ABSTRACT
BACKGROUND: Ovarian cancer (OC) remains one of the most challenging and deadly malignancies facing women today. While PARP inhibitors (PARPis) have transformed the treatment landscape for women with advanced OC, many patients will relapse and the PARPi-resistant setting is an area of unmet medical need. Traditional immunotherapies targeting PD-1/PD-L1 have failed to show any benefit in OC. The CD47/TSP-1 axis may be relevant in OC. We aimed to describe changes in CD47 expression with platinum therapy and their relationship with immune features and prognosis. METHODS: Tumor and blood samples collected from OC patients in the CHIVA trial were assessed for CD47 and TSP-1 before and after neoadjuvant chemotherapy (NACT) and multiplex analysis was used to investigate immune markers. Considering the therapeutic relevance of targeting the CD47/TSP-1 axis, we used the CD47-derived TAX2 peptide to selectively antagonize it in a preclinical model of aggressive ovarian carcinoma. RESULTS: Significant reductions in CD47 expression were observed post NACT. Tumor patients having the highest CD47 expression profile at baseline showed the greatest CD4+ and CD8+ T-cell influx post NACT and displayed a better prognosis. In addition, TSP-1 plasma levels decreased significantly under NACT, and high TSP-1 was associated with a worse prognosis. We demonstrated that TAX2 exhibited a selective and favorable biodistribution profile in mice, localizing at the tumor sites. Using a relevant peritoneal carcinomatosis model displaying PARPi resistance, we demonstrated that post-olaparib (post-PARPi) administration of TAX2 significantly reduced tumor burden and prolonged survival. Remarkably, TAX2 used sequentially was also able to increase animal survival even under treatment conditions allowing olaparib efficacy. CONCLUSIONS: Our study thus (1) proposes a CD47-based stratification of patients who may be most likely to benefit from postoperative immunotherapy, and (2) suggests that TAX2 is a potential alternative therapy for patients relapsing on PARP inhibitors.
Subject(s)
Biomarkers, Tumor , CD47 Antigen , Ovarian Neoplasms , Thrombospondin 1 , CD47 Antigen/metabolism , Female , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Biomarkers, Tumor/metabolism , Animals , Mice , Thrombospondin 1/metabolism , Prognosis , Cell Line, Tumor , Neoadjuvant Therapy , Xenograft Model Antitumor Assays , Middle Aged , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
Background: Inhibitor of NF-κB kinase-interacting protein (IKIP) is known to promote proliferation of glioblastoma (GBM) cells, but how it affects migration and invasion by those cells is unclear. Methods: We compared levels of IKIP between glioma tissues and normal brain tissue in clinical samples and public databases. We examined the effects of IKIP overexpression and knockdown on the migration and invasion of GBM using transwell and wound healing assays, and we compared the transcriptomes under these different conditions to identify the molecular mechanisms involved. Results: Based on data from our clinical samples and from public databases, IKIP was overexpressed in GBM tumors, and its expression level correlated inversely with survival. IKIP overexpression in GBM cells inhibited migration and invasion in transwell and wound healing assays, whereas IKIP knockdown exerted the opposite effects. IKIP overexpression in GBM cells that were injected into mouse brain promoted tumor growth but inhibited tumor invasion of surrounding tissue. The effects of IKIP were associated with downregulation of THBS1 mRNA and concomitant inhibition of THBS1/FAK signaling. Conclusions: IKIP inhibits THBS1/FAK signaling to suppress migration and invasion of GBM cells.
Subject(s)
Brain Neoplasms , Cell Movement , Focal Adhesion Kinase 1 , Glioblastoma , Neoplasm Invasiveness , Signal Transduction , Thrombospondin 1 , Humans , Glioblastoma/pathology , Glioblastoma/metabolism , Glioblastoma/genetics , Animals , Mice , Brain Neoplasms/pathology , Brain Neoplasms/metabolism , Brain Neoplasms/genetics , Cell Line, Tumor , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Focal Adhesion Kinase 1/metabolism , Focal Adhesion Kinase 1/genetics , Down-Regulation , Gene Expression Regulation, Neoplastic , Cell ProliferationABSTRACT
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.
Subject(s)
CD47 Antigen , Erythropoiesis , Spleen , Thrombospondin 1 , Animals , CD47 Antigen/metabolism , CD47 Antigen/genetics , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Spleen/metabolism , Mice , Mice, Knockout , Gene Expression Regulation , Mice, Inbred C57BL , Erythroid Precursor Cells/metabolismABSTRACT
Atherosclerosis is rare in internal thoracic arteries (ITA) even in patients with severe atherosclerotic coronary artery (ACA) disease. To explore cellular differences, ITA SMC from 3 distinct donors and ACA SMC from 3 distinct donors were grown to sub-confluence and growth arrested for 48 h. Proliferation and thrombospondin-1 (TSP1) production were determined using standard techniques. ITA SMC were larger, grew more slowly and survived more passages than ACA SMC. ACA SMC had a more pronounced proliferative response to 10% serum than ITA SMC. Both ACA SMC and ITA SMC proliferated in response to exogenous TSP1 (12.5 µg/ml and 25 µg/ml) and platelet derived growth factor-BB (PDGF-BB; 20 ng/ml) but TSP1- and PDGF-BB-induced proliferation were partially inhibited by anti-TSP1 antibody A4.1, microRNA-21(miR-21)-3p inhibitors and miR-21-5p inhibitors in each of the 3 ACA SMC lines, but not in any of the ITA SMC lines. PDGF-BB stimulated TSP1 production in ACA SMC but not in ITA SMC but there was no increase in TSP1 levels in conditioned media in either SMC type. In summary, there are significant differences in morphology, proliferative capacity and in responses to TSP1 and PDGF-BB in SMC derived from ITA compared to SMC derived from ACA.
Subject(s)
Becaplermin , Cell Proliferation , Coronary Vessels , Myocytes, Smooth Muscle , Thrombospondin 1 , Becaplermin/metabolism , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Humans , Cell Proliferation/drug effects , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/drug effects , Coronary Vessels/metabolism , Coronary Vessels/pathology , Coronary Vessels/drug effects , MicroRNAs/genetics , MicroRNAs/metabolism , Mammary Arteries/metabolism , Mammary Arteries/drug effects , Coronary Artery Disease/metabolism , Coronary Artery Disease/pathology , Atherosclerosis/metabolism , Atherosclerosis/pathology , Cells, Cultured , MaleABSTRACT
BACKGROUND: Myocardial infarction (MI) is a major cause of mortality and morbidity worldwide. The intercellular communication in post-infarction angiogenesis remains unclear. METHODS: In this study, we explored the role and mechanism of action of M2 macrophage-derived exosomes (M2-exos) in angiogenesis after MI. M2-exos were harvested and injected intramyocardially at the onset of MI. Two distinct endothelial cells (ECs) were cultured with M2-exos to explore the direct effects on angiogenesis. RESULTS: We showed that M2-exos improved cardiac function, reduced infarct size, and enhanced angiogenesis after MI. Moreover, M2-exos promoted angiogenesis in vitro; the molecules loaded in the vesicles were responsible for its proangiogenic effects. We further validated that higher abundance of miR-132-3p in M2-exos, which recapitulate their functions, was required for the cardioprotective effects exerted by M2-exos. Mechanistically, miR-132-3p carried by M2-exos down-regulate the expression of THBS1 through direct binding to its 3´UTR and the proangiogenic effects of miR-132-3p were largely reversed by THBS1 overexpression. CONCLUSION: Our findings demonstrate that M2-exos promote angiogenesis after MI by transporting miR-132-3p to ECs, and by binding to THBS1 mRNA directly and negatively regulating its expression. These findings highlight the role of M2-exos in cardiac repair and provide novel mechanistic understanding of intercellular communication in post-infarction angiogenesis.
Subject(s)
Exosomes , Macrophages , MicroRNAs , Myocardial Infarction , Neovascularization, Physiologic , Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Myocardial Infarction/therapy , Myocardial Infarction/genetics , Exosomes/metabolism , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Macrophages/metabolism , Mice , Male , Humans , Endothelial Cells/metabolism , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Mice, Inbred C57BL , AngiogenesisABSTRACT
Microplastics (MPs) pose a significant threat to livestock health. Yet, the roles of polystyrene MPs (PS-MPs) on meat quality and skeletal muscle development in pigs have not been fully determined. To investigate the effect of PS-MPs on skeletal muscle, piglets were given diets supplementation with 0 mg/kg (CON group), 75 mg/kg (75 mg/kg PS-MPs group), and 150 mg/kg PS-MPs (150 mg/kg PS-MPs group), respectively. The results indicated that the average daily gain (ADG) of piglets in the 150 mg/kg PS-MPs group was significantly lower than that in the CON group. No significant differences were observed in the final body weight and ADG between the CON group and the 75 mg/kg PS-MPs group. Piglets in the 150 mg/kg PS-MPs group exhibited decreased meat redness index and type I muscle fiber density. Metabolomic analysis revealed that the contents of meat flavor compounds carnosine, beta-alanine, palmitic acid, and niacinamide in muscle were lower in the 150 mg/kg PS-MPs group than in the CON group. Additionally, piglets subjected to 150 mg/kg PS-MPs exhibited impaired muscle angiogenesis. Further analysis indicated that PS-MPs exposure up-regulated thrombospondin 1 (THBS1) expression by inhibiting THBS1 mRNA and protein degradation, thereby disrupting skeletal muscle angiogenesis. These findings indicate that PS-MPs exposure adversely affects meat quality and hinders skeletal muscle angiogenesis in pigs, providing deeper insights into the detrimental effects of PS-MPs on meat quality and skeletal muscle development.
Subject(s)
Microplastics , Muscle, Skeletal , Polystyrenes , Pork Meat , Thrombospondin 1 , Animals , Male , Angiogenesis , Animal Feed , Carnosine/pharmacology , Food Contamination/analysis , Food Quality , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Neovascularization, Physiologic/drug effects , Swine , Thrombospondin 1/metabolism , Pork Meat/analysisABSTRACT
Osimertinib induces a marked response in non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) gene mutations. However, acquired resistance to osimertinib remains an inevitable problem. In this study, we aimed to investigate osimertinib-resistant mechanisms and evaluate the combination therapy of afatinib and chemotherapy. We established osimertinib-resistant cell lines (PC-9-OR and H1975-OR) from EGFR-mutant lung adenocarcinoma cell lines PC-9 and H1975 by high exposure and stepwise method. Combination therapy of afatinib plus carboplatin (CBDCA) and pemetrexed (PEM) was effective in both parental and osimertinib-resistant cells. We found that expression of thrombospondin-1 (TSP-1) was upregulated in resistant cells using cDNA microarray analysis. We demonstrated that TSP-1 increases the expression of matrix metalloproteinases through integrin signaling and promotes tumor invasion in both PC-9-OR and H1975-OR, and that epithelial-to-mesenchymal transition (EMT) was involved in H1975-OR. Afatinib plus CBDCA and PEM reversed TSP-1-induced invasion ability and EMT changes in resistant cells. In PC-9-OR xenograft mouse models (five female Balb/c-Nude mice in each group), combination therapy strongly inhibited tumor growth compared with afatinib monotherapy (5 mg/kg, orally, five times per week) or CBDCA (75 mg/kg, intraperitoneally, one time per week) + PEM (100 mg/kg, intraperitoneally, one time per week) over a 28-day period. These results suggest that the combination of afatinib plus CBDCA and PEM, which effectively suppresses TSP-1 expression, may be a promising option in EGFR-mutated NSCLC patients after the acquisition of osimertinib resistance.
Subject(s)
Acrylamides , Afatinib , Aniline Compounds , Antineoplastic Combined Chemotherapy Protocols , Carcinoma, Non-Small-Cell Lung , Drug Resistance, Neoplasm , ErbB Receptors , Lung Neoplasms , Mutation , Thrombospondin 1 , Humans , Thrombospondin 1/genetics , Thrombospondin 1/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , Drug Resistance, Neoplasm/genetics , Animals , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Afatinib/pharmacology , Afatinib/therapeutic use , ErbB Receptors/genetics , Mice , Cell Line, Tumor , Acrylamides/pharmacology , Acrylamides/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aniline Compounds/pharmacology , Aniline Compounds/therapeutic use , Xenograft Model Antitumor Assays , Carboplatin/pharmacology , Carboplatin/therapeutic use , Female , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Mice, Nude , Gene Expression Regulation, Neoplastic/drug effects , Mice, Inbred BALB C , Indoles , PyrimidinesSubject(s)
Glioblastoma , Thrombospondin 1 , Humans , Glioblastoma/metabolism , Glioblastoma/genetics , Glioblastoma/pathology , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/therapeutic use , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/genetics , AnimalsABSTRACT
BACKGROUND AND STUDY AIMS: Immunotherapy has emerged as a hot topic in cancer treatment in recent years and has also shown potential in the treatment of Helicobacter pylori-associated gastric cancer. However, there is still a need to identify potential immunotherapy targets. MATERIAL AND METHODS: We used the GSE116312 dataset of Helicobacter pylori-associated gastric cancer to identify differentially expressed genes, which were then overlapped with immune genes from the ImmPort database. The identified immune genes were used to classify gastric cancer samples and evaluate the relationship between classification and tumor mutations, as well as immune infiltration. An immune gene-based prognostic model was constructed, and the expression levels of the genes involved in constructing the model were explored in the tumor immune microenvironment. RESULTS: We successfully identified 60 immune genes and classified gastric cancer samples into two subtypes, which showed differences in prognosis, tumor mutations, immune checkpoint expression, and immune cell infiltration. Subsequently, we constructed an immune prognostic model consisting of THBS1 and PDGFD, which showed significant associations with macrophages and fibroblasts. CONCLUSION: We identified abnormal expression of THBS1 and PDGFD in cancer-associated fibroblasts (CAFs) within the tumor immune microenvironment, suggesting their potential as therapeutic targets.
Subject(s)
Helicobacter Infections , Helicobacter pylori , Platelet-Derived Growth Factor , Stomach Neoplasms , Thrombospondin 1 , Tumor Microenvironment , Stomach Neoplasms/microbiology , Stomach Neoplasms/immunology , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Humans , Tumor Microenvironment/immunology , Tumor Microenvironment/genetics , Helicobacter pylori/immunology , Helicobacter pylori/genetics , Helicobacter Infections/immunology , Helicobacter Infections/complications , Thrombospondin 1/genetics , Prognosis , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Cancer-Associated Fibroblasts/immunology , Cancer-Associated Fibroblasts/metabolism , Mutation , LymphokinesABSTRACT
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.
Subject(s)
Hypertension, Pulmonary , Macrophages , Animals , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/parasitology , Hypertension, Pulmonary/immunology , Hypertension, Pulmonary/pathology , Mice , Macrophages/immunology , Macrophages/parasitology , Phenotype , Schistosoma mansoni/immunology , Mice, Inbred C57BL , Schistosomiasis/immunology , Schistosomiasis/complications , Schistosomiasis/parasitology , Disease Models, Animal , Schistosomiasis mansoni/immunology , Schistosomiasis mansoni/parasitology , Schistosomiasis mansoni/complications , Schistosomiasis mansoni/pathology , Thrombospondin 1/genetics , Thrombospondin 1/metabolism , Monocytes/immunology , Receptors, CCR2/genetics , Receptors, CCR2/metabolism , Female , Schistosoma/immunology , Schistosoma/physiology , Lung/immunology , Lung/parasitology , Lung/pathologyABSTRACT
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.
Subject(s)
Abortion, Habitual , Decidua , Endometrium , Stromal Cells , Thrombospondin 1 , Female , Thrombospondin 1/genetics , Thrombospondin 1/metabolism , Humans , Mice , Abortion, Habitual/genetics , Abortion, Habitual/metabolism , Animals , Pregnancy , Decidua/metabolism , Endometrium/metabolism , Endometrium/pathology , Stromal Cells/metabolism , AdultABSTRACT
Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However, the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection, skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors, ligands, ion channels and metabolites to generate injury-specific neuroimmune interactomes, we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts.
Subject(s)
Nociceptors , Pain , Animals , Mice , Pain/immunology , Pain/metabolism , Nociceptors/metabolism , Transcriptome , Mice, Inbred C57BL , Inflammation/immunology , Male , Macrophages/immunology , Macrophages/metabolism , Disease Models, Animal , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Skin/immunology , Skin/metabolism , Skin/pathology , Zymosan , Single-Cell Analysis , Neuroimmunomodulation , Gene Expression Profiling , Neutrophils/immunology , Neutrophils/metabolismABSTRACT
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.
Subject(s)
CD47 Antigen , Heart Transplantation , Mice, Inbred C57BL , Animals , CD47 Antigen/antagonists & inhibitors , CD47 Antigen/metabolism , CD47 Antigen/immunology , Mice , Male , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Organ Preservation/methods , Myocardial Reperfusion Injury/drug therapy , Myocardial Reperfusion Injury/immunology , Myocardial Reperfusion Injury/metabolism , Thrombospondin 1/metabolism , Oxidative Stress/drug effects , Disease Models, Animal , Apoptosis/drug effectsABSTRACT
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.
Subject(s)
Inflammation , Subtilisins , Thrombospondin 1 , Animals , Male , Mice , Autophagy/drug effects , Endothelial Cells/metabolism , Endothelial Cells/drug effects , Gene Expression Regulation/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Inflammasomes/metabolism , Inflammation/pathology , Inflammation/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Subtilisins/metabolism , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Mice, Inbred C57BLSubject(s)
Receptor, IGF Type 1 , Thrombospondin 1 , Humans , Thrombospondin 1/genetics , Thrombospondin 1/metabolism , Receptor, IGF Type 1/metabolism , Receptor, IGF Type 1/genetics , Male , Female , Oncogene Proteins, Fusion/genetics , Soft Tissue Neoplasms/pathology , Soft Tissue Neoplasms/geneticsABSTRACT
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.
Subject(s)
MAP Kinase Kinase Kinases , Necroptosis , Nedd4 Ubiquitin Protein Ligases , Pre-Eclampsia , Thrombospondin 1 , Trophoblasts , Ubiquitination , Humans , Pre-Eclampsia/metabolism , Pre-Eclampsia/genetics , Female , Pregnancy , Trophoblasts/metabolism , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Kinase Kinases/genetics , Necroptosis/genetics , Nedd4 Ubiquitin Protein Ligases/metabolism , Nedd4 Ubiquitin Protein Ligases/genetics , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Adult , Placenta/metabolismABSTRACT
Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor ß (TGFß)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFß-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFß-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFß-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.
Subject(s)
Activating Transcription Factor 4 , Muscle, Skeletal , Muscular Atrophy , Signal Transduction , Smad2 Protein , Smad3 Protein , Thrombospondin 1 , Transforming Growth Factor beta , Animals , Male , Mice , Activating Transcription Factor 4/metabolism , Autophagy , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Thrombospondin 1/metabolism , Thrombospondin 1/genetics , Transforming Growth Factor beta/metabolismABSTRACT
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.
Subject(s)
AMP-Activated Protein Kinases , Anthraquinones , Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Human Umbilical Vein Endothelial Cells , Nitric Oxide Synthase Type III , Signal Transduction , Thrombospondin 1 , Animals , Humans , Anthraquinones/pharmacology , Signal Transduction/drug effects , AMP-Activated Protein Kinases/metabolism , Thrombospondin 1/metabolism , Nitric Oxide Synthase Type III/metabolism , Male , Rats , Mice , Rats, Sprague-Dawley , Endothelium, Vascular/drug effects , Glucose/metabolism , Mice, Inbred C57BLABSTRACT
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.