatment of GC.

The profile of platelet α-granule released molecules affects postoperative liver regeneration.

Platelets promote liver regeneration through site-specific serotonin release from dense granules, triggering proliferative signaling in hepatocytes. However, the effects of factors derived from platelet α-granules on liver regeneration are unclear, because α-granules contain bioactive molecules with opposing functions. Because α-granule molecules are stored in separate compartments, it has been suggested that platelets selectively release their α-granule content dependent on the environmental stimulus. Therefore, we investigated the pattern of circulating α-granule molecules during liver regeneration in 157 patients undergoing partial hepatectomy. We measured plasma levels of α-granule-derived factors in the liver vein at the end of liver resection, as well as on the first postoperative day. We observed a rapid accumulation of platelets within the liver after induction of liver regeneration. Platelet count and P-selectin (a ubiquitous cargo of α-granules) were not associated with postoperative liver dysfunction. However, low plasma levels of vascular endothelial growth factor (VEGF), but high levels of thrombospondin 1 (TSP-1), predicted liver dysfunction after resection. Patients with an unfavorable postoperative α-granule release profile (high TSP-1/low VEGF) showed substantially worse postoperative clinical outcomes. The unfavorable postoperative α-granule release profile was associated with increased postoperative portal venous pressure and von Willebrand factor antigen levels as a marker for intrahepatic endothelial dysfunction. The postoperative profile of circulating platelet-derived factors correlates with the ability of the remnant liver to regenerate. Portal venous pressure and intrahepatic endothelial dysfunction might account for the selective granule release profile. Selective modulation of platelet α-granule release in patients may represent an attractive target for therapeutic interventions to improve liver regeneration and clinical outcomes after partial hepatectomy.

Interleukin-17 potently increases non-small cell lung cancer growth.

The aim of the present study was to explore the effects of interleukin (IL)-17 on the growth and metastasis of tumors that were subcutaneously implanted into C57BL/6 mice. Lewis lung carcinoma (LLC) cells were subcutaneously injected into C57BL/6 mice followed by intraperitoneal injection of mouse recombinant IL-17 protein (IL-17 groups) or phosphate-buffered saline (control groups). Tumor growth and metastasis were assessed by measuring the size and weight of tumors and cervical lymph nodes, respectively. Cytokine expression in tumor masses was quantified by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. CCR2-positive macrophage infiltration in tumor masses was detected by flow cytometric analysis. The proliferation and migration of LLC cells, stimulated by the IL-17 protein were detected by Cell Counting kit (CCK)-8 and wound scratch assays in vitro. Tumors were grafted into the C57BL/6 mice. The mice that were intraperitoneally injected with IL-17 exhibited significantly larger tumors compared with the control mice. After day 7 of injection and beyond, the weight of cervical lymph nodes in IL-17 groups was higher than that in the control mice. It was also demonstrated that the number of CCR2-positive macrophages that infiltrated the tumor masses in the IL-17 groups was higher than that of the control mice. CD34 expression in vascular endothelial cells was also higher in tumors grafted in IL-17 mice than those grafted in control mice. Furthermore, the tumor tissue mRNA and protein expression levels of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, MMP-9 and tumor necrosis factor-α were greater in mice from the IL-17 group than the control mice, while levels of migration inhibitory factor and thrombospondin-1 were lower in mice from the IL-17 group than in the control. IL-17 also increased the migration of LLC cells in vitro. In conclusion, IL-17 exhibited the ability to promote tumor growth by increasing angiogenesis, metastasis and increasing CCR2+ macrophage infiltration into tumors.

Elevated Plasma Thrombospondin-1 (TSP1) Levels Are Correlated with Oxidative Stress and Worse Cognitive Function in Sickle Cell Disease

IntroductionCognitive impairment (CI) is a serious complication of sickle cell disease (SCD) with a prevalence of more than 40% (Vichinsky, JAMA 2010). It is responsible for functional limitations in schooling in children and in occupation and compliance with therapy in adults, and is therefore recognized as both a medical and social devastation of SCD. While the pathophysiology of SCD-related cognitive impairment is not fully elucidated, SCD patients are exposed to high levels of chronic inflammation and oxidative stress, which are responsible for cognitive impairment in other vascular diseases, suggesting that these factors may also play a role in SCD. Thrombospondin-1 (TSP1) is a secreted protein that stands at the crossroads of multiple inflammatory and oxidative pathways crucial to the SCD pathology (Isenberg, PNAS 2005, JBC 2006, Blood 2008, Csanyi, ATVB 2012; Yao, JASN 2014). Importantly, TSP1 levels are elevated in patients with SCD and correlate with an increased risk of vaso-occlusive complications in adults (Novelli, AJH 2012) and a higher burden of silent cerebral infarcts in children (Faulcon, BJH 2013). Herein, we explored the association between TSP1, oxidative stress and cognitive function in a cohort of patients with SCD.MethodsThirty-four SCD patients (HbSS, HbSC and HbS/β-thalassemia) >18 years and in steady state were recruited from the UPMC Adult Sickle Cell Program outpatient clinic (Pittsburgh, PA). Upon obtaining informed consent, patients underwent a blood draw and the Hopkins Verbal Learning Test - Revised (HVLT-R), a test of episodic memory whose scores are associated with cerebral small vessel disease in SCD patients (Novelli, PRN 2015). Platelet-poor plasma was collected and stored for analysis by ELISA of TSP1 and the oxidized derivative of deoxyguanosine, 8-hydroxy-2-deoxy guanosine (8-OH2dG), a marker of oxidative stress. Statistical analyses were completed using SAS 9.3.ResultsSCD patients had a mean age of 35 (22-65), were 65% female, 66% had greater than high school education, and 65% had HbSS disease, 30% had HbSC disease, and 5% had S/β-thalassemia. Elevated plasma TSP1 levels correlated inversely with HVLT-R retention score (standardized b=-0.457, p=0.007). Conversely, elevated plasma TSP1 levels positively correlated with increased levels of plasma 8-OH2dG (standardized b=0.361, p<0.001). The association between TSP1 and HVLT-R was potentiated after adjustment for 8-OH2dG (standardized b=-0.762, p<0.001). Moreover, the association between 8-OH2dG and cognitive function was also significant after adjusting for TSP1 (standardized b=-0.633, p=0.015).DiscussionElevated plasma TSP1 levels are associated with poorer episodic memory in SCD patients. This is the first evidence of a link between a plasma protein associated with vascular disease and cognitive function in adults with SCD. TSP1 levels were also positively correlated with 8-OH2dG, and adding 8-OH2dG to the model of TSP1 and HVTL-R retention appeared to increase the detrimental effect of TSP1. These results suggest that the effect of 8-OH2dG on cognitive function becomes evident only in the presence of higher TSP1 levels and implicate oxidative stress as intermediary to the effect of TSP1 on cognitive function in SCD. [Display omitted] DisclosuresIsenberg:RCTI, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313; Vasculox, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313.

Identification of a new locus and validation of previously reported loci showing differential methylation associated with smoking. The REGICOR study

Smoking increases the risk of many diseases and could act through changes in DNA methylation patterns. The aims of this study were to determine the association between smoking and DNA methylation throughout the genome at cytosine-phosphate-guanine (CpG) site level and genomic regions. A discovery cross-sectional epigenome-wide association study nested in the follow-up of the REGICOR cohort was designed and included 645 individuals. Blood DNA methylation was assessed using the Illumina HumanMethylation450 BeadChip. Smoking status was self-reported using a standardized questionnaire. We identified 66 differentially methylated CpG sites associated with smoking, located in 38 genes. In most of these CpG sites, we observed a trend among those quitting smoking to recover methylation levels typical of never smokers. A CpG site located in a novel smoking-associated gene (cg06394460 in LNX2) was hypomethylated in current smokers. Moreover, we validated two previously reported CpG sites (cg05886626 in THBS1, and cg24838345 in MTSS1) for their potential relation to atherosclerosis and cancer diseases, using several different approaches: CpG site methylation, gene expression, and plasma protein level determinations. Smoking was also associated with higher THBS1 gene expression but with lower levels of thrombospondin-1 in plasma. Finally, we identified differential methylation regions in 13 genes and in four non-coding RNAs. In summary, this study replicated previous findings and identified and validated a new CpG site located in LNX2 associated with smoking.

Role of smooth muscle Nox4-based NADPH oxidase in neointimal hyperplasia

Elevated levels of reactive oxygen species (ROS) in the vascular wall play a key role in the development of neointimal hyperplasia. Nox4-based NADPH oxidase is a major ROS generating enzyme in the vasculature, but its roles in neointimal hyperplasia remain unclear. ObjectiveOur purpose was to investigate the role of smooth muscle cell (SMC) Nox4 in neointimal hyperplasia. Approach and resultsMice overexpressing a human Nox4 mutant form, carrying a P437H dominant negative mutation (Nox4DN) and driven by SM22α promoter, to achieve specific expression in SMC, were generated in a FVB/N genetic background. After wire injury-induced endothelial denudation, Nox4DN had significantly decreased neointima formation compared with non-transgenic littermate controls (NTg). ROS production, serum-induced proliferation and migration, were significantly decreased in aortic SMCs isolated from Nox4DN compared with NTg. Both mRNA and protein levels of thrombospondin 1 (TSP1) were significantly downregulated in Nox4DN SMCs. Downregulation of TSP1 by siRNA decreased cell proliferation and migration in SMCs. Similar to Nox4DN, downregulation of Nox4 by siRNA significantly decreased TSP1 expression level, cell proliferation and migration in SMCs. ConclusionsDownregulation of smooth muscle Nox4 inhibits neointimal hyperplasia by suppressing TSP1, which in part can account for inhibition of SMC proliferation and migration.

Plasma thrombospondin-1 and clinical outcomes in traumatic brain injury.

Thrombospondin-1 (TSP-1) acts as an anti-angiogenic factor, and its expression in rat brain is upregulated after intracerebral hemorrhage. The current study was designed to investigate the change of plasma TSP-1 levels and assess the prognostic predictive effect of plasma TSP-1 level and it is associated with head trauma severity in the patients with severe traumatic brain injury (STBI). The plasma TSP-1 levels of 134 patients and 134 healthy controls were measured using enzyme-linked immunosorbent assay. The relationships between plasma TSP-1 levels and trauma severity reflected by Glasgow Coma Scale (GCS) scores as well as between plasma TSP-1 levels and short-term and long-term clinical outcomes were analyzed using multivariate analysis. Plasma TSP-1 levels were statistically significantly higher in patients than in healthy controls. The multivariate analysis demonstrated close association of TSP-1 with GCS scores and also identified TSP-1 as an independent predictor for 1-week mortality, 6-month mortality, and 6-month unfavorable outcome. Plasma TSP-1 levels had high prognostic predictive value based on receiver operating characteristic curve. The difference between its prognostic predictive value and GCS scores was not statistically significant. Plasma TSP-1 levels are elevated and are highly associated with head trauma severity and short-term and long-term outcomes of STBI. TSP-1 may be a good prognostic biomarker of STBI.

P82: Integrin receptors and their ligands as potential biomarkers in preoperative diagnosis of papillary thyroid carcinoma

Papillary thyroid cancer (PTC) is the most common malignancy of the endocrine system. The most frequent genetic alteration in PTC is the BRAF V600E mutation, which affects the activation of several intracellular signaling pathways. As a result, changes in the expression levels of cell membrane integrin receptors and their ligands – extracellular matrix proteins – osteopontin (OPN) and thrombospondin -1 (TSP1) are observed. This process increases the metastatic potential of tumor cells. Thus, integrin receptors and their ligands are potential biomarkers of an aggressive PTC phenotype. The aim of our study was to compare the gene expression profile of integrins ITGA2, ITGA3, ITGAV, ITGA6, ITGA9, ITGB1, ITGB3 and their ligands OPNa, OPNb, and TSP1 in PTC with different BRAF V600E mutation status. Intraoperative thyroid tissue samples from 41 patients diagnosed with PTC (n = 26), diffuse nodular nontoxic goiter (n = 10) and follicular adenoma (n = 5) were analyzed to evaluate the expression levels of the investigated genes by real time RT-PCR. Fluorescent immunohistochemistry (IHC) was used to confirm the PCR results and to estimate the amount of protein products. For IHC, frozen and paraffin sections were used. The BRAF V600E mutation was determined using allele-specific amplification. Nonparametric criteria (Kruskal Wallis, Wilcoxon and Mann–Whitney tests) were used to evaluate group differences. P values of less than 0.05 were considered statistically significant. The BRAF V600E mutation was observed in 12 PTC samples, which corresponds to 46% of PTC cases. An increase of gene expression level of ITGA3 (2.9-fold, p = 0.014), ITGAV (1.9-fold, p = 0.038), ITGB1 (1.7-fold, p = 0.026), OPNb (2.5-fold, p = 0.0001) and TSP1 (3.2-fold, p = 0.017) was identified in the PTC tissues, and a high gene expression level of OPNb (5.9-fold, p = 0.003) and TSP1 (12.1-fold, p = 0.005) was identified in the tissue samples of lymph node metastases compared to the conventionally normal tissue. In the samples with advanced cancer (T3, T4, TNM) the expression levels of ITGA3, ITGA6 and ITGA9 were higher compared to the T1 samples. MRNA levels of ITGA3 and ITGAV were significantly higher in the PTC BRAF V600E positive samples than in the BRAF V600E negative samples. Elevated levels of OPNa (11.4-fold, p = 0.0112), OPNb (10.2-fold, p = 0.0216) and TSP1 (33.5-fold, p = 0.0005) genes were observed in the follicular adenoma samples compared to the PTC tissues. For ITGA2 and ITGB3 there was a significant increase of expression in the PTC tissues compared to the benign thyroid tumors (8.9-fold, p = 0.019 and 38.4-fold, p = 0.014, respectively). We also studied the distribution and localization of integrins ITGA2 and ITGB3 in the thyroid tissues by IHC. In normal thyroid tissues ITGA2 and ITGB3 were located mainly in the follicular membrane. In the PTC tissue samples another location of the integrins was registered: ITGA2 was located mainly in the papillary structures, whereas ITGB3 was seen both at the basal and apical surfaces of thyrocytes. Follicular adenoma was characterized by a uniform distribution of both ITGA2 and ITGB3. The observed changes in the expression levels of the studied genes indicate their potential role in tumor progression and a possible impact on their expression of the mutant product of the BRAF gene. Integrins and their ligands, OPN and TSP1, could be considered potential markers in determining the prognosis and treatment of PTC.

Serum levels of TSP-1, NF-κB and TGF-β1 in polycystic ovarian syndrome (PCOS) patients in northern China suggest PCOS is associated with chronic inflammation.

The objective of this study was to determine the levels of thrombospondin-1 (TSP-1), transforming growth factor-β1 (TGF-β1) and nuclear factor kappaβ (NF-κβ) in polycystic ovarian syndrome (PCOS) patients with and without insulin resistance and after treatment with cyproterone acetate/ethinyloestradiol with or without concomitant metformin. Prospective. Patients with PCOS and healthy women were recruited. Patients were subdivided into obese and nonobese based on body mass index. Patients with PCOS were also grouped according to homoeostasis model assessment-insulin resistance (HOMA-IR) ≥ 2·69 or <2·69, and by PCOS phenotype. Patients with PCOS-IR were treated with a 6-month course of cyproterone acetate/ethinyloestradiol with or without concomitant metformin. Inflammatory markers were examined at baseline, and after 6 months of treatment. A total of 445 women with PCOS (mean age 25·9 ± 2·7 years; 298 obese, 147 nonobese) and 213 normal controls (mean age 24·9 ± 3·0 years) were included. Regardless of obesity status, testosterone, free androgen index (FAI), luteinizing hormone/follicle-stimulating hormone (LH/FSH) ratio, HOMA-IR, TSP-1 and NF-κB in the PCOS groups were significantly higher than in the control group, whereas TSP-1 was lower in the PCOS groups (all, P < 0·05). Patients with PCOS without IR had lower TSP-1 levels than control patients (P < 0·05). Treatment with cyproterone acetate/ethinyloestradiol with addition of metformin reduced the level of NF-κB, TGF-β1 and HOMA-IR and increased the level of TSP-1. These results support the association between PCOS and chronic inflammation.

Human umbilical cord blood-derived mesenchymal stem cells improve functional recovery through thrombospondin1, pantraxin3, and vascular endothelial growth factor in the ischemic rat brain.

Cell therapy is a potential therapeutic method for cerebral ischemia, which remains a serious problem. In the search for more effective therapeutic methods, many kinds of stem cells from various tissues have been developed and tested as candidate therapeutic agents. Among them, human umbilical cord blood (hUCB)-derived mesenchymal stem cells (MSCs) are widely used for cell therapy because of their genetic flexibility. To confirm that they are effective and understand how they affect ischemic neural cells, hUCB-MSCs were directly administered ipsilaterally into an ischemic zone induced by middle cerebral artery occlusion (MCAO). We found that the neurobehavioral performance of the hUCB-MSC group was significantly improved compared with that of the vehicle-injected control group. The infarct was also remarkably smaller in the hUCB-MSC group. Additionally, hUCB-MSC transplantation resulted in a greater number of newly generated cells and angiogenic and tissue repair factors and a lower number of inflammatory events in the penumbra zone. To determine why these events occurred, hUCB-MSCs were assayed under hypoxic and normoxic conditions in vitro. The results showed that hUCB-MSCs exhibit higher expression levels of thrombospondin1, pantraxin3, and vascular endothelial growth factor under hypoxic conditions than under normoxic conditions. These results were found to be correlated with our in vivo immunofluorescent staining results. On the basis of these findings, we suggest that hUCB-MSCs may have a beneficial effect on cerebral ischemia, especially through angiogenesis, neurogenesis, and anti-inflammatory effects, and thus could be used as a therapeutic agent to treat neurological disorders such as cerebral ischemia.

Abstract 1548: Baseline serum levels of osteopontin and thrombospondin-1 predict shorter overall survival in primary resected non-small cell lung cancer

Abstract Background: Osteopontin and thrombospondin-1 are extracellular matrix proteins secreted by both stromal and tumor cells. These proteins may play a key role in the tumor microenvironment for cancer development and metastasis. This study focused on the prognostic value of circulating osteopontin and thrombospondin-1 in primary resected NSCLC patients. Methods: Between January and December of 2012, serums were collected from 171 patients prior to primary surgery and from age-matched cancer-free individuals (n = 20). At baseline, circulating osteopontin and thrombospondin-1 levels were measured by ELISA. Tissue protein expression in primary tumor samples was determined by immunohistochemical analysis. Results: High levels of serum osteopontin and thrombospondin-1 levels were found in the cohort study. The difference between patients and healthy individuals was statistically significant (p&amp;lt;0.0001). Serum levels of osteopontin and thrombospondin-1 were independent prognostic factors of overall survival in multivariate analysis (HR 1.71, 95% CI 1.04-2.82, p = 0.04 for an increase in osteopontin of 50 ng/mL; HR 0.18, 95% CI 0.04-0.87, p = 0.03 for an increase in thrombospondin-1 of 10 ng/mL) similarly to their combination (HR is 1.31, 95% CI 1.03-1.67, p = 0.03 for an increase in osteopontin/thrombospondin-1 ratio of 6 ng/mL). Osteopontin and thrombospondin-1 levels were inversely correlated to survival rates. Conclusions: Baseline osteopontin and thrombospondin-1 serum levels, and even more importantly their combination, can serve as strong independent prognostic factors to predict overall survival in primary resected NSCLC patients, and therefore reflect the aggressiveness of the tumor. Citation Format: Mathieu Rouanne, Julien Adam, Aïcha Goubar, Angélique Robin, Caroline Ohana, Emilie Louvet, Jiemin Cormier, Olaf Mercier, Peter Dorfmüller, Soly Fattal, Vincent Thomas de Montpreville, Elie Fadel, Benjamin Besse, Ken André Olaussen, Christian Auclair, Jean-Charles Soria. Baseline serum levels of osteopontin and thrombospondin-1 predict shorter overall survival in primary resected non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1548. doi:10.1158/1538-7445.AM2015-1548

Inflammatory Cytokine-Mediated Regulation of Thrombospondin-1 and CD36 in Conjunctival Cells.

Increased expression of transforming growth factor-β2 (TGF-β2) is reported in the conjunctiva of dry eye patients with no increase of anti-inflammatory activity of TGF-β2. Our aim was to compare the expression of molecules involved in TGF-β2 activation, thrombospondin-1 (TSP-1) and CD36, during murine and human conjunctival inflammation. Human conjunctival tissue from cadaveric donors, human conjunctival epithelial primary cells and fibroblasts, and murine conjunctivas were immunostained for TSP-1, CD36, or TGF-β2. Inflamed conjunctival tissues were obtained from C57BL/6 wild-type (WT) mice induced to develop experimental dry eye (EDE) with 10 days of desiccating conditions and scopolamine injections and TSP-1-deficient (TSP1(-/-)) mice, which spontaneously develop Sjögren's syndrome-associated conjunctival inflammation with age. Immunostaining intensities were compared using ImageJ software. Cultures of human conjunctival fibroblasts were stimulated with IL-1β and both secreted protein and message levels of TSP-1, CD36, and TGF-β2 were analyzed. TSP-1 and CD36 were detectable in human and murine conjunctival tissues as well as primary conjunctival epithelial cells and fibroblasts. Increased conjunctival immunostaining of TGF-β2 and reduced CD36 were detected in EDE mice compared with WT mice. Interestingly, increased TGF-β2 and CD36 conjunctival immunostaining was detected in TSP1(-/-) mice. The expression of TSP-1 and CD36 was downregulated in IL-1β-stimulated conjunctival fibroblasts at both the protein and message level, while active TGF-β2 was undetected. The absence or reduced expression of either of the molecules involved in TGF-β2 activation supports proinflammatory conditions in the conjunctiva. Changes in TSP-1 and CD36 may serve as potential biomarkers of conjunctival inflammation.

Expression of thrombospondin-1 by tumor cells in patient-derived ovarian carcinoma xenografts

Purpose: Thrombospondin-1 (TSP-1), a major regulator of cell interaction with the environment, is often deregulated in cancers, including ovarian carcinoma. Both the tumor and the host cells can release TSP-1 in the tumor microenvironment. The relative contribution of the two sources in determining TSP-1 levels in ovarian cancer remains to be elucidated. This study was designed to investigate the expression of tumor TSP-1 in a panel of 29 patient-derived ovarian adenocarcinoma xenografts (PDX), using analytical tools specific for human (tumor-derived) rather than murine (host-derived) TSP-1.Methodology: Human-specific microarray and ELISA were used to measure tumor TSP-1 expression and plasma levels.Results: Tumor-derived TSP-1 was heterogeneously expressed in PDX. Expression was higher in the corresponding original patient's tumor, where stroma-derived TSP-1 is also analyzed, indicating that both the tumor and the host contribute to TSP-1 production. TSP-1 was differentially expressed according to tumor grade, but not affected by p53 expression or mutational status. Findings were confirmed in an external gene expression dataset (101 patients). In a functional enrichment analysis, TSP-1 correlated with genes related to angiogenesis, cell motility, communication and shape. Plasma TSP-1, detectable in 10/11 PDX, was not associated to its expression in the tumor. The possible association of plasma TSP-1 with p53 mutations and response to chemotherapy warrants further investigation.Conclusions: Ovarian carcinoma PDX are a useful tool to investigate the relative contribution of stroma and tumor cells in the production of tumor associated factors, in relation to the tumor behavior, molecular properties and response to therapy.

Decreased serum thrombospondin-1 levels in pancreatic ductal adenocarcinoma patients: an early indicator of disease or diabetes mellitus development?

Introduction: Identification of serum biomarkers enabling earlier diagnosis of pancreatic ductal adenocarcinoma (PDAC) could improve outcome. Aims: Serum protein profiles in patients with pre-clinical disease and at diagnosis were investigated. Patients & methods: Serum from cases up to 4 years prior to diagnosis of PDAC and controls enrolled on the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS, n=174) were studied, alongside samples from patients diagnosed with PDAC, chronic pancreatitis, benign biliary disease and from healthy subjects (n=199). Isobaric tags for relative and absolute quantification (iTRAQ) enabled comparisons of pooled serum from a test set (n=150). Validation was undertaken using multiple reaction monitoring (MRM) and/or western blotting in all 373 human samples and in a KPC mouse model. Results: iTRAQ identified thrombospondin-1 (TSP-1) as down-regulated pre-clinically and in diagnosed patients. MRM confirmed significant down-regulation of TSP-1 up to 24 months prior to diagnosis. A combination of TSP-1 and CA19-9 (AUC= 0.86), significantly outperformed both markers alone (0.69 & 0.77 respectively). TSP-1 was also decreased in PDAC patients compared to healthy controls (P<0.05), patients with benign biliary obstruction (P<0.01) and in serum of KPC mice with PDAC. Low levels of TSP-1 correlated with poorer survival pre-clinically (P<0.05) and at diagnosis (P<0.02). Reduced TSP-1 levels were observed in PDAC patients with a confirmed diabetes mellitus (P<0.04). The decrease in TSP-1 compared to healthy controls was only observed in PDAC patients with diabetes (P<0.009). Conclusion: Circulating TSP-1 levels decrease during the development of PDAC. The influence of diabetes mellitus on biomarker behavior should be considered in future studies.

Maintenance chemotherapy in children with ALL exerts metronomic-like thrombospondin-1 associated anti-endothelial effect.

Maintenance chemotherapy is an important part of the treatment of ALL in children. It relies on the long-term oral administration of daily low-dose mercaptopurin and weekly low-dose methotrexate. Although it has been used in the clinic for decades, its mechanisms of action remain unclear. Here, we investigated different angiogenic and immune biomarkers to gain insights into the mechanisms of action of maintenance therapy in children with ALL. We thus monitored circulating endothelial cells (CEC), endothelial progenitor cells (EPC) and endothelial microparticles (EMP), pro-angiogenic factors (VEGF, VEGFR-1 and Ang-2), anti-angiogenic factor thrombospondin-1 (THBS1) and regulatory T lymphocytes (Treg) in 47 children with ALL during the maintenance phase of their treatment (at treatment initiation and after 6, 12 and 18 months). We observed a statistically significant decrease in EPC and EMP counts throughout the maintenance phase associated with a significant increase in THBS1 levels. No significant change was detected in other angiogenic markers or in Treg numbers.The results presented here indicate that maintenance therapy in children with ALL exerts its antitumor activity at least in part through anti-angiogenic effects, similar to those induced by metronomic chemotherapy. Larger studies are now warranted to validate these findings and determine their clinical implications.