Thrombospondin 1 enhances systemic inflammation and disease severity in acute-on-chronic liver failure.

BACKGROUND: The key role of thrombospondin 1 (THBS1) in the pathogenesis of acute-on-chronic liver failure (ACLF) is unclear. Here, we present a transcriptome approach to evaluate THBS1 as a potential biomarker in ACLF disease pathogenesis. METHODS: Biobanked peripheral blood mononuclear cells (PBMCs) from 330 subjects with hepatitis B virus (HBV)-related etiologies, including HBV-ACLF, liver cirrhosis (LC), and chronic hepatitis B (CHB), and normal controls (NC) randomly selected from the Chinese Group on the Study of Severe Hepatitis B (COSSH) prospective multicenter cohort underwent transcriptome analyses (ACLF = 20; LC = 10; CHB = 10; NC = 15); the findings were externally validated in participants from COSSH cohort, an ACLF rat model and hepatocyte-specific THBS1 knockout mice. RESULTS: THBS1 was the top significantly differentially expressed gene in the PBMC transcriptome, with the most significant upregulation in ACLF, and quantitative polymerase chain reaction (ACLF = 110; LC = 60; CHB = 60; NC = 45) was used to verify that THBS1 expression corresponded to ACLF disease severity outcome, including inflammation and hepatocellular apoptosis. THBS1 showed good predictive ability for ACLF short-term mortality, with an area under the receiver operating characteristic curve (AUROC) of 0.8438 and 0.7778 at 28 and 90 days, respectively. Enzyme-linked immunosorbent assay validation of the plasma THBS1 using an expanded COSSH cohort subjects (ACLF = 198; LC = 50; CHB = 50; NC = 50) showed significant correlation between THBS1 with ALT and γ-GT (P = 0.01), and offered a similarly good prognostication predictive ability (AUROC = 0.7445 and 0.7175) at 28 and 90 days, respectively. ACLF patients with high-risk short-term mortality were identified based on plasma THBS1 optimal cut-off value (< 28 µg/ml). External validation in ACLF rat serum and livers confirmed the functional association between THBS1, the immune response and hepatocellular apoptosis. Hepatocyte-specific THBS1 knockout improved mouse survival, significantly repressed major inflammatory cytokines, enhanced the expression of several anti-inflammatory mediators and impeded hepatocellular apoptosis. CONCLUSIONS: THBS1 might be an ACLF disease development-related biomarker, promoting inflammatory responses and hepatocellular apoptosis, that could provide clinicians with a new molecular target for improving diagnostic and therapeutic strategies.

THBS1 and THBS2 Enhance the In Vitro Proliferation, Adhesion, Migration and Invasion of Intrahepatic Cholangiocarcinoma Cells.

In intrahepatic cholangiocarcinoma (iCCA), thrombospondin 1 (THBS1) and 2 (THBS2) are soluble mediators released in the tumor microenvironment (TME) that contribute to the metastatic spreading of iCCA cells via a lymphatic network by the trans-differentiation of vascular endothelial cells to a lymphatic-like phenotype. To study the direct role of THBS1 and THBS2 on the iCCA cells, well-established epithelial (HuCCT-1) and mesenchymal (CCLP1) iCCA cell lines were subjected to recombinant human THBS1 and THBS2 (rhTHBS1, rhTHBS2) for cellular function assays. Cell growth, cell adhesion, migration, and invasion were all enhanced in both CCLP1 and HuCCT-1 cells by the treatment with either rhTHBS1 or rhTHBS2, although they showed some variability in their intensity of speeding up cellular processes. rhTHBS2 was more intense in inducing invasiveness and in committing the HuCCT-1 cells to a mesenchymal-like phenotype and was therefore a stronger enhancer of the malignant behavior of iCCA cells compared to rhTHBS1. Our data extend the role of THBS1 and THBS2, which are not only able to hinder the vascular network and promote tumor-associated lymphangiogenesis but also exacerbate the malignant behavior of the iCCA cells.

PP2A Affects Angiogenesis via Its Interaction with a Novel Phosphorylation Site of TSP1.

Alterations in angiogenic properties play a pivotal role in the manifestation and onset of various pathologies, including vascular diseases and cancer. Thrombospondin-1 (TSP1) protein is one of the master regulators of angiogenesis. This study unveils a novel aspect of TSP1 regulation through reversible phosphorylation. The silencing of the B55α regulatory subunit of protein phosphatase 2A (PP2A) in endothelial cells led to a significant decrease in TSP1 expression. Direct interaction between TSP1 and PP2A-B55α was confirmed via various methods. Truncated TSP1 constructs were employed to identify the phosphorylation site and the responsible kinase, ultimately pinpointing PKC as the enzyme phosphorylating TSP1 on Ser93. The biological effects of B55α-TSP1 interaction were also analyzed. B55α silencing not only counteracted the increase in TSP1 expression during wound closure but also prolonged wound closure time. Although B55α silenced cells initiated tube-like structures earlier than control cells, their spheroid formation was disrupted, leading to disintegration. Cells transfected with phosphomimic TSP1 S93D exhibited smaller spheroids and reduced effectiveness in tube formation, revealing insights into the effects of TSP1 phosphorylation on angiogenic properties. In this paper, we introduce a new regulatory mechanism of angiogenesis by reversible phosphorylation on TSP1 S93 by PKC and PP2A B55α.

Scleral remodeling during myopia development in mice eyes: a potential role of thrombospondin-1.

BACKGROUND: Scleral extracellular matrix (ECM) remodeling plays a crucial role in the development of myopia, particularly in ocular axial elongation. Thrombospondin-1 (THBS1), also known as TSP-1, is a significant cellular protein involved in matrix remodeling in various tissues. However, the specific role of THBS1 in myopia development remains unclear. METHOD: We employed the HumanNet database to predict genes related to myopic sclera remodeling, followed by screening and visualization of the predicted genes using bioinformatics tools. To investigate the potential target gene Thbs1, we utilized lens-induced myopia models in male C57BL/6J mice and performed Western blot analysis to detect the expression level of scleral THBS1 during myopia development. Additionally, we evaluated the effects of scleral THBS1 knockdown on myopia development through AAV sub-Tenon's injection. The refractive status and axial length were measured using a refractometer and SD-OCT system. RESULTS: During lens-induced myopia, THBS1 protein expression in the sclera was downregulated, particularly in the early stages of myopia induction. Moreover, the mice in the THBS1 knockdown group exhibited alterations in myopia development in both refraction and axial length changed compared to the control group. Western blotting analysis confirmed the effectiveness of AAV-mediated knockdown, demonstrating a decrease in COLA1 expression and an increase in MMP9 levels in the sclera. CONCLUSION: Our findings indicate that sclera THBS1 levels decreased during myopia development and subsequent THBS1 knockdown showed a decrease in scleral COLA1 expression. Taken together, these results suggest that THBS1 plays a role in maintaining the homeostasis of scleral extracellular matrix, and the reduction of THBS1 may promote the remodeling process and then affect ocular axial elongation during myopia progression.

Determination of vWF, ADAMTS-13 and Thrombospondin-1 in Venous Thromboembolism and Relating Them to the Presence of Factor V Leiden Mutation.

Thrombophilia in venous thromboembolism (VTE) is multifactorial. Von Willebrand factor (vWF) plays a major role in primary hemostasis. While elevated vWF levels are well documented in VTE, findings related to its cleaving protease (ADAMTS-13) are contradicting. The aim of this study was to determine vWF, ADAMTS-13, and the multifactorial Thrombospondin-1 (TSP-1) protein levels in patients after 3-6 months following an unprovoked VTE episode. We also explored a possible association with factor V Leiden (FVL) mutation. vWF, ADAMTS-13 and TSP-1 were analyzed using ELISA kits in 60 VTE patients and 60 controls. Patients had higher levels of vWF antigen (P = .021), vWF collagen-binding activity (P = .008), and TSP-1 protein (P < .001) compared to controls. ADAMTS-13 antigen was lower in patients (P = .046) compared to controls but ADAMTS-13 activity was comparable between the two groups (P = .172). TSP-1 showed positive correlation with vWF antigen (rho = 0.303, P = .021) and negative correlation with ADAMTS-13 activity (rho = -0.244, P = .033) and ADAMTS-13 activity/vWF antigen ratio (rho = -0.348, P = .007). A significant association was found between the presence of FVL mutation and VTE (odds ratio (OR): 9.672 (95% confidence interval (CI) 2.074-45.091- P = .004), but no association was found between the mutation and the studied proteins (P > .05). There appears to be an imbalance between vWF and ADAMTS-13 in VTE patients even after 3-6 months following the onset of VTE. We report that the odds of developing VTE in carriers of FVL mutation are 9.672 times those without the mutation, but the presence of this mutation is not associated with the studied proteins.

Thrombospondin-1 promotes liver fibrosis by enhancing TGF-ß action in hepatic stellate cells.

Insulin resistance in adipose tissue is thought to be a key contributor to the pathogenesis of various metabolic disorders including metabolic dysfunction-associated steatotic liver disease/metabolic dysfunction-associated steatohepatitis (MASLD/MASH), but the mechanism underlying this contribution to MASLD/MASH has remained unknown. We previously showed that dysregulation of the PDK1-FoxO1 signaling axis in adipocytes plays a role in the development of MASLD/MASH by analysis of adipocyte-specific PDK1 knockout (A-PDK1KO) and adipocyte-specific PDK1/FoxO1 double-knockout (A-PDK1/FoxO1DKO) mice. We here focused on the role of the extracellular matrix protein thrombospondin-1 (TSP-1) as a secreted factor whose expression in adipose tissue is increased in A-PDK1KO mice and normalized in A-PDK1/FoxO1DKO mice. Genetic ablation of TSP-1 markedly ameliorated liver fibrosis in A-PDK1KO mice fed a high-fat diet. With regard to the potential mechanism of this effect, TSP-1 augmented the expression of fibrosis-related genes induced by TGF-ß in LX-2 human hepatic stellate cells. We also showed that TSP-1 expression and secretion were negatively regulated by insulin signaling via the PDK1-FoxO1 axis in cultured adipocytes. Our results thus indicate that TSP-1 plays a key role in the pathogenesis of liver fibrosis in MASH. Regulation of TSP-1 expression by PDK1-FoxO1 axis in adipocytes may provide a basis for targeted therapy of hepatic fibrosis in individuals with MASH.

Pericardial Adipose Tissue Thrombospondin-1 Associates With Antiangiogenesis in Ischemic Heart Disease.

Accumulation of ectopic pericardial adipose tissue has been associated with cardiovascular complications which, in part, may relate to adipose-derived factors that regulate vascular responses and angiogenesis. We sought to characterize adipose tissue microvascular angiogenic capacity in subjects who underwent elective cardiac surgeries including aortic, valvular, and coronary artery bypass grafting. Pericardial adipose tissue was collected intraoperatively and examined for angiogenic capacity. Capillary sprouting was significantly blunted (twofold, p <0.001) in subjects with coronary artery disease (CAD) (age 60 ± 9 years, body mass index [BMI] 32 ± 4 kg/m2, low-density lipoprotein cholesterol [LDL-C] 95 ± 46 mg/100 ml, n = 29) compared with age-, BMI-, and LDL-C matched subjects without angiographic obstructive CAD (age 59 ± 10 y, BMI 35 ± 9 kg/m2, LDL-C 101 ± 40 mg/100 ml, n = 12). For potential mechanistic insight, we performed mRNA expression analyses using quantitative real-time polymerase chain reaction and observed no significant differences in pericardial fat gene expression of proangiogenic mediators vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietin-1 (angpt1), or anti-angiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin. In contrast, mRNA expression of anti-angiogenic thrombospondin-1 (TSP-1) was significantly upregulated (twofold, p = 0.008) in CAD compared with non-CAD subjects, which was confirmed by protein western-immunoblot analysis. TSP-1 gene knockdown using short hairpin RNA lentiviral delivery significantly improved angiogenic deficiency in CAD (p <0.05). In conclusion, pericardial fat in subjects with CAD may be associated with an antiangiogenic profile linked to functional defects in vascularization capacity. Local paracrine actions of TSP-1 in adipose depots surrounding the heart may play a role in mechanisms of ischemic heart disease.

CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis.

BACKGROUND: TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 (cluster of differentiation) and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis. METHODS: Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC Cd47 in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms. RESULTS: Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF (vascular endothelial growth factor)-C-induced AKT and eNOS activation in LEC and attenuated NO (nitric oxide) production. Further, CD47 silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV (adeno-associated virus) 8-PCSK9-injected LEC-specific Cd47 knockout mice (Cd47ΔLEC) had reduced atherosclerosis in both aorta and aortic root compared with control mice (Cd47ΔWT). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in Cd47ΔLEC mice in comparison to controls. CONCLUSIONS: These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC Cd47 attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.

Depleted uranium induces thyroid damage through activation of ER stress via the thrombospondin 1-PERK pathway.

Depleted uranium (DU) can cause damage to the body, but its effects on the thyroid are unclear. The purpose of this study was to investigate the DU-induced thyroid damage and its potential mechanism in order to find new targets for detoxification after DU poisoning. A model of acute exposure to DU was constructed in rats. It was observed that DU accumulated in the thyroid, induced thyroid structure disorder and cell apoptosis, and decreased the serum T4 and FT4 levels. Gene screening showed that thrombospondin 1 (TSP-1) was a sensitive gene of DU, and the expression of TSP-1 decreased with the increase of DU exposure dose and time. TSP-1 knockout mice exposed to DU had more severe thyroid damage and lower serum FT4 and T4 levels than wild-type mice. Inhibiting the expression of TSP-1 in FRTL-5 cells aggravated DU-induced apoptosis, while exogenous TSP-1 protein alleviated the decreased viability in FRTL-5 cells caused by DU. It was suggested that DU may caused thyroid damage by down-regulating TSP-1. It was also found that DU increased the expressions of PERK, CHOP, and Caspase-3, and 4-Phenylbutyric (4-PBA) alleviated the DU-induced FRTL-5 cell viability decline and the decrease levels of rat serum FT4 and T4 caused by DU. After DU exposure, the PERK expression was further up-regulated in TSP-1 knockout mice, and the increased expression of PERK was alleviated in TSP-1 over-expressed cells, as well as the increased expression of CHOP and Caspase-3. Further verification showed that inhibition of PERK expression could reduce the DU-induced increased expression of CHOP and Caspase-3. These findings shed light on the mechanism that DU may activate ER stress via the TSP 1-PERK pathway, thereby leading to thyroid damage, and suggest that TSP-1 may be a potential therapeutic target for DU-induced thyroid damage.

Novel thrombospondin-1 transcript exhibits distinctive expression and activity in thyroid tumorigenesis.

Thrombospondin 1 (TSP1) is known for its cell-specific functions in cancer progression, such as proliferation and migration. It contains 22 exons that may potentially produce several different transcripts. Here, we identified TSP1V as a novel TSP1-splicing variant produced by intron retention (IR) in human thyroid cancer cells and tissues. We observed that TSP1V functionally inhibited tumorigenesis contrary to TSP1 wild-type, as identified in vivo and in vitro. These activities of TSP1V are caused by inhibiting phospho-Smad and phospho-focal adhesion kinase. Reverse transcription polymerase chain reaction and minigene experiments revealed that some phytochemicals/non-steroidal anti-inflammatory drugs enhanced IR. We further found that RNA-binding motif protein 5 (RBM5) suppressed IR induced by sulindac sulfide treatment. Additionally, sulindac sulfide reduced phospho-RBM5 levels in a time-dependent manner. Furthermore, trans-chalcone demethylated TSP1V, thereby preventing methyl-CpG-binding protein 2 binding to TSP1V gene. In addition, TSP1V levels were significantly lower in patients with differentiated thyroid carcinoma than in those with benign thyroid nodule, indicating its potential application as a diagnostic biomarker in tumor progression.

[Expression of CD47 and its ligands in pregnant mice infected with Toxoplasma gondii during pregnancy].

OBJECTIVE: To investigate the dynamic expression of cluster of differentiation 47 (CD47) and its ligands signaling regulatory protein α (SIRPα) and thrombospondin-1 (TSP-1) in mice infected with Toxoplasma gondii in the second and third trimesters. METHODS: C57BL/6J mice (6 to 8 weeks old) were used for modeling T. gondii infection in the first trimester, and the pregnant mice were randomly divided into the normal control and infection groups, of 10 mice in each group. Pregnant mice in the infection group were intraperitoneally injected with 150 T. gondii tachyzoites on gestational day (Gd) 6.5, while pregnant mice in the normal control group were intraperitoneally injected with the same volume of physiological saline at the same time. The uterine and placental specimens were collected from all pregnant mice on Gd12.5 and Gd18.5, and the pregnant outcomes were recorded. The pathological damages of mouse uterine and placental specimens were observed using hematoxylin-eosin (HE) staining on Gd12.5 and Gd18.5. The relative expression of CD47, SIRPα, TSP-1, surface antigen 1 (SAG1), interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-13 mRNA was quantified in mouse uterine and placental specimens using real-time fluorescence quantitative PCR (qPCR) assay, and the CD47, SIRPα, TSP-1 expression was determined in mouse uterine and placental specimens using immunohistochemical staining. RESULTS: As compared with those in the normal control group, the pregnant mice in the infection group showed back arching, bristling, trembling and listlessness during pregnancy, and several mice presented virginal bleeding and abortion. Pathological examinations showed inflammatory cell infiltration, congestion and necrosis in uterine and placental specimens of pregnant mice in the infection group, a higher abortion rate of pregnant mice was seen in the infection group than in the normal control group on Gd12.5 (χ2 = 20.405, P < 0.001) and Gd18.5 (χ2 = 28.644, P < 0.001). qPCR assay showed significant differences in the expression of CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 genes in mouse placental specimens between the normal control and infection groups on Gd12.5 and Gd18.5 [F' (F) = 37.511, 29.337, 97.343, 53.755, 67.188, 21.145, 8.658 and 13.930, all P values < 0.001]. Higher CD47, SIRPα and TSP-1 gene expression was quantified in mouse placental specimens in the infection group than in the normal control group on Gd12.5 (all P values < 0.01), and lower CD47, SIRPα and TSP-1 gene expression was quantified in the infection group than in the normal control group on Gd18.5 (all P values < 0.001), while higher SAG1 gene expression was detected in placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01). In addition, higher INF-γ and IL-2 expression and lower IL-4 and IL-13 expression was detected in mouse placental specimens in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001), and there were significant differences in the CD47, SIRPα, TSP-1, SAG1, INF-γ, IL-2, IL-4 and IL-13 gene expression in uterine specimens of pregnant mice between the normal control and infection groups on Gd12.5 and Gd18.5 [H(F' and F) = 14.951, 25.977, 18.711, 48.595, 39.318, 14.248 and 15.468, all P values < 0.01], and higher CD47 and TSP-1 expression was detected in mouse uterine specimens in the infection group than in the control group on Gd12.5 and Gd18.5 (all P values < 0.01); however, no significant difference was found in the SIRPα expression (P > 0.05). Higher SAG1 expression was detected in uterine specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (both P values < 0.01), and higher INF-γ and IL-2 gene expression and lower IL-4 and IL-13 gene expression was found in the placental specimens of pregnant mice in the infection group than in the normal control group on Gd12.5 and Gd18.5 (all P values < 0.001). Spearman correlation analysis showed that the CD47 gene expression correlated positively with IFN-γ (rs = 0.735, P < 0.05) and IL-2 (rs = 0.655, P < 0.05) and negatively with IL-4 (rs = -0.689, P < 0.05) and IL-13 expression (rs = -0.795, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd12.5, and the CD47 gene expression correlated negatively with IFN-γ (rs = -0.745, P < 0.05) and IL-2 expression (rs = -0.816, P < 0.05) and positively with IL-4 (rs = 0.704, P < 0.05) and IL-13 (rs = 0.802, P < 0.05) in the placental specimens of pregnant mice in the infection group on Gd18.5. Immunohistochemical staining showed mild CD47, SIRPα and TSP-1 expression in uterine and placental specimens of pregnant mice in the normal control group on Gd12.5 and Gd18.5, strong CD47, SIRPα and TSP-1 expression in the placental specimens of pregnant mice in the infection group on Gd12.5 and strong CD47 and TSP-1 expression in the uterine specimens of pregnant mice in the infection group on Gd12.5. CONCLUSIONS: T. gondii infection in the first trimester may cause abnormal expression of CD47 and its ligands SIRPα and TSP-1 in the maternal-fetal interface of pregnant mice in the second and third trimesters, which may be associated with the immune escape of T. gondii at the maternal-fetal interface.

Baicalin inhibited both the Furin/TGFß1/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 TGFß1/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/TGFß1/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.

Latexin regulates sex dimorphism in hematopoiesis via gender-specific differential expression of microRNA 98-3p and thrombospondin 1.

Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate to all blood cell types. HSCs and their differentiated progeny show sex/gender differences. The fundamental mechanisms remain largely unexplored. We previously reported that latexin (Lxn) deletion increased HSC survival and repopulation capacity in female mice. Here, we find no differences in HSC function and hematopoiesis in Lxn knockout (Lxn-/-) male mice under physiologic and myelosuppressive conditions. We further find that Thbs1, a downstream target gene of Lxn in female HSCs, is repressed in male HSCs. Male-specific high expression of microRNA 98-3p (miR98-3p) contributes to Thbs1 suppression in male HSCs, thus abrogating the functional effect of Lxn in male HSCs and hematopoiesis. These findings uncover a regulatory mechanism involving a sex-chromosome-related microRNA and its differential control of Lxn-Thbs1 signaling in hematopoiesis and shed light on the process underlying sex dimorphism in both normal and malignant hematopoiesis.

The long noncoding RNA THBS1-AS1 promotes cardiac fibroblast activation in cardiac fibrosis by regulating TGFBR1.

Cardiac fibrosis is associated with an adverse prognosis in cardiovascular disease that results in a decreased cardiac compliance and, ultimately, heart failure. Recent studies have identified the role of long noncoding RNA (lncRNA) in cardiac fibrosis. However, the functions of many lncRNAs in cardiac fibrosis remain to be characterized. Through a whole-transcriptome sequencing and bioinformatics analysis on a mouse model of pressure overload-induced cardiac fibrosis, we screened a key lncRNA termed thrombospondin 1 antisense 1 (THBS1-AS1), which was positively associated with cardiac fibrosis. In vitro functional studies demonstrated that the silencing of THBS1-AS1 ameliorated TGF-ß1 effects on cardiac fibroblast (CF) activation, and the overexpression of THBS1-AS1 displayed the opposite effect. A mechanistic study revealed that THBS1-AS1 could sponge miR-221/222 to regulate the expression of TGFBR1. Moreover, under TGF-ß1 stimulation, the forced expression of miR-221/222 or the knockdown TGFBR1 significantly reversed the THBS1-AS1 overexpression induced by further CF activation. In vivo, specific knockdown of THBS1-AS1 in activated CFs significantly alleviated transverse aorta constriction-induced (TAC-induced) cardiac fibrosis in mice. Finally, we demonstrated that the human THBS1-AS1 can also affect the activation of CFs by regulating TGFBR1. In conclusion, this study reveals that lncRNA THBS1-AS1 is a potentially novel regulator of cardiac fibrosis and may serve as a target for the treatment of cardiac fibrosis.

Thrombospondin-1, CD47, and SIRPα display cell-specific molecular signatures in human islets and pancreata.

Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα), and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint-blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Fresh islets and pancreatic tissue from nondiabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression were determined. CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, whereas SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by ß cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Human pancreatic exocrine and endocrine tissue expressed CD47, whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.NEW & NOTEWORTHY CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.

O-fucosylation of thrombospondin type I repeats is dispensable for trafficking thrombospondin 1 to platelet secretory granules.

Thrombospondin 1 (THBS1) is a secreted extracellular matrix glycoprotein that regulates a variety of cellular and physiological processes. THBS1's diverse functions are attributed to interactions between the modular domains of THBS1 with an array of proteins found in the extracellular matrix. THBS1's three Thrombospondin type 1 repeats (TSRs) are modified with O-linked glucose-fucose disaccharide and C-mannose. It is unknown whether these modifications impact trafficking and/or function of THBS1 in vivo. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) and is sequentially extended to the disaccharide by ß3glucosyltransferase (B3GLCT). The C-mannose is added by one or more of four C-mannosyltransferases. O-fucosylation by POFUT2/B3GLCT in the endoplasmic reticulum has been proposed to play a role in quality control by locking TSR domains into their three-dimensional fold, allowing for proper secretion of many O-fucosylated substrates. Prior studies showed the siRNA knockdown of POFUT2 in HEK293T cells blocked secretion of TSRs 1-3 from THBS1. Here we demonstrated that secretion of THBS1 TSRs 1-3 was not reduced by CRISPR-Cas9-mediated knockout of POFUT2 in HEK293T cells and demonstrated that knockout of Pofut2 or B3glct in mice did not reduce the trafficking of endogenous THBS1 to secretory granules of platelets, a major source of THBS1. Additionally, we demonstrated that all three TSRs from platelet THBS1 were highly C-mannosylated, which has been shown to stabilize TSRs in vitro. Combined, these results suggested that POFUT2 substrates with TSRs that are also modified by C-mannose may be less susceptible to trafficking defects resulting from the loss of the glucose-fucose disaccharide.

MiR-3612 targeting THBS1 suppresses nasopharyngeal carcinoma progression by PI3K/AKT signaling pathway.

BACKGROUND: MicroRNA-3612 (miR-3612) is considered a tumor suppressor in different cancers. Nonetheless, its function in nasopharyngeal carcinoma (NPC) has yet to be uncovered. METHODS: NPC cells and tissues were tested by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis and western blotting to quantify the expressions of miR-3612 and Thrombospondin 1 (THBS1). Cell Counting Kit-8 (CCK-8) and scratch experiments were carried out to evaluate the migration and proliferation of NPC cells. NPC cell adhesion was also assessed. The predicted interaction of miR-3612 with THBS1 was verified by means of a luciferase reporter assay. In vivo experiments were also conducted to examine how miR-3612 overexpression affects in vivo tumorigenicity. Lastly, phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway status was assessed via western blotting. RESULTS: MiR-3612 was downregulated in NPC cells and tissues, whereas THBS1 expression showed an opposite trend. The MiR-3612 mimic inhibited the NPC cell proliferation, adhesion, and migration and also inactivated the PI3K/AKT signaling pathway. Furthermore, miR-3612 mimic also hampered NPC tumorigenesis in vivo. MiR-3612 targeted THBS1 and downregulated THBS1 expression. THBS1 offset the miR-3612-overexpression-induced repression of the migration, adhesion, and proliferation of NPC cells via the activation of the PI3K/AKT pathway. CONCLUSION: MiR-3612 retarded NPC cell migration, adhesion, and proliferation by targeting THBS1 and inactivating the PI3K/AKT signaling pathway. This provides a novel therapeutic approach for NPC intervention.

PRSS2 remodels the tumor microenvironment via repression of Tsp1 to stimulate tumor growth and progression.

The progression of cancer from localized to metastatic disease is the primary cause of morbidity and mortality. The interplay between the tumor and its microenvironment is the key driver in this process of tumor progression. In order for tumors to progress and metastasize they must reprogram the cells that make up the microenvironment to promote tumor growth and suppress endogenous defense systems, such as the immune and inflammatory response. We have previously demonstrated that stimulation of Tsp-1 in the tumor microenvironment (TME) potently inhibits tumor growth and progression. Here, we identify a novel tumor-mediated mechanism that represses the expression of Tsp-1 in the TME via secretion of the serine protease PRSS2. We demonstrate that PRSS2 represses Tsp-1, not via its enzymatic activity, but by binding to low-density lipoprotein receptor-related protein 1 (LRP1). These findings describe a hitherto undescribed activity for PRSS2 through binding to LRP1 and represent a potential therapeutic strategy to treat cancer by blocking the PRSS2-mediated repression of Tsp-1. Based on the ability of PRSS2 to reprogram the tumor microenvironment, this discovery could lead to the development of therapeutic agents that are indication agnostic.

Dysregulated thrombospondin 1 and miRNA-29a-3p in severe COVID-19.

Although nearly a fifth of symptomatic COVID-19 patients suffers from severe pulmonary inflammation, the mechanism of developing severe illness is not yet fully understood. To identify significantly altered genes in severe COVID-19, we generated messenger RNA and micro-RNA profiling data of peripheral blood mononuclear cells (PBMCs) from five COVID-19 patients (2 severe and 3 mild patients) and three healthy controls (HC). For further evaluation, two publicly available RNA-Seq datasets (GSE157103 and GSE152418) and one single-cell RNA-Seq dataset (GSE174072) were employed. Based on RNA-Seq datasets, thrombospondin 1 (THBS1) and interleukin-17 receptor A (IL17RA) were significantly upregulated in severe COVID-19 patients' blood. From single-cell RNA-sequencing data, IL17RA level is increased in monocytes and neutrophils, whereas THBS1 level is mainly increased in the platelets. Moreover, we identified three differentially expressed microRNAs in severe COVID-19 using micro-RNA sequencings. Intriguingly, hsa-miR-29a-3p significantly downregulated in severe COVID-19 was predicted to bind the 3'-untranslated regions of both IL17RA and THBS1 mRNAs. Further validation analysis of our cohort (8 HC, 7 severe and 8 mild patients) showed that THBS1, but not IL17RA, was significantly upregulated, whereas hsa-miR-29a-3p was downregulated, in PBMCs from severe patients. These findings strongly suggest that dysregulated expression of THBS1, IL17RA, and hsa-miR-29a-3p involves severe COVID-19.