ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque.

INTRODUCTION: The N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin αvß3. OBJECTIVES: ANGPTL3 might home to plaque where it directly regulates macrophage function via integrin αvß3 for atherosclerosis progression. METHODS: Ldlr-/- mice on a high-fat diet and ApoE-/- mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE-/- mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3-/-ApoE-/- mice and ApoE-/- littermates. THP-1 cells were exposed to 0 or 50 µg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin ß3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system. RESULTS: Angptl3 overexpression increased atherosclerotic progression and CD68+ macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG+ cells were identified in plaque of gene transferred ApoE-/- mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1ß and tumour necrosis factor-α in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin ß3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-κB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05). CONCLUSIONS: Targeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.

Farnesoid X Receptor Protects Murine Lung against IL-6-promoted Ferroptosis Induced by Polyriboinosinic-Polyribocytidylic Acid.

Various infections trigger a storm of proinflammatory cytokines in which IL-6 acts as a major contributor and leads to diffuse alveolar damage in patients. However, the metabolic regulatory mechanisms of IL-6 in lung injury remain unclear. Polyriboinosinic-polyribocytidylic acid [poly(I:C)] activates pattern recognition receptors involved in viral sensing and is widely used in alternative animal models of RNA virus-infected lung injury. In this study, intratracheal instillation of poly(I:C) with or without an IL-6-neutralizing antibody model was combined with metabonomics, transcriptomics, and so forth to explore the underlying molecular mechanisms of IL-6-exacerbated lung injury. We found that poly(I:C) increased the IL-6 concentration, and the upregulated IL-6 further induced lung ferroptosis, especially in alveolar epithelial type II cells. Meanwhile, lung regeneration was impaired. Mechanistically, metabolomic analysis showed that poly(I:C) significantly decreased glycolytic metabolites and increased bile acid intermediate metabolites that inhibited the bile acid nuclear receptor farnesoid X receptor (FXR), which could be reversed by IL-6-neutralizing antibody. In the ferroptosis microenvironment, IL-6 receptor monoclonal antibody tocilizumab increased FXR expression and subsequently increased the Yes-associated protein (YAP) concentration by enhancing PKM2 in A549 cells. FXR agonist GW4064 and liquiritin, a potential natural herbal ingredient as an FXR regulator, significantly attenuated lung tissue inflammation and ferroptosis while promoting pulmonary regeneration. Together, the findings of the present study provide the evidence that IL-6 promotes ferroptosis and impairs regeneration of alveolar epithelial type II cells during poly(I:C)-induced murine lung injury by regulating the FXR-PKM2-YAP axis. Targeting FXR represents a promising therapeutic strategy for IL-6-associated inflammatory lung injury.

Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization.

The COVID-19 pandemic has fostered major advances in vaccination technologies1-4; however, there are urgent needs for vaccines that induce mucosal immune responses and for single-dose, non-invasive administration4-6. Here we develop an inhalable, single-dose, dry powder aerosol SARS-CoV-2 vaccine that induces potent systemic and mucosal immune responses. The vaccine encapsulates assembled nanoparticles comprising proteinaceous cholera toxin B subunits displaying the SARS-CoV-2 RBD antigen within microcapsules of optimal aerodynamic size, and this unique nano-micro coupled structure supports efficient alveoli delivery, sustained antigen release and antigen-presenting cell uptake, which are favourable features for the induction of immune responses. Moreover, this vaccine induces strong production of IgG and IgA, as well as a local T cell response, collectively conferring effective protection against SARS-CoV-2 in mice, hamsters and nonhuman primates. Finally, we also demonstrate a mosaic iteration of the vaccine that co-displays ancestral and Omicron antigens, extending the breadth of antibody response against co-circulating strains and transmission of the Omicron variant. These findings support the use of this inhaled vaccine as a promising multivalent platform for fighting COVID-19 and other respiratory infectious diseases.

PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction.

Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is strongly associated with non-alcoholic steatohepatitis and advanced fibrosis; however, the underlying mechanisms remain largely unknown. In this study, we investigated the effect of PNPLA3-I148M on the activation of hepatic stellate cell line LX-2 and the progression of liver fibrosis. Immunofluorescence staining and enzyme-linked immunosorbent assay were used to detect lipid accumulation. The expression levels of fibrosis, cholesterol metabolism, and mitochondria-related markers were measured via real-time PCR or western blotting. Electron microscopy was applied to analyze the ultrastructure of the mitochondria. Mitochondrial respiration was measured by a Seahorse XFe96 analyzer. PNPLA3-I148M significantly promoted intracellular free cholesterol aggregation in LX-2 cells by decreasing cholesterol efflux protein (ABCG1) expression; it subsequently induced mitochondrial dysfunction characterized by attenuated ATP production and mitochondrial membrane potential, elevated ROS levels, caused mitochondrial structural damage, altered the oxygen consumption rate, and decreased the expression of mitochondrial-function-related proteins. Our results demonstrated for the first time that PNPLA3-I148M causes mitochondrial dysfunction of LX-2 cells through the accumulation of free cholesterol, thereby promoting the activation of LX-2 cells and the development of liver fibrosis.

CDetection.v2: One-pot assay for the detection of SARS-CoV-2.

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

Exendin-4 attenuates atherosclerosis progression via controlling hematopoietic stem/progenitor cell proliferation.

Beyond glycemic control, applications of glucagon-like peptide-1 receptor (GLP-1r) agonists (GLP-1 RAs) inhibit inflammation and plaque development in murine atherosclerotic models. However, whether they modulate hematopoietic stem/progenitor cells (HSPCs) to prohibit skewed myelopoiesis in hypercholesteremia remains unknown. In this study, GLP-1r expression in fluorescence-activated cell sorting (FACS)-sorted wild-type HSPCs was determined by capillary western blotting. Bone marrow cells (BMCs) of wild-type or GLP-1r-/- mice were transplanted into lethally irradiated low-density lipoprotein receptor deficient (LDLr-/-) recipients followed by high-fat diet (HFD) for chimerism analysis by FACS. In parallel, LDLr-/- mice were placed on HFD for 6 weeks and then treated with saline or Exendin-4 (Ex-4) for another 6 weeks. HSPC frequency and cell cycle were analyzed by FACS, and intracellular metabolite levels were assessed by targeted metabolomics. The results demonstrated that HSPCs expressed GLP-1r and transplantation of GLP-1r-/- BMCs resulted in skewed myelopoiesis in hypercholesterolemic LDLr-/- recipients. In vitro, Ex-4 treatment of FACS-purified HSPCs suppressed cell expansion and granulocyte production induced by LDL. In vivo, Ex-4 treatment inhibited plaque progression, suppressed HSPC proliferation, and modified glycolytic and lipid metabolism in HSPCs of hypercholesteremic LDLr-/- mice. In conclusion, Ex-4 could directly inhibit HSPC proliferation induced by hypercholesteremia.

Dendric nanostructures for SARS-CoV-2 proteins detection based on surface enhanced infrared absorption spectroscopy.

Infrared spectroscopy is a non-destructive and rapid characterization tool that can distinguish different viral proteins by spectral details. However, traditional infrared spectroscopy has insufficient absorption signal intensity contrast when measuring low-concentration samples. In this work, surface enhanced infrared absorption (SEIRA) spectroscopy is proposed by deploying a novel nanostructure array as SEIRA substrates. An array of gold dendric nanostructures are designed and fabricated with a precision resonance control to achieve surface enhancement covering a broadband molecular "finger-print" region. The spectral positions of the multiple resonances accurately correspond to the characteristic absorption peaks of the SARS-CoV-2 proteins. An approach for SARS-CoV-2 protein detection based on SEIRA spectroscopy is then proposed. A low concentration detection of 40 µg/ml diluted SARS-CoV-2 nucleocapsid protein is experimentally demonstrated and the enhancement factor (EF) achieved is in good agreement with simulation results. The SEIRA methodology based on broadband resonance nanostructure design provides a systematic approach for sensitive, non-destructive and rapid protein molecular detection, which could be extended to various kind of molecular characterization and biomedical diagnostics.

Recent Insights into Neutrophil Extracellular Traps in Cardiovascular Diseases.

Neutrophils are primary effector cells of the innate immune system. Emerging evidence has consistently shown that activated neutrophils produce and release neutrophil extracellular traps (NETs) that play roles in immunity and non-infectious diseases. NETs are composed of DNA and proteins and serve as a structural platform for pathogen sequestration and degradation. In contrast to their protective role during pathogenic infection, NETs are pathologically involved in cardiovascular disease (CVD). In this review, we introduce the formation, release, and clearance of NETs and the regulatory mechanisms of NETs formation, followed by an overview of the clinical evidence for the involvement of NETs in CVD. Because atherosclerosis is a fundamental part of the pathogenesis of CVD, we chose to focus on the mechanisms by which NETs promote endothelial cell damage and collaborate with macrophages and platelets to accelerate plaque progression and thrombosis. Finally, we present options for clinical intervention to inhibit NETs production and release in the treatment of CVD. In conclusion, this review integrates the latest findings and provides new insights into NETs, which represent a novel biomarker and therapeutic target in clinical practice.

m7G regulator-mediated methylation modification patterns define immune cell infiltration and patient survival.

Numerous studies have demonstrated the important roles of epigenetic modifications in tumorigenesis, progression and prognosis. However, in hepatocellular carcinoma, the potential link between N7-methylguanosine (m7G) modification and molecular heterogeneity and tumor microenvironment (TME) remains unclear. Method: We performed a comprehensive evaluation of m7G modification patterns in 816 hepatocellular carcinoma samples based on 24 m7G regulatory factors, identified different m7G modification patterns, and made a systematic correlation of these modification patterns with the infiltration characteristics of immunocytes. Then, we built and validated a scoring tool called m7G score. Results: In this study, we revealed the presence of three distinct m7G modification patterns in liver cancer, with remarkable differences in the immunocyte infiltration characteristics of these three subtypes. The m7G scoring system of this study could assess m7G modification patterns in individual hepatocellular carcinoma patients, could predict TME infiltration characteristics, genetic variants and patient prognosis. We also found that the m7G scoring system may be useful in guiding patients' clinical use of medications. Conclusions: This study revealed that m7G methylation modifications exerted a significant role in formation of TME in hepatocellular carcinoma. Assessing the m7G modification patterns of single patients would help enhance our perception of TME infiltration characteristics and give significant insights into immunotherapy efficacy.

The purine metabolite inosine monophosphate accelerates myelopoiesis and acute pancreatitis progression.

Hyperglycemia-induced myelopoiesis and atherosclerotic progression occur in mice with type I diabetes. However, less is known about the effects of metabolites on myelopoesis in type 2 diabetes. Here, we use fluorescence-activated cell sorting to analyze the proliferation of granulocyte/monocyte progenitors (GMP) in db/db mice. Using targeted metabolomics, we identify an increase in inosine monophosphate (IMP) in GMP cells of 24-week-old mice. We show that IMP treatment stimulates cKit expression, ribosomal S6 activation, GMP proliferation, and Gr-1+ granulocyte production in vitro. IMP activates pAkt in non-GMP cells. In vivo, using an established murine acute pancreatitis (AP) model, administration of IMP-treated bone marrow cells enhances the severity of AP. This effect is abolished in the presence of a pAkt inhibitor. Targeted metabolomics show that plasma levels of guanosine monophosphate are significantly higher in diabetic patients with AP. These findings provid a potential therapeutic target for the control of vascular complications in diabetes.

The protective roles of augmenter of liver regeneration in hepatocytes in the non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) occurs in 25% of the global population and manifests as lipid deposition, hepatocyte injury, activation of Kupffer and stellate cells, and steatohepatitis. Predominantly expressed in hepatocytes, the augmenter of liver regeneration (ALR) is a key factor in liver regulation that can alleviate fatty liver disease and protect the liver from abnormal liver lipid metabolism. ALR has three isoforms (15-, 21-, and 23-kDa), amongst which 23-kDa ALR is the most extensively studied. The 23-kDa ALR isoform is a sulfhydryl oxidase that resides primarily in the mitochondrial intermembrane space (IMS), whereby it protects the liver against various types of injury. In this review, we describe the role of ALR in regulating hepatocytes in the context of NAFLD. We also discuss questions about ALR that remain to be explored in the future. In conclusion, ALR appears to be a promising therapeutic target for treating NAFLD.

Landscape of Peripheral Blood Mononuclear Cells and Soluble Factors in Severe COVID-19 Patients With Pulmonary Fibrosis Development.

Resulting from severe inflammation and cell destruction, COVID-19 patients could develop pulmonary fibrosis (PF), which remains in the convalescent stage. Nevertheless, how immune response participates in the pathogenesis of PF progression is not well defined. To investigate that question, 12 patients with severe COVID-19 were included in the study. Peripheral mononuclear cell (PBMC) samples were collected shortly after their admission and proceeded for single-cell RNA sequencing (scRNA-seq). After 14 days of discharge, the patients were revisited for chest CT scan. PF index (FI) was computed by AI-assisted CT images. Patients were categorized into FIhi and FIlo based on median of FI. By scRNA-seq analysis, our data demonstrated that frequency of CD4+ activated T cells and Treg cells were approximately 3-fold higher in FIhi patients compared with FIlo ones (p < 0.034 for all). By dissecting the differentially expressed genes, we found an overall downregulation of IFN-responsive genes (STAT1, IRF7, ISG15, ISG20, IFIs, and IFITMs) and S100s alarmins (S100A8, S100A9, S100A12, etc.) in all T-cell clusters, and cytotoxicity-related genes (GZMB, PRF1, and GNLY) in CTLs and γδ T cells in the FIhi cohort, compared with FIlo subjects. The GSEA analysis illustrated decreased expression of genes enriched in IFN signaling, innate immune response, adaptive immune response in T cells, NK cells, and monocytes in FIhi patients compared with FIlo ones. In conclusion, these data indicated that the attenuated IFN-responsive genes and their related signaling pathways could be critical for PF progression in COVID-19 patients.

Adipocyte-derived PGE2 is required for intermittent fasting-induced Treg proliferation and improvement of insulin sensitivity.

The intermittent fasting (IF) diet has profound benefits for diabetes prevention. However, the precise mechanisms underlying IF's beneficial effects remain poorly defined. Here, we show that the expression levels of cyclooxygenase-2 (COX-2), an enzyme that produces prostaglandins, are suppressed in white adipose tissue (WAT) of obese humans. In addition, the expression of COX-2 in WAT is markedly upregulated by IF in obese mice. Adipocyte-specific depletion of COX-2 led to reduced fractions of CD4+Foxp3+ Tregs and a substantial decrease in the frequency of CD206+ macrophages, an increase in the abundance of γδT cells in WAT under normal chow diet conditions, and attenuation of IF-induced antiinflammatory and insulin-sensitizing effects, despite a similar antiobesity effect in obese mice. Mechanistically, adipocyte-derived prostaglandin E2 (PGE2) promoted Treg proliferation through the CaMKII pathway in vitro and rescued Treg populations in adipose tissue in COX-2-deficient mice. Ultimately, inactivation of Tregs by neutralizing anti-CD25 diminished IF-elicited antiinflammatory and insulin-sensitizing effects, and PGE2 restored the beneficial effects of IF in COX-2-KO mice. Collectively, our study reveals that adipocyte COX-2 is a key regulator of Treg proliferation and that adipocyte-derived PGE2 is essential for IF-elicited type 2 immune response and metabolic benefits.

Three doses of an inactivation-based COVID-19 vaccine induces cross-neutralizing immunity against the SARS CoV-2 Omicron variant.

The immunity potency upon natural infection or vaccination is the main concern for the vaccine strategy of severe acute respiratory syndrome coronavirus 2 (SARS COV-2 variant), especially the recently reported Omicron variant (B.1.1.529). In this study, 200 recipients immunized with three doses of a COVID-19-inactivated vaccine were enrolled, whose serum samples were collected within 2 months after the third immunization. The neutralizing activity of sera against the pseudotyped Omicron variant, prototype, and Delta variant was determined. Our results demonstrated that the positive neutralization activity was 95.5% for the Omicron variant, 99.5% for the prototype, and 98.5% for the Delta variant. The geometric mean titers (GMT) for the Omicron variant was 49 and maintained sustained immune levels for 2 months, which decreased by 4.9-fold and 3.0-fold compared with the prototype (GMT, 239) and Delta variant (GMT, 148), respectively. In summary, our study demonstrated that three doses of a COVID-19-inactivated vaccine effectively yielded potent cross-neutralizing activity against the Omicron variant at 2 months after the third vaccination.