Thrombospondin-1 Levels Research Articles

Detection of lymph node metastasis in colon cancer by ectopically expressed fibroblast markers FOXQ1 and THBS2.

Approximately 25% of colon cancer (CC) patients having curative surgery will relapse. Therefore, it is crucial to identify patients with increased recurrence risk to offer them adjuvant chemotherapy. Three markers with prominent expression in fibroblasts: forkhead box Q1 (FOXQ1), matrix metalloproteinase-11 (MMP11), and thrombospondin-2 (THBS2), and the fibroblast expressed chemokine CXCL12 were selected for studies because of the critical role of fibroblasts in the microenvironment of the tumor. The expression levels of the biomarkers were assessed in primary CC tumors, lymph nodes of CC patients and controls, and CC cell lines at mRNA and protein levels by real-time qRT-PCR and immunohistochemistry, respectively. FOXQ1, MMP11, and THBS2 mRNAs were expressed at significantly higher levels in primary tumors compared to normal colon (P=0.002, P<0.0001, and P<0.0001, respectively). In contrast, CXCL12 mRNA levels were higher in normal colon tissue. FOXQ1, MMP11, and THBS2 levels were also expressed at significantly higher levels in metastasis-positive lymph nodes compared to both metastasis-negative- and control nodes (P<0.0001/P=0.002, P<0.0001/P<0.0001, and P<0.0001/P<0.0001, respectively). Immuno-morphometry revealed that 30-40% of the tumor cells expressed FOXQ1, MMP11, and THBS2. FOXQ1 and THBS2 were barely detected in normal colon epithelium (P<0.0001), while MMP11 was expressed in normal colon epithelium at high levels. We conclude that CC tumor cells show ectopic expression of FOXQ1 and THBS2 possibly making these tumor cells independent of fibroblast cell support. The high expression levels of these two biomarkers in metastatic lymph nodes suggest that they are potential indicators of patients at risk for recurrence.

Inhibitory receptor CD47 binding to plasma TSP1 suppresses NK-cell IFN-γ production via activating the JAK/STAT3 pathway during HIV infection

BackgroundNatural 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.MethodsFresh 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.ResultsCD47 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.ConclusionsThe 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.

Relationship between the thrombospondin-1/Toll-like receptor 4 (TSP1/TLR4) pathway and vitamin D levels in obese and normal weight subjects with different metabolic phenotypes

Thrombospondin-1 (TSP1) contributes to obesity-associated inflammation via activating Toll-like receptor 4 (TLR4). The regulatory role of vitamin D on this pathway has been suggested. This study aimed to investigate the relationship between TSP1/TLR4 pathway and vitamin D in obese and normal weight subjects with different metabolic phenotypes. Thirty obese and thirty normal weight men were selected. Anthropometric parameters and serum TSP1, TLR4, TNF-α, vitamin D, and metabolic profile were determined. Metabolic phenotypes of obese and normal weight subjects were determined. Findings revealed enhanced TSP1/TLR4/TNF-α levels and reduced 25(OH)D levels in obese compared to normal weight subjects and metabolically unhealthy compared to metabolically healthy subjects. TSP1 correlated positively with parameters of unhealthy metabolic profile. TSP1, TLR4 and TNF-α levels significantly negatively correlated with vitamin D levels. In conclusion, vitamin D might exert a regulatory role on TSP1/TLR4 pathway, providing a potential mechanism that links hypovitaminosis D with risk of metabolic dysfunction.

Thrombospondin 2 is a novel biomarker of essential hypertension and associated with nocturnal Na+ excretion and insulin resistance

ABSTRACT Background Thrombospondins (TSPs) play important roles in several cardiovascular diseases. However, the association between circulating (plasma) thrombospondin 2 (TSP2) and essential hypertension remains unclear. The present study was aimed to investigate the association of circulating TSP2 with blood pressure and nocturnal urine Na+ excretion and evaluate the predictive value of circulating TSP2 in subjects with hypertension. Methods and Results 603 newly diagnosed essential hypertensive subjects and 508 healthy subjects were preliminarily screened, 47 healthy subjects and 40 newly diagnosed essential hypertensive subjects without any chronic diseases were recruited. The results showed that the levels of circulating TSP2 were elevated in essential hypertensive subjects. The levels of TSP2 positively associated with systolic blood pressure (SBP), diastolic blood pressure (DBP), and other clinical parameters, including homeostasis model assessment of insulin resistance (HOMA-IR), brachial-ankle pulse wave velocity, and serum triglycerides, but negatively associated with nocturnal urine Na+ concentration and excretion and high-density lipoprotein cholesterol. Results of multiple linear regressions showed that HOMA-IR and nocturnal Na+ excretion were independent factors related to circulating TSP2. Mantel–Haenszel chi-square test displayed linear relationships between TSP2 and SBP (χ2 = 35.737) and DBP (χ2 = 26.652). The area under receiver operating characteristic curve (AUROC) of hypertension prediction was 0.901. Conclusion Our study suggests for the first time that the circulating levels of TSP2 may be a novel potential biomarker for essential hypertension. The association between TSP2 and blood pressure may be, at least in part, related to the regulation of renal Na+ excretion, insulin resistance, and/or endothelial function.

Obesity-Induced Platelet Mitofusin-1 Expression Causes Vascular Dysfunction

Background: The growing epidemic of obesity is strongly linked to cardiovascular disease (CVD). Obesity leads to endothelial dysfunction, an instigating event that drives vascular injury and subsequent CVD, but the mechanisms by which obesity causes endothelial dysfunction remain unclear. Platelets circulate proximal to the endothelium and are traditionally thought to contribute to obesity-associated vasculopathy through thrombotic activation. However, platelets are metabolically active and release vaso-modulatory molecules into their environment and the role of these functions have not been examined in the context of obesity-induced vasculopathy. We previously showed that alterations in platelet mitochondrial function regulate platelet degranulation. Further, it has been reported that thrombospondin 1 (TSP1), a platelet factor that propagates endothelial dysfunction, is elevated in the plasma of obese humans. We hypothesized that obesity/weight gain modulates platelet mitochondrial function, which stimulates the release of TSP1 from platelets to propagate vascular dysfunction. Methods: Platelets were isolated from lean and obese humans and mitochondrial structure and function measured. To induce diet-induced obesity wildtype (WT) mice were fed with a high fat diet (HFD, 60 kcal% fat) for 10 weeks, and low fat diet (LFD, 10 kcal% fat)- fed WT mice were used as controls. Platelets and plasma from the mice were screened for mitochondrial proteins and plasma TSP-1 was measured by ELISA. A murine model with platelet-specific deletion of mitofusin-1 (MFN1; pltMFN1KO mice), a mitochondrial GTP-ase that increases mitochondrial function was generated and fed either HFD or LFD for 4-10 weeks. Further, endothelial-dependent- and independent- vascular relaxation was measured in both groups by stimulating aortic rings with acetylcholine (Ach) or sodium nitroprusside (SNP) using wire myography. Additionally, platelet-specific TSP1 knockout (plt-TSP1KO) mice were generated and fed with HFD for 10 weeks and measured plasma TSP1 levels. Results: Platelets from obese subjects showed increased levels of MFN1 compared to lean subjects. WT mice fed with HFD had increased platelet MFN1 and plasma TSP1 levels compared to LFD-fed WT mice. Platelet-specific deletion of MFN1 in mice resulted in reduced plasma TSP1 levels compared to control PF4-cre mice after HFD challenge. We found that HFD caused impaired Ach-stimulated vasorelaxation (indicative of endothelial dysfunction) in control PF4-cre mice, and this effect was attenuated in pltMFN1KO mice. Further, we found that HFD-induced increases in plasma TSP1 levels were attenuated in plt-TSP1KO mice, indicating platelets are the major source of TSP1 in HFD induced obesity model. Conclusion: These data demonstrate that obesity/weight gain induces platelet MFN1 expression, which drives TSP1 release from platelets to propagate vascular dysfunction. Our findings elucidate a novel platelet-centric mechanism underlying obesity-associated vasculopathy and have implications for the targeting of non-thrombotic platelet function as a therapeutic strategy for obesity-induced vasculopathy.

Relationship between elevated circulating thrombospondin-1 levels and vascular complications in diabetes mellitus.

Thrombospondin-1 (TSP-1) participates in a series of physiological and pathological processes by binding to various receptors regulating cell proliferation, adhesion and apoptosis. Elevated circulating TSP-1 is linked with diabetic vascular complications (DVC). This study aimed to determine the relationship between circulating TSP-1 levels and DVC. A comprehensive search of PubMed, Embase, Web of Science and CNKI databases was carried out. A meta-analysis was carried out to compare circulating TSP-1 levels between diabetes patients without vascular complications (DNVC), diabetes patients with DVC and non-diabetes patients. The correlation between TSP-1 and metabolic parameters was also analyzed. Subgroup analysis was carried out according to complication type, defined as diabetic retinopathy, diabetic nephropathy and diabetic cardiovascular disease (DCVD). A total of eight studies were included. Compared with non-diabetes patients, diabetic patients, including DNVC and DVC, had significantly higher circulating TSP-1 levels (standardized mean difference [SMD] 2.660, 95% CI 1.17-4.145, P = 0.000). DNVC had significantly higher circulating TSP-1 levels than non-diabetes patients (SMD 3.613, 95% CI 1.607-5.619, P = 0.000). DVC had significantly higher TSP-1 levels than DNVC (SMD 0.568, 95% CI 0.100-1.036, P = 0.017). TSP-1 was significantly positively correlated with fasting plasma glucose (overall Fisher's z = 0.696, 95% CI 0.559-0.833) and HbA1c (overall Fisher's z = 0.849, 95% CI 0.776-0.923). Elevated circulating TSP-1 levels are closely related to DVC, especially in diabetic nephropathy and diabetic cardiovascular disease. Circulating TSP-1 detection might be helpful in the timely diagnosis and treatment of DVC.

Associations of potential plasma biomarkers with suicide attempt history, current suicidal ideation and subsequent suicidal events in patients with depression: A discovery study

A growing body of evidences suggests that suicidal ideation (SI) and suicidal behaviors have biological bases. However, no biological marker is currently available to evaluate the suicide risk in individuals with SI or suicide attempt (SA). Moreover, the current risk assessment techniques poorly predict future suicidal events. The aim of this study was to examine the association of 39 new and already described peripheral cells and proteins (implicated in the immune system, oxidative stress and plasticity) with lifetime SA, past month SA, current SI, and future suicidal events (visit to the Emergency Department for SI or SA) in 266 treatment-seeking individuals with mood disorders. Equal parts of patients with and without past history of SA were recruited. All individuals at inclusion gave blood, were evaluated for SA recency, current SI, and were followed for two years afterwards. The 39 peripheral blood cellular and protein markers were entered separately for each outcome in Elastic Net models with 10-fold cross-validation, followed by single-analyte covariate-adjusted regression analyses for pre-selected analytes. Past month SA was associated with increased plasma levels of thrombospondin-2 and C-reactive protein, whereas current SI was associated with lower plasma serotonin levels. These associations were robust to adjustments for key covariates and corrections for multiple testing. The Cox proportional hazards regression showed that higher levels of thrombospondin-1 and of platelet-derived growth factor-AB predicted a future suicidal event. These two associations remained after adjustment for sex, age, and SA history, and outperformed the predictive value of past SA. Thrombospondins and platelet-derived growth factors have never been investigated in the context of suicide. Altogether, our results highlight the involvement in the suicidal process of platelet biological response and plasticity modifiers and also of inflammatory factors. They also suggest that SI and SA may have different biological correlates and that biomarkers associated with past SA or current SI do not automatically also predict future events.

Uncovering pharmacological mechanisms of Phellinus linteus on focal segmental glomeruloscleosis rats through tandem mass tag-based quantitative proteomic analysis, network pharmacology analysis and experimental validation.

To explore the underlying molecular mechanism of (). We used a tandem mass tag-based quantitative proteomic method to determine the differentially expressed proteins. Network pharmacology analysis was used to analysis the main components of and construct the compound-target network. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the analyses results. The expression levels of thrombospondin-1 (TSP-1) and transforming growth factor (TGF)-β1/Smad3 signaling pathway proteins were significantly upregulated in focal segmental glomeruloscleosis (FSGS) rats. The reduced the expression levels of TSP-1 and TGF-β1 signaling pathway proteins. Network pharmacology analysis revealed that protocatechualdehyde was the main active component. Subsequent and experiments validated the results of proteomic and network pharmacology analyses. Our results suggested that may inhibit renal sclerosis by inhibiting TSP-1-activated TGF-β1 signaling and may have potential applications in the treatment of FSGS.

Circ_0058063 regulates cell vitality and proliferation in oesophageal squamous-cell carcinomas.

Oesophageal squamous-cell carcinoma (ESCC) is a malignant tumor of the digestive system with a poor prognosis. Recent studies have shown the promoting effect of hsa_circ_0058063 (circ_0058063) on ESCC, but the potential regulatory mechanisms of circ_0058063 in ESCC remain largely unclear. The levels of circ_0058063, microRNA-4319 (miR-4319) and mRNA of thrombospondin-1 (THBS1) were indicated by quantitative real-time polymerase chain reactionin ESCC tissues and cells. Meanwhile, the level of THBS1 was quantified by western blot analysis. In addition, the cell functions were examined by CCK8 assay, Edu assay, flow cytometry assay and transwell assay. Furthermore, the interplay between miR-4319 and circ_0058063 or THBS1 was detected by dual-luciferase reporter assay. Finally, anin vivo experiment was implemented to confirm the effect of circ_0058063. The level of circ_0058063 and THBS1 were increased, and the miR-4319 level was decreased in ESCC tissues in contrast to that in normal tissues and cells. For functional analysis, circ_0058063 deficiency inhibited cell vitality, cell proliferation, migration and invasion in ESCC cells, whereas promoted cell apoptosis. Moreover, miR-4319 was confirmed to repress the progression of ESCC cells by suppressing THBS1. In mechanism, circ_0058063 acted as a miR-4319 sponge to regulate the level of THBS1. Besides, circ_0058063 knockdown also attenuated tumour growth in vivo.Circ_0058063 facilitates the development of ESCC through increasing THBS1 expression by regulating miR-4319, which also offered an underlying targeted therapy for ESCC treatment.

Relationship of thrombospondin-1 and thrombospondin-2 with hematological, biochemical and inflammatory markers in COVID-19 patients

Abstract Objectives Roles of thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) in tissue repair and inflammation are well-documented, but the association of their serum expressions with the pathogenesis of COVID-19 remains unclear. We investigate the roles of TSP-1 and TSP-2 in COVID-19. Methods 106 SARS-CoV-2 infected patients and 23 healthy people were enrolled in our study. COVID-19 patients were divided into two groups as non-severe and severe. TSP-1 and TSP-2 concentrations were measured with an enzyme-linked Immunosorbent Assay, and blood markers were analyzed with routine laboratory techniques. Results COVID-19 patients had significantly higher TSP-1 and TSP-2 levels than healthy controls. TSP-1 and TSP-2 positively correlated with inflammatory markers, including ESR, CRP, PCT, ferritin, and biochemical parameters such as ALT, AST, BUN, CK, and LDH. In addition, TSP-1 and TSP-2 were negatively correlated with hematological markers such as LYM, EOS, and HGB. Receiver operating characteristic analyses revealed that COVID-19 may be predicted with TSP-1 levels over 189.94 ng/mL and TSP-2 levels higher than 0.70 ng/mL. Conclusions Our analysis suggests that TSP-1 and TSP-2 expressions at the systemic level may have clinical importance for COVID-19.

1292-P: Serum Thrombospondin-2 Level as a Novel Liver Fibrosis Biomarker That Changes with Liver Stiffness Improvement in Patients with Type 2 Diabetes

Introduction: High baseline circulating thrombospondin-2 (TSP2) level was recently found to associate with both the development and progression of liver fibrosis in patients with co-existing nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Here, we investigated whether circulating TSP2 levels changed with improvement in liver fibrosis. Methods: Serum TSP2 levels were measured in all participants of the DISTINCTION study (NCT 03959501), a randomized 1:1 open-label intervention study, at baseline and after treatment with either 10mg dapagliflozin (N=30) or 100mg sitagliptin daily (N=30) for 24 weeks. Liver stiffness (LS) and controlled attenuation parameter (CAP) were measured using vibration-controlled transient elastography for evaluation of hepatic fibrosis and steatosis, respectively. Results: Among these 60 participants with similar clinical characteristics at baseline (mean HbA1c 8.9%, CAP 289 dB/m and LS 5.9 kPa), despite similar HbA1c lowering, treatment with dapagliflozin, but not sitagliptin, led to significant improvements in body weight (BW) (p=0.012), CAP (p=0.015) and LS (p=0.011) after 24 weeks. Serum TSP2 level significantly decreased from 3.07 to 2.90 ng/mL (p=0.012), whereas no significant change in TSP2 was observed with sitagliptin. In Pearson correlation analysis, change in serum TSP2 levels positively correlated with change in LS in the dapagliflozin group (r=0.388, p=0.034), but not with changes in BW, CAP or HbA1c after dapagliflozin treatment. Conclusion: Serum TSP2 level decreased with LS after dapagliflozin treatment, and was independent of improvements in BW, glycemic control and liver steatosis, further supporting the potential of serum TSP2 level as a novel liver fibrosis biomarker in type 2 diabetes. Disclosure J.H.C. Mak: None. H. Fong: None. Y. Wong: None. K.C. Tan: None. S. Lam: None. C. Lee: None.

CD47 expression in ovarian cancer: Dynamic correlation with lymphocyte and macrophage features as well as thrombospondin-1 (TSP-1) under neoadjuvant chemotherapy.

5569 Background: To date, the results of immunotherapy strategies to treat ovarian cancer (OC) have been unfulfilling. A more comprehensive knowledge of the intricate immune tumor microenvironment (iTME) as well as the effects of chemotherapy on immune features may reveal novel immune targets. CD47 is overexpressed in OC and correlates with shorter survival and immune evasion via interaction with circulating ligands such as TSP-1. Little is known about how the CD47 receptor or its ligand TSP-1 changes under chemotherapy or their association with other features of iTME. In the randomized CHIVA trial, we therefore investigated CD47 expression in OC patients at diagnosis and after neoadjuvant chemotherapy (NACT) and assessed its relationship to other iTME features, including circulating level of thrombospondin-1 (TSP-1). Methods: Tumour samples were collected from 105 patients from the CHIVA trial, a GINECO-GINEGEPS study. CD47 expression was assessed by IHC in paired samples before and after NACT and scored by H-score: staining intensity (0, +1, +2, +3) x % cells positive (0-300). At baseline, we defined a CD47high (highest quartile, score above 240) and CD47low population (lowest quartile, score under 150). CD4+, CD8+, CD68+, CD163+, FoxP3+ cells were assessed by IF and scored as number of positive cells. The Pearson correlation coefficient (r) was used to determine the linear correlation between CD47 expression and other immune markers. Circulating TSP-1 was quantified by ELISA on paired plasma samples pre- and post-NACT. Results: While CD47high does not correlate with CD4 and CD8 expression at baseline, a correlation with CD8 (r=0.59, p=0.02) and a trend with CD4 (r=0.49, p=0.05) is observed after NACT. These linear correlations do not exist in the CD47low population. In addition, NACT induces a 2-fold decrease in FoxP3+ cells, but only within the CD47high group (p=0.028). Thus, both the CD4+/FoxP3+ (p=0.001) and CD8+/FoxP3+ (p=0.005) ratios were increased by 2-fold under NACT, exclusively in OC patients with CD47high expression. Interestingly, these correlations appear to be T-cell specific as no correlation exist when considering CD68 and CD163 before or after NACT, neither for CD47high nor CD47low. In parallel, we determined that the plasma level of TSP-1, a preferential CD47 agonist, was very high (&gt;1500 ng/mL) in OC patients and decreased by about 2-fold after NACT. Interestingly, a pattern emerges in which the highest level of circulating TSP-1 is associated with the CD47lowpopulation. Conclusions: Our data suggest that OC patients with the highest CD47 expression profile at baseline have greatest lymphocyte influx post-NACT and may be most likely to benefit from post-operative immunotherapy. Whether blocking the immune-suppressive CD47:TSP1 interaction in this subset would provide an alternative strategy merits investigation. Clinical trial information: NCT01583322 .

Antifibrotic Agent Mediated Tumor Microenvironment Modulation and Improved Nanomedicine Delivery in Solid Tumor.

The unique physiology of tumors limits the efficacy of chemotherapeutics. In efforts to improve the effectiveness of the existing chemotherapy drugs, nanomedicine emerged as a new hope but proved inadequate due to the transport barriers present within the tumor tissues, which limits the potential of nanomedicine. Dense collagen networks in fibrotic tissues contribute to hindering the penetration of molecular- or nano-scale medicine through tumor interstitium. In the present study, human serum albumin (HSA)-based nanoparticles (NPs) were developed for gemcitabine (GEM) and losartan (LST), which could offer secreted protein acids rich in cysteine (SPARC) and enhanced permeability and retention effect (EPR)-mediated drug accumulation in tumors. Also, the tumor microenvironment (TME) modulation approach using LST was coupled to investigate the impact on antitumor efficacy. GEM-HSA NPs and LST-HSA NPs were prepared by the desolvation-cross-linking method and characterized for size, potential, morphology, drug loading, drug-polymer interactions, and hemocompatibility. For investigating the efficacy of prepared NPs, cytotoxicity and mechanisms of cell death were elucidated in vitro by using various assays. Intracellular uptake studies of prepared HSA NPs indicated their uptake and cytoplasmic localization. Furthermore, in vivo studies demonstrated significantly improved anticancer efficacy of GEM-HSA NPs in combination with LST pretreatment. Extended LST treatment further improved the anticancer potential. It was shown that the improved efficacy of the nanomedicine was correlated with the reduced thrombospondin-1 (TSP-1) and collagen level in tumor tissue upon LST pretreatment. Moreover, this approach exhibited augmented nanomedicine accumulation in the tumor, and hematological, biochemical, and tissue histology indicated the safety profile of this combination regimen. Concisely, the undertaken study demonstrated the potential of the triple targeting (SPARC, EPR, TME modulation) approach for augmented efficacy of chemotherapeutics.

Abstract 2510: Pentraxin-3 associated up-regulation of genes involved in motility, angiogenesis, and inflammation in glioblastoma

Abstract Glioblastoma, a malignant brain cancer, is highly invasive and resistant to multimodal treatment partly due to distorted vasculature and exacerbated inflammation in its tumor microenvironment. The glycoprotein pentraxin-3 (PTX3), a modulator of inflammation, is correlated with the severity of some cancers. However, the mechanism responsible for the oncogenic role of PTX3 in GBM malignancy remains unclear. Our previous study examined the role of PTX3 in GBM growth, angiogenesis, and invasion using in vitro glioblastoma cells and in vivo/pre-clinical GBM models, proteomic profiling, molecular and biochemical approaches. Over-expression of PTX3 in U87 brain tumor cells correlated with cell cycle progression, increased migratory potential, and survival under hypoxic conditions. PTX3 over-expression increased blood vessel forming ability of endothelial cells. Importantly, PTX3 over-expression increased tumor growth, micro-vessel density, and invasion in brains of an orthotopic mouse model. To understand the molecular mechanisms associated with PTX3 mediated tumor aggressiveness, we further examined the effects of PTX3 on mRNA levels of genes involved in motility/invasion, angiogenesis, and inflammation using pathway specific gene arrays. PTX3 over expression was associated with the 5-fold upregulation of hypoxia-inducible factor (HIF)-1α, which in turn transactivates several genes essential for tumor growth and survival. Indeed, higher PTX3 expression led to increased mRNA levels of genes involved in motility, angiogenesis, and inflammation. The upregulated motility associated genes included ARHGEF7, RAC1, and PAK, a protein kinase involved in intracellular signaling pathways downstream of integrins. Inflammatory genes that were increased in expression included IL-1beta, CCL3, CCL5, and CXCL5 that amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway. The upregulated pro-angiogenic genes included interleukin-8 (IL-8), matrix metalloproteinase-2 (MMP2), and vascular endothelial growth factor (VEGF), expression of which correlates with GBM malignancy. Interestingly PTX3 decreased the levels of thrombospondin-1 that inhibits angiogenesis. Thus, PTX3 appears to trigger multiple signaling pathways that enhance the aggressive behavior of tumor cells. Taken together, our study indicates that targeted therapy such as blocking PTX3 may reduce the aggressiveness of glioblastomas through regulation of multiple molecular pathways. Citation Format: Umadevi V. Wesley, Ian Sutton, Dylan E. Nelson, Paul A. Clark, Robert J. Dempsey. Pentraxin-3 associated up-regulation of genes involved in motility, angiogenesis, and inflammation in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2510.

Neutrophil extracellular trap formation in anti-neutrophil cytoplasmic antibody-associated and large-vessel vasculitis.

Levels of neutrophil extracellular traps (NETs) were measured in plasma of healthy controls (HC, n=30) and patients with granulomatosis with polyangiitis (GPA, n=123), microscopic polyangiitis (MPA, n=61), Takayasu's arteritis (TAK, n=58), and giant cell arteritis (GCA, n=68), at times of remission or activity and correlated with levels of the platelet-derived thrombospondin-1 (TSP-1). Levels of NETs were elevated during active disease in patients with GPA (p<0.0001), MPA (p=0.0038), TAK (p<0.0001), and GCA (p<0.0001), and in remission for GPA, p<0.0001, MPA, p=0.005, TAK, p=0.03, and GCA, p=0.0009. All cohorts demonstrated impaired NET degradation. Patients with GPA (p=0.0045) and MPA (p=0.005) had anti-NET IgG antibodies. Patients with TAK had anti-histone antibodies (p<0.01), correlating with presence of NETs. Levels of TSP-1 were increased in all patients with vasculitis, and associated with NET formation. NET formation is a common process in vasculitides. Targeting NET formation or degradation could be potential therapeutic approaches for vasculitides.

Baicalin inhibited both the Furin/TGFβ1/Smad3/TSP-1 pathway in endothelial cells and the AKT/Ca2+/ROS pathway in platelets to ameliorate inflammatory coagulopathy

Inflammatory coagulopathy is resulted from endothelial dysfunction and platelet hyperactivation in inflammatory diseases. In this study, the effects of baicalin, an active component of the traditional Chinese medicine Huangqin, on inflammatory coagulopathy were observed both in vivo and in vitro. In LPS-induced rats, baicalin ameliorated coagulation indexes, inhibited platelet hyperactivation and decreased the expression of thrombospondin-1 (TSP-1) in vessels. In cultured endothelial cells, baicalin decreased the expression of TSP-1 and collagen as well as the TNF-α-induced increase in the levels of TSP-1 and ICAM-1. Baicalin could significantly decrease the platelet adhesion on endothelial cells treated with TNF-α. Baicalin also could inhibit the increase of ROS level and the activation of the NLRP3/Caspase-1/GSDMD pathway in TNF-α-induced endothelial cells. Furin was found to be the direct target of baicalin in HUVECs. Knockdown of Furin using siRNA could ameliorate the effects of baicalin on the activation of TGFβ1/Smad3 pathway, TSP-1 expression and the adhesion of platelets on TNF-α-treated endothelial cells. At the same time, baicalin inhibited platelet aggregation induced by collagen or combination of collagen and TSP-1 peptide. Collagen-induced Ca2+ mobilization, ROS level increase, AKT1 phosphorylation, platelet degranulation and TSP-1 release could be all inhibited by baicalin. In all, baicalin ameliorated endothelial dysfunction by inhibiting Furin/TGFβ1/Smad3/TSP-1 pathway and also ameliorated platelet activation by inhibiting AKT-related pathway. Both the inhibiting effects of baicalin on endothelial dysfunction and platelet activation might contribute to its ameliorating effects on inflammatory coagulopathy.

Circulating thrombospondin-2 level for identifying individuals with rapidly declining kidney function trajectory in type 2 diabetes: a prospective study of the Hong Kong West Diabetes Registry.

Thrombospondin-2 (TSP2) is a matricellular protein with tissue expression induced by hyperglycaemia. TSP2 has been implicated in non-diabetic renal injury in preclinical studies and high circulating levels were associated with worse kidney function in cross-sectional clinical studies. Therefore, we investigated the prospective associations of circulating TSP2 level with kidney function decline and the trajectories of estimated glomerular filtration rate (eGFR) in type 2 diabetes. Baseline serum TSP2 level was measured in 5471 patients with type 2 diabetes to evaluate its association with incident eGFR decline, defined as≥40% sustained eGFR decline, using multivariable Cox regression analysis. Among participants with relatively preserved kidney function (Baseline eGFR≥60ml/min/1.73m2), joint latent class modelling was employed to identify three different eGFR trajectories. Their associations with baseline serum TSP2 was evaluated using multinomial logistic regression analysis. The predictive performance of serum TSP2 level was examined using time-dependent c-statistics and calibration statistics. Over a median follow-up of 8.8 years, 1083 patients (19.8%) developed eGFR decline. Baseline serum TSP2 level was independently associated with incident eGFR decline (HR 1.21, 95%CI 1.07-1.37, P=0.002). With internal validation, incorporating serum TSP2 to a model of clinical risk factors including albuminuria led to significant improvement in c-statistics from 83.9 to 84.4 (P<0.001). Among patients with eGFR≥60ml/min/1.73m2, baseline serum TSP2 level was independently associated with a rapidly declining eGFR trajectory (HR 1.63, 95%CI 1.26-2.10, P<0.001). Serum TSP2 level was independently associated with incident eGFR decline, particularly a rapidly declining trajectory, in type 2 diabetes.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Thrombospondin-2 as a Predictive Biomarker for Hepatocellular Carcinoma after Hepatitis C Virus Elimination by Direct-Acting Antiviral.

We evaluated the value of secreted glycoprotein thrombospondin-2 (TSP-2) to predict hepatocellular carcinoma (HCC) occurrence in chronic hepatitis C (CHC) patients after Hepatitis C virus (HCV) elimination by direct-acting antiviral agents (DAAs). A total of 786 CHC patients without an HCC history who achieved a sustained virological response (SVR) with DAAs were randomly assigned 2:1, with 524 patients as the derivation cohort and 262 patients as the validation cohort. Serum TSP-2 levels at the end of treatment were measured by enzyme-linked immunosorbent assay (ELISA). In the derivation cohort, the cumulative HCC rate was significantly higher in the high TSP-2 group than in the low TSP-2 group. Multivariate Cox proportional hazards analysis revealed that TSP-2, α-fetoprotein (AFP), and the fibrosis-4 (FIB-4) index were independent HCC risk factors. The area under the receiver operating characteristic curve (AUROC) of the score calculated from these three factors (AFT score) for predicting HCC was 0.83, which was significantly higher than that of each factor alone (TSP-2: 0.70, AFP: 0.72, FIB-4: 0.69). The AFT score was used to stratify patients according to the risk of HCC occurrence in the validation cohort. Lastly, in patients with a FIB-4 index < 3.25, the serum TSP-2 levels could be used to identify those patients with a high risk of HCC occurrence. Serum TSP-2 levels are a predictive biomarker of HCC occurrence in CHC patients after HCV elimination by DAA treatment. The AFT score using TSP-2, AFP, and the FIB-4 index may identify those who require HCC surveillance.

Clinical phenotyping of plasma thrombospondin-2 reveals relationship to right ventricular structure and function in pulmonary hypertension.

Converging evidence from proteogenomic analyses prioritises thrombospondin-2 (TSP2) as a potential biomarker for idiopathic or heritable pulmonary arterial hypertension (PAH). We aimed to assess TSP2 levels in different forms of pulmonary hypertension (PH) and to define its clinical phenotype. Absolute concentrations of TSP2 were quantified in plasma samples from a prospective single-centre cohort study including 196 patients with different forms of PH and 16 disease controls (suspected PH, but normal resting pulmonary haemodynamics). In an unbiased approach, TSP2 levels were related to 152 clinical variables. Concentrations of TSP2 were increased in patients with PH versus disease controls (p<0.001 for group comparison). The discriminatory ability of TSP2 levels to distinguish between patients and controls was superior to that of N-terminal pro-brain natriuretic peptide (p=0.0023 for comparison of areas under the curve). Elevation of TSP2 levels was consistently found in subcategories of PAH, in PH due to lung disease and due to left heart disease. Phenotypically, TSP2 levels were robustly related to echocardiographic markers that indicate the right ventricular (RV) response to chronically increased afterload with increased levels in patients with impaired systolic function and ventriculoarterial uncoupling. Focusing on PAH, increased TSP2 levels were able to distinguish between adaptive and maladaptive RV phenotypes (area under the curve 0.87, 95% CI 0.76-0.98). The study indicates that plasma TSP2 levels inform on the presence of PH and associate with clinically relevant RV phenotypes in the setting of increased afterload, which may provide insight into processes of RV adaptability.