Journal of Molecular Medicine

Peng, Xin; Hou, Lei; Wu, Xue; Liu, Zhengqi; Wang, Yun; Zeng, Ping; Yang, Ying; Ma, Wukai; Yang, Peng

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02361-0pmid: 37656227

Primary Sjögren’s syndrome (pSS) is an autoimmune disease represented by exocrine gland epithelial cell lesions. However, the mechanism underlying these lesions remains unclear. This study analyzed the plasma exosomes of pSS patients using proteomics and revealed the presence of 24 differentially expressed proteins (DEPs) involved in the primary biological processes and signaling pathways related to ferroptosis. The DEPs enriched in the ferroptosis-related items were represented by downregulated ceruloplasmin (CP) and transferrin (TF). CC analysis of GO enrichment showed that CP and TF were localized at the apical plasma membrane, which is currently found only in epithelial cells. PPI analysis indicated that these exosomal DEPs formed a clustering network containing CP and TF. Among them, C5, C9, Haptoglobin (HP), and SERPING1 interacted directly with CP and TF. Notably, the expression of these proteins significantly decreased in both the pSS and secondary Sjögren’s syndrome (sSS) plasma exosomes but not in non-autoimmune sicca syndrome (nSS). In addition, their expression levels were significantly different in the exosomes and plasma. More importantly, the plasma and salivary exosomes of pSS patients contain higher levels of exocrine gland epithelial autoantigens SSA and SSB than those of healthy controls, and epithelial cells with positive labial glands biopsy (LGB) were more susceptible to ferroptosis than those with negative LGB. The results indicated that ferroptosis may be closely related to SS epithelial cell lesions.Key messages• pSS plasma exosomes contain epithelial cell–derived proteins involved in ferroptosis.• Complement C5 and C9 may be new molecules involved in ferroptosis and play a crucial role in pSS epithelial cell pathology.• The serum exosomes from pSS patients, not nSS patients, contain ferroptosis-related proteins.• The changes in the ferroptosis-related protein content in the exosomes can better reflect the state of the epithelial cell lesions than those in the plasma.

Cairoli, Victoria; Valle-Millares, Daniel; Terrón-Orellano, María C.; Luque, Daniel; Ryan, Pablo; Dominguez, Lourdes; Martín-Carbonero, Luz; De los Santos, Ignacio; De Matteo, Elena; Ameigeiras, Beatriz; Briz, Verónica; Casciato, Paola; Preciado, María Victoria; Valva, Pamela; Fernández-Rodríguez, Amanda

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02367-8pmid: 37704856

Hepatitis C virus (HCV) coinfection with human immunodeficiency virus (HIV) has a detrimental impact on disease progression. Increasing evidence points to extracellular vesicles (EVs) as important players of the host-viral cross-talk. The microRNAs (miRNAs), as essential components of EVs cargo, are key regulators of normal cellular processes and also promote viral replication, viral pathogenesis, and disease progression. We aimed to characterize the plasma-derived EVs miRNA signature of chronic HCV infected and HIV coinfected patients to unravel the molecular mechanisms of coinfection. EVs were purified and characterized from 50 plasma samples (21 HCV mono- and 29 HCV/HIV co-infected). EV-derived small RNAs were isolated and analyzed by massive sequencing. Known and de novo miRNAs were identified with miRDeep2. Significant differentially expressed (SDE) miRNA identification was performed with generalized linear models and their putative dysregulated biological pathways were evaluated. Study groups were similar for most clinical and epidemiological characteristics. No differences were observed in EVs size or concentration between groups. Therefore, HCV/HIV co-infection condition did not affect the concentration or size of EVs but produced a disturbance in plasma-derived EVs miRNA cargo. Thus, a total of 149 miRNAs were identified (143 known and 6 de novo) leading to 37 SDE miRNAs of which 15 were upregulated and 22 downregulated in HCV/HIV co-infected patients. SDE miRNAs regulate genes involved in inflammation, fibrosis, and cancer, modulating different biological pathways related to HCV and HIV pathogenesis. These findings may help to develop new generation biomarkers and treatment strategies, in addition to elucidate the mechanisms underlying virus–host interaction.Key messagesHCV and HCV/HIV displayed similar plasma-EV size and concentration.EVs- derived miRNA profile was characterized by NGS.37 SDE miRNAs between HCV and HCV/HIV were observed.SDE miRNAs regulate genes involved in inflammation, fibrosis and cancer.

Freiholtz, David; Bergman, Otto; Pradhananga, Sailendra; Lång, Karin; Poujade, Flore-Anne; Granath, Carl; Olsson, Christian; Franco-Cereceda, Anders; Sahlén, Pelin; Eriksson, Per; Björck, Hanna M.

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02370-zpmid: 37698712

Degenerative ascending aortic aneurysm (AscAA) is a silent and potentially fatal disease characterized by excessive vascular inflammation and fibrosis. We aimed to characterize the cellular and molecular signature for the fibrotic type of endothelial mesenchymal transition (EndMT) that has previously been described in degenerative AscAA. Patients undergoing elective open-heart surgery for AscAA and/or aortic valve repair were recruited. Gene expression in the intima-media of the ascending aorta was measured in 22 patients with non-dilated and 24 with dilated aortas, and candidate genes were identified. Protein expression was assessed using immunohistochemistry. Interacting distal gene enhancer regions were identified using targeted chromosome conformation capture (HiCap) in untreated and LPS-treated THP1 cells, and the associated transcription factors were analyzed. Differential expression analysis identified SPP1 (osteopontin) as a key gene in the signature of fibrotic EndMT in patients with degenerative AscAA. The aortic intima-media expression of SPP1 correlated with the expression of inflammatory markers, the level of macrophage infiltration, and the aortic diameter. HiCap analysis, followed by transcription factor binding analysis, identified ETS1 as a potential regulator of SPP1 expression under inflammatory conditions. In conclusion, the present findings suggest that SPP1 may be involved in the development of the degenerative type of AscAA.Key messagesIn the original manuscript titled “SPP1/osteopontin, a driver of fibrosis and inflammation in degenerative ascending aortic aneurysm?” by David Freiholtz, Otto Bergman, Saliendra Pradhananga, Karin Lång, Flore-Anne Poujade, Carl Granath, Christian Olsson, Anders Franco-Cereceda, Pelin Sahlén, Per Eriksson, and Hanna M Björck, we present novel findings on regulatory factors on osteopontin (SPP1) expression in immune cells involved in degenerative ascending aortic aneurysms (AscAA).The central findings convey:SPP1 is a potential driver of the fibrotic endothelial-to-mesenchymal transition in AscAA.SPP1/osteopontin expression in AscAA is predominately by immune cells.ETS1 is a regulatory transcription factor of SPP1 expression in AscAA immune cells.

Ye, Zirui; Zhang, Nana; Lei, Hong; Yao, Huimin; Fu, Jingya; Zhang, Nan; Xu, Lexuan; Zhou, Guxiang; Liu, Zhijun; Lv, Yi

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02363-y

Necroptosis has received increasing attention and is extensively studied as a recently discovered mode of cell death distinct from necrosis and apoptosis. It is a programmed cell death with a necrotic morphology that occurs in various biological processes, including inflammation, immune response, embryonic development, and metabolic abnormalities. Necroptosis is indispensable in maintaining tissue homeostasis in vivo and closely correlates with the occurrence and development of various diseases. First, we outlined the etiology of necroptosis and how it affects the onset and development of prevalent liver diseases in this review. Additionally, we reviewed the therapeutic strategy by targeting the necroptosis pathway in related liver diseases. We conclude that the necroptosis signaling pathway is critical in the physiological control of liver diseases’ onset, progression, and prognosis. It will likely be used as a therapeutic target in the future. Further research is required to determine the mechanisms governing the necroptosis signaling pathway and the effector molecules.

He, Ting; Bai, Xiaozhi; Li, Yan; Zhang, Dongliang; Xu, Zhigang; Yang, Xuekang; Hu, Dahai; Han, Juntao

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02364-xpmid: 37707556

Macrophage is a critical regulator in wound healing and scar formation, and SIRT1 is related to macrophage activation and polarization, while the specific mechanism is still unclear. To explore the specific effects of SIRT1 in scarring, we established a skin incision mouse model and LPS-induced inflammation cell model. The expression of SIRT1 in tissue and macrophage was detected, and the level of SIRT1 was changed to observe the downstream effects. LPS-induced macrophages with or without SIRT1 deficiency were used for TMT-based quantitative proteomic analysis. SIRT1 was suppressed in scar while increased in macrophages of scar tissue. And macrophages were proven to be necessary for wound healing. In the early stage of wound healing, knockout of SIRT1 in macrophage could greatly strengthen inflammation and finally promote scarring. NADH-related activities and oxidoreductase activities were differentially expressed in TMT-based quantitative proteomic analysis. We confirmed that ROS production and NOX2 level were elevated after LPS stimulation while the Nrf2 pathway and the downstream proteins, such as Nqo-1 and HO-1, were suppressed. In contrast, the suppression of SIRT1 strengthened this trend. The NF-κB pathway was remarkably activated compared with the control group. Insufficient increase of SIRT1 in macrophage leads to over activated oxidative stress and activates NF-κB pathways, which then promotes inflammation in wound healing and scarring. Further increasing SIRT1 in macrophages could be a promising method to alleviate scarring.Key messagesSIRT1 was suppressed in scar while increased in macrophages of scar tissue.Inhibition of SIRT1 in macrophage leads to further activated oxidative stress.SIRT1 is negatively related to oxidative stress in macrophage.The elevation of SIRT1 in macrophage is insufficient during scarring.

Beuchel, Carl; Dittrich, Julia; Becker, Susen; Kirsten, Holger; Tönjes, Anke; Kovacs, Peter; Stumvoll, Michael; Loeffler, Markus; Teren, Andrej; Thiery, Joachim; Isermann, Berend; Ceglarek, Uta; Scholz, Markus

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02362-zpmid: 37672078

Investigating the cross talk of different omics layers is crucial to understand molecular pathomechanisms of metabolic diseases like obesity. Here, we present a large-scale association meta-analysis of genome-wide whole blood and peripheral blood mononuclear cell (PBMC) gene expressions profiled with Illumina HT12v4 microarrays and metabolite measurements from dried blood spots (DBS) characterized by targeted liquid chromatography tandem mass spectrometry (LC–MS/MS) in three large German cohort studies with up to 7706 samples. We found 37,295 associations comprising 72 amino acids (AA) and acylcarnitine (AC) metabolites (including ratios) and 8579 transcripts. We applied this catalogue of associations to investigate the impact of associating transcript-metabolite pairs on body mass index (BMI) as an example metabolic trait. This is achieved by conducting a comprehensive mediation analysis considering metabolites as mediators of gene expression effects and vice versa. We discovered large mediation networks comprising 27,023 potential mediation effects within 20,507 transcript-metabolite pairs. Resulting networks of highly connected (hub) transcripts and metabolites were leveraged to gain mechanistic insights into metabolic signaling pathways. In conclusion, here, we present the largest available multi-omics integration of genome-wide transcriptome data and metabolite data of amino acid and fatty acid metabolism and further leverage these findings to characterize potential mediation effects towards BMI proposing candidate mechanisms of obesity and related metabolic diseases. Key messagesThousands of associations of 72 amino acid and acylcarnitine metabolites and 8579 genes expand the knowledge of metabolome-transcriptome associations.A mediation analysis of effects on body mass index revealed large mediation networks of thousands of obesity-related gene-metabolite pairs.Highly connected, potentially mediating hub genes and metabolites enabled insight into obesity and related metabolic disease pathomechanisms.

Ahmad, Firdos; Marzook, Hezlin; Gupta, Anamika; Aref, Aseel; Patil, Kiran; Khan, Amir Ali; Saleh, Mohamed A.; Koch, Walter J.; Woodgett, James R.; Qaisar, Rizwan

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02373-wpmid: 37707557

Reperfusion after acute myocardial infarction further exaggerates cardiac injury and adverse remodeling. Irrespective of cardiac cell types, loss of specifically the α isoform of the protein kinase GSK-3 is protective in chronic cardiac diseases. However, the role of GSK-3α in clinically relevant ischemia/reperfusion (I/R)-induced cardiac injury is unknown. Here, we challenged cardiomyocyte-specific conditional GSK-3α knockout (cKO) and littermate control mice with I/R injury and investigated the underlying molecular mechanism using an in vitro GSK-3α gain-of-function model in AC16 cardiomyocytes post-hypoxia/reoxygenation (H/R). Analysis revealed a significantly lower percentage of infarct area in the cKO vs. control hearts post-I/R. Consistent with in vivo findings, GSK-3α overexpression promoted AC16 cardiomyocyte death post-H/R which was accompanied by an induction of reactive oxygen species (ROS) generation. Consistently, GSK-3α gain-of-function caused mitochondrial dysfunction by significantly suppressing mitochondrial membrane potential. Transcriptomic analysis of GSK-3α overexpressing cardiomyocytes challenged with hypoxia or H/R revealed that NOD-like receptor (NLR), TNF, NF-κB, IL-17, and mitogen-activated protein kinase (MAPK) signaling pathways were among the most upregulated pathways. Glutathione and fatty acid metabolism were among the top downregulated pathways post-H/R. Together, these observations suggest that loss of cardiomyocyte-GSK-3α attenuates cardiac injury post-I/R potentially through limiting the myocardial inflammation, mitochondrial dysfunction, and metabolic derangement. Therefore, selective inhibition of GSK-3α may provide beneficial effects in I/R-induced cardiac injury and remodeling.Key messagesGSK-3α promotes cardiac injury post-ischemia/reperfusion (I/R).GSK-3α regulates inflammatory and metabolic pathways post-hypoxia/reoxygenation (H/R).GSK-3α overexpression upregulates NOD-like receptor (NLR), TNF, NF-kB, IL-17, and MAPK signaling pathways in cardiomyocytes post-H/R.GSK-3α downregulates glutathione and fatty acid metabolic pathways in cardiomyocytes post-H/R.

Cheng, Haiyan; Zhang, Li; Yang, Shen; Ren, Qinghua; Chang, Saishuo; Jin, Yaqiong; Mou, Wenjun; Qin, Hong; Yang, Wei; Zhang, Xianwei; Zhang, Wancun; Wang, Huanmin

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02372-x

This study aimed to analyze the clinical characteristics, cell types, and molecular characteristics of the tumor microenvironment to better predict the prognosis of neuroblastoma (NB). The gene expression data and corresponding clinical information of 498 NB patients were obtained from the Gene Expression Omnibus (GEO: GSE62564) and ArrayExpress (accession: E-MTAB-8248). The relative cell abundances were estimated using single-sample gene set enrichment analysis (ssGSEA) with the R gene set variation analysis (GSVA) package. We performed Cox regression analyses to identify marker genes indicating cell subsets and combined these with prognostically relevant clinical factors to develop a new prognostic model. Data from the E-MTAB-8248 cohort verified the predictive accuracy of the prognostic model. Single-cell RNA-seq data were analyzed by using the R Seurat package. Multivariate survival analysis for each gene, using clinical characteristics as cofactors, identified 34 prognostic genes that showed a significant correlation with both event-free survival (EFS) and overall survival (OS) (log-rank test, P value < 0.05). The pathway enrichment analysis revealed that these prognostic genes were highly enriched in the marker genes of NB cells with mesenchymal features and protein translation. Ultimately, USP39, RPL8, IL1RAPL1, MAST4, CSRP2, ATP5E, International Neuroblastoma Staging System (INSS) stage, age, and MYCN status were selected to build an optimized Cox model for NB risk stratification. These samples were divided into two groups using the median of the risk score as a cutoff. The prognosis of samples in the poor prognosis group (PP) was significantly worse than that of samples in the good prognosis group (GP) (log-rank test, P value < 0.0001, median EFS: 640.5 vs. 2247 days, median OS: 1279.5 vs. 2519 days). The risk model was also regarded as a prognostic indicator independent of MYCN status, age, and stage. Finally, through scRNA-seq data, we found that as an important prognostic marker, USP39 might participate in the regulation of RNA splicing in NB. Our study established a multivariate Cox model based on gene signatures and clinical characteristics to better predict the prognosis of NB and revealed that mesenchymal signature genes of NB cells, especially USP39, were more abundant in patients with a poor prognosis than in those with a good prognosis.Key messagesOur study established a multivariate Cox model based on gene signatures and clinical characteristics to better predict the prognosis of NB and revealed that mesenchymal signature genes of NB cells, especially USP39, were more abundant in patients with a poor prognosis than in those with a good prognosis.USP39, RPL8, IL1RAPL1, MAST4, CSRP2, ATP5E, International Neuroblastoma Staging System (INSS) stage, age, and MYCN status were selected to build an optimized Cox model for NB risk stratification.These samples were divided into two groups using the median of the risk score as a cutoff. The prognosis of samples in the poor prognosis group (PP) was significantly worse than that of samples in the good prognosis group (GP).Finally, through scRNA-seq data, we found that as an important prognostic marker, USP39 might participate in the regulation of RNA splicing in NB.

Lee, Yun Sun; Kim, Hyung Sun; Kim, Hyo Jung; Kang, Hyeon Woong; Lee, Da Eun; Kim, Myeong Jin; Hong, Woosol Chris; Kim, Ju Hyun; Kim, Minsoo; Cheong, Jae-Ho; Park, Joon Seong

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02369-6pmid: 37737908

Gemcitabine is considered a standard treatment for pancreatic cancer, but developing drug resistance greatly limits the effectiveness of chemotherapy and increases the rate of recurrence. Lysyl oxide-like 2 (LOXL2) is highly expressed in pancreatic cancer and is involved in carcinogenesis and EMT regulation. However, studies on the role of LOXL2 in drug resistance are limited. Here, we investigated the mechanism of LOXL2 induction and the effect of LOXL2 on EMT and CSC in gemcitabine-resistant pancreatic cancer. Glucose metabolism was activated in gemcitabine-resistant pancreatic cancer cells, and NF-κB signaling was regulated accordingly. Activated NF-κB directly induces transcription by binding to the promoters of LOXL2 and ZEB1. The EMT process was significantly inhibited by the coregulation of ZEB1 and LOXL2. In addition, LOXL2 inhibition reduced the expression of cancer stemness markers and stemness by regulating MAPK signaling activity. LOXL2 inhibits tumor growth of gemcitabine-resistant pancreatic cancer cells and increases the sensitivity to gemcitabine in mouse models.Key messagesWe identified a specific mechanism for inducing LOXL2 overexpression in gemcitabine-resistant pancreatic cancer. Taken together, our results suggest LOXL2 has an important regulatory role in maintaining gemcitabine resistance and may be an effective therapeutic target to treat pancreatic cancer.

Yu, Lei; Qin, Jianxin; Xing, Jiajun; Dai, Zihao; Zhang, Tingting; Wang, Feng; Zhou, Jin; Zhang, Xiaobai; Chen, Xia; Gu, Yunjuan

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02357-w

As time goes by, the morbidity of diabetes mellitus continues to rise, and the economic burden of diabetic foot ulcers as a common and serious complication of diabetes is increasing. However, currently there is no unified clinical treatment strategy for this complication, and the therapeutic efficacy is unsatisfactory. Recent studies have revealed that biological effects of exosomes involved in multiple stages of the process of wound closure are similar to source cells. Compared with source cells, exosomes possess lowly immunogenicity, highly stability and easily stored, etc. Accumulating evidence confirmed that exosomes promote diabetic wound healing through various pathways such as promoting angiogenesis, collagen fiber deposition, and inhibiting inflammation. The superior therapeutic efficacy of exosomes in accelerating diabetic cutaneous wound healing has attracted an increasing attention. Notably, the molecular mechanisms of exosomes vary among different sources in the chronic wound closure of diabetes. This review focuses on the specific roles and mechanisms of different cell- or tissue-derived exosomes relevant to wound healing. Additionally, the paper provides an overview of the current pre-clinical and clinical applications of exosomes, illustrates their special advantages in wound repair. Furthermore, we discuss the potential obstacles and various solutions for future research on exosomes in the management of diabetic foot ulcer. The aim is to offer novel insights and approaches for the treatment of diabetic foot ulcer.

de Paula, Carla Peres; de Oliveira da Silva, João Pedro Maia; Romanello, Karen Simone; Bernardo, Victoria Simões; Torres, Flaviene Felix; da Silva, Danilo Grünig Humberto; da Cunha, Anderson Ferreira

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02368-7

The red blood cells (RBCs) are essential to transport oxygen (O2) and nutrients throughout the human body. Changes in the structure or functioning of the erythrocytes can lead to several deficiencies, such as hemolytic anemias, in which an increase in reactive oxidative species generation is involved in the pathophysiological process, playing a significant role in the severity of several clinical manifestations. There are important lines of defense against the damage caused by oxidizing molecules. Among the antioxidant molecules, the enzyme peroxiredoxin (Prx) has the higher decomposition power of hydrogen peroxide, especially in RBCs, standing out because of its abundance. This review aimed to present the recent findings that broke some paradigms regarding the three isoforms of Prxs found in RBC (Prx1, Prx2, and Prx6), showing that in addition to their antioxidant activity, these enzymes may have supplementary roles in transducing peroxide signals, as molecular chaperones, protecting from membrane damage, and maintenance of iron homeostasis, thus contributing to the overall survival of human RBCs, roles that seen to be disrupted in hemolytic anemia conditions.

Albrecht, Martin; Hummitzsch, Lars; Rusch, Rene; Eimer, Christine; Rusch, Melanie; Heß, Katharina; Steinfath, Markus; Cremer, Jochen; Fändrich, Fred; Berndt, Rouven; Zitta, Karina

2023 Journal of Molecular Medicine

doi: 10.1007/s00109-023-02374-9

Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EVMreg) with an average size of 7.5 μm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EVMreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EVMreg were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EVMreg. Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EVMreg was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B–positive cells (P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 106 Mreg/ml) led to a reduction of T-cell activation (number of granzyme B–positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EVMreg (P < 0.05 for 3.2 × 106 L-EVMreg/ml). A differential analysis of the effects of Mreg and L-EVMreg on CD4+ and CD8+ T-cells showed an inhibition of CD4+ T-cells by Mreg (P < 0.01) and L-EVMreg (P < 0.05 for 1.6 × 106 L-EVMreg/ml; P < 0.01 for 3.2 × 106 L-EVMreg/ml). A moderate inhibition of CD8+ T-cells was observed by Mreg (P < 0.05) and by L-EVMreg (P < 0.01 for 1.6 × 106 L-EVMreg/ml and 3.2 × 106 L-EVMreg/ml). PS was restricted to confined regions of the Mreg surface, while L-EVMreg showed strong signals for PS in the exoplasmic leaflet. L-EVMreg attenuate CD3/CD28-mediated activation of CD4+ and CD8+ T-cells. L-EVMreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity.Key messagesMreg release large extracellular vesicles (L-EVMreg) with an average size of 7.5 µmL-EVMreg exhibit phosphatidylserine positivityL-EVMreg suppress CD4+ and CD8+ T-cellsL-EVMreg hold clinical potential in T-cell-related diseases