Interstitial macrophage phenotypes in Schistosoma-induced pulmonary hypertension.

Background: Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-ß, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1. Methods: Mice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression. Results: Flow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation. Conclusion: Schistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-ß and causes PH.

CD47 blockade reduces ischemia/reperfusion injury in murine heart transplantation and improves donor heart preservation.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is an important cause of early dysfunction and exacerbation of immune rejection in transplanted hearts. The integrin-related protein CD47 exacerbates myocardial ischemia-reperfusion injury by inhibiting the nitric oxide signaling pathway through interaction with thrombospondin-1 (TSP-1). In addition, the preservation quality of the donor hearts is a key determinant of transplant success. Preservation duration beyond four hours is associated with primary graft dysfunction. We hypothesized that blocking the CD47-TSP-1 system would attenuate ischemia-reperfusion injury in the transplanted heart and, thus, improve the preservation of donor hearts. METHODS: We utilized a syngeneic mouse heart transplant model to assess the effect of CD47 monoclonal antibody (CD47mAb) to treat MIRI. Donor hearts were perfused with CD47mAb or an isotype-matched control immunoglobulin (IgG2a) and were implanted into the abdominal cavity of the recipients after being stored in histidine-tryptophan-ketoglutarate (HTK) solution at 4 °C for 4 h or 8 h. RESULTS: At both the 4-h and 8-h preservation time points, mice in the experimental group perfused with CD47mAb exhibited prolonged survival in the transplanted heart, reduced inflammatory response and oxidative stress, significantly decreased inflammatory cell infiltration, and fewer apoptosis-related biomarkers. CONCLUSION: The application of CD47mAb for the blocking of CD47 attenuates MIRI as well as improves the preservation and prognosis of the transplanted heart in a murine heart transplant model.

Nattokinase attenuates endothelial inflammation through the activation of SRF and THBS1.

Natto contains a potent fibrinolytic enzyme called nattokinase (NK), which has thrombolytic, antihypertensive, antiatherosclerotic and lipid-lowering effects. Although NK has been recognized for its beneficial effect on humans with atherosclerotic cardiovascular disease (ASCVD), the underlying mechanisms involved in vascular inflammation-atherosclerosis development remain largely unknown. The current study aimed to explore the effects of NK on gene regulation, autophagy, necroptosis and inflammasome in vascular inflammation. The transcriptional profiles of NK in endothelial cells (ECs) by RNA sequencing (RNA-seq) revealed that NK affected THBS1, SRF and SREBF1 mRNA expression. In Q-PCR analysis, SRF and THBS1 were upregulated but SREBF1 was unaffected in ECs treated with NK. NK treatment induced autophagy and inhibited NLRP3 inflammasome and necroptosis in ECs. Furthermore, the inhibition of SRF or THBS1 by siRNA suppressed autophagy and enhanced the NLRP3 inflammasome and necroptosis. In a mouse model, NK reduced vascular inflammation by activating autophagy and inhibiting NLRP3 inflammasome and necroptosis. Our findings provide the first evidence that NK upregulates SRF and THBS1 genes, subsequently increasing autophagy and decreasing necroptosis and NLRP3 inflammasome formation to reduce vascular inflammation. Therefore, NK could serve as nutraceuticals or adjuvant therapies to reduce vascular inflammation and possible atherosclerosis progression.

CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Protective effect of alizarin on vascular endothelial dysfunction via inhibiting the type 2 diabetes-induced synthesis of THBS1 and activating the AMPK signaling pathway.

BACKGROUND: In this study, we investigated the protective effects of alizarin (AZ) on endothelial dysfunction (ED). AZ has inhibition of the type 2 diabetes mellitus (T2DM)-induced synthesis of thrombospondin 1 (THBS1). Adenosine 5'-monophosphate- activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. PURPOSE: The aim of this study was to investigate the ameliorative effect of AZ on vascular injury caused by T2DM and to reveal the potential mechanism of AZ in high glucose (HG)-stimulated human umbilical vein endothelial cells (HUVECs) and diabetic model rats. STUDY DESIGN: HUVECs, rats and AMPK-/- transgenic mice were used to investigate the mitigating effects of AZ on vascular endothelial dysfunction caused by T2DM and its in vitro and in vivo molecular mechanisms. METHODS: In type 2 diabetes mellitus rats and HUVECs, the inhibitory effect of alizarin on THBS1 synthesis was verified by immunohistochemistry (IHC), immunofluorescence (IF) and Western blot (WB) so that increase endothelial nitric oxide synthase (eNOS) content in vitro and in vivo. In addition, we verified protein interactions with immunoprecipitation (IP). To probe the mechanism, we also performed AMPKα2 transfection. AMPK's pivotal role in AZ-mediated prevention against T2DM-induced vascular endothelial dysfunction was tested using AMPKα2-/- mice. RESULTS: We first demonstrated that THBS1 and AMPK are targets of AZ. In T2DM, THBS1 was robustly induced by high glucose and inhibited by AZ. Furthermore, AZ activates the AMPK signaling pathway, and recoupled eNOS in stressed endothelial cells which plays a protective role in vascular endothelial dysfunction. CONCLUSIONS: The main finding of this study is that AZ can play a role in different pathways of vascular injury due to T2DM. Mechanistically, alizarin inhibits the increase in THBS1 protein synthesis after high glucose induction and activates AMPKα2, which increases NO release from eNOS, which is essential in the prevention of vascular endothelial dysfunction caused by T2DM.

Stromal thrombospondin 1 suppresses angiogenesis in oral submucous fibrosis.

A decline in mucosal vascularity is a histological hallmark of oral submucous fibrosis (OSF), a premalignant disease that is largely induced by betel quid chewing. However, the lack of available models has challenged studies of angiogenesis in OSF. Here, we found that the expression of thrombospondin 1 (THBS1), an endogenous angiostatic protein, was elevated in the stroma of tissues with OSF. Using a fibroblast-attached organoid (FAO) model, the overexpression of THBS1 in OSF was stably recapitulated in vitro. In the FAO model, treatment with arecoline, a major pathogenic component in areca nuts, enhanced the secretion of transforming growth factor (TGF)-ß1 by epithelial cells, which then promoted the expression of THBS1 in fibroblasts. Furthermore, human umbilical vein endothelial cells (HUVECs) were incorporated into the FAO to mimic the vascularized component. Overexpression of THBS1 in fibroblasts drastically suppressed the sprouting ability of endothelial cells in vascularized FAOs (vFAOs). Consistently, treatment with arecoline reduced the expression of CD31 in vFAOs, and this effect was attenuated when the endothelial cells were preincubated with neutralizing antibody of CD36, a receptor of THBS1. Finally, in an arecoline-induced rat OSF model, THBS1 inhibition alleviated collagen deposition and the decline in vascularity in vivo. Overall, we exploited an assembled organoid model to study OSF pathogenesis and provide a rationale for targeting THBS1.

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.

TSP-1 increases autophagy level in cartilage by upregulating HSP27 which delays progression of osteoarthritis.

This study aimed to determine whether Thrombospondin-1 (TSP-1) can be used as a biomarker to diagnose early osteoarthritis (OA) and whether it has a chondroprotective effect against OA. We examined TSP-1 expression in cartilage, synovial fluid, and serum at different time points after anterior cruciate ligament transection (ACLT) surgery in rats. Subsequently, TSP-1 was overexpressed or silenced to detect its effects on extracellular matrix (ECM) homeostasis, autophagy level, proliferation and apoptosis in chondrocytes. Adenovirus encoding TSP-1 was injected into the knee joints of ACLT rats to test its effect against OA. Combined with proteomic analysis, the molecular mechanism of TSP-1 in cartilage degeneration was explored. Intra-articular injection of an adenovirus carrying the TSP-1 sequence showed chondroprotective effects against OA. Moreover, TSP-1 expression decreases with OA progression and can effectively promote cartilage proliferation, inhibit apoptosis, and helps to sustain the balance between ECM anabolism and catabolism. Overexpression of TSP-1 also can increase autophagy by upregulating Heat Shock Protein 27 (HSP27, hspb1), thereby enhancing its effect as a stimulator of autophagy. TSP-1 is a hopeful strategy for OA treatment.

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.

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

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

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.

Thrombospondin 1 and Reelin act through Vldlr to regulate cardiac growth and repair.

Adult mammalian cardiomyocytes have minimal cell cycle capacity, which leads to poor regeneration after cardiac injury such as myocardial infarction. Many positive regulators of cardiomyocyte cell cycle and cardioprotective signals have been identified, but extracellular signals that suppress cardiomyocyte proliferation are poorly understood. We profiled receptors enriched in postnatal cardiomyocytes, and found that very-low-density-lipoprotein receptor (Vldlr) inhibits neonatal cardiomyocyte cell cycle. Paradoxically, Reelin, the well-known Vldlr ligand, expressed in cardiac Schwann cells and lymphatic endothelial cells, promotes neonatal cardiomyocyte proliferation. Thrombospondin1 (TSP-1), another ligand of Vldlr highly expressed in adult heart, was then found to inhibit cardiomyocyte proliferation through Vldlr, and may contribute to Vldlr's overall repression on proliferation. Mechanistically, Rac1 and subsequent Yap phosphorylation and nucleus translocation mediate the regulation of the cardiomyocyte cell cycle by TSP-1/Reelin-Vldlr signaling. Importantly, Reln mutant neonatal mice displayed impaired cardiomyocyte proliferation and cardiac regeneration after apical resection, while cardiac-specific Thbs1 deletion and cardiomyocyte-specific Vldlr deletion promote cardiomyocyte proliferation and are cardioprotective after myocardial infarction. Our results identified a novel role of Vldlr in consolidating extracellular signals to regulate cardiomyocyte cell cycle activity and survival, and the overall suppressive TSP-1-Vldlr signal may contribute to the poor cardiac repair capacity of adult mammals.

THBS1-Mediated Degradation of Collagen via the PI3K/AKT Pathway Facilitates the Metastasis and Poor Prognosis of OSCC.

Oral squamous cell carcinoma (OSCC) is a prevalent form of malignant tumor, characterized by a persistently high incidence and mortality rate. The extracellular matrix (ECM) plays a crucial role in the initiation, progression, and diverse biological behaviors of OSCC, facilitated by mechanisms such as providing structural support, promoting cell migration and invasion, regulating cell morphology, and modulating signal transduction. This study investigated the involvement of ECM-related genes, particularly THBS1, in the prognosis and cellular behavior of OSCC. The analysis of ECM-related gene data from OSCC samples identified 165 differentially expressed genes forming two clusters with distinct prognostic outcomes. Seventeen ECM-related genes showed a significant correlation with survival. Experimental methods were employed to demonstrate the impact of THBS1 on proliferation, migration, invasion, and ECM degradation in OSCC cells. A risk-prediction model utilizing four differentially prognostic genes demonstrated significant predictive value in overall survival. THBS1 exhibited enrichment of the PI3K/AKT pathway, indicating its potential role in modulating OSCC. In conclusion, this study observed and verified that ECM-related genes, particularly THBS1, have the potential to influence the prognosis, biological behavior, and immunotherapy of OSCC. These findings hold significant implications for enhancing survival outcomes and providing guidance for precise treatment of OSCC.

Electroacupuncture activates angiogenesis by regulating the PI3K/Pten/Thbs1 signaling pathway to promote the browning of adipose tissue in HFD-induced obese mice.

This study investigated the effect of electroacupuncture (EA) on the browning of white adipose tissue (WAT) via angiogenesis and its potential mechanism in obese mice. Four-week-old male C56BL/6 mice were randomly divided into a high-fat diet (HFD) and a normal chow diet (ND) group. After 12 weeks, HFD mice were randomly divided into two groups to receive or not receive EA for 3 weeks. After EA treatment, body weight, adipocyte size, serum glucose (GLU), triacylglycerol (TG), cholesterol (CHO), leptin (Lep), monocyte chemoattractant protein-1 (MCP-1), WAT browning-related genes, angiogenesis-related genes, and the PI3K/Pten/Thbs1 signaling pathway were evaluated. The results indicated that EA significantly reduced body weight, adipocyte size, and serum concentrations of GLU, TG, CHO, Lep and MCP-1 and promoted WAT browning. Angiogenesis and the PI3K/Pten/Thbs1 signaling pathway were all activated by EA intervention. The expression levels were consistent with the results of RNA-seq and confirmed via qRTPCR and WB. Our study showed that EA may activate angiogenesis via the PI3K/Pten/Thbs1 signaling pathway in WAT, thereby promoting the browning and thermogenesis of adipose tissue.

Evaluating von Willebrand factor and ADAMTS13 levels in thalassemia major patients and assessing a possible association with Thrombospondin-1.

INTRODUCTION: Alterations in the endothelium and endothelial adhesion proteins such as von Willebrand factor (vWF) play major roles in hypercoagulability in thalassemia. vWF protein release leads to platelet aggregation and thrombi formation at the site of vascular injury. It is then degraded by the proteolytic enzyme ADAMTS13. Thrombospondin-1 is a multifactorial glycoprotein, which was reported to compete with ADAMTS13 for sites of vWF proteolysis. In this study, levels of vWF, ADAMTS13, and TSP-1 proteins were determined in ß-thalassemia major patients. A possible association between TSP-1 and vWF and ADAMTS-13 was also evaluated. METHODS: The study was conducted on 80 ß-thalassemia major patients and 80 age and sex matched healthy controls. The 80 patients were sub-divided into two groups; splenectomised and non-splenectomised. vWF, ADAMTS13 and TSP-1 plasma level were measured using ELISA technique. RESULTS: There was no significant difference in vWF and TSP-1 levels between patients and controls (p > 0.05). However, ADAMTS13 levels and ADAMTS13 activity/vWF antigen ratio were significantly higher in patients compared to controls (p < 0.05). VWF antigen and TSP-1 level were significantly higher in splenectomised patients (p = 0.025 and p < 0.001, respectively). We also observed a significant decrease in ADAMTS13 activity/vWF antigen ratio among splenectomised compared to non- splenectomised patients (p = 0.019). Correlation analysis showed a significant negative correlation between TSP-1 and vWF Collagen Binding Activity (r = -0.394, p = 0.021) and a positive correlation with ADAMTS13 activity/vWF antigen ratio (r = 0.356, p = 0.039) in splenectomised compared to non- splenectomised patients. CONCLUSION: Our findings highlight the adequacy of patient management protocols for ß-TM in Kuwait as patients presented with comparable levels of platelets, vWF and TSP-1 compared to normal controls. The reported increase in ADAMTS13 in patients may be required to maintain normal levels of vWF. Although no active thrombotic episodes were reported at the time of the study, the significant rise in platelets, vWF:Ag and TSP-1 levels in splenectomised patients may indicate a tendency towards hypercoagulability. Monitoring of splenectomised patients is recommended.

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

OBJECTIVE: To explore the underlying molecular mechanism of (). METHODS: We used a tandem mass tag-based quantitative proteomic method to determine the differentially expressed proteins. Network pharmacology analysis was used to analysis the main components of and construct the compound-target network. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the analyses results. RESULTS: The expression levels of thrombospondin-1 (TSP-1) and transforming growth factor (TGF)-ß1/Smad3 signaling pathway proteins were significantly upregulated in focal segmental glomeruloscleosis (FSGS) rats. The reduced the expression levels of TSP-1 and TGF-ß1 signaling pathway proteins. Network pharmacology analysis revealed that protocatechualdehyde was the main active component. Subsequent and experiments validated the results of proteomic and network pharmacology analyses. CONCLUSIONS: Our results suggested that may inhibit renal sclerosis by inhibiting TSP-1-activated TGF-ß1 signaling and may have potential applications in the treatment of FSGS.