Thrombospondin-1 Expression Research Articles

Tumor-stroma TGF-β1-THBS2 feedback circuit drives pancreatic ductal adenocarcinoma progression via integrin αvβ3/CD36-mediated activation of the MAPK pathway.

The pancreatic ductal adenocarcinoma (PDAC) microenvironment contains dense desmoplastic stroma dominated by cancer-associated fibroblasts (CAFs) and is crucial to cancer development and progression. Several studies have revealed that thrombospondin 2 (THBS2) is a valuable serological-marker in PDAC. However, the detailed mechanism of the cancer-stroma interactome remains unclear. Here we showed that elevated THBS2 expression in PDAC was predominantly restricted to stroma and correlated with tumor progression and poor prognosis by quantitative proteomics and immunohistochemistry analyses. RNA in situ hybridization confirmed that CAFs but not neoplastic cells expressed THBS2 in precancerous lesions and its levels gradually increased with disease progression in genetically engineered mouse models. Mechanistically, cancer cell-secreted TGF-β1 activated CAFs to induce THBS2 expression via the p-Smad2/3 pathway. Consequently, CAF-derived THBS2 bound to the membrane receptors integrin αvβ3/CD36 and activated the MAPK pathway in PDAC cells to promote tumor growth and adhesion in vitro and in vivo. Inhibition of integrin αvβ3, CD36, MEK and JNK rescued THBS2-induced malignant phenotypes. In conclusion, the TGF-β1-THBS2-integrin αvβ3/CD36-MAPK cascade forms a complex feedback circuit to mediate reciprocal interactions of pancreatic cancer cells-CAFs. THBS2 may act as a novel therapeutic-target to block the cancer-stroma communication.

Transforming growth factor-β1/Thrombospondin-1/CD47 axis mediates dysfunction in CD34+ cells derived from diabetic older adults

Aging with diabetes is associated with impaired vasoprotective functions and decreased nitric oxide (NO) generation in CD34+ cells. Transforming growth factor- β1 (TGF-β1) is known to regulate hematopoietic functions. This study tested the hypothesis that transforming growth factor- β1 (TGF-β1) is upregulated in diabetic CD34+ cells and impairs NO generation via thrombospondin-1 (TSP-1)/CD47/NO pathway. CD34+ cells from nondiabetic (ND) (n=58) or diabetic older adults (DB) (both type 1 and type 2) (n=62) were isolated from peripheral blood. TGF-β1 was silenced by using an antisense delivered as phosphorodiamidate morpholino oligomer (PMO-TGF-β1). Migration and proliferation in response to stromal-derived factor-1α (SDF-1α) were evaluated. NO generation and eNOS phosphorylation were determined by flow cytometry. CD34+ cells from older, but not younger, diabetics have higher expression of TGF-β1 compared to that observed in cells derived from healthy individuals (P<0.05, n=14). TSP-1 expression was higher (n=11) in DB compared to ND cells. TGFβ1-PMO decreased the secretion of TGF-β1, which was accompanied with decreased TSP-1 expression. Impaired proliferation, migration and NO generation in response to SDF-1α in DB cells were reversed by TGF-β1-PMO (n=6). TSP-1 inhibited migration and proliferation of nondiabetic CD34+ cells that was reversed by CD47-siRNA, which also restored these responses in diabetic CD34+ cells. TSP-1 opposed SDF-1α-induced eNOS phosphorylation at Ser1177 that was reversed by CD47-siRNA. These results infer that increased TGF-β1 expression in CD34+ cells induces dysfunction in CD34+ cells from diabetic older adults via TSP-1/CD47-dependent inhibition of NO generation.

THBS2 is Closely Related to the Poor Prognosis and Immune Cell Infiltration of Gastric Cancer.

Background: The potential functions of Thrombospondin 2 (THBS2) in the progression and immune infiltration of gastric cancer (GC) remain unclear. The purpose of this study was to clarify the role of THBS2 in GC prognosis and the relationship between THBS2 and GC immune cell infiltration. Material and Methods: The differential expression levels of THBS2 in the GC and cancer-adjacent tissues were identified using the TCGA databases and verified using real-time polymerase chain reaction (PCR), immunohistochemical staining and two datasets from Gene Expression Omnibus (GEO). THBS2 related differential expressed genes (DEGs) were identified and used for further functional enrichment analysis and Gene Set Enrichment Analysis (GSEA). Furthermore, a THBS2-related immune infiltration analysis was also performed. Kaplan-Meier and Cox regression analyses were utilized to illustrate the effects of THBS2 on the prognosis and clinical variables of GC. Finally, a nomogram was constructed to predict the survival probability of patients with GC. Results: The THBS2 expression in GC was significantly higher than that in cancer-adjacent tissues (p < 0.001), which was verified using real-time PCR, immunohistochemical staining and datasets from GEO. The 599 identified DEGs were primarily enriched in pathways related to tumorigenesis and tumor progression, including the focal adhesion pathway, signaling by vascular endothelial growth factor, and Wnt signaling. THBS2 expression was positively correlated with the enrichment of the macrophages (r = 0.590, p < 0.001), which was also confirmed by immunohistochemistry; however, negatively correlated with the enrichment of Th17 cells (r = 0.260, p < 0.001). The high expression of THBS2 was significantly correlated with the pathological grade (p < 0.01), histological grade (p < 0.05), histological type (p < 0.05), T stage (p < 0.001), and poor overall survival (OS) (P = 0.003) of GC. The constructed nomogram can well predict the 1-, 3-, and 5-years OS probability of patients with GC (C-index [95% confidence interval] = 0.725 [0.701–0.750]). Conclusion: THBS2 is closely related to the poor prognosis and immune infiltration of gastric cancer.

Neuronal GPR81 regulates developmental brain angiogenesis and promotes brain recovery after a hypoxic ischemic insult

Perinatal hypoxic/ischemic (HI) brain injury is a major clinical problem with devastating neurodevelopmental outcomes in neonates. During HI brain injury, dysregulated factor production contributes to microvascular impairment. Glycolysis-derived lactate accumulated during ischemia has been proposed to protect against ischemic injury, but its mechanism of action is poorly understood. Herein, we hypothesize that lactate via its G-protein coupled receptor (GPR81) controls postnatal brain angiogenesis and plays a protective role after HI injury. We show that GPR81 is predominantly expressed in neurons of the cerebral cortex and hippocampus. GPR81-null mice displayed a delay in cerebral microvascular development linked to reduced levels of various major angiogenic factors and augmented expression of anti-angiogenic Thrombospondin-1 (TSP-1) in comparison to their WT littermates. Coherently, lactate stimulation induced an increase in growth factors (VEGF, Ang1 and 2, PDGF) and reduced TSP-1 expression in neurons, which contributed to accelerating angiogenesis. HI injury in GPR81-null animals curtailed vascular density and consequently increased infarct size compared to changes seen in WT mice; conversely intracerebroventricular lactate injection increased vascular density and diminished infarct size in WT but not in GPR81-null mice. Collectively, we show that lactate acting via GPR81 participates in developmental brain angiogenesis, and attenuates HI injury by restoring compromised microvasculature.

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells.

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.

High Expression of THBS1 Leads to a Poor Prognosis in Papillary Thyroid Cancer and Suppresses the Anti-Tumor Immune Microenvironment.

Objectives: To study the role of thrombospondin-1 (THBS1) in papillary thyroid cancer (PTC) prognosis and the immune microenvironment. Methods: A retrospective cohort study was designed, and data from The Cancer Genome Atlas database and PTC tissues from Fudan University Shanghai Cancer Center were used. Weighted gene co-expression network analysis was performed to build a THBS1-immune-related gene prognostic index (T-I index). Results: High THBS1 expression was correlated with advanced TNM stage, higher recurrence risk, and shorter progression-free interval. High THBS1 expression correlated with MAPK and PD1 pathways indicating a tumor promoting and immunity-inhibiting tendency. The T-I index showed a powerful capacity to predict progression-free survival and immunotherapy benefit. Conclusion: High expression of THBS1 leads to a poor prognosis in PTCs and suppresses the anti-tumor immune microenvironment.

MiR-375 and miR-5691 exert anti-fibroproliferative effects on hypertrophic scar fibroblasts by suppressing thrombospondin 1 expression

Background: Hypertrophic scar (HS) is characterized by the hyperproliferation of fibroblasts and the excessive deposition of extracellular matrix (ECM). Thrombospondin 1 (THBS1) is a component of the ECM, which has been implicated in HS formation. Objectives: This study aimed to explore whether miR-375/miR-5691 could modulate HS formation by targeting THBS1. Methods: The expression levels of miR-375/miR-5691/THBS1 in HS and normal skin tissues were measured by quantitative reverse transcription-polymerase chain reaction. 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di-phenytetrazoliumromide and Western blot assays were performed on fibroblasts isolated from HS tissues (HSFBs) to determine cell proliferation and the expression levels of proliferating cell nuclear antigen (PCNA), apoptosis-related proteins (caspase3/9, cleaved caspase3/9, Bax, and Bcl-2), and ECM-related proteins. The binding sites between THBS1 and miR-375/miR-5691 were predicted by the TargetScan. Dual-luciferase reporter and anti-Ago2 immunoprecipitation assays were applied to confirm the interactions between THBS1 and miR-375/miR-5691. Results: The expression levels of both miR-375 and miR-5691 were downregulated in HS tissues and HSFBs, which were negatively correlated with THBS1 expression levels. The overexpression of miR-375/miR-5691 inhibited cell proliferation and ECM production, and promoted apoptosis of HSFBs, while silencing of miR-375/miR-5691 led to an opposite result. In the mechanism analysis, THBS1 was confirmed as the direct target gene of miR-375/miR-5691. Furthermore, rescue experiments showed that the suppressed growth of HSFBs and ECM production induced by silencing of THBS1 was reversed by miR-375/miR-5691 inhibitors. Conclusion: MiR-375/miR-5691 was downregulated in HS tissues, and it could suppress the hyperproliferation and ECM production of HSFBs by targeting THBS1.

High Level of Staufen1 Expression Confers Longer Recurrence Free Survival to Non-Small Cell Lung Cancer Patients by Promoting THBS1 mRNA Degradation.

Stau1 is a pluripotent RNA-binding protein that is responsible for the post-transcriptional regulation of a multitude of transcripts. Here, we observed that lung cancer patients with a high Stau1 expression have a longer recurrence free survival. Strikingly, Stau1 did not impair cell proliferation in vitro, but rather cell migration and cell adhesion. In vivo, Stau1 depletion favored tumor progression and metastases development. In addition, Stau1 depletion strongly impaired vessel maturation. Among a panel of candidate genes, we specifically identified the mRNA encoding the cell adhesion molecule Thrombospondin 1 (THBS1) as a new target for Staufen-mediated mRNA decay. Altogether, our results suggest that regulation of THBS1 expression by Stau1 may be a key process involved in lung cancer progression.

Thrombospondin-1 aggravates colonic mucosal inflammatory injuries via promoting the differentiation of CD11c+ macrophages with lysosomal activity limited in colitis.

BackgroundIncreased CD11c+ Mφ aggravates colonic mucosal injuries in ulcerative colitis (UC) with TSP1 protein increased. The thrombospondin-1 (TSP1) protein which could activate Mφ is closely related to the colonic mucosal damage in UC. Here, we investigated the role of TSP1 in the differentiation of CD11c+ Mφ and the mechanism.MethodsWe analyzed the population characteristics of TSP1 genes using the Genotype-Tissue Expression (GTEx) database, and human serum TSP1 protein was detected with ELISA. DSS-induced colitis rats were used to explore the effects of TSP1 on colonic mucosal inflammation. We analyzed the serum cytokines and tissue histopathology to evaluate the severity of UC. Furthermore, we analysed the main source of TSP1 in colon tissue. In vitro, lamina propria mononuclear cells (LPMC) and CD11c+ lamina propria macrophages (LPMP) was isolated from model rats in vivo. The target of TSP1 protein was assessed by LSKL, CD36 and CD47 interfering plasmids. The proteins, the lysosome, lysosomal activity and Cathepsin E activity, and the migration were detected by western blotting, test kits and Transwell.ResultsThe expression of TSP1 was significantly higher in younger, male, and in the rectum and sigmoid than that in older, females, and colon tissues, and was closely related to the severity of UC. Compared with normal rats, the worse disease activity index (DAI) score, more histological damage, CD11c+ Mφ infiltration, and increased expression of several proinflammatory cytokines was displayed in colitis rats with the elevation of serum TSP1 protein. In vitro, TSP1 protein derived from cmMφ and endothelial cells promoted the migration and the differentiation of CD11c+ Mφ via binding on CD36, rather than the cell proliferation. Furthermore, PRKCQ/NF-κB signaling pathway was activated by CD36. However, the effect of TSP1 protein could be reversed by LSKL in vivo, and LSKL and anti-TSP1 antibody in vitro.ConclusionsTSP1 promotes the migration and the differentiation of CD11c+ LPMP with lysosomal activity limited via activating the CD36-PRKCQ/NF-κB signaling pathway, which aggravates the colonic mucosal inflammatory injuries in UC.

MicroRNA 1228 Mediates the Viability of High Glucose-Cultured Renal Tubule Cells through Targeting Thrombospondin 2 and PI3K/AKT Signaling Pathway

Aim: The present study aimed to elucidate the potential function of microRNA 1228 (miR-1228) on the high glucose (HG)-damaged human renal proximal tubule cells (HK-2) and the underlying mechanism. Methods: The datasets GSE47185 and GSE51674 were downloaded from the Gene Expression Omnibus database for mining differently expressed mRNAs and miRNAs, respectively. Bioinformatics online tools were applied to predict the binding sites between miR-1228 and thrombospondin 2 (THBS2), which was confirmed by dual-luciferase assay. Real-time quantitative polymerase chain reaction was used to detect the mRNA level of miR-1228/THBS2. Western blot was used to detect the protein level of THBS2 and the PI3K/AKT signaling pathway-associated markers. HK-2 cells were cultured in HG (30 mM) to mimic hyperglycemia. Cell counting kit 8 and flow cytometry assays were utilized to determine the cell proliferation and apoptosis. Results: The expression of THBS2 was significantly upregulated in diabetic nephropathy (DN) based on bioinformatics tools and identified as a direct target of miR-1228. miR-1228 was downregulated in DN and HG-damaged HK-2 cells. HG notably reduced HK-2 cell proliferation. This negative effect was attenuated by transfecting with an miR-1228 mimic and aggravated by transfecting with an miR-1228 inhibitor. However, under basal condition, there was no significant effect on the HK-2 cell proliferation among blank control, mimic, and inhibitor groups. Overexpression of THBS2 abolished the elevating effect of the miR-1228 mimic on the HG-damaged HK-2 cell proliferation, while restored the inhibitory effects of the miR-1228 mimic on the cell apoptosis. On the contrary, the suppressive effects on the proliferation and the enhancive effects on the apoptosis by silencing miR-1228 in HK-2 cells stimulated with HG can be weakened by recommendation of THBS2 small interference RNAs. Furthermore, we also found that HG significantly enhanced the phosphorylation levels of PI3K and AKT. In terms of overexpression and knockdown experiments, Western blot analysis further revealed that miR-1228 inhibited the activation of the PI3K/AKT signaling pathway in HG-damaged HK-2 cells by regulating THBS2. Conclusion: The findings illustrated that miR-1228 improved survivability and inhibited apoptosis in HK-2 cells stimulated with HG partly by restraining the activation of the PI3K/AKT signaling pathway.

CTIM-06. CLINICAL EFFICACY AND BIOMARKER ASSESSMENT OF VT1021, A CD36/CD47 DUAL-TARGETING AGENT, IN RECURRENT GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults and recurs after treatment in &amp;gt;90% of all patients. Prognosis of patients with recurrent GBM (rGBM) is poor with a median progression free survival of ~1.8 months and median overall survival of 8-10 months. VT1021, a cyclic peptide derived from prosaposin, induces the expression of thrombospondin-1 (TSP-1) in myeloid derived suppressor cells (MDSCs), which are heavily recruited to the tumor microenvironment. Following completion of the Phase 1 Dose Escalation study and determination of the RP2D, an expansion study was conducted in a heavily pretreated rGBM population. The results of the expansion study reveal that VT1021 is safe and well tolerated in rGBM. Pharmacodynamic response to VT1021 was observed by induction of TSP-1 in circulating MDSCs and in the tumor microenvironment. VT1021 demonstrated significant single agent activity. Among 22 evaluable GBM subjects, 3 had complete response (CR), 1 had partial response (PR), and 7 had stable disease (SD) with an average study duration of over 120 days. The overall disease control rate (DCR) was 50%. Among the 3 CR, 2 showed complete radiological regression of the target lesion, the third was found to have no evidence of disease upon pathological examination following surgery. Nine of the 19 (47%) evaluable subjects with available biopsy samples showed high expression levels of both CD36 AND CD47. Among these 9 subjects, 3 achieved CR, representing an overall response rate of 33.3%, with another 3 patients achieving SD for a DCR of 67%. In conclusion, VT1021 demonstrates promising single-agent clinical activity in rGBM, particularly in subjects with high expression levels of CD36 and CD47. Additional clinical studies have been planned to further investigate the efficacy of VT1021 in rGBM and other solid tumor indications.

MiR-1290 contributes to oncogenesis and angiogenesis via targeting of THBS1, DKK3 and, SCAI.

Introduction: Colorectal cancer (CRC) is the third most common cancer in the world with high mortality, hence, understanding the molecular mechanisms involved in the tumor progression are important for CRC diagnosis and treatment. MicroRNAs (miRNAs) are key gene expression regulators that can function as tumor suppressors or oncogenes in tumor cells, and modulate angiogenesis as a critical process in tumor metastasis. MiR-1290 has been demonstrated as an onco-miRNA in various types of cancer, however, the role of miR-1290 in CRC is not fully understood. This study aimed to investigate the oncogenic and angiogenic potential of miR-1290 in CRC. Methods: Lenti-miR-1290 was transduced into HCT116, SW480, and human umbilical vein endothelial cells (HUVECs). By bioinformatics analysis, we identified thrombospondin 1 (THBS1) as a novel predicted target for miR-1290. Quantitative real-time PCR, western blotting, and luciferase reporter assay were used to demonstrate suppression of miR-1290 target genes including THBS1, Dickkopf Wnt signaling pathway inhibitor 3 (DKK3), and suppressor of cancer cell invasion (SCAI) in HCT116 and HUVECs. Cell cycle analysis, proliferation, migration and, tube formation were determined by flow cytometry, MTT, wound healing, and tube formation assays, respectively. Results: MiR-1290 significantly decreased the expression of THBS1, DKK3, and SCAI. We demonstrated that miR-1290 enhanced proliferation, migration, and angiogenesis partially through suppression of THBS1, DKK3, and SCAI in CRC. Conclusion: These results suggest a novel function of miR-1290 which may contribute to tumorigenesis and angiogenesis in CRC.

A2A receptor agonists and P2Y12 receptor antagonists modulate expression of thrombospondin-1 (TSP-1) and its secretion from Human Microvascular Endothelial Cells (HMEC-1).

To address the problem of resistance to standard antiplatelet therapy in some patients, our team proposed a purinoceptor-dependent dual therapy. Its efficacy is also determined by the condition of the vascular endothelium which, by secreting numerous factors, is involved in hemostasis. Among them, thrombospondin-1 is important in the context of thrombotic events. Therefore we sought to determine if the novel dual purinoceptor-dependent concept is associated with TSP-1 changes in vascular endothelial cells. TSP-1 expression in human microvascular endothelial cells was determined at transcriptional and protein level. We performed real-time PCR, the Western blot analysis and ELISA test. We found that TSP-1 mRNA and protein expression levels significantly changed in response to P1R agonists treatment. Furthermore, we have observed that co-administration of selective A2AR agonists (UK-432,097 or MRE0094) with P2Y12R antagonists altered TSP-1 expression levels, and the direction of these changes was not synergistic. MRE0094 applied with ARC69931MX or R-138727 increased mRNA expression from 39 to 56 or 57%, respectively (*P<0.05 vs. MRE0094; ***P<0.001 vs. control). Also, in the case of the P2Y12R antagonists used together with UK-432,097, there was an increase from 53 to 71 and 70% (*P<0.05 vs. UK-432,097; ***P<0.001 vs. control). The observed trends in gene expression were reflected in the protein expression and the level of its secretion from HMEC-1. The article presents evidence which proves that the purinoceptor-dependent concept is associated with TSP-1 changes in endothelial cells (EC). Moreover, Human Microvascular Endothelial Cells treatment applied together with agonists (MRE0094 or UK-432,097) and P2Y12R antagonist did not result in any synergistic effect, implicating a possible crosstalk between G proteins in GPCRs dependent signaling. Therefore, we suggest that understanding of the specific mechanism underlying TSP-1 alterations in EC in the context of the dual purinoceptor-dependent approach is essential for antiplatelet therapies and should be the subject of future research.

MRNA Expression of thrombospondin 1, 2 and 3 from proximal to distal in human abdominal aortic aneurysm - preliminary report.

Abdominal aortic aneurysm is a process involving the disruption and reconstruction of the extracellular matrix and the apoptosis of smooth muscle cells under the strong influence of the immune system. Thrombospondins are proteins that influence a wide range of cell-matrix interactions. While THBS1 and THBS2 are widely studied, the effects of THBS3 on extracellular matrix and vascular cells are poorly understood. Additionally, it is not known whether expression of these genes' changes along the aneurysm tissue. Here we analyzed the expression of THBSs mRNA isolated from the harvested tissues along the aneurysm divided into three zones based on their morphology. Total mRNA was isolated from 13 male patients undergoing scheduled open aortic repair, with each aneurysm divided into a proximal part, an aneurysm bag, and a distal part with border tissue as a control. Two step real-time PCR analysis with random hexamers was performed, which allowed the detection of significantly increased expression of all analyzed thrombospondins, especially THBS3, at the control tissue. Overexpression of THBSs may have a destabilizing effect on the structure of the extracellular matrix by affecting both the matrix producing cells and by inhibiting the activity of matrix proteins.

Exosomes derived from LPS-stimulated human thymic mesenchymal stromal cells enhance inflammation via thrombospondin-1.

Inflammatory response mediated by immune cells is either directly or indirectly regulated by mesenchymal stromal cells (MSCs). Accumulating evidence suggests that thrombospondin-1 (TSP-1) is highly expressed in response to inflammation. In this work, we isolated and identified human thymic mesenchymal stromal cells (tMSCs) and detected the expression of TSP-1. We found that tMSCs expressed TSP-1 and Poly (I:C) or LPS treatment promoted the expression of TSP-1. Further, we isolated and identified exosomes originating from tMSCs (MEXs). Notably, exosomes derived from LPS-pretreated tMSCs (MEXsLPS) promoted the polarization of macrophages to M1-like phenotype and IL-6, TNF-α secretion as well as the pro-inflammatory differentiation of CD4+T cells into Th17 cells. Upon silencing the expression of TSP-1 in tMSCs, the pro-inflammatory effects of MEXsLPS were suppressed. Therefore, these findings uncovered TSP-1 as the principal factor in MEXsLPS pro-inflammatory regulation.

MiR-29a-3p/THBS2 Axis Regulates PAH-Induced Cardiac Fibrosis.

Pulmonary artery hypertension (PAH) pathology involves extracellular matrix (ECM) remodeling in cardiac tissues, thus promoting cardiac fibrosis progression. miR-29a-3p reportedly inhibits lung progression and liver fibrosis by regulating ECM protein expression; however, its role in PAH-induced fibrosis remains unclear. In this study, we aimed to investigate the role of miR-29a-3p in cardiac fibrosis progression in PAH and its influence on ECM protein thrombospondin-2 (THBS2) expression. The diagnostic and prognostic values of miR-29a-3p and THBS2 in PAH were evaluated. The expressions and effects of miR-29a-3p and THBS2 were assessed in cell culture, monocrotaline-induced PAH mouse model, and patients with PAH. The levels of circulating miR-29a-3p and THBS2 in patients and mice with PAH decreased and increased, respectively. miR-29a-3p directly targets THBS2 and regulates THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis. The circulating levels of miR-29a-3p and THBS2 were correlated with PAH diagnostic parameters, suggesting their independent prognostic value. miR-29a-3p targeted THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis, indicating miR-29a-3p acts as a messenger with promising therapeutic effects.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

Abstract P153: Upregulation Of Thrombospondin-1 Associates With Accelerated Atherosclerosis And Reduced Smooth Muscle Cell Differentiation In Metabolic Syndrome

Hyperglycemia and obesity, characteristic of metabolic syndrome (MetS), are important risk-factors for atherosclerosis. MetS patients manifest increased vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of VSMC phenotypic transition, critical for evolution of atherosclerosis. We previously reported that high glucose and high leptin independently upregulate a potent proatherogenic matricellular protein, Thrombospondin-1 (TSP-1), expression in VSMC. The goal of the present study was to interrogate the role of TSP-1 in SMC de-differentiation and development of atherosclerosis in MetS. We utilized a mouse model of combined MetS and atherosclerosis (KKAy +/- /ApoE -/- ) generated by crossing obese hyperglycemic agouti KKAy +/- mice with atherosclerotic ApoE -/- . Upon weaning (4 wks age), male yellow KKAy +/- /ApoE -/- and age-matched black KKAy -/- /ApoE -/- littermates on standard lab diet were monitored monthly for body weight and blood glucose. At 24 wks age, mice were harvested after overnight fasting; plasma, aorta and heart were collected for biochemical, molecular and lesion studies. Prior to harvest, mice were subjected to treadmill exercise test at an incline with progressively increasing speed until exhaustion. At 16 wks age, yellow KKAy +/- /ApoE -/- mice showed significant increase in body weight and random blood glucose levels (&lt;0.0001) vs. black KKAy -/- /ApoE -/- littermates, validating the MetS phenotype. Peak oxygen consumption (VO 2max ), maximum running speed and total run time until exhaustion were significantly reduced in MetS KKAy +/- /ApoE -/- mice (p&lt;0.003 vs. non-MetS KKAy -/- /ApoE -/- ), suggesting impaired cardiovascular fitness. Aortic root morphometry revealed 4-fold increase in lesion lipid burden in MetS vs non-MetS mice. This was accompanied with reduced LMOD (SM contractile marker) and SRF (transcriptional activator of SM contractile genes) expression in aortic vessels of MetS mice (p&lt;0.04 vs. non-MetS mice). Notably, lesion abundance and reduced SMC differentiation associated with increased TSP-1 expression in the aortic vasculature of MetS mice. Together, our data suggest a putative role of TSP-1 in SMC de-differentiation and atherosclerotic lesion formation in metabolic syndrome.

Prognostic and Immunological Role of THBS2 in Colorectal cancer

Objective Thrombospondin 2 (THBS2) acts as oncogenic or tumor suppressive gene in diverse cancers. Here we studied the prognostic and immunological role of THBS2 in colorectal cancer (CRC) using bioinformatic analysis. Methods The genetic and protein expression of THBS2 in CRC were explored across several databases, including ONCOMINE, GEPIA2, TIMER 2.0, UALCAN and HPA databases. Correlation between THBS2 expression and clinical features in CRC was assessed using UALCAN tool. Prognostic analysis was performed using GEPIA2 and PrognoScan. Immune infiltration correlation with THBS2 in CRC was investigated with TIMER 2.0 and TISIDB. THBS2 binding and correlated genes were analyzed using String, GEPIA2, and TIMER 2.0. Results THBS2 was significantly higher in CRC across multiple databases. Age and histological subtype were correlated with THBS2 level. High THBS2 expression correlated with short overall and disease-free survival. THBS2 expression was positively correlated with immune infiltrates in CRC. Moreover, extracellular matrix structural constituent and organization, PI3K-Akt pathway, were involved in the functional mechanisms of THBS2. Conclusions THBS2 correlates with poor prognosis and immune infiltration in CRC. THBS2 may act as a prognostic and immunological biomarker for CRC.

Transcriptomics Identify Thrombospondin-2 as a Biomarker for NASH and Advanced Liver Fibrosis.

Background and AimsNAFLD is the most common liver disease worldwide. NASH, the progressive form of NAFLD, and advanced fibrosis are associated with poor outcomes. We searched for their noninvasive biomarkers.Approach and ResultsGlobal RNA sequencing of liver tissue from 98 patients with biopsy‐proven NAFLD was performed. Unsupervised hierarchical clustering well distinguished NASH from nonalcoholic fatty liver (NAFL), and patients with NASH exhibited molecular abnormalities reflecting their pathological features. Transcriptomic analysis identified proteins up‐regulated in NASH and/or advanced fibrosis (stage F3‐F4), including matricellular glycoprotein thrombospondin‐2 (TSP‐2), encoded by the thrombospondin 2 (THBS2) gene. The intrahepatic THBS2 expression level showed the highest areas under the receiver operating characteristic curves (AUROCs) of 0.915 and 0.957 for diagnosing NASH and advanced fibrosis, respectively. THBS2 positively correlated with inflammation and ballooning according to NAFLD activity score, serum aspartate aminotransferase and hyaluronic acid (HA) levels, and NAFLD Fibrosis Score (NFS). THBS2 was associated with extracellular matrix and collagen biosynthesis, platelet activation, caspase‐mediated cleavage of cytoskeletal proteins, and immune cell infiltration. Serum TSP‐2 expression was measured in 213 patients with biopsy‐proven NAFLD, was significantly higher in NASH than in NAFL, and increased parallel to fibrosis stage. The AUROCs for predicting NASH and advanced fibrosis were 0.776 and 0.856, respectively, which were comparable to Fibrosis‐4 index, serum HA level, and NFS in advanced fibrosis diagnosis. Serum TSP‐2 level and platelet count were independent predictors of NASH and advanced fibrosis. Serum TSP‐2 levels could stratify patients with NAFLD according to the risk of hepatic complications, including liver cancer and decompensated cirrhotic events.ConclusionsTSP‐2 may be a useful biomarker for NASH and advanced fibrosis diagnosis in patients with NAFLD.