Thrombospondin-1 Levels Research Articles

Plasma thrombospondin 1 as a predictor of postoperative liver dysfunction.

Liver regeneration following liver resection involves a complex interplay of growth factors and their antagonists. Thrombospondin 1 has recently been identified as a critical inhibitor of liver regeneration by the activation of transforming growth factor β1 in mice, and preliminary data seem to confirm its relevance in humans. This study aimed to confirm these observations in an independent validation cohort. Perioperative circulating levels of thrombospondin 1 were measured in patients undergoing liver resection between January 2012 and September 2013. Postoperative liver dysfunction was defined according to the International Study Group of Liver Surgery and classification of morbidity was based on the criteria by Dindo et al. In 85 patients (44 major and 41 minor liver resections), plasma levels of thrombospondin 1 increased 1 day after liver resection (mean 51·6 ng/ml before surgery and 68·3 ng/ml on postoperative day 1; P = 0·001). Circulating thrombospondin 1 concentration on the first postoperative day specifically predicted liver dysfunction (area under the receiver operating characteristic (ROC) curve 0·818, P = 0·003) and was confirmed as a significant predictor in multivariable analysis (Exp(B) 1·020, 95 per cent c.i. 1·005 to 1·035; P = 0·009). Patients with a high thrombospondin 1 concentration (over 80 ng/ml) on postoperative day 1 more frequently had postoperative liver dysfunction than those with a lower level (28 versus 2 per cent) and severe morbidity (44 versus 15 per cent), and their length of hospital stay was more than doubled (19·7 versus 9·9 days). Thrombospondin 1 may prove a helpful clinical marker to predict postoperative liver dysfunction as early as postoperative day 1.

Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions

Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-β2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-β2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-β2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.

Glioma-Derived Thrombospondin-1 Modulates Cd14+ Cell Tolerogenic Properties

Thrombospondin-1 (TSP1) plays a role in the immune tolerance, and is involved in the pathogenesis of glioma. This study aims to investigate the role of the glioma-derived TSP1 in the induction of the tumor immune tolerance. The results showed that the primary human glioma cells expressed high levels of TSP1. Glioma cells enhanced the expression of transforming growth factor (TGF)-β in CD4+ CD16− naïve monocytes (Mos). The TGF-β+ Mos showed inhibitory effect on CD8+ T cell proliferation. We conclude that glioma cell-derived TSP1 facilitates the induction of TGF-β in Mos. The TSP1 may be a potential therapeutic target of glioma.

Circulating thrombospondine-2 in patients with moderate-to-severe chronic heart failure due to coronary artery disease.

Chronic heart failure (CHF) remains a leading cause of morbidity and mortality. In the current study, we aimed to evaluate the predictive value of circulating thrombospondin-2 (TSP-2) for cumulative survival in patients with ischemic CHF due to coronary artery disease (CAD). The results showed that during a median follow-up of 2.18 years, 21 participants died and 106 subjects were hospitalized repeatedly. The median circulating levels of TSP-2 in patients who survived and those who died were 0.63 ng/mL (95%CI=0.55-0.64 ng/mL) and 1.03 ng/mL (95% CI=0.97-1.07 ng/mL) (P<0.001). Circulating TSP-2 independently predicted all-cause mortality (OR=1.27; 95%CI=1.08-1.59; P=0.002), CHF-related death (OR=1.16; 95%CI=1.02-1.50; P<0.001), and also CHF-related rehospitalization (OR=1.12; 95%CI=1.07-1.25; P<0.001). In conclusion, among CAD patients with symptomatic CHF, increased circulating TSP-2 is correlated with increased 3-year CHF-related death, all-cause mortality, and risk for recurrent hospitalization.

Circulating thrombospondin-2 in patients with moderate-to-severe chronic heart failure due to coronary artery disease

Chronic heart failure (CHF) remains a leading cause of morbidity and mortality. In the current study, we aimed to evaluate the predictive value of circulating thrombospondin-2 (TSP-2) for cumulative survival in patients with ischemic CHF due to coronary artery disease (CAD). The results showed that during a median follow-up of 2.18 years, 21 participants died and 106 subjects were hospitalized repeatedly. The median circulating levels of TSP-2 in patients who survived and those who died were 0.63 ng/mL (95%CI = 0.55-0.64 ng/mL) and 1.03 ng/mL (95% CI = 0.97-1.07 ng/mL) (P<0.001). Circulating TSP-2 independently predicted all-cause mortality (OR = 1.27; 95%CI = 1.08–1.59; P = 0.002), CHF-related death (OR = 1.16; 95%CI = 1.02–1.50; P<0.001), and also CHF-related rehospitalization (OR = 1.12; 95%CI = 1.07–1.25; P<0.001). In conclusion, among CAD patients with symptomatic CHF, increased circulating TSP-2 is correlated with increased 3-year CHF-related death, all-cause mortality, and risk for recurrent hospitalization.

Association of Plasma Thrombospondin-1 Level with Cardiovascular Disease and Mortality in Hemodialysis Patients.

Thrombospondin-1 (TSP-1) is known to be involved in the regulation of angiogenesis, inflammation, and vascular function. Clinical studies have demonstrated its correlation with peripheral artery disease, coronary artery disease, and pulmonary hypertension. In this study, we explored its potential roles in the background of end-stage renal disease (ESRD). A total of 140 ESRD outpatients (ages 61.0 ± 12.4 years) were prospectively followed for 34 ± 7 months. Their TSP-1 levels were analyzed from pre-hemodialysis blood sample. Cardiovascular survey included ankle- brachial index (ABI), echocardiography and Tl-201 dipyridamole single-photon emission computed tomography (SPECT). Plasma TSP-1 levels were higher in those patients with preexisting clinical evidence of cardiovascular disease (CVD) than those without (p = 0.002). TSP-1 concentrations were also correlated with ABI, left ventricular ejection fraction, and scar burden in SPECT. Stepwise logistic regression analysis revealed that TSP-1 level was independently associated with the presence of CVD, with an odds ratio of 1.38 [95% confidence interval (CI), 1.09-1.75, p = 0.008]. In survival analyses, 31 patients (22%) died during the follow-up, 16 (52%) arising from cardiovascular causes. Cox hazards analysis revealed that the patients with TSP-1 levels in the highest tertile had a 5.32- and 6.75-fold higher risk for all-cause and cardiovascular mortality than those in the lowest tertile. This predictive value for all-cause mortality still persisted after multivariate adjustment (hazard ratio, 8.71; 95% CI, 1.36-55.68; p = 0.02). This study hallmarks the association of elevated TSP-1 level with CVD and adverse outcome among hemodialysis patients. Thrombospondin-1; End-stage renal disease; Cardiovascular disease; Mortality.

Curcumin protects against lipopolysaccharide-induced vasoconstriction dysfunction via inhibition of thrombospondin-1 and transforming growth factor-β1.

Sepsis is a complex syndrome characterized by the development of progressive dysfunction in multiple organs. The aim of the present study was to investigate the protective effect of curcumin against lipopolysaccharide (LPS)-induced vasoconstrictive dysfunction, and to investigate the possible underlying mechanism. Male Sprague-Dawley rats were randomly divided into the following groups: Control, sepsis and curcumin. A sepsis model was established by an intraperitoneal (i.p.) injection of 5 mg/kg LPS. Thoracic aortic rings obtained from the rats were mounted in an organ bath and the vasoconstriction of the rings was recorded. In addition, the serum E-selectin levels were determined by an enzyme-linked immunosorbent assay. The expression levels of thrombospondin (TSP)-1 and transforming growth factor (TGF)-β1 in the aortic tissue were detected by immunohistochemistry. Vasoconstriction of the aortic rings was found to significantly decrease in the sepsis rats when compared with the control group. However, curcumin (10 or 20 mg/kg, i.p.) prevented the vasoconstrictive dysfunction induced by LPS. The serum level of E-selectin and the expression levels of TSP-1 and TGF-β1 significantly increased in the sepsis rats when compared with the control group rats; however, the levels decreased significantly following treatment with curcumin (10 or 20 mg/kg). Furthermore, hematoxylin and eosin staining revealed that curcumin alleviated the LPS-induced damage in the aortic tunica intima and tunica media. Therefore, the results indicated that curcumin alleviates LPS-induced vasoconstrictive dysfunction in the thoracic aorta of rats. In addition, the inhibition of TSP-1 and TGF-β1 expression may be involved in the mechanism underlying this protective effect.

Disturbed angiogenic activity of adipose-derived stromal cells obtained from patients with coronary artery disease and diabetes mellitus type 2.

BackgroundMultipotent mesenchymal stem/stromal cells (MSC) including adipose-derived stromal cells (ADSC) have been successfully applied for cardiovascular diseases treatment. Their regenerative potential is considered due to the multipotency, paracrine activity and immunologic privilege. However, therapeutic efficacy of autologous MSC for myocardial ischemia therapy is modest. We analyzed if ADSC properties are attenuated in patients with chronic diseases such as coronary artery disease (CAD) and diabetes mellitus type 2 (T2DM).Methods and resultsADSC were isolated from subcutaneous fat tissue of patients without established cardiovascular diseases and metabolic disorders (control group, n = 19), patients with CAD only (n = 32) and patients with CAD and T2DM (n = 28). ADSC phenotype (flow cytometry) was CD90+/CD73+/CD105+/CD45−/CD31− and they were capable of adipogenic and osteogenic differentiation. ADSC morphology and immunophenotype were similar for all patients, but ADSC from patients with CAD and T2DM had higher proliferation activity and shorter telomeres compared to control patients.ADSC conditioned media stimulated capillary-like tubes formation by endothelial cells (EA.hy926), but this effect significantly decreased for patients with CAD (p = 0.03) and with CAD + T2DM (p = 0.017) compared to the control group. Surprisingly we revealed significantly higher secretion of some pro-angiogenic factors (ELISA) by ADSC: vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) for patients with CAD and HGF and placental growth factor (PlGF) for patients with CAD + T2DM. Among angiogenesis inhibitors such as thrombospondin-1, endostatin and plasminogen activator inhibitor-1 (PAI-1) level of PAI-1 in ADSC conditioned media was significantly higher for patients with CAD and CAD + T2DM compared to the control group (p < 0.01). Inhibition of PAI-1 in ADSC conditioned media by neutralizing antibodies partially restored ADSC angiogenic activity (p = 0.017).ConclusionsADSC angiogenic activity is significantly declined in patients with CAD and T2DM, which could restrict the effectiveness of autologous ADSC cell therapy in these cohorts of patients. This impairment might be due to the disturbance in coordinated network of pro- and anti-angiogenic growth factors secreted by ADSC. Changes in ADSC secretome differ between patients with CAD and T2DM and further investigation are necessary to reveal the MSC-involved mechanisms of cardiovascular and metabolic diseases and develop novel approaches to their correction using the methods of regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0337-4) contains supplementary material, which is available to authorized users.

Abstract 15515: Sexual Dimorphism in Cardiac Response to Intrauterine Growth Restriction (IUGR)

Background: Poor fetal nutrient availability predisposes to adult cardiovascular disease (CVD). We have developed a fetal baboon model of maternal undernutrition resulting in Intrauterine Growth Restriction (IUGR) in both male and female fetuses (Li, et al, J Endocrinol, 2013). MicroRNAs are small non-coding RNAs that regulate gene expression through mRNA degradation and translational repression. Hypothesis: We hypothesized that IUGR is accompanied by cardiac dysfunction and dysregulation of cardiac miRNAs. Methods: Six control pregnant baboons ate ad libitum, and six nutrient restricted pregnant baboons ate a globally reduced diet (70% of control feed) from 0.16 gestation (G) through pregnancy resulting in fetal IUGR compared to controls (p&lt;0.05). No differences in heart weight were observed. Fetuses (3 males and 3 females per group) were euthanized at C-section at 0.9G. Cardiac sections were studied for morphological changes and fibrosis using H&amp;E and Masson’s trichrome staining respectively. Total left ventricular RNA was isolated and miRNA array was performed. Results: While no differences in myofiber orientation were observed, IUGR male but not female fetuses showed a reverse correlation between birth weight and accumulation of fibrotic tissue in the heart (r=-0.831, p=0.01). We next investigated the expression of the extracellular matrix proteins thrombospondin-1 (TSP-1) and connective tissue growth factor (CTGF). A reverse correlation between birth weight and cardiac TSP-1 levels (r=-0.77, p=0.03) was found in male but not female fetuses. No changes in CTGF were observed. Sexual dimorphism was observed in fetal cardiac miRNA expression within both IUGR and control groups. In male IUGR group, the increase in levels of TSP-1 was associated with more than 50% reduction in expression of its targeting miRNA-19a, reduction in which has been implicated in CVD. Other CVD-related and differentially expressed miRNAs in male IUGR included miRNA-23b, 27b, 99b, 143, 181a, and 378-3p. Summary: IUGR resulting from decreased maternal nutrition is associated with sexual dimorphism in cardiac structure and miRNA expression. If the changes observed in male IUGR fetuses persists postnatally they may program offspring for higher CVD risk later in life.

Abstract 13720: High Serum Levels of Thrombospondin-2 Correlate with Poor Prognosis of Patients with Heart Failure with Preserved Ejection Fraction

Introduction: Thrombospondin-2 (TSP-2) is highly expressed in hypertensive heart. Interstitial fibrosis is frequently observed in hypertensive heart, and it is a characteristic feature of heart failure with preserved ejection fraction (HFpEF). Hypothesis: High TSP-2 serum levels reflect disease severity and can predict poor prognosis of patients with HFpEF. Methods: Serum TSP-2 levels were measured by ELISA in 150 patients with HFpEF. HFpEF was defined as clinical signs or symptom of HF, left ventricular ejection fraction &gt;50%, B-type natriuretic peptide (BNP) &gt;100 pg/ml or E/e’ &gt;15. The endpoints of this study were mortality rate, HF-related hospitalization, stroke and non-fatal myocardial infarction. Results: The median serum TSP-2 level was 19.2 (14.4-26.0) ng/ml in all study participants. Serum TSP-2 levels correlated with the New York Heart Association (NYHA) functional class. Then the patients were divided into high and low TSP-2 groups based on the median value. Pulmonary capillary wedge pressure, and circulating levels of BNP and high-sensitivity cardiac troponin T were significantly higher in the high TSP-2 group than low TSP-2 group. Whereas, cardiac index was significantly lower in the high TSP-2 group. Kaplan-Meier survival curve showed high risk of adverse cardiovascular events in the high TSP-2 group, and that the combination of high TSP-2 and high BNP (&gt;100 pg/ml) was associated with the worst event-free survival rate. Univariate Cox proportional hazard analysis showed that high levels of TSP-2, NYHA functional class, presence of atrial fibrillation, high levels of BNP, left atrium dilatation were associated with future adverse events. Stepwise multivariate Cox proportional hazard analysis identified BNP (ln(BNP)) (hazard ratio [HR]: 1.98; confidence interval [CI]: 1.26-3.10, p=0.003) and TSP-2 (HR: 2.59; CI: 1.09-6.14, p=0.031) as independent predictors of risk of death and cardiovascular events. Conclusions: Circulating TSP-2 correlates with disease severity in patients with HFpEF. TSP-2 is a potentially useful predictor of future adverse cardiovascular events in patients with HFpEF.

Host deficiency in caveolin-2 inhibits lung carcinoma tumor growth by impairing tumor angiogenesis.

Caveolin-2 (Cav-2), a member of caveolin protein family, is largely different from better known caveolin-1 (Cav-1) and thus might play distinct functions. Here, we provide the first genetic evidence suggesting that host-expressed Cav-2 promotes subcutaneous tumor growth and tumor-induced neovascularization using two independent syngeneic mouse models. Host deficiency in Cav-2 resulted in defective and reduced growth of subcutaneously implanted Lewis lung carcinoma (LLC) and B16-F10 melanoma tumors, respectively. Consistent with the defective growth, LLC and B16-F10 melanoma tumors implanted into Cav-2 KO mice displayed reduced microvascular density (MVD) determined by IHC with anti-CD31 antibodies, suggesting impaired pathologic angiogenesis. Additional studies involving LLC tumors extracted from Cav-2 KO mice just 10 days after implantation determined reduced cell proliferation, massive necrotic cell death, and fibrosis. In contrast with day 10, only MVD but not cell proliferation and survival was reduced in the earliest palpable LLC tumors extracted 6 days after implantation into Cav-2 KO mice, suggesting that impaired angiogenesis is the causative factor. Mechanistically, impaired LLC tumor growth and angiogenesis in Cav-2 KO mice was associated with increased expression levels of antiangiogenic thrombospondin-1 and inhibited S1177 phosphorylation of endothelial nitric oxide synthase. Taken together, our data suggest that host deficiency in Cav-2 impairs tumor-induced angiogenesis, leading to compromised tumor cell survival/proliferation manifested by the defective tumor growth. In conclusion, host-expressed Cav-2 may promote tumor growth via supporting tumor-induced angiogenesis. Thus, Cav-2 expressed in tumor microenvironment may potentially become a novel target for cancer therapy.

Abstract LB-104: Reverting the angiogenic switch of glioblastoma with a nanopolyplex based on the molecular fingerprint of tumor dormancy

Abstract Small,microscopic, avascular and therefore asymptomatic tumors can remain in their dormant stage for a considerable period of time depending on numerous processes. One crucial mechanism underlying the transformation from a dormant to a fast-growing phenotype is the ability of tumor cells to induce angiogenesis, a phenomenon termed “angiogenic switch”. We used the aggressive tumor-forming U-87 MG human glioblastoma cell line to identify and isolate a clone which generates dormant microscopic tumors (1). This clone was isolated using single-cell clone and identified by gene expression signature of dormant tumors (2). While both cell lines share a similar growth rate in vitro, we found profound differences in tumor growth patterns when injected into mice. Furthermore, both cell lines exhibit major differences in their angiogenic potential and in expression patterns of genes involved in angiogenesis regulation. Two of the major dissimilarities were found in thrombospondin-1 (TSP-1) and epidermal growth factor receptor (EGFR) expression levels. The dormant avascular tumor-generating cell line (U-87-D) expresses significantly higher levels of TSP-1 and lower levels of EGFR compared to the fast-growing angiogenic tumor-generating parental cell line (U-87-F). It has been previously demonstrated that TSP-1 is a key endogenous angiogenesis inhibitor, whose expression is lost during the malignant transformation. EGFR is a major modulator of tumorigenicity in glioblastoma and therefore, is considered an attractive potential target for glioblastoma therapy. Following the identification of a tumor dormancy gene signature, we induced the upregulation of TSP-1 signaling, using a TSP-1 peptidomimetic (TSP-1 PM), and the downregulation of EGFR, using a dendritic nanocarrier entrapping siRNA (polyglycerol amine (PG-NH2)-siEGFR). We evaluated the ability of this combination therapy to reverse the fast-growing angiogenic phenotype of U-87-F to the dormant avascular phenotype of U-87-D. Mice bearing established U-87-F tumors (50 mm3) received TSP-1 PM (50 mg/kg/day every day) and PG-NH2-siEGFR (2 mg/kg twice a week) for 14 days. This combination therapy exhibited anti-angiogenic and anti-tumorigenic activity. It remarkably decreased tumor volume by 99.5% compared with the control on day 25 post treatment initiation, to a volume of ∼1 mm3.Immunohistochemistry analysis of TSP-1 PM-treated tumors revealed reduced abnormal vasculature, increased αSMA expression and decreased VEGF expression. We concluded that TSP-1 PM in combination with EGFR-siRNA present a promising treatment for advanced glioblastoma promoting a dormant phenotype.

Head and Neck Cancers: Why Is It Better to HPV than Not to HPV?

Objectives: Dissect the impact of human papillomavirus (HPV) on angiogenesis in head and neck cancers (HNSCC). Methods: We analyzed 18 newly diagnosed HNSCC patients and controls. HPV tumor status was ascertained by p16 staining. Eleven pro- and anti-angiogenic factors were quantified in sera using a multiplex assay. Angiogenic factors were analyzed in the tumor tissue using immunohistochemistry. Results: We found that circulating levels of anti-angiogenic factor endostatin were higher in all HNSCC patients compared with healthy subjects irrespective of HPV status. Notably, we detected higher levels of the proangiogenic factors angiopoetin-1 and vascular endothelial growth factor (VEGF) in the circulation of HPV-negative patients compared with patients with HPV-positive tumors. Interestingly, levels of thrombospondin-2, an antiangiogenic factor, were lower in the circulation of patients with HPV-positive compared with HPV-negative tumors and healthy controls. We next analyzed these angiogenic factors in the tumor tissue. Interestingly, while HPV-negative tumors exhibited VEGF expression both in tumor tissue and stromal cells, only a fraction of HPV-positive tumors exhibited VEGF expression in tumor tissue. Angiopoietin-1 was expressed in tumor cells as well as endothelial cells in the stroma. Another interesting finding was that macrophages in HNSCC had an M2 “alternatively activated” phenotype and appeared to be the major source of endostatin in the stroma. Conclusions: Our novel results indicate that angiogenic factors are differentially expressed in the circulation and tumor tissue in HPV-positive and HPV-negative HNSCC and could potentially confer a more aggressive phenotype to HPV-negative tumors.

Phase Ii Trial of Metronomic Oral Vinorelbine As First-Line Treatment in Elderly Patients with Advanced Non-Small Cell Lung Cancer (Move Trial)

ABSTRACT Aim: Metronomic oral vinorelbine could be a safe option for elderly patient with advanced non small cell lung cancer (NSCLC). Metronomic administration of chemotherapy leads to a cytostatic action shifting treatment target from cancer cell to tumor angiogenesis. Methods: 43 chemotherapy naive elderly (≥70 yrs) PS 0-2 patients with stage IIIB-IV NSCLC were prospectively recruited. Median age was 80 yrs (M/F 36/7) with predominantly squamous histology. PS distribution was 0-1(16)/2(27) with a median of 3 serious co-morbid illnesses. Study treatment consisted of oral vinorelbine 50mg three times weekly (Monday-Wednesday-Friday) continuously. Primary endpoints were overall response rate (ORR), clinical benefit (CB – disease response + stabilization >12 weeks) and safety. Health-related QoL (HRQoL) was also assessed. We conducted an exploratory time-course analysis of VEGF and thrombospondin-1 (TSP1) serum levels in a subgroup of patients. Results: Patients received a median of 5 (range 1-21) cycles with a total of 272 cycles delivered. ORR was 18.6% with 7 partial and 1 complete responses; 17/43 experienced stable disease lasting more than 12 weeks leading to an overall CB of 58.1%. Median time to progression was 5 (range 2-21) and median overall survival 9 (range 3-29) months. Treatment was well tolerated with rare G3/4 toxicity. Regardless of severity main toxicities observed were anemia in 44%, fatigue in 32.4%, and diarrhoea 10.5%. HRQoL scores did not significantly vary. Baseline VEGF levels were lower and showed a rapid increase during treatment in non-responders pts only while TSP1 levels did not change. Conclusions: Metronomic oral vinorelbine is safe in elderly patients with advanced NSCLC with an interesting activity mainly consisting in long-term disease stabilization coupled with an optimal patient compliance. Disclosure: All authors have declared no conflicts of interest.

High serum levels of thrombospondin-2 correlate with poor prognosis of patients with heart failure with preserved ejection fraction

Thrombospondin-2 (TSP-2) is highly expressed in hypertensive heart. Interstitial fibrosis is frequently observed in hypertensive heart, and it is a characteristic feature of heart failure with preserved ejection fraction (HFpEF). We tested here the hypothesis that high TSP-2 serum levels reflect disease severity and can predict poor prognosis of patients with HFpEF. Serum TSP-2 levels were measured by ELISA in 150 patients with HFpEF. HFpEF was defined as left ventricular ejection fraction ≥ 50%, B-type natriuretic peptide (BNP) ≥ 100 pg/ml or E/e' ≥ 15. The endpoints were mortality rate, HF-related hospitalization, stroke and non-fatal myocardial infarction. The median serum TSP-2 level was 19.2 (14.4-26.0) ng/ml. Serum TSP-2 levels were associated with the New York Heart Association (NYHA) functional class. Circulating levels of BNP and high-sensitivity troponin T were positively correlated with serum TSP-2 levels. Kaplan-Meier survival curve showed high risk of adverse cardiovascular events in the high TSP-2 group (>median value), and that the combination of high TSP-2 and high BNP (≥ 100 pg/ml) was associated with the worst event-free survival rate. Multivariate Cox proportional hazard analysis identified TSP-2 as independent predictor of risk of death and cardiovascular events. Circulating TSP-2 correlates with disease severity in patients with HFpEF. TSP-2 is a potentially useful predictor of future adverse cardiovascular events in patients with HFpEF.

Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.

Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma.

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

Active von Willebrand factor predicts 28-day mortality in patients with systemic inflammatory response syndrome

AbstractEndothelial dysfunction contributes to the pathology of systemic inflammatory response syndrome (SIRS). However, endothelial biomarkers are not routinely evaluated in this setting. Here, 275 patients with SIRS and plasma levels of von Willebrand factor (VWF), thrombospondin-1, myeloperoxidase, ADAMTS-13, and active VWF (aVWF) were studied in relation to 28-day mortality. On admission, aVWF levels were higher in nonsurvivors vs survivors (0.69 vs 0.47 µg/mL, P = .019). Patients in the highest tertile of aVWF levels had a lower cumulative survival (86% vs 75%, P = .017) and twofold increased hazard ratio (HR). When adjusted for the Acute Physiology and Chronic Health Evaluation IV (APACHE-IV) score, this difference remained significant (HR 1.82, 95% confidence interval, 1.03-3.3). On admission, no significant differences were measured for the other proteins. These observations suggest that the stimulated release of VWF is not predictive for mortality in patients with SIRS, opposite of the processing of VWF after release. aVWF could be used with the APACHE-IV score to stratify SIRS patients at high mortality risk.

Progenitor cell fate: fine tuning by blood vessels and oxygen (342.2)

Bone regeneration is a complex physiological process that progresses rapidly through distinct cellular and tissue types before ultimately restoring normal bone structure and function. Recent work has begun to demonstrate the essential mechanistic role of the vasculature ‐ and in turn tissue oxygenation ‐ in regulating multiple aspects of bone healing, including influencing progenitor cell differentiation. Dr. Hankenson is a mesenchymal progenitor cell biologist, whom also studies bone regeneration. His most recent work has shown that when expression of the matricellular protein thrombospondin‐2 (TSP2) is disrupted, enhanced vascularization of the early regenerate tissue (soft callus) during the fracture healing process results in differential mesenchymal progenitor cell fate through the regulation of tissue oxygenation. TSP2 is a potent angiogenesis inhibitor highly expressed during tissue regeneration. Under ischemic conditions, enhanced callus vascularization expedites healing. Computational models that consider alterations in TSP2 levels, vascularization, and oxygen gradients in the callus are highly predictive of observed experimental outcomes. These studies will be used as a starting point to discuss the role of tissue oxygenation in regulating mesenchymal cell fate and how these fate decisions impact bone regeneration.

Identification of Potential Serum Biomarkers for Rheumatoid Arthritis by High-Resolution Quantitative Proteomic Analysis

The aim of this study was to find serum biomarkers of rheumatoid arthritis (RA) by high-resolution proteomic analysis. Low-abundance proteins from pooling serum sample of early RA patients and healthy controls were enriched using ProteoMiner™ enrichment kits. The enriched proteins were separated on SDS-PAGE, digested by trypsin, labeled with tandem mass tag (TMT) reagents, and desalted by C18 stage tip column. Then, the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with nano-LC combined with Orbitrap Q Exactive mass spectrometer, and experiments were carried out three times using different specimens, and differentially expressed proteins were screened by intensity ratios of identified peptides. Enzyme-linked immunosorbent assays (ELISAs) were performed to confirm differentially expressed proteins. Twenty-six proteins were found differentially expressed in RA serum by high-resolution proteomic analysis. Among them, levels of thrombospondin-1, ficolin-2, isoform 10 of fibronectin, and apolipoprotein E were higher in RA patients than in healthy controls (RA/healthy control (HC) ≥ 1.5, p<0.05), while levels of angiotensinogen, paraoxonase/arylesterase 1, isoform E of proteoglycan 4, and plasminogen were significantly lower (RA/HC ≤ 0.67, p<0.05). Further study by ELISA showed a higher level of ficolin-2 in the serum of RA patients compared to healthy controls; the level of ficolin-2 was found to be positively correlated with swollen joint counts (SJCs), disease activity score (DAS28), rheumatoid factor, and IgM in RA patients and with DAS28 and IgM in early RA patients through statistical analysis. The results of this study suggest that ficolin-2, as a newly screened biomarker by high-resolution quantitative proteomic analysis, offers the potentiality to become a diagnostic or disease evaluation tool in RA.