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Porphyromonas gingivalis is a gram-negative anaerobic bacterium linked to periodontal disease. Remarkably, P. gingivalis thrives in an inflamed environment rich in activated neutrophils. Toll-like receptor 2 (TLR2) recognition is required for P. gingivalis to evade innate immune killing; however, the mechanisms through which P. gingivalis uncouples host inflammation from bactericidal activity are only partially known. Since integrin activation and alternative signaling are implicated in P. gingivalis TLR2-mediated immune escape, we explored the role of CD47, a widely expressed integrin-associated protein known to suppress phagocytosis and implicated as an interacting partner with other innate immune receptors. We found that CD47 associates with TLR2, and blocking CD47 leads to decreased intracellular P. gingivalis survival in macrophages in a manner dependent on the bacterial major fimbria. In vivo, CD47 knock-out mice cleared P. gingivalis more efficiently than wild-type mice. Next, we found increased expression and secretion of the CD47 ligand thrombospondin-1 (TSP-1) following P. gingivalis infection. Secreted TSP-1 broadly protected P. gingivalis and other periodontitis-associated bacterial species from neutrophil bactericidal activity. Therefore, CD47-TLR2 cosignaling in response to P. gingivalis induces TSP-1 that in turn suppresses neutrophil activity, an effect that can explain how species such as P. gingivalis survive in an inflamed environment and cause dysbiosis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Background: Fine particulate matter (PM 2.5 ) induces ocular surface toxicity through pyroptosis, oxidative stress, autophagy, and inflammatory responses. However, the precise molecular pathways through which PM 2.5 causes corneal damage remain unclear. This study aims to investigate the underlying mechanisms by exposing human corneal epithelial cells (HCECs) to PM 2.5 ., Methods: After the morphology and chemical composition analysis of the PM samples, we conducted both in vivo and in vitro experiments to investigate PM 2.5 -induced corneal epithelial damage. We assessed corneal barrier function in HCECs using transepithelial electrical resistance (TEER) assays. To explore the molecular mechanisms of PM 2.5 -induced corneal epithelial damage, we performed whole-transcriptome resequencing, quantitative RT-PCR, and western blotting in vitro. In addition, we analyzed mouse corneas exposed to concentrated ambient PM 2.5 through immunofluorescence staining to observe the resulting changes in corneal epithelial protein expression in vivo., Results: Our results showed significant impairment of corneal epithelial barrier function in PM 2.5 -treated HCECs, as indicated by decreased TEER values. The expression of thrombospondin-1 (THBS1) and claudin-1, both key factors for maintaining corneal epithelial barrier integrity, was markedly reduced at the gene and protein levels in both in vitro and in vivo PM 2.5 exposure models. Moreover, the levels of tight junction-associated proteins, including occludin, zonula occludens-1 (ZO-1) and ZO-2, essential components of the corneal epithelial barrier, were significantly diminished in PM 2.5 -treated HCECs., Conclusion: PM 2.5 exposure leads to corneal epithelium damage by disrupting tight junction proteins and THBS1 expression. These findings provide insight into potential pathways for PM 2.5 -induced ocular toxicity and underscore the need for protective strategies against such environmental pollutants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Thrombospondin-1 (TSP-1) is a key protein product of activated platelets which is significantly increased in sickle cell vaso-occlusive crisis (VOC) and other inflammatory conditions. This study aims to determine TSP-1 levels and their relationship with platelet indices and inflammation., Objective: To evaluate TSP-1 levels in sickle cell anaemia (SCA) patients and assess their relationship with platelet indices and inflammation., Methods: This hospital-based cross-sectional comparative study was conducted in North Western Nigeria from July to December 2022. The study included 120 participants: 80 adults with SCA (40 in VOC) and 40 in steady state (SS)), matched with 40 healthy controls (HbAA). TSP-1 levels were measured using ELISA and haematological parameters were obtained from an automated analyzer. Analysis of variance was used to compare groups and determinants of TSP-1 variability were analyzed using multiple logistic regression. Statistical significance was at p ≤0.05., Results: The mean TSP-1 level was significantly higher in VOC patients (406.0 ± 164.6 ng/mL) compared to SS patients (312.4 ± 145.6 ng/mL) and HbAA controls (192.3 ± 121.2 ng/mL) (p < 0.001). In the SS group, TSP-1 negatively correlated with mean platelet volume (MPV; r = -0.38, p = 0.02) and platelet-large cell ratio (P-LCR; r = -0.36, p = 0.02). White blood cell count (WBC) was the only predictor of TSP-1 variability (β = 10.301, t = 3.843, p < 0.001)., Conclusion: Elevation of TSP-1 during VOC indicates its potential as a biomarker for crisis episodes. In SS patients, TSP-1 is associated with platelet indices, suggesting its role in platelet activation. WBC is a key predictor of TSP-1 variability, emphasizing the influence of inflammation., Competing Interests: The Authors declare that no competing interest exists, (Copyright © 2024 by West African Journal of Medicine.)

Aims/introduction: We investigated the relationship of circulating TSP-1 mRNA and miR-194 with diabetic kidney disease's degree., Materials and Methods: We enrolled 167 hospitalized type 2 diabetes patients in the endocrinology department. Patients were split into three groups according to urinary microalbumin: A, B and C. The control group comprised healthy outpatients (n = 163). The quantities of microribonucleic acid (miR)-194 and thrombospondin-1 (TSP-1) messenger ribonucleic acid (mRNA) in the participants' circulation were measured using a quantitative real-time polymerase chain reaction., Results: Circulating TSP-1 mRNA (P = 0.024) and miR-194 (P = 0.029) expressions significantly increased in type 2 diabetes patients. Circulating TSP-1 mRNA (P = 0.040) and miR-194 (P = 0.007) expression levels differed significantly among the three groups; circulating TSP-1 mRNA expression increased with urinary microalbumin. However, miR-194 declined in group B and increased in group C. Circulating TSP-1 mRNA was positively correlated with cystatin-c (r = 0.281; P = 0.021) and microalbumin/creatinine ratio (UmALB/Cr; r = 0.317; P = 0.009); miR-194 was positively correlated with UmALB/Cr (r = 0.405; P = 0.003). Stepwise multivariate linear regression analysis showed cystatin-c (β = 0.578; P = 0.021) and UmALB/Cr (β = 0.001; P = 0.009) as independent factors for TSP-1 mRNA; UmALB/Cr (β = 0.005; P = 0.028) as an independent factor for miR194. Areas under the curve for circulating TSP-1 mRNA and miR194 were 0.756 (95% confidence interval 0.620-0.893; sensitivity 0.69 and specificity 0.71, P < 0.01) and 0.584 (95% confidence interval 0.421-0.748; sensitivity 0.54 and specificity 0.52, P < 0.01), respectively., Conclusions: Circulating TSP-1 mRNA and miR-194 expressions significantly increased in type 2 diabetes patients. The microalbumin group had lower levels of miR-194 (a risk factor that is valuable for type 2 diabetes kidney disease evaluation)., (© 2024 The Author(s). Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Inappropriate endometrial stromal decidualization has been implied as an important reason of many pregnancy-related complications, such as unexplained recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. Here, we observed that thrombospondin-1, an adhesive glycoprotein, was significantly downregulated in endometrial decidual cells from patients with unexplained recurrent spontaneous abortion. The immortalized human endometrial stromal cell line was used to investigate the possible THBS1-mediated regulation of decidualization. In vitro experiments found that the expression level of THBS1 increased with the normal decidualization process. Knockdown of THBS1 could decrease the expression levels of prolactin and insulin-like growth factor binding protein-1, two acknowledged human decidualization markers, whereas THBS1 overexpression could reverse these effects. The RNA sequencing results demonstrated that the extracellular regulated protein kinases signaling pathway was potentially affected by the knockdown of THBS1. We further confirmed that the regulation of THBS1 on decidualization was achieved through the ERK signaling pathway by the treatment of inhibitors. Moreover, knockdown of THBS1 in pregnant mice could impair decidualization and result in an increased fetus resorption rate. Altogether, our study demonstrated a crucial role of THBS1 in the pathophysiological process of unexplained recurrent spontaneous abortion and provided some new insights into the research of pregnancy-related complications., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47 -/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47 -/- spleens but significantly depleted in Thbs1 -/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119 - CD34 + progenitors and Ter119 + CD34 - committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1 -/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen., Competing Interests: RB, TM, MC, SK, DR No competing interests declared

Preeclampsia (PE) is considered as a disease of placental origin. However, the specific mechanism of placental abnormalities remains elusive. This study identified thrombospondin-1 (THBS1) is downregulated in preeclamptic placentae and negatively correlated with blood pressure. Functional studies show that THBS1 knockdown inhibits proliferation, migration, and invasion and increases the cycle arrest and apoptosis rate of HTR8/SVneo cells. Importantly, THBS1 silencing induces necroptosis in HTR8/SVneo cells, accompanied by the release of damage-associated molecular patterns (DAMPs). Necroptosis inhibitors necrostatin-1 and GSK'872 restore the trophoblast survival while pan-caspase inhibitor Z-VAD-FMK has no effect. Mechanistically, the results show that THBS1 interacts with transforming growth factor B-activated kinase 1 (TAK1), which is a central modulator of necroptosis quiescence and affects its stability. Moreover, THBS1 silencing up-regulates the expression of neuronal precursor cell-expressed developmentally down-regulated 4 (NEDD4), which acts as an E3 ligase of TAK1 and catalyzes K48-linked ubiquitination of TAK1 in HTR8/SVneo cells. Besides, THBS1 attenuates PE phenotypes and improves the placental necroptosis in vivo. Taken together, the down-regulation of THBS1 destabilizes TAK1 by activating NEDD4-mediated, K48-linked TAK1 ubiquitination and promotes necroptosis and DAMPs release in trophoblast cells, thus participating in the pathogenesis of PE., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Epilepsy is a neural network disorder caused by uncontrolled neuronal hyperexcitability induced by an imbalance between excitatory and inhibitory networks. Abnormal synaptogenesis plays a vital role in the formation of overexcited networks. Recent evidence has confirmed that thrombospondin-1 (TSP-1), mainly secreted by astrocytes, is a critical cytokine that regulates synaptogenesis during epileptogenesis. Furthermore, numerous studies have reported that TSP-1 is also involved in other processes, such as angiogenesis, neuroinflammation, and regulation of Ca 2+ homeostasis, which are closely associated with the occurrence and development of epilepsy. In this review, we summarize the potential contributions of TSP-1 to epilepsy development., (© 2024. Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.)

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 Ca 2+ 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest Dr. Michailidou received Advisory Board fees from ChemoCentryx. Dr. Khalidi received clinical trial support from BMS, Sanofi and Abbvie, travel support from Astra Zeneca, and Advisory Board fee from Roche. Dr. Koening served on the advisory board for Chemocentryx and Genentech. Dr. Specks reports receiving funds for the following activities: Consulting: AstraZeneca, ChemoCentryx. Research Support: AstraZeneca, GlaxoSmithKline, Bristol-Myers Squibb, Genentech/Roche, InflaRx. Dr. Sreih works at Bristol-Myers Squibb and owns Astra Zeneca and Alexion Stocks. Dr. Warrington received clinical trial support from Eli Lilly and Kiniksa. Dr. Monach received consulting fees from Kiniksa, Celgene/BMC, and ChemoCentryx. Dr. Merkel reports receiving funds for the following activities: Consulting and Research Support: AbbVie, AstraZeneca, Boeringher-Ingelheim, Bristol-Myers Squibb, ChemoCentryx, Forbius, Genentech/Roche, Genzyme/Sanofi, GlaxoSmithKline, InflaRx, Takeda. Consulting only: CSL Behring, Dynacure, EMDSerono, Janssen, Kiniksa, Kyverna, Magenta, MiroBio, Neutrolis, Novartis, Pfizer, Sparrow, Talaris. Royalties: UpToDate. Dr. Lood received research funding from Pfizer, Gilead Sciences, Boehringer Ingelheim, Redd Pharma, Amytryx, and Eli Lilly., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Matricellular proteins comprise a diverse group of molecular entities secreted into the extracellular space. They interact with the extracellular matrix (ECM), integrins, and other cell-surface receptors, and can alter matrix strength, cell attachment to the matrix, and cell-cell adhesion. A founding member of this group is thrombospondin-1 (TSP-1), a high molecular-mass homotrimeric glycoprotein. Given the importance of the matrix and ECM remodeling in the lung following injury, TSP-1 has been implicated in a number of lung pathologies. This review examines the role of TSP-1 as a damage controller in the context of lung inflammation, injury resolution, and repair in noninfectious and infectious models. This review also discusses the potential role of TSP-1 in human diseases as it relates to lung inflammation and injury.

Objective: To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism., Methods: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture., Results: The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK., Conclusion: TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.

Thrombospondin-1 (TSP1) is generally assumed to suppress the growth of osteosarcoma through inhibiting angiogenesis; however, it is unclear whether TSP1 could affect the antitumor immunity against osteosarcoma. We aimed to explore the immune-related tumor-promoting effects of TSP1 and decipher its underlying mechanism. First, we identified that TSP1 regulated programmed death-ligand 1 (PD-L1) expression, which was related to the CD8 + T cells anergy in osteosarcoma cells. The exact role of PD-L1 in the immunosuppressive effect of TSP1 was then further confirmed by the addition of the PD-L1 neutralizing Ab. With the addition of PD-L1 neutralizing Abs during cocultivation, the inhibition of CD8 + T cells was abolished to a certain extent. Further mechanistic investigations showed that TSP1-induced PD-L1 upregulation was achieved by activation of the signal transducer and activator of transcription 3 (STAT3) pathway. In vivo experiments also indicated that TSP1 overexpression could promote the growth of primary lesions, whereas TSP1 knockdown effectively inhibits the growth of the primary lesion as well as lung metastasis by restoring the antitumor immunity. Thrombospondin-1 knockdown combined with PD-L1 neutralizing Ab achieved a more pronounced antitumor effect. Taken together, our study showed that TSP1 upregulates PD-L1 by activating the STAT3 pathway and, therefore, impairs the antitumor immunity against osteosarcoma., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Objectives: Tumor vasculature is structurally abnormal, with anatomical deformities, reduced pericyte coverage and low tissue perfusion. As a result of this vascular dysfunction, tumors are often hypoxic, which is associated with an aggressive tumor phenotype, and reduced delivery of therapeutic compounds to the tumor. We have previously shown that a peptide containing the thrombospondin-1 type I repeats (3TSR) specifically targets tumor vessels and induces vascular normalization in a mouse model of epithelial ovarian cancer (EOC). However, due to its small size, 3TSR is rapidly cleared from circulation. We now introduce a novel construct with the 3TSR peptide fused to the C-terminus of each of the two heavy chains of the Fc region of human IgG1 (Fc3TSR). We hypothesize that Fc3TSR will have greater anti-tumor activity in vitro and in vivo compared to the native compound., Methods: Fc3TSR was evaluated in vitro using proliferation and apoptosis assays to investigate differences in efficacy compared to native 3TSR. In light of the multivalency of Fc3TSR, we also investigate whether it induces greater clustering of its functional receptor, CD36. We also compare the compounds in vivo using an orthotopic, syngeneic mouse model of advanced stage EOC. The impact of the two compounds on changes to tumor vasculature morphology was also investigated., Results: Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR. This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer., Conclusion: The development of Fc3TSR which is greater in size, stable in circulation and enhances receptor activation compared to 3TSR, facilitates its translational potential as a therapy in the treatment of metastatic advanced stage ovarian cancer., Competing Interests: Declaration of Competing Interest J.P. and J.L. are co-inventors on US patent US20140271641A1 for the treatment of ovarian cancer with 3TSR., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sepsis often causes myocardial injury with a high mortality. The aim of the present study was to investigate the effects of thrombospondin‑1 (THBS1) on myocardial cell injury, oxi‑dative stress and apoptosis in sepsis. The expression of THBS1 mRNA in lipopolysaccharide (LPS)‑induced mouse primary cardiomyocytes was detected by reverse transcription‑quantitative PCR (RT‑qPCR). A eukaryotic small interfering (si)RNA expression vector was constructed and transfected into myocardial cells to knockdown THBS1 mRNA expression, which was confirmed by RT‑qPCR. Four in vitro experimental groups were used: i) Control, ii) LPS, iii) THBS1 siRNA (siTHBS1) and iv) siTHBS + LPS. ELISA was used to detect cardiac troponin I (cTnI), pro‑brain natriuretic peptide (proBNP), reactive oxygen species (ROS), caspase‑3, IL‑6 and TNF‑α. In vivo mouse sepsis models were also established, and H&E, TUNEL and caspase‑3 staining were used to evaluate myocardial cell injury and apoptosis. Clinical samples were collected to analyze the serum THBS1 levels and to associate this with the prognosis of patients with sepsis‑induced myocardial injury. The expression level of THBS1 mRNA in myocardial cells induced by LPS was increased, and the serum THBS1 level in patients with myocardial injury in sepsis was also significantly increased compared with patients without sepsis‑induced myocardial injury. In the siTHBS1‑treated mice with myocardial injury, the levels of cTnI and proBNP were significantly decreased, the levels of the inflammatory cytokines IL‑6 and TNF‑α were significantly decreased, ROS were significantly decreased, caspase‑3 was significantly decreased, and myocardial cell apoptosis was also reduced, compared with the LPS group. Data from the present study suggested that THBS1 may be closely related to the biological behavior of myocardial cells and may be a therapeutic target for myocardial injury in sepsis.

(1) Background: Chronic increases in blood flow, as in cardiovascular diseases, induce outward arterial remodeling. Thrombospondin-1 (TSP-1) is known to interact with matrix proteins and immune cell-surface receptors, but its contribution to flow-mediated remodeling in the microcirculation remains unknown. (2) Methods: Mesenteric arteries were ligated in vivo to generate high- (HF) and normal-flow (NF) arteries in wild-type (WT) and TSP-1-deleted mice (TSP-1 -/- ). After 7 days, arteries were isolated and studied ex vivo. (3) Results: Chronic increases in blood flow induced outward remodeling in WT mice (increasing diameter from 221 ± 10 to 280 ± 10 µm with 75 mmHg intraluminal pressure) without significant effect in TSP-1 -/- (296 ± 18 to 303 ± 14 µm), neutropenic or adoptive bone marrow transfer mice. Four days after ligature, pro inflammatory gene expression levels (CD68, Cox2, Gp91phox, p47phox and p22phox) increased in WT HF arteries but not in TSP-1 -/- mice. Perivascular neutrophil accumulation at day 4 was significantly lower in TSP-1 -/- than in WT mice. (4) Conclusions: TSP-1 origin is important; indeed, circulating TSP-1 participates in vasodilation, whereas both circulating and tissue TSP-1 are involved in arterial wall thickness and diameter expansion.

Backgrounds and Aims: 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., Methods and Results: 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 A2 A R agonists (UK-432,097 or MRE0094) with P2Y 12 R 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 P2Y 12 R 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., Conclusion: 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 P2Y 12 R 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease in reproductive women, and the endocrine levels are also affected by diseases. The aim of this study was to determine the effect of thrombospondin-1 (TSP-1) on PCOS rat model., Methods: We established the PCOS rat model, the serum hormones including TSP-1 expression were determined and morphological characteristics were investigated to evaluate the model. These above endocrine and morphological features were investigated again to evaluate the effect of TSP-1 treatment., Results: In the PCOS model group, the serum hormones change (higher luteinizing hormone, testosterone and estrogen) and decreased TSP-1 expression levels were found compared with the control group. Besides, the morphological characteristics of PCOS were also observed in the model group. After TSP-1 treatment, the higher TSP-1, ANGPT2, PDGFB and PDGFD expression levels, the lower LH and T levels, decreased vessel density as well as VEGFA and ANGPT1 expression levels were found compared with the control group, and the ovary morphological changes were also observed in the TSP-1 experimental group., Conclusions: TSP-1 delivery system might be an alternative therapy for PCOS treatment.

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., (© 2021 The Author(s).)

Objectives: Atrial remodeling is the main developmental cause of atrial arrhythmias (AA), which may induce atrial fibrillation, atrial flutter, atrial tachycardia, and frequent premature atrial beats in acute myocardial infarction (AMI) patients. Thrombospondin-1 (TSP-1) has been shown to play an important role in inflammatory and fibrotic processes, but its role in atrial arrhythmias is not well described. The purpose of this study was to investigate the role of TSP-1 in AMI patients with atrial arrhythmias., Methods: A total of 219 patients with AMI who underwent percutaneous coronary intervention and with no previous arrhythmias were included. TSP-1 were analyzed in plasma samples. Patients were classified into 2 groups, namely, with and without AA during the acute phase of MI. Continuous electrocardiographic monitoring was used for AA diagnosis in hospital., Results: Twenty-four patients developed AA. Patients with AA had higher TSP-1 levels (29.01 ± 25.87 μg/mL vs 18.36 ± 10.89 μg/mL, p < 0.001) than those without AA. AA patients also tended to be elderly (65.25 ± 9.98 years vs 57.47 ± 10.78 years, p < 0.001), had higher Hs-CRP (39.74 ± 43.50 mg/L vs 12.22 ± 19.25 mg/L, p < 0.001) and worse heart function. TSP-1 (OR 1.033; 95% CI 1.003-1.065, p = 0.034), Hs-CRP (OR 1.023; 95% CI 1.006-1.041, p = 0.008), age (OR 1.067; 95% CI 1.004-1.135, p = 0.038) and LVDd (OR 1.142; 95% CI 1.018-1.282, p = 0.024) emerged as independent risk factors for AA in AMI patients., Conclusion: TSP-1 is a potential novel indicator of atrial arrhythmias during AMI., (© 2021. The Author(s).)

Purpose: Conjunctival squamous cell carcinoma (SCC) is primarily treated with surgical resection. SCC has various stages, and local recurrence is common. The purpose of this study was to investigate thrombospondin-1 expression and its association with prognosis., Methods: In this retrospective study, a gene expression array along with immunohistochemistry were performed for the evaluation of thrombospondin-1 expression, localization, as well as Ki67 labeling cell indices in carcinoma in situ (Tis) and advanced conjunctival SCC (Tadv). The presence or absence and intensity of cytoplasmic and nuclear staining in tumor cells were also divided into groups with a score of 0-3 and semi-quantitatively analyzed to investigate intracellular staining patterns. The association between thrombospondin-1 expression and tumor progression in a series of 31 conjunctival SCCs was further investigated., Results: All 31 patients in the cohort (100%) were East Asian. A simple comparison between Tis and Tadv demonstrated significant differences in expressions of 45 genes, including thrombospondin-1 (p < 0.01). In this cohort, 30/31 tumors were positive (96%) for thrombospondin-1. Furthermore, thrombospondin-1 intracellular staining pattern analysis scores were 2.12 and 0.96 for nuclear and cytoplasmic staining, respectively, with a significant difference observed between Tis and Tadv (p < 0.01). Alteration of the Ki67 labeling index was significantly correlated with that of the thrombospondin-1 cytoplasmic score (p = 0.030). Furthermore, univariate Cox regression analysis showed a significant correlation between thrombospondin-1 staining and progression-free survival (p = 0.026) and final orbital exenteration (p = 0.019)., Conclusions: The present results demonstrated that thrombospondin-1 is a potential molecular target in the pathology of conjunctival SCC, in addition to serving as a prognostic factor., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lysophosphatidic acid (LPA), a brain membrane-derived lipid mediator, plays important roles including neural development, function, and behavior. In the present study, the effects of LPA on astrocyte-derived synaptogenesis factor thrombospondins (TSPs) production were examined by real-time PCR and western blotting, and the mechanism underlying this event was examined by pharmacological approaches in primary cultured rat cortical astrocytes. Treatment of astrocytes with LPA increased TSP-1 mRNA, and TSP-2 mRNA, but not TSP-4 mRNA expression. TSP-1 protein expression and release were also increased by LPA. LPA-induced TSP-1 production were inhibited by AM966 a LPA 1 receptor antagonist, and Ki16425, LPA 1/3 receptors antagonist, but not by H2L5146303, LPA 2 receptor antagonist. Pertussis toxin, Gi/o inhibitor, but not YM-254890, Gq inhibitor, and NF499, Gs inhibitor, inhibited LPA-induced TSP-1 production, indicating that LPA increases TSP-1 production through Gi/o-coupled LPA 1 and LPA 3 receptors. LPA treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). LPA-induced TSP-1 mRNA expression was inhibited by U0126, MAPK/ERK kinase (MEK) inhibitor, but not SB202190, p38 MAPK inhibitor, or SP600125, JNK inhibitor. However, LPA-induced TSP-1 protein expression was diminished with inhibition of all three MAPKs, indicating that these signaling molecules are involved in TSP-1 protein production. Treatment with antidepressants, which bind to astrocytic LPA 1 receptors, increased TSP-1 mRNA and protein production. The current findings show that LPA/LPA 1/3 receptors signaling increases TSP-1 production in astrocytes, which could be important in the pathogenesis of affective disorders and could potentially be a target for the treatment of affective disorders., (© 2020 International Society for Neurochemistry.)

The balance of angiogenic factors, including vascular endothelial growth factor (VEGF), and angiostatic factors, like thrombospondin-1 (TSP-1) and endostatin, controls striated muscle angiogenic responses to exercise training. The effect of age on circulating levels of these factors following a bout of exercise is unclear. The authors hypothesized that older adults would have lower circulating VEGF but higher TSP-1 and endostatin after exercise compared with young adults. Ten young and nine older participants cycled for 45 min at 60% estimated HRmax. Serum [VEGF], [TSP-1], and [endostatin] obtained before (PREX), immediately after (POSTX0), and 3 hr after (POSTX3) exercise were analyzed. [VEGF] increased in older adults only from PREX to POSTX0 (p < .05). [TSP-1] increased in both age groups (p < .05). There was no effect of age or exercise on [endostatin]. In conclusion, immediately after exercise, both groups had a similar increase in [TSP-1], but [VEGF] increased in older adults only.

Platelet function is developmentally regulated. Healthy neonates do not spontaneously bleed, but their platelets are hypo-reactive to several agonists. The mechanisms underlying immature platelet function in neonates are incompletely understood. This critical issue remains challenging for the establishment of age-specific reference ranges. In this study, we evaluated platelet reactivity of five pediatric age categories, ranging from healthy full-term neonates up to adolescents (11-18 years) in comparison to healthy adults (>18 years) by flow cytometry. We confirmed that platelet hypo-reactivity detected by fibrinogen binding, P-selectin, and CD63 surface expression was most pronounced in neonates compared to other pediatric age groups. However, maturation of platelet responsiveness varied with age, agonist, and activation marker. In contrast to TRAP and ADP, collagen-induced platelet activation was nearly absent in neonates. Granule secretion markedly remained impaired at least up to 10 years of age compared to adults. We show for the first time that neonatal platelets are deficient in thrombospondin-1, and exogenous platelet-derived thrombospondin-1 allows platelet responsiveness to collagen. Platelets from all pediatric age groups normally responded to the C-terminal thrombospondin-1 peptide RFYVVMWK. Thus, thrombospondin-1 deficiency of neonatal platelets might contribute to the relatively impaired response to collagen, and platelet-derived thrombospondin-1 may control distinct collagen-induced platelet responses.

Interleukin-36γ (IL-36γ), a member of the IL-1 cytokine superfamily, amplifies lung inflammation and impairs host defense during acute pulmonary Pseudomonas aeruginosa infection. To be fully active, IL-36γ is cleaved at its N-terminal region by proteases such as neutrophil elastase (NE) and cathepsin S (CatS). However, it remains unclear whether limiting extracellular proteolysis restrains the inflammatory cascade triggered by IL-36γ during P. aeruginosa infection. Thrombospondin-1 (TSP-1) is a matricellular protein with inhibitory activity against NE and the pathogen-secreted Pseudomonas elastase LasB-both proteases implicated in amplifying inflammation. We hypothesized that TSP-1 tempers the inflammatory response during lung P. aeruginosa infection by inhibiting the proteolytic environment required for IL-36γ activation. Compared to wild-type (WT) mice, TSP-1-deficient (Thbs1 -/- ) mice exhibited a hyperinflammatory response in the lungs during P. aeruginosa infection, with increased cytokine production and an unrestrained extracellular proteolytic environment characterized by higher free NE and LasB, but not CatS activity. LasB cleaved IL-36γ proximally to M 19 at a cleavage site distinct from those generated by NE and CatS, which cleave IL-36γ proximally to Y 16 and S 18 , respectively. N-terminal truncation experiments in silico predicted that the M 19 and the S 18 isoforms bind the IL-36R complex almost identically. IL-36γ neutralization ameliorated the hyperinflammatory response and improved lung immunity in Thbs1 -/- mice during P. aeruginosa infection. Moreover, administration of cleaved IL-36γ induced cytokine production and neutrophil recruitment and activation that was accentuated in Thbs1 -/- mice lungs. Collectively, our data show that TSP-1 regulates lung neutrophilic inflammation and facilitates host defense by restraining the extracellular proteolytic environment required for IL-36γ activation. IMPORTANCE pulmonary infection can lead to exaggerated neutrophilic inflammation and tissue destruction, yet host factors that regulate the neutrophilic response are not fully known. IL-36γ is a proinflammatory cytokine that dramatically increases in bioactivity following N-terminal processing by proteases. Here, we demonstrate that thrombospondin-1, a host matricellular protein, limits N-terminal processing of IL-36γ by neutrophil elastase and the Pseudomonas aeruginosa -secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1 Pseudomonas aeruginosa -secreted protease LasB. Thrombospondin-1-deficient mice (Thbs1 -/- ) exhibit a hyperinflammatory response following infection. Whereas IL-36γ neutralization reduces inflammatory cytokine production, limits neutrophil activation, and improves host defense in Thbs1 -/- mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36γ in the lung by restraining the extracellular proteolytic environment.-/- mice. Our findings indicate that thrombospondin-1 guards against feed-forward neutrophilic inflammation mediated by IL-36γ in the lung by restraining the extracellular proteolytic environment., (Copyright © 2021 Peñaloza et al.)

Recently, a growing body of evidence has suggested that abnormal ovarian angiogenesis, secondary to the imbalance between various angiogenic markers, is involved in the pathogenesis of PCOS, and this has led to the use of various interventions (such as Diane-35) to restore the normal ovarian angiogenesis. Therefore, we conducted the current investigation to determine the role of such markers (endothelial growth factor (VEGF), endostatin (ES), and thrombospondin-1 (TSP-1)) in the pathogenesis of PCOS along with the associated changes in ovarian blood flow in patients with PCOS compared to healthy controls, both before and after a course of oral contraception. A total of 381 patients with PCOS and 98 healthy females of childbearing age were recruited from July 2014 to June 2017 at the Reproductive Center of the Second Affiliated Hospital of Harbin Medical University. The serum levels of VEGF, ES, and TSP-1 were determined by enzyme-linked immunosorbent assay, while ovarian perfusion was measured by the pulsatility index (PI) and resistance index (RI) by using transvaginal color Doppler ultrasound. Repeated analyses were carried out after 3 months of Diane-35 treatment. Post-treatment serum levels of luteinizing hormone (LH)/follicle stimulating hormone (FSH) ratio of patients with PCOS decreased significantly (P <0.05). The RI values of most PCOS patients increased after treatment (P<0.05), while PI was significantly increased in all patients (P<0.05). However, variable changes in the serum levels of TSP-1, VEGF, and ES after treatment were observed. Serum VEGF levels showed a negative correlation with serum LH/FSH ratio, T concentration, and ES (P <0.05), while ES levels were negatively correlated with serum T concentrations only (P<0.05). The markers of angiogenesis (VEGF, ES, and TSP-1) were expressed differently among PCOS patients, who also responded differently to the same course of Diane-35 treatment. This field still warrants further investigation to reach a more definitive conclusion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Liu, Chen, Lu, Wang, Wang, Liu, Li, Du, Liang, Gong and Wang.)

Thrombospondin-1 (TSP1) is involved in corneal wound healing caused by chemical injury. Herein, we examined the effects of TSP1 on hypoxia-induced damages and wound-healing activity in human corneal epithelial (HCE) cells. Exosomal protein expression was determined using liquid chromatography-tandem mass spectrometry, and HCE cell migration and motility were examined through wound-healing assay and time-lapse microscopy. Reestablishment of cell junctions by TSP1 was assessed through confocal microscopy and 3D image reconstruction. Our results show that CoCl 2 -induced hypoxia promoted HCE cell death by paraptosis. TSP1 protected these cells against paraptosis by attenuating mitochondrial membrane potential depletion, swelling and dilation of endoplasmic reticulum and mitochondria, and mitochondrial fission. Exosomes isolated from HCE cells treated with TSP1 contained wound healing-associated proteins that were taken up by HCE cells to promote tissue remodeling and repair. TSP1 protected HCE cells against hypoxia-induced damages and inhibited paraptosis progression by promoting cell migration, cell-cell adhesion, and extracellular matrix remodeling. These findings indicate that TSP1 ameliorates hypoxia-induced paraptosis in HCE cells and promotes wound healing and remodeling by regulating exosomal protein expression. TSP1 may, therefore, play important roles in the treatment of hypoxia-associated corneal diseases., (© 2020 Federation of American Societies for Experimental Biology.)

Instantaneous blood coagulation after bioengineered liver transplantation is a major issue, and the key process in its prevention is the construction of the endothelial vascular bed on biomimetic scaffolds. However, the specific molecules involved in the regulation of the vascular bed formation remain unclear. Syndecan-4 is a type I transmembrane glycoprotein commonly expressed in the human body; its receptor has been reported as critical for optimal cell adhesion and initiation of intracellular signaling, indicating its promising application in vascular bed formation. In the current study, bioinformatics analysis and in vitro experiments were performed to evaluate whether syndecan-4 promoted endothelial cell migration and functional activation. Exogenous syndecan-4-overexpressing endothelial cells were perfused into the decellularized liver scaffold, which was assessed by Masson's trichrome staining. Western blotting and qRT-PCR were used to evaluate the effects of syndecan-4 on the thrombospondin 1 (THBS1) stability. We found that syndecan-4 promoted the adhesion of vascular endothelial cells and facilitated cell migration and angiogenesis. Furthermore, syndecan-4 overexpression resulted in a well-aligned endothelium on the decellularized liver scaffolds. Mechanistically, syndecan-4 destabilized THBS1 at the protein level. Therefore, our data revealed that syndecan-4 promoted the biological activity of endothelial cells on the bionic liver vascular bed through THBS1. These findings provide scientific evidences for solving transient blood coagulation after bionic liver transplantation.

Background: Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of both human and mouse models of dysferlinopathy suggest dysferlin deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patients with two missense mutations in exon 44., Methods: To assess proteasome inhibition we treated dysferlin deficient myotubes with EB1089, a vitamin D3 analog, oprozomib and ixazomib. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury and fusion index was also measured with the different treatments. Data were analyzed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p ≤ 0.05 was considered statistically significant., Results: Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on the repair of muscle membrane after injury., Conclusions: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-β-structure of misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN - -denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.

Blood-brain barrier (BBB) disruption contributes to neurodegenerative diseases. Loss of tight junction (TJ) proteins in cerebral endothelial cells (ECs) is a leading cause of BBB breakdown. We recently reported that miR-195 provides vasoprotection, which urges us to explore the role of miR-195 in BBB integrity. Here, we found cerebral miR-195 levels decreased with age, and BBB leakage was significantly increased in miR-195 knockout mice. Furthermore, exosomes from miR-195-enriched astrocytes increased endothelial TJ proteins and improved BBB integrity. To decipher how miR-195 promoted BBB integrity, we first demonstrated that TJ proteins were metabolized via autophagic-lysosomal pathway and the autophagic adaptor p62 was necessary to promote TJ protein degradation in cerebral ECs. Next, proteomic analysis of exosomes revealed miR-195-suppressed thrombospondin-1 (TSP1) as a major contributor to BBB disruption. Moreover, TSP1 was demonstrated to activate selective autophagy of TJ proteins by increasing the formation of claudin-5-p62 and ZO1-p62 complexes in cerebral ECs while TSP1 impaired general autophagy. Delivering TSP1 antibody into the circulation showed dose-dependent reduction of BBB leakage by 20%-40% in 25-month-old mice. Intravenous or intracerebroventricular injection of miR-195 rescued TSP1-induced BBB leakage. Dementia patients with BBB damage had higher levels of serum TSP1 compared to those without BBB damage (p = 0.0015), while the normal subjects had the lowest TSP1 (p < 0.0001). Taken together, the study implies that TSP1-regulated selective autophagy facilitates the degradation of TJ proteins and weakens BBB integrity. An adequate level of miR-195 can suppress the autophagy-lysosome pathway via a reduction of TSP1, which may be important for maintaining BBB function., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Human adipose tissue derived mesenchymal stem cells (hAD-MSC S ) with suppressive immunogenicity, homing to injury, inflammatory, and cancer sites can be suitable for gene therapy. PiggyBac (PB) is a type of transposon vector applied in mammalian systems and could overcome some limitations of other transposon and viral vectors. In this study, the therapeutic potential hAD-MSCs expressing thrombospondin-1 (TSP-1) is assessed through tail vein injection in C57BL/6 models bearing melanoma mice. Twenty days after injection, antiangiogenic effects and number of activated T. cells are assessed by Immunohistochemistry (IHC) method. Apoptosis value is analyzed by tunnel assay. Mice survival and numbers of nodules in mice lungs also are assessed. By western blotting, value of TSP-1, Bax and Bcl2 expression are assessed. The result revealed that hAD-MSCs . TSP-1 can inhibit angiogenesis and induce apoptosis and activated T. cells in a significant manner in C57BL/6 mice models bearing melanoma. Survival also significantly increased and number of nodules decreased, value of Bax and TSP-1 expression increased and value of Bcl2 expression decreased. In conclusion, our result showed that hAD-MSC. TSP-1 can be applied as an effective delivery vehicle in lung metastatic melanoma therapy., (Copyright © 2020 International Alliance for Biological Standardization. All rights reserved.)

CD47 serves as a ligand for signaling regulatory protein α (SIRPα) and as a receptor for thrombospondin-1 (TSP-1). Although CD47, TSP-1, and SIRPα are thought to be involved in the clearance of aged red blood cells (RBCs), aging-associated changes in the expression and interaction of these molecules on RBCs have been elusive. Using direct stochastic optical reconstruction microscopy (dSTORM)-based imaging and quantitative analysis, we can report that CD47 molecules on young RBCs reside as nanoclusters with little binding to TSP-1, suggesting a minimal role for TSP-1/CD47 signaling in normal RBCs. On aged RBCs, CD47 molecules decreased in number but formed bigger and denser clusters, with increased ability to bind TSP-1. Exposure of aged RBCs to TSP-1 resulted in a further increase in the size of CD47 clusters via a lipid raft-dependent mechanism. Furthermore, CD47 cluster formation was dramatically inhibited on thbs1 -/- mouse RBCs and associated with a significantly prolonged RBC lifespan. These results indicate that the strength of CD47 binding to its ligand TSP-1 is predominantly determined by the distribution pattern and not the amount of CD47 molecules on RBCs, and offer direct evidence for the role of TSP-1 in phagocytosis of aged RBCs. This study provides clear nanoscale pictures of aging-associated changes in CD47 distribution and TSP-1/CD47 interaction on the cell surface, and insights into the molecular basis for how these molecules coordinate to remove aged RBCs., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Background: Thrombospondin-1 (TSP-1), a Ca 2+ -binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca 2+ dynamics, survival, and deformability of human red blood cells (RBCs)., Methods: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca 2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer., Results: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca 2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca 2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca 2+ - and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index)., Conclusions: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca 2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract.

p97 has recently emerged as a therapeutic target for cancer due to its essential functions in protein homeostasis. CB-5083 is a first-in-class, potent and selective ATP-competitive p97 inhibitor that induces proteotoxic stress in cancer cells. Potential mechanisms regulating the sensitivity of cells to p97 inhibition remain poorly studied. Here, we demonstrate that Thrombospondin-1 (THBS1) is a CB-5083-upregulated gene that helps confer resistance of HCT116 cells to CB-5083. Our immunoblotting and immunofluorescence data showed that CB-5083 significantly increases the steady-state abundance of THBS1. Blockade of THBS1 induction sensitized cells to CB-5083-mediated growth inhibition. Suppression of THBS1 caused an increase of CB-5083-induced sub-G1 population and caspase 3/7 activity suggesting that its function is linked to the survival of cancer cells in response to p97 inhibition. Altogether our data provide new evidence that THBS1 is important for the susceptibility of cells to p97 inhibition.

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth, including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix-cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvβ1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin β1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo., Competing Interests: The authors declare no competing interest.

Aim: Thrombospondin-1 (TSP-1) is an astrocyte-derived synaptogenesis-related factor. It was previously reported to be increased by chronic treatment of electroconvulsive seizure, a model of electroconvulsive therapy (ECT), in rat hippocampus. The aim of this study was to examine whether serum levels of TSP-1 are associated with depression and ECT., Methods: Serum TSP-1 levels of major depressive disorder (MDD) patients (n = 36) and age- and gender-matched healthy controls (n = 36) were measured by TSP-1 ELISA. MDD patients were diagnosed according to the Diagnostics and Statistical Manual of Mental Disorders-IV-TR and underwent ECT. MDD patients were also analyzed for serum TSP-1 levels pre- and post-ECT. Evaluation of symptoms was obtained using the Hamilton Rating Scale for Depression., Results: Serum TSP-1 levels showed significant decreases specific to female MDD patients. However, TSP-1 did not change pre- and post-ECT, did not correlate with symptoms, nor was not affected by the dose of antidepressants., Conclusion: Serum TSP-1 is a possible female-specific factor that reflects depressive trait, but not state., (© 2019 The Authors. Neuropsychopharmacology Reports published by John Wiley & Sons Australia, Ltd on behalf of the Japanese Society of Neuropsycho Pharmacology.)

Previous studies have suggested that thrombospondin-1 (TSP-1) regulates the transforming growth factor beta 1 (TGF-β1)/phosphorylated Smad2/3 (pSmad2/3) pathway. Moreover, TSP-1 is closely associated with epilepsy. However, the role of the TSP-1-regulated TGF-β1/pSmad2/3 pathway in seizures remains unclear. In this study, changes in this pathway were assessed following kainic acid (KA)-induced status epilepticus (SE) in rats. The results showed that increases in the TSP-1/TGF-β1/pSmad2/3 levels spatially and temporally matched the increases in glial fibrillary acidic protein (GFAP)/chondroitin sulfate (CS56) levels following KA administration. Inhibition of TSP-1 expression by small interfering RNA or inhibition of TGF-β1 activation with a Leu-Ser-Lys-Leu peptide significantly reduced the severity of KA-induced acute seizures. These anti-seizure effects were accompanied by decreased GFAP/CS56 expression and Smad2/3 phosphorylation. Moreover, inhibiting Smad2/3 phosphorylation with ponatinib or SIS3 also significantly reduced seizure severity, alongside reducing GFAP/CS56 immunoreactivity. These results suggest that the TSP-1-regulated TGF-β1/pSmad2/3 pathway plays a key role in KA-induced SE and astrogliosis, and that inhibiting this pathway may be a potential anti-seizure strategy.

Rationale : Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease. Methods : We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results in vitro and using a model of SV carotid interposition in pigs. Results : Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22α) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-β-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-β-dependent pathway, and validated in a saphenous vein into carotid interposition pig model. Conclusions : Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Fabry disease (FD) is a recessive X-linked lysosomal storage disorder caused by α-galactosidase A (GLA) deficiency. Although the mechanism is unclear, GLA deficiency causes an accumulation of globotriaosylceramide (Gb3), leading to vasculopathy., Methods: To explore the relationship between the accumulation of Gb3 and vasculopathy, induced pluripotent stem cells generated from four Fabry patients (FD-iPSCs) were differentiated into vascular endothelial cells (VECs). Genome editing using CRISPR-Cas9 system was carried out to correct the GLA mutation or to delete Thrombospondin-1 (TSP-1). Global transcriptomes were compared between wild-type (WT)- and FD-VECs by RNA-sequencing analysis., Findings: Here, we report that overexpression of TSP-1 contributes to the dysfunction of VECs in FD. VECs originating from FD-iPSCs (FD-VECs) showed aberrant angiogenic functionality even upon treatment with recombinant α-galactosidase. Intriguingly, FD-VECs produced more p-SMAD2 and TSP-1 than WT-VECs. We also found elevated TSP-1 in the peritubular capillaries of renal tissues biopsied from FD patients. Inhibition of SMAD2 signaling or knock out of TSP-1 (TSP-1 -/- ) rescues normal vascular functionality in FD-VECs, like in gene-corrected FD-VECs. In addition, the enhanced oxygen consumption rate is reduced in TSP-1 -/- FD-VECs., Interpretation: The overexpression of TSP-1 secondary to Gb3 accumulation is primarily responsible for the observed FD-VEC dysfunction. Our findings implicate dysfunctional VEC angiogenesis in the peritubular capillaries in some of the complications of Fabry disease., Funding: This study was supported by grant 2018M3A9H1078330 from the National Research Foundation of the Republic of Korea., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Thrombospondin-1, an extracellular matrix protein, is the first identified natural angiogenesis inhibitor. Thrombospondin-1 participates in a great number of physiological and pathological processes, including cell-cell and cell-matrix interactions via a number of cell receptors, including CD36 and CD47, which plays a vital role in mediating inflammation and performs a promoting effect in pulmonary arterial vasculopathy and diabetes. Thrombospondin-1 consists of six domains, which combine with different molecules and participate in various functions in cancers, serving as a critical member in diverse pathways in cancers. Thrombospondin-1 works as a cancer promotor in some pathways but as a cancer suppressor in others, which makes it highly possible that its erroneous functioning might lead to opposite effects. Therefore, subdividing the roles of thrombospondin-1 and distinguishing them in cancers are necessary. Complex structure and multiple roles take disadvantage of the research and application of thrombospondin-1. Compared with the whole thrombospondin-1 protein, each thrombospondin- 1 active peptide performs an uncomplicated structure and, nevertheless, a specific role. In other words, various thrombospondin-1 active peptides may function differently. For instance, thrombospondin-1 could both promote and inhibit glioblastoma, which is significantly inhibited by the three type I repeats, a thrombospondin-1 active peptide but promoted by the fragment 167-569, a thrombospondin-1 active peptide consisting of the procollagen homology domain and the three type I repeats. Further studies of the functions of thrombospondin-1 active peptides and applying them reasonably are necessary. In addition to mediating cancerogenesis, thrombospondin-1 is also affected by cancer development, as reflected by its expression in plasma and the cancer tissue. Therefore, thrombospondin-1 may be a potential biomarker for pre-clinical and clinical application. This review summarizes findings on the multiple roles of thrombospondin-1 in cancer processes, with a focus on its use as a potential therapeutic target., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Transforming growth factor-β1 (TGF-β1) is a profibrotic cytokine which induces mesothelial cell mesothelial-to-mesenchymal transition (MMT) and peritoneal fibrosis in patients receiving treatment of peritoneal dialysis. Because thrombospondin-1 (TSP-1) is able to activate latent TGF-β1 in vivo, we investigated whether blockade of TSP-1 could modulate mesothelial cell MMT and ameliorate peritoneal fibrosis., Methods: Human pleural mesothelial cells (Met-5A cells) were treated with TSP-1 and addition of TGF-β1 neutralizing antibody to assess the effect of TSP-1 on MMT. Furthermore, TSP-1 blocking peptide Leu-Ser-Lys-Leu (LSKL) was applied to Met-5A cells treated with 4.25% d-glucose to determine its function in high glucose-induced MMT. Consequently, a uremic dialysate injection rat model was set up to confirm the results in vivo., Results: Exposure of Met-5A cells to TSP-1 increased TGF-β1 secretion, expression and bioactivity, triggered Smad3 phosphorylation, upregulated the expression of mesenchymal molecules including fibronectin, collagen type III, α-smooth muscle actin, Snail, and decreased calretinin expression. The effect was partially attenuated by TGF-β1 neutralizing antibody. TSP-1 expression in Met-5A cells was increased by 4.25% d-glucose, followed by increased secretion and bioactivity of TGF-β1, the onset of Smad3 phosphorylation and induction of MMT. LSKL significantly attenuated high glucose-mediated mesothelial cell MMT and ameliorated peritoneal fibrosis in uremic rats receiving dextrose dialysate injection., Conclusions: Taken together, these data demonstrated that TSP-1 contributes to mesothelial cell MMT by activating TGF-β1/Smad3 signaling pathway and blockade of TSP-1 attenuates high glucose-mediated mesothelial cell MMT and peritoneal fibrosis., (© 2019 Wiley Periodicals, Inc.)

Background: Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer associated with a high mortality. Long non-coding RNAs (lncRNAs) have recently emerged as regulators in the development and progression of several cancers, and therefore represent an opportunity to uncover new targets for therapy. In the present study, we aimed to investigate the potential effect of lncRNA BZRAP1-AS1 on the angiogenesis of HCC., Methods: Microarray-based data analysis was initially employed to screen genes and lncRNAs that are differentially expressed in HCC and the candidate BZRAP1-AS1 was identified as a hit. The expression of BZRAP1-AS1 and thrombospondin-1 (THBS1) in HCC tissues and cells were then determined using RT-qPCR. The gene methylation level was measured by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) assays. Next, the interactions between BZRAP1-AS1, DNA methyltransferase 3B (DNMT3b), and THBS1 were assessed by RIP, RNA pull-down and ChIP assays. Finally, the roles of BZRAP1-AS1, DNMT3b and THBS1 in angiogenesis in vitro as well as tumorigenesis in vivo were evaluated by a battery of the gain- and loss-of function experiments., Results: BZRAP1-AS1 was identified as a highly expressed lncRNA in HCC tissues and cells. Down-regulation of BZRAP1-AS1 in HCC cells inhibited HUVEC proliferation, migration and angiogenesis. By interacting with DNMT3b, BZRAP1-AS1 induced methylation of the THBS1 promoter and inhibited the transcription of THBS1, resulting in promoted angiogenesis of HUVECs. Moreover, silencing of BZRAP1-AS1 repressed the angiogenesis as well as the tumor growth of HCC in vivo via up-regulating THBS1., Conclusion: This study provides evidence that angiogenesis in HCC is hindered by silencing of BZRAP1-AS1. Thus, BZRAP1-AS1 may be a promising marker for the treatment of HCC.

Purpose: The aim of the study was to investigate whether maternal serum TSP-1 level was associated with PE., Materials and Methods: In our case control study, 84 pregnant women in the third trimester were included. Forty-one of them were healthy and 43 of them were with the diagnosis of PE. The diagnosis was based on the definitions of the National High Blood Pressure Education Program working Group on High Blood Pressure in Pregnancy. Preeclamptic patients were divided into two subgroups as mild and severe. Blood pressure (BP) of pregnant women were obtained in left-side lying position using a mercury sphygmomanometer after at least 10 minutes of rest. Ten milliliters of venous blood was taken from every pregnant women and dispensed into lithium heparin and serum was obtained. Samples were stored at -80 °C until analyzed. Serum TSP-1 level was measured using enzyme-linked immunosorbent assay (ELISA). All tests were two-tailed and p < .05 was considered to be statistically significant., Results: TSP-1 level was significantly lower in PE group than in controls (p = .003). Platelet counts were similar in two groups (p = .26). TSP-1 levels were significantly lower in severe PE than in mild PE cases. According to the subgroup analysis, TSP-1 level was found significantly lower in severe preeclampsia group compared to control group (p = .015)., Conclusions: In light of the association between endothelial dysfunction and preeclampsia, we claim that lower levels of TSP-1 which is released mostly from endothelial cells seem to reflect disease severity in PE. Our study reveals that maternal serum TSP-1 levels decrease in pregnant women presenting with PE and TSP-1 may be a new biomarker for the detection of PE and even severity of it. Further studies especially prospective ones with greater numbers of cases are needed.

The pathogenesis of Cushing's disease, which is caused by pituitary corticotroph adenoma, remains to be studied. Secreted angioinhibitory factor thrombospondin-1 (TSP-1) is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions and is associated with platelet aggregation, angiogenesis and tumorigenesis. We have found that the expression of TSP-1 is significantly lower in human pituitary corticotroph tumours compared with normal adenohypophysis. This study aims to elucidate the role of TSP-1 in regulating the tumour function of pituitary adenomas. Forced overexpression of TSP-1 in a murine AtT20 pituitary corticotroph tumour cell line decreased corticotroph precursor hormone proopiomelanocortin (POMC) transcription and adrenocorticotropic hormone (ACTH) secretion. Functional studies showed that TSP-1 overexpression in pituitary adenoma cells suppressed proliferation, migration and invasion. We have demonstrated that TSP-1 is a direct target of miR-449c. Further study showed that miR-449c activity enhanced tumorigenesis by directly inhibiting TSP-1 expression. Low expression of lncTHBS1, along with low expression of TSP-1, was associated with the high expression of miR-449c in Cushing's disease patients. Furthermore, RNA-immunoprecipitation associates miR-449c with lncTHBS1 suggesting that lncTHBS1 might be a negative regulator of miR-449c. Taken together, this study has demonstrated that lncTHBS1 might function as competing endogenous RNA for miR-449c, which could suppress the development of Cushing's disease., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Pulmonary hypertension (PH) is a leading cause of death in sickle cell disease (SCD) patients. Hemolysis and oxidative stress contribute to SCD-associated PH. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and, by interacting with its receptor CD47, limits vasodilation of distal pulmonary arteries ex vivo. We hypothesized that the TSP1-CD47 interaction may promote PH in SCD. We found that TSP1 and CD47 are upregulated in the lungs of Berkeley (BERK) sickling (Sickle) mice and patients with SCD-associated PH. We then generated chimeric animals by transplanting BERK bone marrow into C57BL/6J ( n = 24) and CD47 knockout (CD47KO, n = 27) mice. Right ventricular (RV) pressure was lower in fully engrafted Sickle-to-CD47KO than Sickle-to-C57BL/6J chimeras, as shown by the reduced maximum RV pressure ( P = 0.013) and mean pulmonary artery pressure ( P = 0.020). The afterload of the sickle-to-CD47KO chimeras was also lower, as shown by the diminished pulmonary vascular resistance ( P = 0.024) and RV effective arterial elastance ( P = 0.052). On myography, aortic segments from Sickle-to-CD47KO chimeras showed improved relaxation to acetylcholine. We hypothesized that, in SCD, TSP1-CD47 signaling promotes PH, in part, by increasing reactive oxygen species (ROS) generation. In human pulmonary artery endothelial cells, treatment with TSP1 stimulated ROS generation, which was abrogated by CD47 blockade. Explanted lungs of CD47KO chimeras had less vascular congestion and a smaller oxidative footprint. Our results show that genetic absence of CD47 ameliorates SCD-associated PH, which may be due to decreased ROS levels. Modulation of TSP1-CD47 may provide a new molecular approach to the treatment of SCD-associated PH.

In this work, an aptamer was used as the target capturing agent and a trimetallic signal amplification strategy based on Ce-MOF@Au and AuPtRu NPs was demonstrated for the sensitive detection of TSP-1. Herein, the synthesized AuPtRu nanocomposite (AuPtRu NPs) not only acts as the catalyst for catalyzing hydrogen peroxide but also acts as a nanocarrier for capturing the -NH 2 termination single strand DNA (S1) to obtain the signal probe (SP, AuPtRu nanocomposite/S1). Then, SP was efficiently linked into TSP-1 aptamers with the addition of complementary linking strands to form M1 (SP/aptamer). The Ce-MOF@Au nanocomposites were obtained by in situ reduction and used as GCE electrode modification materials. The -NH 2 -modified capture probe (CP) DNA was immobilized on the surface of Ce-MOF@Au nanocomposites for hybridizing SP. In the presence of the target TSP-1, the aptamer recognizes the target and binds strongly so that SP is released from the prepared M1 and then hybridized with CP. When the detection solution contains an electrochemical matrix of H 2 O 2 , AuPtRu NPs can oxidize H 2 O 2 to obtain an enhanced signal. Furthermore, the proposed aptasensor has a very low LOD of 0.13 fg mL -1 TSP-1 in the detection range of 1 fg mL -1 to 10 ng mL -1 . Moreover, the proposed platform also has application implications for other potential targets., (Copyright © 2019 Elsevier B.V. All rights reserved.)