LXRα Promotes Abdominal Aortic Aneurysm Formation Through UHRF1 Epigenetic Modification of miR-26b-3p.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. METHODS: Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3-knockout and vascular smooth muscle cell-specific Nr1h3-knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl2; 0.5 mol/L; 42 days). RESULTS: Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl2-treated mice. Global or vascular smooth muscle cell-specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1, an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl2-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. CONCLUSIONS: Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

Identification and experimental validation of PYCARD as a crucial PANoptosis-related gene for immune response and inflammation in COPD.

Chronic inflammatory and immune responses play key roles in the development and progression of chronic obstructive pulmonary disease (COPD). PANoptosis, as a unique inflammatory cell death modality, is involved in the pathogenesis of many inflammatory diseases. We aim to identify critical PANoptosis-related biomarkers and explore their potential effects on respiratory tract diseases and immune infiltration landscapes in COPD. Total microarray data consisting of peripheral blood and lung tissue datasets associated with COPD were obtained from the GEO database. PANoptosis-associated genes in COPD were identified by intersecting differentially expressed genes (DEGs) with genes involved in pyroptosis, apoptosis, and necroptosis after normalizing and removing the batch effect. Furthermore, GO, KEGG, PPI network, WGCNA, LASSO-COX, and ROC curves analysis were conducted to screen and verify hub genes, and the correlation between PYCARD and infiltrated immune cells was analyzed. The effect of PYCARD on respiratory tract diseases and the potential small-molecule agents for the treatment of COPD were identified. PYCARD expression was verified in the lung tissue of CS/LPS-induced COPD mice. PYCARD was a critical PANoptosis-related gene in all COPD patients. PYCARD was positively related to NOD-like receptor signaling pathway and promoted immune cell infiltration. Moreover, PYCARD was significantly activated in COPD mice mainly by targeting PANoptosis. PANoptosis-related gene PYCARD is a potential biomarker for COPD diagnosis and treatment.

Identification of ferroptosis-related key genes associated with immune infiltration in sepsis by bioinformatics analysis and in vivo validation.

OBJECTIVES: Sepsis is a life-threatening infectious disease in which an immune inflammatory response is triggered. The potential effect of ferroptosis-related genes (FRGs) in inflammation of sepsis remained unclear. We focused on identifying and validating core FRGs and their association with immune infiltration in blood from currently all patients with sepsis. METHODS: All current raw data of septic blood were obtained from Gene Expression Omnibus. After removing the batch effect merging into a complete dataset and obtaining Diferentially expressed genes (DEGs). Common cross-talk genes were identified from DEGs and FRGs. WGCNA, GO, KEGG, PPI, GESA, ROC curves, and LASSO regression analysis were performed to indentify and validate key genes based on external septic datasets. Infiltrated immune cells in 2 hub genes (MAPK14 and ACSL4) were conducted using CIBERSORT algorithm and Spearman correlation analysis. Further, the expressions of 2 core FRGs were verified in the LPS-induced ALI and cardiac injury sepsis mice. RESULTS: MAPK14 and ACSL4 were identified, mostly enriched in T cell infiltration through NOD-like receptor signaling pathway according to the high or low 2 hub genes expression. The upregulated 2 ferroptosis-related genes were validated in LPS-induced ALI and cardiac injury mice, accompanied by upregulation of the NLRP3 pathway. CONCLUSION: MAPK14 and ACSL4 could become robustly reliable and promising biomarkers for sepsis by regulating ferroptosis through the NLRP3 pathway, which is mainly associated with T-cell infiltration.

Endothelial ELABELA improves post-ischemic angiogenesis by upregulating VEGFR2 expression.

BACKGROUND: Post-ischemic angiogenesis is critical for perfusion recovery and tissue repair. ELABELA (ELA) plays an essential role in embryonic heart development and vasculogenesis. However, the mechanism of ELA on post-ischemic angiogenesis is poorly characterized. METHODS: We first assessed ELA expression after hind limb ischemia (HLI) in mice. We then established a HLI model in tamoxifen-inducible endothelial-ELA-specific knockout mice (ELAECKO) and assessed the rate of perfusion recovery, capillary density, and VEGFR2 pathway. Knockdown of ELA with lentivirus or siRNA and exogenous addition of ELA peptides were employed to analyze the effects of ELA on angiogenic capacity and VEGFR2 pathway in endothelial cells in vitro. The serum levels of ELA in healthy people and patients with type 2 diabetes mellitus (T2DM) and diabetic foot ulcer (DFU) were detected by a commercial ELISA kit. RESULTS: In murine HLI models, ELA was significantly up-regulated in the ischemic hindlimb. Endothelial-specific deletion of ELA impaired perfusion recovery and angiogenesis. In physiologic conditions, no significant difference in VEGFR2 expression was found between ELAECKO mice and ELAWT mice. After ischemia, the expression of VEGFR2, p-VEGFR2, and p-AKT was significantly lower in ELAECKO mice than in ELAWT mice. In cellular experiments, the knockdown of ELA inhibited endothelial cell proliferation and tube formation, and the addition of ELA peptides promoted proliferation and tube formation. Mechanistically, ELA upregulated the expression of VEGFR2, p-VEGFR2, and p-AKT in endothelial cells under hypoxic conditions. In clinical investigations, DFU patients had significantly lower serum levels of ELA compared to T2DM patients. CONCLUSION: Our results indicated that endothelial ELA is a positive regulator of post-ischemic angiogenesis via upregulating VEGFR2 expression. Targeting ELA may be a potential therapeutic option for peripheral arterial diseases.

Sodium dichloroacetate improves migration ability by suppressing LPS-induced inflammation in HTR-8/SVneo cells via the TLR4/NF-κB pathway.

Objectives: Inadequate cytotrophoblast migration and invasion are speculated to result in preeclampsia, which is a pro-inflammatory condition. Sodium dichloroacetate (DCA) exerts anti-inflammatory actions. Thus,we sought to investigate the effect of DCA on the migration function of the lipopolysaccharide (LPS)-stimulated human-trophoblast-derived cell line (HTR-8/SVneo). Materials and Methods: HTR-8/SVneo cells were treated with LPS to suppress cell migration. Cell migration was examined by both scratch wound healing assay and transwell migration assay. Western blotting was used to analyze the expression levels of toll-like receptor-4 (TLR4), nuclear factor-κB (NF-κB), TNF-α, IL-1ß, and IL-6 in the cells. Results: DCA reversed LPS-induced inhibition of migration in HTR-8/SVneo cells. Furthermore, DCA significantly suppressed LPS-induced activation of TLR4, phosphorylation of NF-κB (p65), translocation of p65 into the nucleus, and the production of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6). Treatment with inhibitors of TLR4 signal transduction (CLI095 or MD2-TLR-4-IN-1) reduced LPS-induced overexpression of pro-inflammatory cytokines, and a synergistic effect was found between TLR4 inhibitors and DCA in HTR-8/SVneo cells. Conclusion: DCA improved trophoblast cell migration function by suppressing LPS-induced inflammation, at least in part, via the TLR4/NF-κB signaling pathway. This result indicates that DCA might be a potential therapeutic candidate for human pregnancy-related complications associated with trophoblast disorder.

O-GlcNAc modification of GSDMD attenuates LPS-induced endothelial cells pyroptosis.

OBJECTIVE: Increased O-linked ß-N-acetylglucosamine (O-GlcNAc) stimulation has been reported to protect against sepsis associated mortality and cardiovascular derangement. Previous studies, including our own research, have indicated that gasdermin-D(GSDMD)-mediated endothelial cells pyroptosis contributes to sepsis-associated endothelial injury. This study explored the functions and mechanisms of O-GlcNAc modification on lipopolysaccharide (LPS)-induced pyroptosis and its effects on the function of GSDMD. METHODS: A LPS-induced septic mouse model administrated with O-GlcNAcase (OGA) inhibitor thiamet-G (TMG) was used to assess the effects of O-GlcNAcylation on sepsis-associated vascular dysfunction and pyroptosis. We conducted experiments on human umbilical vein endothelial cells (HUVECs) by challenging them with LPS and TMG to investigate the impact of O-GlcNAcylation on endothelial cell pyroptosis and implications of GSDMD. Additionally, we identified potential O-GlcNAcylation sites in GSDMD by utilizing four public O-GlcNAcylation site prediction database, and these sites were ultimately established through gene mutation. RESULTS: Septic mice with increased O-GlcNAc stimulation exhibited reduced endothelial injury, GSDMD cleavage (a marker of pyroptosis). O-GlcNAc modification of GSDMD mitigates LPS-induced pyroptosis in endothelial cells by preventing its interaction with caspase-11 (a human homologous of caspases-4/5). We also identified GSDMD Serine 338 (S338) as a novel site of O-GlcNAc modification, leading to decreased association with caspases-4 in HEK293T cells. CONCLUSIONS: Our findings identified a novel post-translational modification of GSDMD and elucidated the O-GlcNAcylation of GSDMD inhibits LPS-induced endothelial injury, suggesting that O-GlcNAc modification-based treatments could serve as potential interventions for sepsis-associated vascular endothelial injury.

Functions, Mechanisms, and therapeutic applications of the inositol pyrophosphates 5PP-InsP5 and InsP8 in mammalian cells.

Water-soluble myo-inositol phosphates have long been characterized as second messengers. The signaling properties of these compounds are determined by the number and arrangement of phosphate groups on the myo-inositol backbone. Recently, higher inositol phosphates with pyrophosphate groups were recognized as signaling molecules. 5-Diphosphoinositol 1,2,3,4,6-pentakisphosphate (5PP-InsP5) is the most abundant isoform, constituting more than 90% of intracellular inositol pyrophosphates. 5PP-InsP5 can be further phosphorylated to 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). These two molecules, 5PP-InsP5 and InsP8, are present in various subcellular compartments, where they participate in regulating diverse cellular processes such as cell death, energy homeostasis, and cytoskeletal dynamics. The synthesis and metabolism of inositol pyrophosphates are subjected to tight regulation, allowing for their highly specific functions. Blocking the 5PP-InsP5/InsP8 signaling pathway by inhibiting the biosynthesis of 5PP-InsP5 demonstrates therapeutic benefits in preclinical studies, and thus holds promise as a therapeutic approach for certain diseases treatment, such as metabolic disorders.

Aging-Associated ALKBH5-m6A Modification Exacerbates Doxorubicin-Induced Cardiomyocyte Apoptosis Via AT-Rich Interaction Domain 2.

BACKGROUND: Chemotherapy-induced cardiovascular disease is a growing concern in the elderly population who have survived cancer, yet the underlying mechanism remains poorly understood. We investigated the role of ALKBH5 (AlkB homolog 5), a primary N6-methyladenosine (m6A) demethylase, and its involvement in m6A methylation-mediated regulation of targets in aging-associated doxorubicin-induced cardiotoxicity. METHODS AND RESULTS: To validate the relationship between doxorubicin-induced cardiotoxicity and aging, we established young and old male mouse models. ALKBH5 expression was modulated through adeno-associated virus 9 (in vivo), Lentivirus, and siRNAs (in vitro) to examine its impact on cardiomyocyte m6A modification, doxorubicin-induced cardiac dysfunction, and remodeling. We performed mRNA sequencing, methylated RNA immunoprecipitation sequencing, and molecular assays to unravel the mechanism of ALKBH5-m6A modification in doxorubicin-induced cardiotoxicity. Our data revealed an age-dependent increase in doxorubicin-induced cardiac dysfunction, remodeling, and injury. ALKBH5 expression was elevated in aging mouse hearts, leading to reduced global m6A modification levels. Through mRNA sequencing and methylated RNA immunoprecipitation sequencing analyses, we identified ARID2 (AT-rich interaction domain 2) as the downstream effector of ALKBH5-m6A modulation in cardiomyocytes. Further investigations revealed that ARID2 modulates DNA damage response and enhances doxorubicin-induced cardiomyocyte apoptosis. CONCLUSIONS: Our findings provide insights into the role of ALKBH5-m6A modification in modulating doxorubicin-induced cardiac dysfunction, remodeling, and cardiomyocyte apoptosis in male mice. These results highlight the potential of ALKBH5-targeted treatments for elderly patients with cancer in clinical settings.

Plasma metabolic fingerprints for large-scale screening and personalized risk stratification of metabolic syndrome.

Direct diagnosis and accurate assessment of metabolic syndrome (MetS) allow for prompt clinical interventions. However, traditional diagnostic strategies overlook the complex heterogeneity of MetS. Here, we perform metabolomic analysis in 13,554 participants from the natural cohort and identify 26 hub plasma metabolic fingerprints (PMFs) associated with MetS and its early identification (pre-MetS). By leveraging machine-learning algorithms, we develop robust diagnostic models for pre-MetS and MetS with convincing performance through independent validation. We utilize these PMFs to assess the relative contributions of the four major MetS risk factors in the general population, ranked as follows: hyperglycemia, hypertension, dyslipidemia, and obesity. Furthermore, we devise a personalized three-dimensional plasma metabolic risk (PMR) stratification, revealing three distinct risk patterns. In summary, our study offers effective screening tools for identifying pre-MetS and MetS patients in the general community, while defining the heterogeneous risk stratification of metabolic phenotypes in real-world settings.

iPSC-derived exosomes promote angiogenesis in naturally aged mice.

Heterochronic parabiosis has shown that aging individuals can be rejuvenated by a youthful circulatory system; however, the underlying mechanisms remain unclear. Here, we evaluated the effect of exosomes isolated from mouse induced pluripotent stem cells (iPSCs) on angiogenesis in naturally aged mice. To achieve this, the angiogenic capacity of aortic ring, the total antioxidant capacity (TAOC), p53 and p16 expression levels of major organs, the proliferation of adherent bone marrow cells, and the function and content of serum exosomes in aged mice administered iPSC-derived exosomes were examined. Additionally, the effect of iPSC-derived exosomes on injured human umbilical vein endothelial cells (HUVECs) was assessed. The angiogenic capacity of aortic rings and clonality of bone marrow cells from young mice were significantly higher than those from aged mice; moreover, the organs of aged mice had a higher expression of aging genes and lower total TAOC. However, in vitro and in vivo experiments showed that the administration of iPSC-derived exosomes significantly improved these parameters in aged mice. The synergistic effect of both in vivo and in vitro treatments of aortic rings with iPSC-derived exosomes improved the angiogenic capacity of aortic rings from aged mice to levels similar to that of young mice. Compared with untreated aged mice, serum exosomal protein content and their promoted effect on endothelial cell proliferation and angiogenesis were significantly higher in untreated young mice and aged mice treated with iPSC-derived exosomes. Overall, these results showed that iPSC-derived exosomes may rejuvenate the body by anti-aging the vascular system.

Induction of KLF2 by Exercise Activates eNOS to Improve Vasodilatation in Diabetic Mice.

Diabetic endothelial dysfunction associated with diminished endothelial nitric oxide (NO) synthase (eNOS) activity accelerates the development of atherosclerosis and cardiomyopathy. However, the approaches to restore eNOS activity and endothelial function in diabetes remain limited. The current study shows that enhanced expression of Krüppel-like factor 2 (KLF2), a shear stress-inducible transcription factor, effectively improves endothelial function through increasing NO bioavailability. KLF2 expression is suppressed in diabetic mouse aortic endothelium. Running exercise and simvastatin treatment induce endothelial KLF2 expression in db/db mice. Adenovirus-mediated endothelium-specific KLF2 overexpression enhances both endothelium-dependent relaxation and flow-mediated dilatation, while it attenuates oxidative stress in diabetic mouse arteries. KLF2 overexpression increases the phosphorylation of eNOS at serine 1177 and eNOS dimerization. RNA-sequencing analysis reveals that KLF2 transcriptionally upregulates genes that are enriched in the cyclic guanosine monophosphate-protein kinase G-signaling pathway, cAMP-signaling pathway, and insulin-signaling pathway, all of which are the upstream regulators of eNOS activity. Activation of the phosphoinositide 3-kinase-Akt pathway and Hsp90 contributes to KLF2-induced increase of eNOS activity. The present results suggest that approaches inducing KLF2 activation, such as physical exercise, are effective to restore eNOS activity against diabetic endothelial dysfunction. ARTICLE HIGHLIGHTS: Exercise and statins restore the endothelial expression of Krüppel-like factor 2 (KLF2), which is diminished in diabetic db/db mice. Endothelium-specific overexpression of KLF2 improves endothelium-dependent relaxation and flow-mediated dilation through increasing nitric oxide bioavailability. KLF2 promotes endothelial nitric oxide synthase (eNOS) coupling and phosphorylation in addition to its known role in eNOS transcription. KLF2 upregulates the expression of several panels of genes that regulate eNOS activity.

S1PR2/Wnt3a/RhoA/ROCK1/ß-catenin signaling pathway promotes diabetic nephropathy by inducting endothelial mesenchymal transition and impairing endothelial barrier function.

AIMS: Hyperglycemia and hyperlipidemia are key factors in the pathogenesis of diabetic nephropathy (DN), and renal fibrosis is the most common pathway leading to the disease. Endothelial mesenchymal transition (EndMT) is a crucial mechanism for the production of myofibroblasts, and impaired endothelial barrier function is one of the mechanisms for the generation of microalbuminuria in DN. However, the specific mechanisms behind these are not yet clear. MAIN METHODS: Protein expression was detected by immunofluorescence, immunohistochemistry and Western blot. Knocking down or pharmacological inhibition of S1PR2 were used to inhibit Wnt3a, RhoA, ROCK1, ß-catenin, and Snail signaling. Changes in cell function were analyzed by CCK-8 method, cell scratching assay, FITC-dextran permeability assay, and Evans blue staining. KEY FINDINGS: Consistent with increased gene expression of S1PR2 in DN patients and mice with kidney fibrosis disease, S1PR2 expression was significantly increased in glomerular endothelial cells of DN mice and HUVEC cells treated with glucolipids. Knocking down or pharmacological inhibition of S1PR2 significantly decreased the expression of Wnt3a, RhoA, ROCK1, and ß-catenin in endothelial cells. Furthermore, inhibition of S1PR2 in vivo reversed EndMT and endothelial barrier dysfunction in glomerular endothelial cells. Inhibition of S1PR2 and ROCK1 in vitro also reversed EndMT and endothelial barrier dysfunction in endothelial cells. SIGNIFICANCE: Our results suggest that the S1PR2/Wnt3a/RhoA/ROCK1/ß-catenin signaling pathway is involved in the pathogenesis of DN by inducing EndMT and endothelial barrier dysfunction.

Characterizing the cellular and molecular variabilities of peripheral immune cells in healthy recipients of BBIBP-CorV inactivated SARS-CoV-2 vaccine by single-cell RNA sequencing.

Over 3 billion doses of inactivated vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been administered globally. However, our understanding of the immune cell functional transcription and T cell receptor (TCR)/B cell receptor (BCR) repertoire dynamics following inactivated SARS-CoV-2 vaccination remains poorly understood. Here, we performed single-cell RNA and TCR/BCR sequencing on peripheral blood mononuclear cells at four time points after immunization with the inactivated SARS-CoV-2 vaccine BBIBP-CorV. Our analysis revealed an enrichment of monocytes, central memory CD4+ T cells, type 2 helper T cells and memory B cells following vaccination. Single-cell TCR-seq and RNA-seq comminating analysis identified a clonal expansion of CD4+ T cells (but not CD8+ T cells) following a booster vaccination that corresponded to a decrease in the TCR diversity of central memory CD4+ T cells and type 2 helper T cells. Importantly, these TCR repertoire changes and CD4+ T cell differentiation were correlated with the biased VJ gene usage of BCR and the antibody-producing function of B cells post-vaccination. Finally, we compared the functional transcription and repertoire dynamics in immune cells elicited by vaccination and SARS-CoV-2 infection to explore the immune responses under different stimuli. Our data provide novel molecular and cellular evidence for the CD4+ T cell-dependent antibody response induced by inactivated vaccine BBIBP-CorV. This information is urgently needed to develop new prevention and control strategies for SARS-CoV-2 infection. (ClinicalTrials.gov Identifier: NCT04871932).

The TRPM7 channel reprograms cellular glycolysis to drive tumorigenesis and angiogenesis.

Cancer or endothelial cells preferably catabolize glucose through aerobic glycolysis rather than oxidative phosphorylation. Intracellular ionic signaling has been shown to regulate glucose metabolism, but the underlying ion channel has yet to be identified. RNA-seq, metabolomics and genetic assay revealed that the TRPM7 channel regulated cellular glycolysis. Deletion of TRPM7 suppressed cancer cell glycolysis and reduced the xenograft tumor burden. Deficiency of endothelial TRPM7 inhibited postnatal retinal angiogenesis in mice. Mechanistically, TRPM7 transcriptionally regulated the solute carrier family 2 member 3 (SLC2A3, also known as GLUT3) via Ca2+ influx-induced calcineurin activation. Furthermore, CREB-regulated transcription coactivator 2 (CRTC2) and CREB act downstream of calcineurin to relay Ca2+ signal to SLC2A3 transcription. Expression of the constitutively active CRTC2 or CREB in TRPM7 knockout cell normalized glycolytic metabolism and cell growth. The TRPM7 channel represents a novel regulator of glycolytic reprogramming. Inhibition of the TRPM7-dependent glycolysis could be harnessed for cancer therapy.

The beneficial effects of alpha-tocopherol on intestinal function and the expression of tight junction proteins in differentiated segments of the intestine in piglets.

Alpha (α)-tocopherol is a major component of dietary vitamin E. Despite being one of the most widely used food supplements in both animals and humans, its role in intestinal functions remains unknown. We were able to examine and accurately demonstrate its permeability effect in vitro and its differentiated effect on tight junction expression in different segments of the intestine in vivo using cultured intestinal porcine epithelial cell line (IPEC-J2) and piglets. A cultured IPEC-J2 demonstrated that α-tocopherol upregulated the expression of tight junction proteins and improved their integrity, with a maximum effect at concentrations ranging from 20 to 40 µmol/L. In vivo data from weaned pigs fed different doses of α-tocopherol for 2 weeks revealed that α-tocopherol effectively increases the expression of tight junction proteins in all sections of the intestinal mucosa, with the highest effect on the duodenum at an optimum dose of 20-50 mg/kg. In contrast, α-tocopherol did not affect intestinal inflammation. These findings suggest that α-tocopherol maintains intestinal integrity and increases the expression of tight junction proteins both in vitro and in vivo.

ASGR1 promotes liver injury in sepsis by modulating monocyte-to-macrophage differentiation via NF-κB/ATF5 pathway.

AIMS: Liver is a pivotal organ for sepsis-induced injury and approximately 40 % of liver injury results from sepsis. During hepatic injury, monocyte-to-macrophage differentiation is a key event because it results in the regulation of immune response. Asialoglycoprotein receptor 1 (ASGR1) is enriched in classical monocyte of peripheral blood mononuclear cells (PBMCs). We aimed to explore the effect of ASGR1 on monocyte-to-macrophage differentiation and the modulation of sepsis-induced liver injury. MAIN METHODS: ASGR1-knockdown/overexpression THP-1 cells and mice bone marrow-derived macrophages (BMDMs) induced by PMA and 30 % L929-cell conditioned medium were utilized to test the impact of ASGR1 on monocyte-to-macrophage differentiation and molecular mechanism respectively. Expression of differentiation specific factors were assessed via flow cytometry and real-time quantitative PCR. RNA-sequencing (RNA-seq) analysis revealed the effect of ASGR1 on monocyte-to-macrophage differentiation. Further, differentiation specific factors ATF5 and NF-κB pathways were examined via Western blot. The interaction between ASGR1 and ATF5 was further examined by co-IP. Finally, LPS-induced ASGR1-knockdown mice sepsis was used to investigate the effect of ASGR1 on monocyte-to-macrophage differentiation, liver injury and survival. KEY FINDINGS: ASGR1 promoted monocyte-to-macrophage differentiation via up-regulating CD68, F4/80 and CD86. Additionally, inhibited-ASGR1 decreased ATF5 expression by suppressing phosphorylation of NF-κB and IKBa in vitro and in vivo. ASGR1-knockdown mice suppressed Ly6Chi inflammatory monocytes in PBMCs, and restrained CD45+CD11bhiF4/80+Ly6Clo monocyte-derived macrophages and CD45+CD11b+F4/80+Ly6C+ inflammatory macrophages in livers. It also suppressed the level of IL-1ß, IL-6, TNF-α and alleviated liver injury and improved survival after sepsis. SIGNIFICANCE: ASGR1 is a negative regulator for sepsis-induced liver injury and survival.

Sema3G activates YAP and promotes VSMCs proliferation and migration via Nrp2/PlexinA1.

BACKGROUND: Diabetes exacerbates neointima formation after vascular procedures, manifested by accelerated proliferation and migration of vascular smooth muscle cells (VSMCs). Semaphorin 3G (Sema3G), secreted mainly from endothelial cells (ECs), regulates various cellular functions and vascular pathologies. However, the function and potential mechanism of ECs-derived Sema3G in VSMCs under diabetic condition remain unclear. OBJECTIVE: To investigate the role and the mechanism of ECs-derived Sema3G in the regulation of VSMCs proliferation and migration. RESULTS: ECs-derived Sema3G promoted human aortic SMCs (HASMCs) cell cycle progression and proliferation. Sema3G upregulated the expression of MMP2 and MMP9, which might explain the increased HASMCs migration by Sema3G. Inhibition of Nrp2/PlexinA1 mitigated the effect of Sema3G on promoting HASMCs proliferation and migration. Mechanistically, Sema3G inhibited LATS1 and activated YAP via Nrp2/PlexinA1. Verteporfin, an FDA-approved YAP pathway inhibitor, counteracted Sema3G-induced cyclin E and cyclin D1 expression. Besides, Sema3G expression was upregulated in ECs of diabetic mouse aortas. Serum Sema3G level was increased in type 2 diabetic patients and mice. Moreover, compared to chow diet-fed mice, high-fat diet (HFD)-fed obese mice showed thicker neointima and higher Sema3G expression in vasculature after femoral injury. CONCLUSIONS: Our results indicated that ECs-derived Sema3G under diabetic condition activated YAP and promoted HASMCs proliferation and migration via Nrp2/PlexinA1. Thus, inhibition of Sema3G may hold therapeutic potential against diabetes-associated intimal hyperplasia.

Low-dose nifedipine rescues impaired endothelial progenitor cell-mediated angiogenesis in diabetic mice.

It is of great clinical significance to develop potential novel strategies to prevent diabetic cardiovascular complications. Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic vascular complications. In the present study we evaluated whether low-dose nifedipine could rescue impaired EPC-mediated angiogenesis and prevent cardiovascular complications in diabetic mice. Diabetes was induced in mice by five consecutive injections of streptozotocin (STZ, 60 mg·kg-1·d-1, i.p.). Diabetic mice were treated with low-dose nifedipine (1.5 mg·kg-1·d-1, i.g.) for six weeks. Then, circulating EPCs in the peripheral blood were quantified, and bone marrow-derived EPCs (BM-EPCs) were prepared. We showed that administration of low-dose nifedipine significantly increased circulating EPCs, improved BM-EPCs function, promoted angiogenesis, and reduced the cerebral ischemic injury in diabetic mice. Furthermore, we found that low-dose nifedipine significantly increased endothelial nitric oxide synthase (eNOS) expression and intracellular NO levels, and decreased the levels of intracellular O2.- and thrombospondin-1/2 (TSP-1/2, a potent angiogenesis inhibitor) in BM-EPCs of diabetic mice. In cultured BM-EPCs, co-treatment with nifedipine (0.1, 1 µM) dose-dependently protected against high-glucose-induced impairment of migration, and suppressed high-glucose-induced TSP-1 secretion and superoxide overproduction. In mice with middle cerebral artery occlusion, intravenous injection of diabetic BM-EPCs treated with nifedipine displayed a greater ability to promote local angiogenesis and reduce cerebral ischemic injury compared to injection of diabetic BM-EPCs treated with vehicle, and the donor-derived BM-EPCs homed to the recipient ischemic brain. In conclusion, low-dose nifedipine can enhance EPCs' angiogenic potential and protect against cerebral ischemic injury in diabetic mice. It is implied that chronic treatment with low-dose nifedipine may be a safe and economic manner to prevent ischemic diseases (including stroke) in diabetes.

Berberine hydrochloride inhibits migration ability via increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Inadequate trophoblasts migration and invasion is considered as an initial event resulting in preeclampsia, which is closely related to oxidative stress. Berberine hydrochloride (BBR), extracted from the traditional medicinal plant Coptis chinensis Franch., exerts a diversity of pharmacological effects, and the crude drug has been widely taken by most Chinese women to treat nausea and vomit during pregnancy. But there is no research regarding its effects on trophoblast cell function. AIM OF THE STUDY: This study aimed to investigate the effect of BBR on human-trophoblast-derived cell line (HTR-8/SVneo) migration ability and its mechanism. MATERIALS AND METHODS: Cell viability was detected by CCK-8 assay. The effect of BBR on cells migration function was examined by scratch wound healing assay and transwell migration assay. Intracellular nitric oxide (NO), superoxide (O2-) and peroxynitrite (ONOO-) levels were measured by flow cytometry. The expression levels of inducible NO synthase (iNOS), eNOS, p-eNOS, MnSOD, CuZnSOD, Rac1, NOX1, TLR4, nuclear factor-κB (NF-κB), p-NFκB, pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) in cells were analyzed by Western blotting. Uric acid sodium salt (UA), the scavenger of ONOO-, PEG-SOD (a specific superoxide scavenger), L-NAME (a NOS inhibitor) and antioxidants (Vit E and DFO) were further used to characterize the pathway of BBR action. RESULTS: 5 µM BBR decreased both the migration distance and the number of migrated cells without affecting cells viability in HTR-8/SVneo cells after 24 h treatment. BBR could increase the level of NO in HTR-8/SVneo cells, and the over-production of NO might be attributable to iNOS, but not eNOS. BBR could increase intracellular O2- levels, and the over-production of O2- is closely related with Rac1 in HTR-8/SVneo cells. The excessive production of NO and O2- further react to form ONOO-, and the increased ONOO- level induced by BBR was blunted by UA. Moreover, UA improved the impaired migration function caused by BBR in HTR-8/SVneo cells. The depressed migration function stimulated by BBR in HTR-8/SVneo cells was diminished by PEG-SOD and L-NAME. Furthermore, BBR increased the expression of IL-6 in HTR-8/SVneo cells, and antioxidants (Vit E and DFO) could decrease the expression of IL-6 and iNOS induced by BBR. CONCLUSIONS: BBR inhibits the cell migration ability through increasing inducible NO synthase and peroxynitrite in HTR-8/SVneo cells, indicating that BBR and traditional Chinese medicines containing a high proportion of BBR should be used with caution in pregnant women.