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Journal of Extracellular Vesicles Apr 2024Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we...
Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we found that calcified apoptotic vesicles (apoVs) led to increased calcification and stiffness of tendon extracellular matrix (ECM), which initiated M2 macrophage polarization and HO progression. Specifically, single-cell transcriptome analyses of different stages of HO revealed that calcified apoVs were primarily secreted by a PROCR fibroblast population. In addition, calcified apoVs enriched calcium by annexin channels, absorbed to collagen I via electrostatic interaction, and aggregated to produce calcifying nodules in the ECM, leading to tendon calcification and stiffening. More importantly, apoV-releasing inhibition or macrophage deletion both successfully reversed HO development. Thus, we are the first to identify calcified apoVs from PROCR fibroblasts as the initiating factor of HO, and might serve as the therapeutic target for inhibiting pathological calcification.
Topics: Humans; Endothelial Protein C Receptor; Extracellular Vesicles; Ossification, Heterotopic; Extracellular Matrix; Fibroblasts
PubMed: 38594791
DOI: 10.1002/jev2.12425 -
Scientific Reports Aug 2023The damaging effects of sleep deprivation (SD) on brain parenchyma have been extensively studied. However, the specific influence of SD on brain pericytes, a primary...
The damaging effects of sleep deprivation (SD) on brain parenchyma have been extensively studied. However, the specific influence of SD on brain pericytes, a primary component of the blood-brain barrier (BBB) and the neurovascular unit (NVU), is still unclear. The present study examined how acute or repeated SD impairs brain pericytes by measuring the cerebrospinal fluid (CSF) levels of soluble platelet-derived growth factor receptor beta (sPDGFRβ) and quantifying pericyte density in the cortex, hippocampus, and subcortical area of the PDGFRβ-P2A-CreER/tdTomato mice, which predominantly express the reporter tdTomato in vascular pericytes. Our results showed that a one-time 4 h SD did not significantly change the CSF sPDGFRβ level. In contrast, repeated SD (4 h/day for 10 consecutive days) significantly elevated the CSF sPDGFRβ level, implying explicit pericyte damages due to repeated SD. Furthermore, repeated SD significantly decreased the pericyte densities in the cortex and hippocampus, though the pericyte apoptosis status remained unchanged as measured with Annexin V-affinity assay and active Caspase-3 staining. These results suggest that repeated SD causes brain pericyte damage and loss via non-apoptosis pathways. These changes to pericytes may contribute to SD-induced BBB and NVU dysfunctions. The reversibility of this process implies that sleep improvement may have a protective effect on brain pericytes.
Topics: Animals; Mice; Pericytes; Brain; Sleep Deprivation; Receptor, Platelet-Derived Growth Factor beta; Brain Injuries; Mice, Transgenic
PubMed: 37550395
DOI: 10.1038/s41598-023-40138-0 -
Frontiers in Immunology 2023Exosomes, organelles measuring 30-200nm, are secreted by various cell types. exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein...
Exosomes, organelles measuring 30-200nm, are secreted by various cell types. exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using exosomes prior to infection by the same pathogen. Human macrophages were stimulated with exosomes and then infected with . Higher levels of IL-1β and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with exosomes induced macrophage production of IL-1β, IL-6, IL-10 and TNF. Inhibition of exosome secretion by prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1β. Our results suggest that exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.
Topics: Humans; Leishmania braziliensis; NLR Family, Pyrin Domain-Containing 3 Protein; Exosomes; Interleukin-6; Macrophages; Leishmania donovani
PubMed: 37841240
DOI: 10.3389/fimmu.2023.1256425 -
International Journal of Medical... 2023Eicosapentaenoic acid (EPA) is an omega-3 fatty acid that protects against cardiovascular diseases in patients with hypertriglyceridemia and may have pleotropic effects...
Eicosapentaenoic acid (EPA) is an omega-3 fatty acid that protects against cardiovascular diseases in patients with hypertriglyceridemia and may have pleotropic effects beyond lowering triglycerides. Many degenerative diseases, such as atherosclerosis and diabetes, are related to cellular senescence as a pathophysiological mechanism. We aimed to examine whether EPA could protect vascular endothelial cells under stress conditions against stress-induced accelerated senescence (SIAS). Cultured human umbilical vein endothelial cells (HUVECs) were exposed to HO as oxidative stress and a high glucose concentration with palmitate as a glucolipotoxic condition. Changes in cell viability, apoptosis, lactate dehydrogenase release, and cell cycle analysis were measured by cell counting kit-8 assay, annexin V/ propidium iodide staining, and enzyme-linked immunosorbent assay, respectively. EPA was applied in stress conditions. The degree of senescence was measured by senescence-associated beta-galactosidase staining and p16 staining using immunofluorescence. Apoptosis and cellular senescence-related proteins were measured by Western blotting. Cultured HUVECs under oxidative and glucolipotoxic stresses revealed accelerated senescence and increased apoptosis. These changes were markedly reversed by EPA administration, and the expressions of apoptosis and cellular senescence-related proteins were reversed by EPA treatment. EPA effectively protects HUVECs against SIAS, which may be one of its pleotrophic effects.
Topics: Humans; Eicosapentaenoic Acid; Hydrogen Peroxide; Human Umbilical Vein Endothelial Cells; Oxidative Stress; Cellular Senescence; Apoptosis; Cells, Cultured
PubMed: 37790848
DOI: 10.7150/ijms.85224 -
Biology Direct Nov 2023Pancreatic cancer is a malignancy with high mortality. Once diagnosed, effective treatment strategies are limited and the five-year survival is extremely poor. Recent...
Overexpression of ZNF488 supports pancreatic cancer cell proliferation and tumorigenesis through inhibition of ferroptosis via regulating SCD1-mediated unsaturated fatty acid metabolism.
BACKGROUND
Pancreatic cancer is a malignancy with high mortality. Once diagnosed, effective treatment strategies are limited and the five-year survival is extremely poor. Recent studies have shown that zinc finger proteins play important roles in tumorigenesis, including pancreatic cancer. However, it remains unknown on the clinical significance, function and underlying mechanisms of zinc finger protein 488 (ZNF488) during the development of pancreatic cancer.
METHODS
The clinical relevance of ZNF488 and stearoyl-CoA desaturase 1 (SCD1) was examined by analyzing the data from The Cancer Genome Atlas (TCGA) and immunohistochemical staining of the tissue microarray. Gain-of-function and loss-of-function experiments were performed by transfecting the cells with overexpressing lentivirus and siRNAs or shRNA lentivirus, respectively. The function of ZNF488 in pancreatic cancer was assessed by CCK8, colony formation, EdU staining, PI/Annexin V staining and xenografted tumorigenesis. Chip-qPCR assay was conducted to examine the interaction between ZNF488 and the promoter sequence of SCD1. Transcription activity was measured by dual luciferase reporter assay. mRNA and protein expression was detected by qRT-PCR and immunoblotting experiment, respectively. Fatty acid was quantified by gas chromatography mass spectrometry.
RESULTS
ZNF488 was overexpressed in pancreatic cancer samples compared with normal tissues. High expression of ZNF488 predicted the poor prognosis of the patients. In vitro, ZNF488 upregulation contributed to the EuU cooperation, proliferation and colony formation of MIAPaCa-2 and PANC-1 cells. Based on PI/Annexin V and trypan blue staining results, we showed that ZNF488 suppressed the ferroptosis and apoptosis of pancreatic cancer cells. Mechanistically, ZNF488 directly interacted with the promoter sequence of SCD1 gene and promoted its transcription activity, which resulted in enhanced palmitoleic and oleic acid production, as well as the peroxidation of fatty acid. In vivo, ZNF488 overexpression promoted the xenograted tumorigenesis of PANC-1, which was reversed by SCD1 knockdown. Importantly, combination of erastin and SCD1 inhibitors A939572 completely blunted the growth of ZNF488 overexpressed MIAPaCa-2 and PANC-1 cells. Usage of A939572 or erastin recovered the sensitivity of pancreatic cancer cells to the treatment of gemcitabine. Lastly, we found a positive correlation between ZNF488 and SCD1 in pancreatic cancer patients based on TCGA and immunohistochemical staining results.
CONCLUSION
Overexpression of ZNF488 suppresses the ferroptosis and apoptosis to support the growth and tumorigenesis of pancreatic cancer through augmentation of SCD1-mediated unsaturated fatty acid metabolism. Combination of SCD1 inhibitors, ferroptosis inducers or gemcitabine could be applied for the treatment of pancreatic cancer with overexpression of ZNF488.
Topics: Humans; Ferroptosis; Cell Line, Tumor; Annexin A5; Carcinogenesis; Pancreatic Neoplasms; Cell Proliferation; Fatty Acids; Gemcitabine; Fatty Acids, Unsaturated; Stearoyl-CoA Desaturase
PubMed: 37986084
DOI: 10.1186/s13062-023-00421-6 -
Cancers Sep 2023(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. The lack of validated disease biomarkers makes timely diagnosis...
(1) Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. The lack of validated disease biomarkers makes timely diagnosis challenging in most cases. Cell membrane and surface proteins play a crucial role in several routes of oncogenesis. The aim of this study was to evaluate the expression of six membrane antigens on PDAC (CA 19-9, mucin 1 and 4 (MUC1, MUC4), mesothelin (MSLN), Annexin A10 (ANXA10), Glypican-1 (GPC-1)) and their correlation with oncologic outcomes. (2) Methods: Immunohistochemical staining for CA 19.9, MUC1, MUC4, MSLN, ANXA10, and GPC-1 of surgical samples of 50 consecutive patients with PDAC was performed. Antigen expression for tumor, ductal, and acinar tissues was classified according to the histo-score (H-score) by two pathologists. (3) Results: Recurrence rate was 47% and 18 patients (36%) deceased (median follow-up 21.5 months). Immunostaining for CA 19-9 and MUC1 showed a significantly higher expression in the neoplastic tissue compared to non-tumor ductal and acinar tissues ( < 0.001). MUC4, MSLN, ANXA10, and GPC-1 were selectively expressed in the neoplastic tissue ( < 0.001). A CA 19-9 H-score value >270 was independently associated with a worse overall survival ( = 0.05) and disease-free survival ( = 0.05). (4) Conclusions: CA 19-9 and MUC1 are highly expressed in PDAC cells. The histological expression of CA 19-9 may predict prognosis. MUC4, MSLN, ANXA10, and GPC-1 are selectively expressed by neoplastic tissue and may represent a potential histological biomarker of disease.
PubMed: 37760554
DOI: 10.3390/cancers15184586 -
Developmental Cell Oct 2023Elucidating the mechanism(s) modulating appropriate tissue size is a critical biological issue. Pancreatic β cells increase during pregnancy via cellular proliferation,...
Elucidating the mechanism(s) modulating appropriate tissue size is a critical biological issue. Pancreatic β cells increase during pregnancy via cellular proliferation, but how β cells promptly decrease to the original amount after parturition remains unclear. Herein, we demonstrate the role and mechanism of macrophage accumulation in this process. In the final stage of pregnancy, HTR1D signaling upregulates murine β cell CXCL10, thereby promoting macrophage accumulation in pancreatic islets via the CXCL10-CXCR3 axis. Blocking this mechanism by administering an HTR1D antagonist or the CXCR3 antibody and depleting islet macrophages inhibited postpartum β cell mass reduction. β cells engulfed by macrophages increased in postpartum islets, but Annexin V administration suppressed this engulfment and the postpartum β cell mass reduction, indicating the accumulated macrophages to phagocytose β cells. This mechanism contributes to both maintenance of appropriate β cell mass and glucose homeostasis promptly adapting to reduced systemic insulin demand after parturition.
Topics: Pregnancy; Female; Mice; Animals; Insulin-Secreting Cells; Parturition; Islets of Langerhans; Insulin; Macrophages; Phagocytosis
PubMed: 37716356
DOI: 10.1016/j.devcel.2023.08.002 -
Renal Failure Dec 2023This study investigated the mechanism of action of ABT-263 in the treatment of neurogenic bladder fibrosis (NBF)and its protective effects against upper urinary tract...
This study investigated the mechanism of action of ABT-263 in the treatment of neurogenic bladder fibrosis (NBF)and its protective effects against upper urinary tract damage (UUTD). Sixty 12-week-old Sprague-Dawley (SD) rats were randomly divided into sham, sham + ABT-263 (50 mg/kg), NBF, NBF + ABT-263 (25 mg/kg, oral gavage), and NBF + ABT-263 (50 mg/kg, oral gavage) groups. After cystometry, bladder and kidney tissue samples were collected for hematoxylin and eosin (HE), Masson, and Sirius red staining, and Western Blotting (WB) and qPCR detection. Primary rat bladder fibroblasts were isolated, extracted, and cultured. After co-stimulation with TGF-β1 (10 ng/mL) and ABT-263 (concentrations of 0, 0.1, 1, 10, and 100 µmol/L) for 24 h, cells were collected. Cell apoptosis was detected using CCK8, WB, immunofluorescence, and annexin/PI assays. Compared with the sham group, there was no significant difference in any physical parameters in the sham + ABT-263 (50 mg/kg) group. Compared with the NBF group, most of the markers involved in fibrosis were improved in the NBF + ABT-263 (25 mg/kg) and NBF + ABT-263 (50 mg/kg) groups, while the NBF + ABT-263 (50 mg/kg) group showed a significant improvement. When the concentration of ABT-263 was increased to 10 µmol/L, the apoptosis rate of primary bladder fibroblasts increased, and the expression of the anti-apoptotic protein BCL-xL began to decrease.ABT-263 plays an important role in relieving NBF and protecting against UUTD, which may be due to the promotion of myofibroblast apoptosis through the mitochondrial apoptosis pathway.
Topics: Rats; Animals; Rats, Sprague-Dawley; Urinary Bladder, Neurogenic; Urinary Tract; Fibrosis
PubMed: 37154092
DOI: 10.1080/0886022X.2023.2194440 -
CNS Neuroscience & Therapeutics Nov 2023Annexin A2 (ANXA2) participates in the pathology of a variety of diseases. Nevertheless, the impact of ANXA2 on epilepsy remains to be clarified.
INTRODUCTION
Annexin A2 (ANXA2) participates in the pathology of a variety of diseases. Nevertheless, the impact of ANXA2 on epilepsy remains to be clarified.
AIMS
Hence, the study aimed at investigating the underlying role of ANXA2 in epilepsy through behavioral, electrophysiological, and pathological analyses.
RESULTS
It was found that ANXA2 was markedly upregulated in the cortical tissues of temporal lobe epilepsy patients (TLE), kainic acid (KA)-induced epilepsy mice, and in a seizure-like model in vitro. ANXA2 silencing in mice suppressed first seizure latency, number of seizures, and seizure duration in behavioral analysis. In addition, abnormal brain discharges were less frequent and shorter in the hippocampal local field potential (LFP) record. Furthermore, the results showed that the frequency of miniature excitatory postsynaptic currents was decreased in ANXA2 knockdown mice, indicating that the excitatory synaptic transmission is reduced. Co-immunoprecipitation (COIP) experiments demonstrated that ANXA2 interacted with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit GluA1. Moreover, ANXA2 knockdown decreased GluA1 expression on the cell surface and its phosphorylation onserine 831 and serine 845, related to the decreased phosphorylation levels mediated by protein kinases A and C (PKA and PKC).
CONCLUSIONS
This study covers a previously unknown and key function of ANXA2 in epilepsy. These findings indicate that ANXA2 can regulate excitatory synaptic activity mediated by AMPAR subunit GluA1 to improve seizure activity, which can provide novel insights for the treatment and prevention of epilepsy.
Topics: Humans; Mice; Animals; Phosphorylation; Annexin A2; Receptors, AMPA; Epilepsy; Seizures
PubMed: 37302990
DOI: 10.1111/cns.14295 -
British Journal of Anaesthesia Sep 2023Major cardiac surgery related blood loss is associated with increased postoperative morbidity and mortality. Platelet dysfunction is believed to contribute to...
BACKGROUND
Major cardiac surgery related blood loss is associated with increased postoperative morbidity and mortality. Platelet dysfunction is believed to contribute to post-cardiopulmonary bypass (CPB)-induced microvascular bleeding. We hypothesised that moderately hypothermic CPB induces platelet dysfunction and that supplemental fibrinogen can restore in vitro thrombus formation.
METHODS
Blood from 18 patients, undergoing first-time elective isolated aortic valve surgery was drawn before CPB, 30 min after initiation of CPB, and after CPB and protamine administration, respectively. Platelet aggregation was quantified by optical aggregometry, platelet activation by flow-cytometric detection of platelet surface expression of P-selectin, annexin V, and activated glycoprotein IIb/IIIa, thrombus formation under flow and effect of supplemental fibrinogen (4 mg ml) on in vitro thrombogenesis.
RESULTS
Post-CPB adenosine-diphosphate and TRAP-6-induced aggregation decreased by 40% and 10% of pre-CPB levels, respectively (P<0.0001). Although CPB did not change glycoprotein IIb/IIIa receptor expression, it increased the percentage of unstimulated P-selectin (1.2% vs 7%, P<0.01) positive cells and annexin V mean fluorescence intensity (15.5 vs 17.2, P<0.05), but decreased percentage of stimulated P-selectin (52% vs 26%, P<0.01) positive cells and annexin V mean fluorescence intensity (508 vs 325, P<0.05). Thrombus area decreased from 6820 before CPB to 5230 after CPB (P<0.05, arbitrary units [a.u.]). Supplemental fibrinogen increased thrombus formation to 20 324 and 11 367 a.u. before CPB and after CPB, respectively (P<0.001), thereby restoring post-CPB thrombus area to levels comparable with or higher than pre-CPB baseline.
CONCLUSIONS
Single valve surgery using moderately hypothermic CPB induces partial platelet dysfunction. Thrombus formation was restored in an experimental study design by ex vivo supplementation of fibrinogen.
Topics: Humans; Cardiopulmonary Bypass; P-Selectin; Fibrinogen; Annexin A5; Platelet Aggregation; Hemostatics; Thrombosis
PubMed: 37087333
DOI: 10.1016/j.bja.2023.03.010