OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVES: To propose a hypothesis that aloe-emodin may inhibit scar tissue fibrosis through thrombospondin-1(THBS1)-PI3K-Akt pathway. METHODS: By cultivating fibroblasts derived from scar tissue after cleft palate surgery in humans, aloe emodin of different concentrations (10, 20, 30, 40 and 50 μmol/L) was added to the cells which activity was detected. At the same time, transcriptome sequencing was performed on scar tissue and cells, and bioinformatics methods were used to explore potential targets and signaling pathways of scar tissue fibrosis. RESULTS: Aloe-emodin had a concentration dependent inhibitory effect on fibroblast proliferation,with the 40 μmol/L concentration group showing the most significant effect. The results of tissue and cell sequencing indicated that differentially expressed genes were significantly enriched in extracellular matrix-receptor interaction pathway, and shared a common differential gene which was THBS1. The ORA analysis results indicated that differentially expressed genes, including THBS1, were significantly enriched in the PI3K-Akt signaling pathway. CONCLUSIONS Aloe emodin may inhibit the PI3K-Akt pathway by downregulating THBS1, thereby reducing the proliferation activity of fibroblasts derived from postoperative palatal scar tissue.
Thrombospondin 1/genetics* ; Humans ; Signal Transduction/drug effects* ; Fibroblasts/cytology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Fibrosis ; Phosphatidylinositol 3-Kinases/metabolism* ; Cicatrix/metabolism* ; Cell Proliferation/drug effects* ; Anthraquinones/pharmacology* ; Cells, Cultured

OBJECTIVE: To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. METHODS: BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV). RESULTS: CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic ( P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group ( P<0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group ( P<0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group ( P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant ( P<0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP ( P<0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group ( P<0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group ( P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group ( P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased ( P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group ( P<0.05) except Tb.Th. CONCLUSION Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.
Animals ; Female ; Ovariectomy ; Mice ; Cell Differentiation ; Osteogenesis ; Mice, Inbred C57BL ; Mesenchymal Stem Cells/cytology* ; Cells, Cultured ; Thrombospondins/metabolism* ; Bone Marrow Cells/metabolism* ; Bone Density ; Cell Proliferation ; Intercellular Signaling Peptides and Proteins/metabolism* ; Coculture Techniques

Objective To investigate the clinical significance of thrombospondin type 1 domain-containing 7A (THSD7A) and neural epidermal growth factor-like 1 protein (NELL1) in phospholipase A2 receptor (PLA2R)-negative membranous nephropathy (MN). Methods A total of 116 PLA2R-negative MN patients treated in Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University from 2014 to 2021 were enrolled in this study.Immunohistochemistry was employed to detect THSD7A and NELL1 in the renal tissue.The pathological characteristics,treatment,and prognosis were compared between positive and negative groups. Results The 116 PLA2R-negative MN patients included 23 THSD7A-positive patients and 9 NELL1-positive patients.One patient was tested positive for both proteins.The THSD7A-positive group showed higher positive rate of IgG4 (P=0.010),more obvious glomerular basement membrane (GBM) thickening (P=0.034),and higher proportion of stage Ⅱ MN and lower proportion of stage I MN (P=0.002) than the THSD7A-negative group.The NELL1-positive group had lower positive rates of C1q and IgG2 (P=0.029,P=0.001),less obvious GBM thickening (P<0.001),more extensive inflammatory cell infiltration (P=0.033),lower proportion of deposits on multi-locations (P=0.001),and lower proportion of atypical MN (P=0.010) than the NELL1-negative group.One patient with THSD7A-positive MN was diagnosed with colon cancer,while none of the NELL1-positive patients had malignancy.Survival analysis suggested that THSD7A-positive MN had worse composite remission (either complete remission or partial remission) of nephrotic syndrome than the negative group (P=0.016),whereas NELL1-positive MN exhibited better composite remission of nephrotic syndrome than the negative group (P=0.015).The MN patients only positive for NELL1 showed better composite remission of nephrotic syndrome than the MN patients only positive for THSD7A (P<0.001). Conclusions THSD7A- and NELL1-positive MN is more likely to be primary MN,and there is no significant malignancy indication.However,it might have a predictive value for the prognosis of MN.
Humans ; Autoantibodies ; Clinical Relevance ; Colonic Neoplasms ; EGF Family of Proteins ; Glomerulonephritis, Membranous/diagnosis* ; Nephrotic Syndrome ; Receptors, Phospholipase A2/metabolism* ; Thrombospondins/metabolism*

OBJECTIVE: To explore the changes of Ⅻ antithrombin (FⅫa-AT), thrombospondin-1 (TSP-1), and lupus anticoagulant (LA) ratio in the peripheral blood factor of patients with systemic lupus erythematosus (SLE) and the clinical value of combined diagnosis of thrombotic events. METHODS: A total of 133 SLE patients treated in Xingtai People's Hospital were selected and divided into simple SLE group (105 cases) and SLE complicated with thrombosis group (28 cases) according to whether thrombotic events occurred, and 102 cases of healthy people in the same period were selected as control. The clinical data of the 3 groups, the level of peripheral blood FⅫa-AT, TSP-1, and LA ratio were compared, the relationship between each peripheral blood index and SLE disease activity index (SLEDAI) score were analyzed. The influencing factors of thrombotic events in SLE patients were analyzed, and the value of each peripheral blood index in the diagnosis of SLE complicated with thrombotic events were evaluated. RESULTS: The proportion of the patients with age ≥60 year, hypertension, and smoking history in SLE complicated with thrombosis group was higher than those in simple SLE group and control group (P＜0.05). The SLEDAI score, peripheral blood FⅫa-AT, TSP-1, LA ratio levels of the patients in SLE complicated with thrombosis group were significantly higher than those in simple SLE group and control group, and the simple SLE group was significantly higher than the control group (P＜0.05). FⅫa-AT, TSP-1, LA ratio in peripheral blood of SLE patients were positively correlated with SLEDAI score (r=0.663, 0.578 and 0.625). Age, blood pressure, smoking history, peripheral blood FⅫa-AT, TSP-1, LA ratio were the important influencing factors of thrombotic events in SLE patients (P＜0.05). The AUC diagnosed by the FⅫa-AT, TSP-1, and LA ratio in peripheral blood was 0.881, the 95% CI was 0.813-0.931, the sensitivity was 82.14%, and the specificity was 91.43%, which was superior to each index alone (P＜0.05). CONCLUSION Peripheral blood FⅫa-AT, TSP-1, LA ratio level changes in SLE patients are significantly related to disease activity, and the combined diagnosis of thrombotic events is more reliable.
Humans ; Lupus Erythematosus, Systemic/complications* ; Risk Factors ; Thrombosis/etiology* ; Thrombospondin 1

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.
Animals ; Apoptosis ; CD36 Antigens/metabolism* ; Caspase 3/metabolism* ; Cell Line ; Humans ; Leukemia, Megakaryoblastic, Acute ; Mice ; Thrombospondin 1/pharmacology*

OBJECTIVES: To evaluate the value of thrombospond in Type I domain-containing 7A (THSD7A) and M-type phospholipase A2 receptor (PLA2R) in primary membranous nephropathy (PMN) and to explore the relationship between their antibody levels and prognosis. METHODS: Renal tissues in 128 patients with membranous nephropathy in the Second Xiangya Hospital of Central South University were collected from February 2015 to August 2017, including 108 patients with primary membranous nephropathy (PMN group) and 20 patients with secondary membranous nephropathy (SMN) (SMN group). Indirect immunofluorescence method was used to detect the expression of PLA2R antigen in kidney tissues,and the glomerular expression of THSD7A antigen was examined by immunohistochemistry and indirect immunofluorescence. The serum levels of anti-PLA2R antibodies and THSD7A antibodies were also detected by ELISA. According to the results of PMN examination,the patients were also divided into a PLA2R-related membranous nephropathy group and a THSD7A-related membranous nephropathy group. RESULTS: The positive rate of PLA2R in the renal tissues in the PMN group was higher than that in the SMN group (78% in the PMN group, 35% in the SMN group, <0.01),while the positive rate of anti-PLA2R antibody in the PMN group was also higher than that in the SMN group (50% in the PMN group, 25% in the SMN group, <0.05).The serum level of anti-PLA2R antibody was positively correlated with 24 h urine protein (=0.254, <0.05) and negatively correlated with serum albumin (=-0.236, <0.05). The expression of THSD7A was positive in glomeruli in 7 cases of the PMN group (6%) by immuno-histochemistry, and which was positive in 1case of the SMN group (5%).The serum levels of anti-THSD7A antibody in the PMN group were higher than those in the SMN group [(0.49±0.26) pg/mL in the PMN group,(0.34±0.27) pg/mL in the SMN group, <0.05]. There was no difference in the clinical characteristics between the PLA2R-related membranous nephropathy group and the THSD7A-related membranous nephropathy group. CONCLUSIONS PLA2R and THSD7A are the target antigen of PMN, and the associated autoantibodies are helpful for the differential diagnosis of PMN. The anti-PLA2R antibody levels can reflect the severity of the disease and evaluate the effect of treatment. The incidence of THSD7A membranous nephropathy is low, and monitoring the serum anti-THSD7A antibody levels can assess patients' condition and predict disease outcome.
Autoantibodies ; Glomerulonephritis, Membranous ; Humans ; Immunohistochemistry ; Receptors, Phospholipase A2 ; Thrombospondins

Diagnosis of thrombotic microangiopathy (TMA) is challenging due to its close association with other forms of microangiopathic hemolytic anemia, such as malignant hypertension and disseminated intravascular coagulation, and because other manifestations including cytopenia and acute kidney injury are manifestations of other medical comorbidities. Further challenges for accurate diagnosis include distinguishing between primary and secondary TMA, as well as between hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). TTP is typically differentiated from HUS by the presence of more severe thrombocytopenia, along with a higher frequency of altered mental status with relatively preserved renal function. However, the clinical course can vary among patients, requiring polymerase chain reaction testing of patient stools for enterohemorrhagic Escherichia coli and a disintegrin and metalloproteinase with thrombospondin type 1 motif 13 (ADAMTS13) assay. To reduce the mortality rate, prompt initiation of plasmapheresis is important in cases where TPP cannot be excluded. Future advances enabling more rapid testing for ADAMTS13 levels will reduce the need for unnecessary plasmapheresis, so that treatment strategy can be more optimized.
Acute Kidney Injury ; Anemia, Hemolytic ; Comorbidity ; Diagnosis ; Diagnosis, Differential ; Disseminated Intravascular Coagulation ; Enterohemorrhagic Escherichia coli ; Hemolytic-Uremic Syndrome ; Humans ; Hypertension, Malignant ; Mortality ; Plasma Exchange ; Plasmapheresis ; Polymerase Chain Reaction ; Purpura, Thrombotic Thrombocytopenic ; Thrombocytopenia ; Thrombospondins ; Thrombotic Microangiopathies

PURPOSE: Accumulating evidence suggests that microRNA-145 (miR-145) plays an important role in osteoarthritis (OA), which is a chronic progressive joint disease. Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes metastasis in cancers and functions as a sponge for miR-145. However, the role of MALAT1/miR-145 in OA pathogenesis has not yet been elucidated. MATERIALS AND METHODS: The expression of MALAT1 and miR-145 was examined by quantitative real-time PCR; the interaction between miR-145, MALAT1 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was verified by luciferase reporter assay. Correlations among MALAT1, miR-145, and ADAMTS5 were analyzed by Spearman rank analysis. Chondrocytes viability and cartilage extracellular matrix (ECM) degradation were investigated with cell viability assay and Western blotting analyzing expression of ADAMTS5, collagen type 2 alpha 1 (COL2A1), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP). RESULTS: MALAT1 was upregulated, and miR-145 was downregulated in OA samples and IL-1β-induced chondrocytes. Mechanically, miR-145 could directly bind to MALAT1 and ADAMTS5. Moreover, miR-145 expression was negatively correlated with MALAT1 and ADAMTS5 expression in OA patients, whereas MALAT1 and ADAMTS5 expression was positively correlated. Functionally, overexpression of MALAT1 inhibited chondrocyte viability and promoted cartilage ECM degradation in IL-1β-induced chondrocytes. In support thereof, MALAT1 silencing and miR-145 upregulation exerted the opposite effect in IL-1β-induced chondrocytes. Moreover, the effect of MALAT1 was counteracted by miR-145 upregulation, and ADAMTS5 restoration could abate miR-145 effects. CONCLUSION: An MALAT1/miR-145 axis contributes to ECM degradation in IL-1β-induced chondrocytes through targeting ADAMTS5, suggesting that MALAT1/miR-145/ADAMTS5 signaling may underlie human OA pathogenesis.
Adenocarcinoma ; Aggrecans ; Blotting, Western ; Cartilage Oligomeric Matrix Protein ; Cartilage ; Cell Survival ; Chondrocytes ; Collagen ; Extracellular Matrix ; Humans ; Joint Diseases ; Luciferases ; Lung ; Neoplasm Metastasis ; Osteoarthritis ; Porifera ; Real-Time Polymerase Chain Reaction ; RNA, Long Noncoding ; Temefos ; Thrombospondins ; Up-Regulation

PURPOSE: Polycystic ovary syndrome (PCOS) is a gynecological endocrine disorder that is characterized by disturbances in ovarian blood flow and angiogenesis. The aim of this study was to determine the association of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) serum levels with the body mass index (BMI) in patients with PCOS compared with healthy subjects. METHODS: The study was conducted with 80 subjects in 3 PCOS groups, including normal weight, overweight, and obese PCOS groups, and a control group of healthy subjects (n=20). The participants in all groups completed a questionnaire comprising sociodemographic and obstetric questions. The PCOS diagnosis in the study subjects was confirmed based on the Rotterdam criteria, BMI was determined according to the World Health Organization guidelines, and the lipid accumulation product index was calculated for all groups. Venous blood samples were collected from all participants after fasting to measure the serum levels of fasting blood glucose (FBG), lipids, insulin, VEGF, TSP-1, and leptin. RESULTS: Our findings showed that the serum VEGF level was significantly higher in the normal BMI PCOS group than that in the control group (P=0.03), and the TSP-1 level was significantly lower in the obese PCOS group than that in the control group (P=0.04). CONCLUSIONS: Our study demonstrated that alterations in VEGF and TSP-1 concentrations are dependent on BMI. Because abnormal ovarian angiogenesis is considered to be the main feature of PCOS, the study of ovarian angiogenic imbalance is proposed as a new tool for PCOS diagnosis and management.
Blood Glucose ; Body Mass Index ; Case-Control Studies ; Diagnosis ; Fasting ; Healthy Volunteers ; Humans ; Insulin ; Leptin ; Lipid Accumulation Product ; Overweight ; Polycystic Ovary Syndrome ; Thrombospondin 1 ; Vascular Endothelial Growth Factor A ; World Health Organization