S-Nitrosylation of Septin2 Exacerbates Aortic Aneurysm and Dissection by Coupling the TIAM1-RAC1 Axis in Macrophages.

BACKGROUND: S-Nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in cardiovascular disease. Aortic aneurysm and dissection are high-risk cardiovascular diseases without an effective cure. The aim of this study was to determine the role of SNO of Septin2 in macrophages in aortic aneurysm and dissection. METHODS: Biotin-switch assay combined with liquid chromatography-tandem mass spectrometry was performed to identify the S-nitrosylated proteins in aortic tissue from both patients undergoing surgery for aortic dissection and Apoe-/- mice infused with angiotensin II. Angiotensin II-induced aortic aneurysm model and ß-aminopropionitrile-induced aortic aneurysm and dissection model were used to determine the role of SNO of Septin2 (SNO-Septin2) in aortic aneurysm and dissection development. RNA-sequencing analysis was performed to recapitulate possible changes in the transcriptome profile of SNO-Septin2 in macrophages in aortic aneurysm and dissection. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation were used to uncover the TIAM1-RAC1 (Ras-related C3 botulinum toxin substrate 1) axis as the downstream target of SNO-Septin2. Both R-Ketorolac and NSC23766 treatments were used to inhibit the TIAM1-RAC1 axis. RESULTS: Septin2 was identified S-nitrosylated at cysteine 111 (Cys111) in both aortic tissue from patients undergoing surgery for aortic dissection and Apoe-/- mice infused with Angiotensin II. SNO-Septin2 was demonstrated driving the development of aortic aneurysm and dissection. By RNA-sequencing, SNO-Septin2 in macrophages was demonstrated to exacerbate vascular inflammation and extracellular matrix degradation in aortic aneurysm. Next, TIAM1 (T lymphoma invasion and metastasis-inducing protein 1) was identified as a SNO-Septin2 target protein. Mechanistically, compared with unmodified Septin2, SNO-Septin2 reduced its interaction with TIAM1 and activated the TIAM1-RAC1 axis and consequent nuclear factor-κB signaling pathway, resulting in stronger inflammation and extracellular matrix degradation mediated by macrophages. Consistently, both R-Ketorolac and NSC23766 treatments protected against aortic aneurysm and dissection by inhibiting the TIAM1-RAC1 axis. CONCLUSIONS: SNO-Septin2 drives aortic aneurysm and dissection through coupling the TIAM1-RAC1 axis in macrophages and activating the nuclear factor-κB signaling pathway-dependent inflammation and extracellular matrix degradation. Pharmacological blockade of RAC1 by R-Ketorolac or NSC23766 may therefore represent a potential treatment against aortic aneurysm and dissection.

Spexin Diminishes Atrial Fibrillation Vulnerability by Acting on Galanin Receptor 2.

BACKGROUND: G protein-coupled receptors play a critical role in atrial fibrillation (AF). Spexin is a novel ligand of galanin receptors (GALRs). In this study, we investigated the regulation of spexin and GALRs on AF and the underlying mechanisms. METHODS: Global spexin knockout (SPX-KO) and cardiomyocyte-specific GALRs knockout (GALR-cKO) mice underwent burst pacing electrical stimulation. Optical mapping was used to determine atrial conduction velocity and action potential duration. Atrial myocyte action potential duration and inward rectifying K+ current (IK1) were recorded using whole-cell patch clamps. Isolated cardiomyocytes were stained with Fluo-3/AM dye, and intracellular Ca2+ handling was examined by CCD camera. A mouse model of AF was established by Ang-II (angiotensin II) infusion. RESULTS: Spexin plasma levels in patients with AF were lower than those in subjects without AF, and knockout of spexin increased AF susceptibility in mice. In the atrium of SPX-KO mice, potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) and sarcolipin (SLN) were upregulated; meanwhile, IK1 current was increased and Ca2+ handling was impaired in isolated atrial myocytes of SPX-KO mice. GALR2-cKO mice, but not GALR1-cKO and GALR3-cKO mice, had a higher incidence of AF, which was associated with higher IK1 current and intracellular Ca2+ overload. The phosphorylation level of CREB (cyclic AMP responsive element binding protein 1) was upregulated in atrial tissues of SPX-KO and GALR2-cKO mice. Chromatin immunoprecipitation confirmed the recruitment of p-CREB to the proximal promoter regions of KCNJ2 and SLN. Finally, spexin treatment suppressed CREB signaling, decreased IK1 current and decreased intracellular Ca2+ overload, which thus reduced the inducibility of AF in Ang-II-infused mice. CONCLUSIONS: Spexin reduces atrial fibrillation susceptibility by inhibiting CREB phosphorylation and thus downregulating KCNJ2 and SLN transcription by GALR2 receptor. The spexin/GALR2/CREB signaling pathway represents a novel therapeutic avenue in the development of agents against atrial fibrillation.

Blocking CTLA-4 promotes pressure overload-induced heart failure via activating Th17 cells.

Targeting cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) with specific antibody offers long-term benefits for cancer immunotherapy but can cause severe adverse effects in the heart. This study aimed to investigate the role of anti-CTLA-4 antibody in pressure overload-induced cardiac remodeling and dysfunction. Transverse aortic constriction (TAC) was used to induce cardiac hypertrophy and heart failure in mice. Two weeks after the TAC treatment, mice received anti-CTLA-4 antibody injection twice a week at a dose of 10 mg/kg body weight. The administration of anti-CTLA-4 antibody exacerbated TAC-induced decline in cardiac function, intensifying myocardial hypertrophy and fibrosis. Further investigation revealed that anti-CTLA-4 antibody significantly elevated systemic inflammatory factors levels and facilitated the differentiation of T helper 17 (Th17) cells in the peripheral blood of TAC-treated mice. Importantly, anti-CTLA-4 mediated differentiation of Th17 cells and hypertrophic phenotype in TAC mice were dramatically alleviated by the inhibition of interleukin-17A (IL-17A) by an anti-IL-17A antibody. Furthermore, the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100, also reversed anti-CTLA-4-mediated cardiotoxicity in TAC mice. Overall, these results suggest that the administration of anti-CTLA-4 antibody exacerbates pressure overload-induced heart failure by activating and promoting the differentiation of Th17 cells. Targeting the CXCR4/Th17/IL-17A axis could be a potential therapeutic strategy for mitigating immune checkpoint inhibitors-induced cardiotoxicity.

CyTOF analysis revealed platelet heterogeneity in breast cancer patients received T-DM1 treatment.

T-DM1 (Trastuzumab Emtansine) belongs to class of Antibody-Drug Conjugates (ADC), where cytotoxic drugs are conjugated with the antibody Trastuzumab to specifically target HER2-positive cancer cells. Platelets, as vital components of the blood system, intricately influence the immune response to tumors through complex mechanisms. In our study, we examined platelet surface proteins in the plasma of patients before and after T-DM1 treatment, categorizing them based on treatment response. We identified a subgroup of platelets with elevated expression of CD63 and CD9 exclusively in patients with favorable treatment responses, while this subgroup was absent in patients with poor responses. Another noteworthy discovery was the elevated expression of CD36 in the platelet subgroups of patients exhibiting inadequate responses to treatment. These findings suggest that the expression of these platelet surface proteins may be correlated with the prognosis of T-DM1 treatment. These indicators offer valuable insights for predicting the therapeutic response to T-DM1 and may become important references in future clinical practice, contributing to a better understanding of the impact of ADC therapies and optimizing personalized cancer treatment strategies.

Doramectin attenuates inflammation, obesity and insulin resistance in food-borne obese mice.

The avermectin derivative doramectin is widely used clinically as an antiparasitic drug and, in addition, doramectin may have a modulatory role in obesity. Adipose tissue macrophage recruitment and polarization play an important role in obesity-induced inflammation and insulin resistance. The aim of this study was to investigate the effects of doramectin on high-fat diet-induced inflammation and macrophage polarization in white adipose tissue of epididymis of obese mice. We found that compared with high-fat diet-fed obese mice, doramectin treatment resulted in a significant decrease in body weight and lipid levels, improved insulin resistance, an increase in the proportion of M2-type macrophages and a decrease in the proportion of M1-type macrophages in the epididymal white adipose tissues, as well as a decrease in the infiltration of inflammatory cells in the adipose tissues. Thus, doramectin can ameliorate high-fat diet-induced obesity and adipose inflammation by affecting macrophage polarization in white adipose tissue.

Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization.

BACKGROUND: The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine. Stem cells can self-organise into microsized organ units, partially modelling tissue function and regeneration. Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases. However, the lack of vasculature limits the utility of dental pulp organoids. AIM: To improve survival and aid in recovery after stem cell transplantation, we demonstrated the three-dimensional (3D) self-assembly of adult stem cell-human dental pulp stem cells (hDPSCs) and endothelial cells (ECs) into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium (CM). METHODS: During culture, primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM. The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids. The biological characteristics of the organoids were analysed, and the regulatory pathways associated with angiogenesis were studied. RESULTS: The combination of these two agents resulted in prevascularized human dental pulp organoids (Vorganoids) that more closely resembled dental pulp tissue in terms of morphology and function. Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis. The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids. CONCLUSION: In this innovative study, we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration, facilitating the development of clinical treatment strategies.

L-fucose and fucoidan alleviate high-salt diet-promoted acute inflammation.

Excessive salt intake is a widespread health issue observed in almost every country around the world. A high salt diet (HSD) has a strong correlation with numerous diseases, including hypertension, chronic kidney disease, and autoimmune disorders. However, the mechanisms underlying HSD-promotion of inflammation and exacerbation of these diseases are not fully understood. In this study, we observed that HSD consumption reduced the abundance of the gut microbial metabolite L-fucose, leading to a more substantial inflammatory response in mice. A HSD led to increased peritonitis incidence in mice, as evidenced by the increased accumulation of inflammatory cells and elevated levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemotactic protein-1 (MCP-1, also known as C-C motif chemokine ligand 2 or CCL2), in peritoneal lavage fluid. Following the administration of broad-spectrum antibiotics, HSD-induced inflammation was abolished, indicating that the proinflammatory effects of HSD were not due to the direct effect of sodium, but rather to HSD-induced alterations in the composition of the gut microbiota. By using untargeted metabolomics techniques, we determined that the levels of the gut microbial metabolite L-fucose were reduced by a HSD. Moreover, the administration of L-fucose or fucoidan, a compound derived from brown that is rich in L-fucose, normalized the level of inflammation in mice following HSD induction. In addition, both L-fucose and fucoidan inhibited LPS-induced macrophage activation in vitro. In summary, our research showed that reduced L-fucose levels in the gut contributed to HSD-exacerbated acute inflammation in mice; these results indicate that L-fucose and fucoidan could interfere with HSD-promotion of the inflammatory response.

Proteomic, single-cell and bulk transcriptomic analysis of plasma and tumor tissues unveil core proteins in response to anti-PD-L1 immunotherapy in triple negative breast cancer.

BACKGROUND: Anti-PD-1/PD-L1 treatment has achieved durable responses in TNBC patients, whereas a fraction of them showed non-sensitivity to the treatment and the mechanism is still unclear. METHODS: Pre- and post-treatment plasma samples from triple negative breast cancer (TNBC) patients treated with immunotherapy were measured by tandem mass tag (TMT) mass spectrometry. Public proteome data of lung cancer and melanoma treated with immunotherapy were employed to validate the findings. Blood and tissue single-cell RNA sequencing (scRNA-seq) data of TNBC patients treated with or without immunotherapy were analyzed to identify the derivations of plasma proteins. RNA-seq data from IMvigor210 and other cancer types were used to validate plasma proteins in predicting response to immunotherapy. RESULTS: A random forest model constructed by FAP, LRG1, LBP and COMP could well predict the response to immunotherapy. The activation of complement cascade was observed in responders, whereas FAP and COMP showed a higher abundance in non-responders and negative correlated with the activation of complements. scRNA-seq and bulk RNA-seq analysis suggested that FAP, COMP and complements were derived from fibroblasts of tumor tissues. CONCLUSIONS: We constructe an effective plasma proteomic model in predicting response to immunotherapy, and find that FAP+ and COMP+ fibroblasts are potential targets for reversing immunotherapy resistance.

A mitochondria-targeting heptamethine cyanine-chlorambucil formulated polymeric nanoparticle to potentiate native tumor chemotherapeutic efficacy.

Chlorambucil (Cbl) is a DNA alkylating drug in the nitrogen mustard family, but the clinical applications of nitrogen mustard antitumor drugs are frequently limited by their poor aqueous solubility, poor cellular uptake, lack of targeting, and severe side effects. Additionally, mitochondria are the energy factories for cells, and tumor cells are more susceptible to mitochondrial dysfunction than some healthy cells, thus making mitochondria an important target for tumor therapy. As a proof-of-concept, direct delivery of Cbl to tumor cells' mitochondria will probably bring about new opportunities for the nitrogen mustard family. Furthermore, IR775 chloride is a small-molecule lipophilic cationic heptamethine cyanine dye with potential advantages of mitochondria targeting, near-infrared (NIR) fluorescence imaging, and preferential internalization towards tumor cells. Here, an amphiphilic drug conjugate was facilely prepared by covalently coupling chlorambucil with IR775 chloride and further self-assembly to form a carrier-free self-delivery theranostic system, in which the two components are both functional units aimed at theranostic improvement. The theranostic IR775-Cbl potentiated typical "1 + 1 > 2" tumor inhibition through specific accumulation in mitochondria, which triggered a remarkable decrease in mitochondrial membrane potential and ATP generation. In vivo biodistribution and kinetic monitoring were achieved by real-time NIR fluorescence imaging to observe its transport inside a living body. Current facile mitochondria-targeting modification with clinically applied drugs was promising for endowing traditional drugs with targeting, imaging, and improved potency in disease theranostics.

Pyroptosis Signature Gene CHMP4B Regulates Microglia Pyroptosis by Inhibiting GSDMD in Alzheimer's Disease.

In this study, we aimed to work through the key genes involved in the process of pyroptosis in Alzheimer's disease (AD) to identify potential biomarkers using bioinformatics technology and further explore the underlying molecular mechanisms. The transcriptome data of brain tissue in AD patients were screened from the GEO database, and pyroptosis-related genes were analyzed. The functions of differential genes were analyzed by enrichment analysis and protein-protein interaction. The diagnostic model was established using LASSO and logistic regression analysis, and the correlation of clinical data was analyzed. Based on single-cell analysis of brain tissues of patients with AD, immunofluorescence and western blotting were used to explore the key cells affected by the hub gene. After GSEA, qRT-PCR, western blotting, LDH, ROS, and JC-1 were used to investigate the potential mechanism of the hub gene on pyroptosis. A total of 15 pyroptosis differentially expressed genes were identified. A prediction model consisting of six genes was established by LASSO and logistic regression analysis, and the area under the curve was up to 0.81. As a hub gene, CHMP4B was negatively correlated with the severity of AD. CHMP4B expression was decreased in the hippocampal tissue of patients with AD and mice. Single-cell analysis showed that CHMP4B was downregulated in AD microglia. Overexpression of CHMP4B reduced the release of LDH and ROS and restored mitochondrial membrane potential, thereby alleviating the inflammatory response during microglial pyroptosis. In summary, CHMP4B as a hub gene provides a new strategy for the diagnosis and treatment of AD.

Whole genome sequencing insight into carbapenem-resistant and multidrug-resistant Acinetobacter baumannii harboring chromosome-borne blaOXA-23.

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat to hospitalized patients as effective therapeutic options are scarce. Based on the genomic characteristics of the CRAB strain AB2877 harboring chromosome-borne blaOXA-23, which was isolated from the bronchoalveolar lavage fluid (BALF) of a patient in a respiratory intensive care unit (RICU), we systematically analyzed antibiotic resistance genes (ARGs) and the genetic context associated with ARGs carried by CRAB strains harboring chromosome-borne blaOXA-23 worldwide. Besides blaOXA-23, other ARGs were detected on the chromosome of the CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme). Several key genetic contexts associated with the ARGs were identified on the chromosome of the CRAB strain AB2877, including (1) the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id (2); the resistance island AbGRI3 harboring armA and mph(E)-msr(E) (3); the Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia; and (4) the structure "ISAba1-blaADC-25." The first two genetic contexts were most common in ST195/1816, followed by ST208/1806. The last two genetic contexts were found most frequently in ST208/1806, followed by ST195/1816.IMPORTANCEThe blaOXA-23 gene can be carried by plasmid or chromosome, facilitating horizontal genetic transfer and increasing carbapenem resistance in healthcare settings. In this study, we focused on the genomic characteristics of CRAB strains harboring the chromosome-borne blaOXA-23 gene, and the important genetic contexts associated with blaOXA-23 and other ARGs were identified, and their prevalent clones worldwide were determined. Notably, although the predominant clonal CRAB lineages worldwide containing the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id was ST195/1816, followed by ST208/1806, the CRAB strain AB2877 in our study belonged to ST208/1806. Our findings contribute to the knowledge regarding the dissemination of CRAB strains and the control of nosocomial infection.

SLC44A2 regulates vascular smooth muscle cell phenotypic switching and aortic aneurysm.

Aortic aneurysm is a life-threatening disease with limited interventions that is closely related to vascular smooth muscle cell (VSMC) phenotypic switching. SLC44A2, a member of the solute carrier series 44 (SLC44) family, remains undercharacterized in the context of cardiovascular diseases. Venn diagram analysis based on microarray and single-cell RNA sequencing identified SLC44A2 as a major regulator of VSMC phenotypic switching in aortic aneurysm. Screening for Slc44a2 among aortic cell lineages demonstrated its predominant location in VSMCs. Elevated levels of SLC44A2 were evident in the aorta of both patients with abdominal aortic aneurysm and angiotensin II-infused (Ang II-infused) Apoe-/- mice. In vitro, SLC44A2 silencing promoted VSMCs toward a synthetic phenotype, while SLC44A2 overexpression attenuated VSMC phenotypic switching. VSMC-specific SLC44A2-knockout mice were more susceptible to aortic aneurysm under Ang II infusion, while SLC44A2 overexpression showed protective effects. Mechanistically, SLC44A2's interaction with NRP1 and ITGB3 activates TGF-ß/SMAD signaling, thereby promoting contractile gene expression. Elevated SLC44A2 in aortic aneurysm is associated with upregulated runt-related transcription factor 1 (RUNX1). Furthermore, low-dose lenalidomide (LEN; 20 mg/kg/day) suppressed aortic aneurysm progression by enhancing SLC44A2 expression. These findings reveal that the SLC44A2-NRP1-ITGB3 complex is a major regulator of VSMC phenotypic switching and provide a potential therapeutic approach (LEN) for aortic aneurysm treatment.

Anti-CTLA-4 m2a Antibody Exacerbates Cardiac Injury in Experimental Autoimmune Myocarditis Mice By Promoting Ccl5-Neutrophil Infiltration.

The risk for suffering immune checkpoint inhibitors (ICIs)-associated myocarditis increases in patients with pre-existing conditions and the mechanisms remain to be clarified. Spatial transcriptomics, single-cell RNA sequencing, and flow cytometry are used to decipher how anti-cytotoxic T lymphocyte antigen-4 m2a antibody (anti-CTLA-4 m2a antibody) aggravated cardiac injury in experimental autoimmune myocarditis (EAM) mice. It is found that anti-CTLA-4 m2a antibody increases cardiac fibroblast-derived C-X-C motif chemokine ligand 1 (Cxcl1), which promots neutrophil infiltration to the myocarditic zones (MZs) of EAM mice via enhanced Cxcl1-Cxcr2 chemotaxis. It is identified that the C-C motif chemokine ligand 5 (Ccl5)-neutrophil subpopulation is responsible for high activity of cytokine production, adaptive immune response, NF-κB signaling, and cellular response to interferon-gamma and that the Ccl5-neutrophil subpopulation and its-associated proinflammatory cytokines/chemokines promoted macrophage (Mφ) polarization to M1 Mφ. These altered infiltrating landscape and phenotypic switch of immune cells, and proinflammatory factors synergistically aggravated anti-CTLA-4 m2a antibody-induced cardiac injury in EAM mice. Neutralizing neutrophils, Cxcl1, and applying Cxcr2 antagonist dramatically alleviates anti-CTLA-4 m2a antibody-induced leukocyte infiltration, cardiac fibrosis, and dysfunction. It is suggested that Ccl5-neutrophil subpopulation plays a critical role in aggravating anti-CTLA-4 m2a antibody-induced cardiac injury in EAM mice. This data may provide a strategic rational for preventing/curing ICIs-associated myocarditis.

Chinese Society of Cardiology guidelines on the diagnosis and treatment of adult fulminant myocarditis.

Fulminant myocarditis is an acute diffuse inflammatory disease of myocardium. It is characterized by acute onset, rapid progress and high risk of death. Its pathogenesis involves excessive immune activation of the innate immune system and formation of inflammatory storm. According to China's practical experience, the adoption of the "life support-based comprehensive treatment regimen" (with mechanical circulation support and immunomodulation therapy as the core) can significantly improve the survival rate and long-term prognosis. Special emphasis is placed on very early identification,very early diagnosis,very early prediction and very early treatment.