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BackgroundMesenchymal stem cell (MSC)-derived exosomes play significant roles in ameliorating cardiac damage after myocardial ischemia-reperfusion (I/R) injury. Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 (Lnc A2M-AS1) was found that might protect against myocardial I/R. However, whether Lnc A2M-AS1 delivery via MSC-derived exosomes could also regulate myocardial I/R injury remains unknown.MethodsExosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Hypoxia/reoxygenation (H/R) treatment in human cardiomyocytes was used to mimic the process of myocardial I/R in vitro. The viability and apoptosis of cardiomyocytes were detected using cell counting kit-8, flow cytometry, and Western blot assays. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using corresponding commercial kits. The quantitative real-time polymerase chain reaction and Western blot were used to determine the expression levels of Lnc A2M-AS1, microRNA (miR)-556-5p, and X-linked inhibitor of apoptosis protein (XIAP). The binding interaction between miR-556-5p and Lnc A2M-AS1 or XIAP was confirmed by the dual-luciferase reporter, RIP and pull-down assays.ResultsExosomes isolated from hMSCs (hMSCs-exo) attenuated H/R-induced apoptosis and oxidative stress in cardiomyocytes. Lnc A2M-AS1 was lowly expressed in AMI patients and H/R-induced cardiomyocytes. Besides, Lnc A2M-AS1 was detectable in hMSCs-exo, exosomes derived from Lnc A2M-AS1-transfected hMSCs weakened H/R-induced apoptosis and oxidative stress, and enhanced the protective action of hMSCs-exo on H/R-induced cardiomyocytes. Further mechanism analysis showed that Lnc A2M-AS1 acted as a sponge for miR-556-5p to increase XIAP expression level. Importantly, miR-556-5p overexpression or XIAP knockdown reversed the action of exosomal Lnc A2M-AS1 on H/R-induced cardiomyocytes.ConclusionLnc A2M-AS1 delivery via MSC-derived exosomes ameliorated H/R-induced cardiomyocyte apoptosis and oxidative stress via regulating miR-556-5p/XIAP, opening a new window into the pathogenesis of myocardial I/R injury.

Purpose Rapid progression in late‐stage is a characteristic of pancreatic cancer (PC), leading to mortality. The critical role of lncRNA A2M‐AS1 (long non‐coding RNA alpha‐2‐macroglobulin antisense RNA 1) is involved in cancer progression, but the upstream regulator of A2M‐AS1 in the PC progression phenotype remains elusive. Methods We conducted an integrated analysis using bioinformatics, in vitro experiments, and in vivo studies. Human PC tissues were analyzed for A2M‐AS1 and p53 expressions. The PANC‐1 and BxPC‐3 cell lines were used for functional assays, including cell proliferation, apoptosis, migration, and invasion assays. The role of p53 in regulating A2M‐AS1 was investigated through overexpression and knockdown studies, along with using a MAPK pathway inhibitor. Results We found that A2M‐AS1 is downregulated in PC tissues and that its high expression correlates with a better prognosis. p53 was identified as a negative regulator of A2M‐AS1, with its knockdown leading to increased A2M‐AS1 expression and decreased PC cell invasiveness. Mechanistically, p53 was shown to bind to the A2M‐AS1 promoter, modulating its transcriptional activity. The MAPK pathway was revealed as a downstream effector of the p53‐A2M‐AS1 axis, with its inhibition reversing the effects on PC cell behavior. Conclusion High A2M‐AS1 expression is associated with a better PC prognosis. A2M‐AS1 overexpression subdues PC cell development. Our study unveils a new mechanism by which p53 decreases A2M‐AS1 expression.

Subarachnoid hemorrhage (SAH) is a disease with high mortality and morbidity, and its pathophysiology is complex but poorly understood. To investigate the potential therapeutic targets post-SAH, the SAH-related feature genes were screened by the combined analysis of transcriptomics and metabolomics of rat cortical tissues following SAH and proteomics of cerebrospinal fluid from SAH patients, as well as WGCNA and machine learning. The competitive endogenous RNAs (ceRNAs) and transcription factors (TFs) regulatory networks of the feature genes were constructed and further validated by molecular biology experiments. A total of 1336 differentially expressed proteins were identified, including 729 proteins downregulated and 607 proteins upregulated. The immune microenvironment changed after SAH and the changement persisted at SAH 7d. Through multi-omics and bioinformatics techniques, five SAH-related feature genes (A2M, GFAP, GLIPR2, GPNMB, and LCN2) were identified, closely related to the immune microenvironment. In addition, ceRNAs and TFs regulatory networks of the feature genes were constructed. The increased expression levels of A2M and GLIPR2 following SAH were verified, and co-localization of A2M with intravascular microthrombus was demonstrated. Multiomics and bioinformatics tools were used to predict the SAH associated feature genes confirmed further through the ceRNAs and TFs regulatory network development. These molecules might play a key role in SAH and may serve as potential biological markers and provide clues for exploring therapeutic options.

Background The study was performed to evaluate whether targeted alpha-2-macroglobulin (A2M) variants have a similar or enhanced function at wild-type (wt)-A2M to attenuate cartilage degeneration in vivo. Methods In and ex-vivo experiment, bovine cartilage explants (BCE) were incubated with TNF-α and IL-1β with or without wt-A2M or A2M variants. Cartilage catabolism was measured in culture supernatant by sulfated glycosaminoglycan (sGAG). In an in-vivo experiment, 2-month-old male Wistar rats (n = 77) were randomly divided into seven groups and treated with different doses of A2M or its variants by intra-articular injection at 24 hours and day 14 after anterior cruciate ligament transection (ACLT), receiving (1) ACLT/PBS; (2) ACLT/wt-A2M (0.153 mg); (3) ACLT/CYT-108 A2M (0.153 mg); (4) ACLT/CYT-108 A2M (0.077 mg); (5) ACLT/CYT-98 A2M (0.153 mg); (6) ACLT/CYT-98 A2M (0.077 mg); or (7) sham/PBS. The joints and synovial lavage were collected 8 weeks after surgery. Fluorescence molecular tomography was used to monitor inflammation in vivo using probes ProSense and MMPSense at 24 hours, and weeks 2, 4, and 6 after surgery. The cartilage damage was quantified using Osteoarthritis Research Society International score and matrix metalloproteinase (MMP)-3, -13, collagen (Col) X, Col 2, Runx2, and aggrecan (Acan) were detected by immunohistochemical analysis (IHC), ELISA, and RT-PCR. Results A2M variants inhibited catabolism in the BCE model by up to 200% compared with wt-A2M. ProSense and MMPSense were dramatically increased in all groups after surgery. Supplemental A2M or its variants reduced ProSense and MMPSense compared with the PBS treatment. Less cartilage damage, lower MMP-13 and Col 2 degraded product, and stronger Col 2 synthesis were detected in animals treated with A2M or its variants compared with PBS-treated animals. A2M and its variants enhanced Col 2 and Acan synthesis, and suppressed MMP-3, MMP-13, Runx2, and Col X production. A2M-108 variant demonstrated less cartilage damage compared with wt-A2M and A2M-98 variant. Conclusion The targeted variants of A2M have a chondroprotective effect similar to wt-A2M. However, A2M-108 variant has enhanced function to attenuate cartilage degeneration compared with wt-A2M.

Fighting external pathogens relies on the tight regulation of the gene expression of the immune system. Ferroptosis, which is a distinct form of programmed cell death driven by iron, is involved in the enhancement of follicular helper T cell function during infection. The regulation of RNA is a key step in final gene expression. The present study aimed to identify the expression level of antisense lncRNAs (A2M-AS1, DBH-AS1, FLVCR1-DT, and NCBP2AS2-1) and FLVCR1 in COVID-19 patients and its relation to the severity of the disease. COVID-19 patients as well as age and gender-matched healthy controls were enrolled in this study. The expression level of the antisense lncRNAs was measured by RT-PCR. Results revealed the decreased expression of A2M-AS1 and FLVCR1 in COVID-19 patients. Additionally, they showed the increased expression of DBH-AS1, FLVCR1-DT, and NCBP2AS2. Both FLVCR1-DT and NCBP2AS2 showed a positive correlation with interleukin-6 (IL-6). DBH-AS1 and FLVCR1-DT had a significant association with mortality, complications, and mechanical ventilation. A significant negative correlation was found between A2M-AS1 and NCBP2AS2-1 and between FLVCR1 and FLVCR1-DT. The study confirmed that the expression level of the antisense lncRNAs was deregulated in COVID-19 patients and correlated with the severity of COVID-19, and that it may have possible roles in the pathogenesis of this disease.

Canine osteosarcoma (OSA) is an aggressive bone neoplasia with high metastatic potential. Metastasis is the main cause of death associated with OSA, and there is no current treatment available for metastatic disease. Proteomic analyses, including matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF/TOF MS), are widely used to select molecular targets and identify proteins that may play a key role in primary tumours and at various steps of the metastatic cascade. The main aim of this study was to identify proteins differently expressed in canine OSA cell lines with different malignancy phenotypes (OSCA-8 and OSCA-32) compared to canine osteoblasts (CnOb). The intermediate aim of the study was to compare canine OSA cell migration capacity and assess its correlation with the malignancy phenotypes of each cell line. Using MALDI-TOF/TOF MS analyses, we identified eight proteins that were significantly differentially expressed (p ≤ 0.05) in canine OSA cell lines compared to CnOb: cilia- and flagella-associated protein 298 (CFAP298), general transcription factor II-I (GTF2I), mirror-image polydactyly gene 1 protein (MIPOL1), alpha-2 macroglobulin (A2M), phosphoglycerate mutase 1 (PGAM1), ubiquitin (UB2L6), ectodysplasin-A receptor-associated adapter protein (EDARADD), and leucine-rich-repeat-containing protein 72 (LRRC72). Using the Simple Western technique, we confirmed high A2M expression in CnOb compared to OSCA-8 and OSCA-32 cell lines (with intermediate and low A2M expression, respectively). Then, we confirmed the role of A2M in cancer cell migration by demonstrating significantly inhibited OSA cell migration by treatment with A2M (both at 10 and 30 mM concentrations after 12 and 24 h) in a wound-healing assay. This study may be the first report indicating A2M’s role in OSA cell metastasis; however, further in vitro and in vivo studies are needed to confirm its possible role as an anti-metastatic agent in this malignancy.

The HUGO Gene Nomenclature Committee (HGNC) assigns unique symbols and names to human genes and its sister project, the Vertebrate Gene Nomenclature Committee (VGNC), names genes across selected vertebrates (chimp, macaque, horse, cattle, pig, dog, cat) in line with their human orthologs. The A2M gene family, a subfamily of the thioester-containing protein (TEP) superfamily, is well conserved across vertebrates and several members of this family have already been characterized as non-specific peptidase inhibitors. Chicken ovostatin, originally termed “ovomacroglobulin”, is an A2M family member that was first identified as being one of the most abundant proteins found in egg white. Two uncharacterized ovostatin homologs have also been reported in human. We wanted to assign standardized nomenclature to the multiple members of the A2M family across a wide range of vertebrate species, to capture the variation within this complex gene family. We constructed a maximum likelihood phylogenetic tree based on a multiple alignment of A2M protein sequences to help assign new nomenclature to previously unnamed A2M family genes, including the ovostatins, in human and across selected vertebrate species. This resulted in the naming of 4 human A2M family pseudogenes and 14 protein coding genes and 4 pseudogenes across VGNC species. An additional 48 genes were also named in model organisms (mouse, rat, xenopus, zebrafish, chicken) by their nomenclature committees based on this phylogenetic analysis.

Background Pre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with inflammatory signaling; however, the pathophysiological mechanism by which A2M is involved in PE development is not yet understood. Methods Human placenta samples, serum, and corresponding clinical data of the participants were collected to study the pathophysiologic mechanism underlying PE. Pregnant Sprague–Dawley rats were intravenously injected with an adenovirus vector carrying A2M via the tail vein on gestational day (GD) 8.5. Human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein endothelial cells (HUVECs), and HTR-8/SVneo cells were transfected with A2M-expressing adenovirus vectors. Results In this study, we demonstrated that A2M levels were significantly increased in PE patient serum, uterine spiral arteries, and feto-placental vasculature. The A2M-overexpression rat model closely mimicked the characteristics of PE (i.e., hypertension in mid-to-late gestation, histological and ultrastructural signs of renal damage, proteinuria, and fetal growth restriction). Compared to the normal group, A2M overexpression significantly enhanced uterine artery vascular resistance and impaired uterine spiral artery remodeling in both pregnant women with early-onset PE and in pregnant rats. We found that A2M overexpression was positively associated with HUASMC proliferation and negatively correlated with cell apoptosis. In addition, the results demonstrated that transforming growth factor beta 1 (TGFβ1) signaling regulated the effects of A2M on vascular muscle cell proliferation described above. Meanwhile, A2M overexpression regressed rat placental vascularization and reduced the expression of angiogenesis-related genes. In addition, A2M overexpression reduced HUVEC migration, filopodia number/length, and tube formation. Furthermore, HIF-1α expression was positively related to A2M, and the secretion of sFLT-1 and PIGF of placental origin was closely related to PE during pregnancy or A2M overexpression in rats. Conclusions Our data showed that gestational A2M overexpression can be considered a contributing factor leading to PE, causing detective uterine spiral artery remodeling and aberrant placental vascularization.

The male reproductive impairment caused by environmental estrogens (EEs) stands as a pivotal research area in environmental toxicology. Alpha2-macroglobulin (A2M) emerges as a promising molecule capable of counteracting oxidative stress induced by EEs. This study conducted exposure experiments spanning PND1 to PND56 employing ICR mice, aiming to delve into the expression patterns of A2M and its modulated IL-6 in the testicular tissue of mice subsequent to diethylstilbestrol (DES) and benzophenone (BP) exposure, while elucidating the pivotal role of ERs in this intricate process. Our findings revealed that upon DES exposure (10 and 100 nM), there was a pronounced upregulation of A2M (mRNA and in situ protein levels) in mouse testicular tissue. Similarly, exposure to BPs (BP-1, BP-2, and BP-3, each at 10 and 1000 nM) exhibited comparable effects and increasing A2M levels in serum. Notably, BP exposure also caused an elevation in IL-6 levels (which could be directly regulated by A2M) within testicular tissue (mRNA and in situ protein). Remarkably, the specific estrogen receptor antagonist ICI 182780 (0.5 mg/kg/day) was effective in reversing the upregulation of both A2M and IL-6 induced by BP exposure. Significantly, the results of theoretical prediction of the potential ERE site in the A2m gene promoter region and ChIP-qPCR experiment provide essential and strong evidence for the key conclusion that A2m is the target gene of ER. Taken together, our study highlights EEs’ ability to regulate A2M expression in the male reproductive system via the ER signaling pathway. This vital insight deepens our understanding of molecular mechanisms protecting against oxidative stress caused by EEs.

Transfusion‐dependent thalassemia (TDT) is a severe inherited anemia characterized by impaired synthesis of hemoglobin chains. Disease progression and TDT severity are potentially linked to oxidative stress and protein damage. This study aimed to explore the expression patterns of ceruloplasmin (CP), α2‐macroglobulin (A2M), and alpha‐2‐HS‐glycoprotein (AHSG) in TDT serum through quantitative proteomic profiling. The results were validated using enzyme‐linked immunosorbent assays (ELISA). The study participants were divided into three groups based on the duration of blood transfusion. Age and gender‐matched normal individuals served as controls. The results revealed the downregulation of these proteins. The reduced levels of these proteins may contribute to tissue damage in TDT patients, primarily due to increased oxidative stress. For example, decreased CP levels can disrupt iron and copper metabolism, leading to heightened oxidative stress and rendering red blood cell membranes more susceptible to rupture due to active oxygen radicals. In summary, CP, A2M, and AHSG association with iron metabolism, inflammation, and oxidative stress underscores their potential relevance in understanding TDT’s pathogenesis and progression. These findings may pave the way for improved diagnostic and therapeutic strategies for TDT patients.