NLRC3 silencing accelerates the invasion of hepatocellular carcinoma cell via IL-6/JAK2/STAT3 pathway activation.

Nucleotide-binding domain, leucine-rich repeat family with a caspase activation and recruitment domain 3 (NLRC3) participates in both immunity and cancer. The aim of this study was to determine the role of NLRC3 in human hepatocellular carcinoma (HCC) and the underlying mechanisms. We collected human liver tissues from nonalcoholic steatohepatitis (NASH), HCC, and adjacent normal tissues to characterize the pattern of NLRC3 expression by real-time quantitative polymerase chain reaction and immunohistochemistry. Then, we used the HCC cell line, HuH-7, transfected with small interfering RNA to silence the NLRC3 expression. 5-Ethynyl-2'-deoxyuridine assay, scratch assay, and transwell invasion assay were used for assessing proliferation, migration, and invasion, respectively. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted to assess cell apoptosis. The expression of NLRC3 was reduced in human HCC tissues, compared with normal liver and nonalcoholic steatohepatitis tissues. After knocking down of NLRC3, the proliferation, migration, and invasion were increased in HuH-7 cells. And flow cytometry and TUNEL assay showed that HuH-7 cell apoptosis was suppressed after NLRC3 knockdown. As for the underlying mechanisms, knockdown of NLRC3 in HuH-7 cells was associated with the activation of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway under interleukin-6 (IL-6) stimulation. NLRC3 expression was downregulated in human HCC tissues. NLRC3 silencing in HuH-7 cells can promote the proliferation, migration, and invasion of hepatocellular carcinoma cells. JAK2/STAT3 pathway activation induced by IL-6 may be the underlying mechanism for HCC when NLRC3 expression is silenced. And the invasion of HuH-7 cells was partially suppressed by the STAT3 specific inhibitor (cryptotanshinone). Therefore, NLRC3 may play a significant role in HCC and might be a therapeutic target for the treatment of HCC.

FNDC5/Irisin inhibits pathological cardiac hypertrophy.

Cardiac hypertrophy is a common pathophysiological process in various cardiovascular diseases, which still has no effective therapies. Irisin is a novel myokine mainly secreted by skeletal muscle and is believed to be involved in the regulation of energy metabolism. In the present study, we found that irisin expression was elevated in hypertrophic murine hearts and serum. Moreover, angiotension II-induced cardiomyocyte hypertrophy was attenuated after irisin administration and aggravated after irisin knockdown in vitro Next, we generated transverse aortic constriction (TAC)-induced cardiac hypertrophy murine model and found that cardiac hypertrophy and fibrosis were significantly attenuated with improved cardiac function assessed by echocardiography after irisin treatment. Mechanistically, we demonstrated that FNDC5 was cleaved into irisin, at least partially, in a disintegrin and metalloproteinase (ADAM) family-dependent manner. ADAM10 was the candidate enzyme responsible for the cleavage. Further, we found irisin treatment activated AMPK and subsequently inhibited activation of mTOR. AMPK inhibition ablated the protective role of irisin administration. In conclusion, we find irisin is secreted in an ADAM family-dependent manner, and irisin treatment improves cardiac function and attenuates pressure overload-induced cardiac hypertrophy and fibrosis mainly through regulating AMPK-mTOR signaling.

NLRC5 deficiency ameliorates diabetic nephropathy through alleviating inflammation.

NOD-like receptor family caspase recruitment domain family domain containing 5 (NLRC5) has important roles in inflammation and innate immunity. NLRC5 was highly expressed in kidney from streptozotocin-induced diabetic mice, db/ db mice and patients with diabetes. Based on that evidence, the present study was designed to explore the roles of NLRC5 in the progression of diabetic nephropathy (DN). We examined kidney injury, including inflammation and fibrosis in Nlrc5 gene knockout ( Nlrc5-/-) and wild-type (WT) diabetic mice. We found that Nlrc5-/- mice developed less-severe diabetic kidney injury compared with WT mice, exhibiting lower albuminuria, less fibronectin and collagen IV expression, and reduced macrophage infiltration but greater levels of podocin and nephrin in the diabetic kidney. The underlying mechanisms were further investigated in vitro with peritoneal macrophages and mesangial cells treated with high glucose. Reduced proinflammatory effect was observed in peritoneal macrophages from Nlrc5-/- mice, associated with NF-κB pathway suppression. Knocking down of NLRC5 in mesangial cells in high-glucose conditions was also associated with reduced NF-κB and TGF-ß/Smad signaling. Taken together, NLRC5 promotes inflammation and fibrosis during DN progression partly through the effects on NF-κB and TGF-ß/Smad pathways. NLRC5 may, therefore, be a promising therapeutic target for DN treatment.-Luan, P., Zhuang, J., Zou, J., Li, H., Shuai, P., Xu, X., Zhao, Y., Kou, W., Ji, S., Peng, A., Xu, Y., Su, Q., Jian, W., Peng, W. NLRC5 deficiency ameliorates diabetic nephropathy through alleviating inflammation.

The Yin-Yang Dynamics of DNA Methylation Is the Key Regulator for Smooth Muscle Cell Phenotype Switch and Vascular Remodeling.

OBJECTIVE: DNA methylation plays an important role in chronic diseases such as atherosclerosis, yet the mechanisms are poorly understood. The objective of our study is to indicate the regulatory mechanisms of DNA methylation in vascular smooth muscle cells (VSMCs) and its roles in atherosclerosis. APPROACH AND RESULTS: In ApoE-/- mice fed a Western diet, DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, significantly attenuated atherosclerotic lesions (20.1±2.2% versus 30.8±7.5%; P=0.016) and suppressed DNA methyltransferase activity and concomitantly decreased global 5-methylcytosine content in atherosclerotic lesions of ApoE-/- mice. Using a carotid ligation model, we found that 5-aza-2'-deoxycytidine also dramatically inhibited neointimal formation (intimal area: 2.25±0.14×104 versus 4.07±0.22×104 µm2; P<0.01). Abnormal methylation status at the promoter of ten-eleven translocation 2, one of the key demethylation enzymes in mammals, was ameliorated after 5-aza-2'-deoxycytidine treatment, which in turn caused an increase in global DNA hydroxymethylation and 5-hydroxymethylcytosine enrichment at the promoter of Myocardin. In vitro, 5-aza-2'-deoxycytidine treatment or DNA methyltransferase 1 knockdown decreased global 5-methylcytosine content and restored Myocardin expression in VSMCs induced by platelet-derived growth factor, thus preventing excessive VSMCs dedifferentiation, proliferation, and migration. Furthermore, DNA methyltransferase 1 binds to ten-eleven translocation 2 promoter and is required for ten-eleven translocation 2 methylation in VSMCs. CONCLUSIONS: The inhibitory effects of DNA demethylation on global 5-methylcytosine content and ten-eleven translocation 2 hypermethylation in atherosclerotic aorta can recover 5-hydroxymethylcytosine enrichment at the Myocardin promoter and prevent VSMC dedifferentiation and vascular remodeling.

Extracellular traps from activated vascular smooth muscle cells drive the progression of atherosclerosis.

Extracellular DNA traps (ETs) represent an immune response by which cells release essential materials like chromatin and granular proteins. Previous studies have demonstrated that the transdifferentiation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. This study seeks to investigate the interaction between CD68+ VSMCs and the formation of ETs and highlight its function in atherosclerosis. Here we show that ETs are inhibited, and atherosclerotic plaque formation is alleviated in male Myh11CrePad4flox/flox mice undergoing an adeno-associated-virus-8 (AAV8) mediating overexpression of proprotein convertase subtilisin/kexin type 9 mutation (PCSK9) injection and being challenged with a high-fat diet. Obvious ETs generated from CD68+ VSMCs are inhibited by Cl-amidine and DNase I in vitro. By utilizing VSMCs-lineage tracing technology and single-cell RNA sequencing (scRNA-seq), we demonstrate that the ETs from CD68+ VSMCs influence the progress of atherosclerosis by regulating the direction of VSMCs' transdifferentiation through STING-SOCS1 or TLR4 signaling pathway.

Identification of Three Significant Genes Associated with Immune Cells Infiltration in Dysfunctional Adipose Tissue-Induced Insulin-Resistance of Obese Patients via Comprehensive Bioinformatics Analysis.

BACKGROUND: Low-grade chronic inflammation in dysfunctional adipose tissue links obesity with insulin resistance through the activation of tissue-infiltrating immune cells. Numerous studies have reported on the pathogenesis of insulin-resistance. However, few studies focused on genes from genomic database. In this study, we would like to explore the correlation of genes and immune cells infiltration in adipose tissue via comprehensive bioinformatics analyses and experimental validation in mice and human adipose tissue. METHODS: Gene Expression Omnibus (GEO) datasets (GSE27951, GSE55200, and GSE26637) of insulin-resistant individuals or type 2 diabetes patients and normal controls were downloaded to get differently expressed genes (DEGs), and GO and KEGG pathway analyses were performed. Subsequently, we integrated DEGs from three datasets and constructed commonly expressed DEGs' PPI net-works across datasets. Center regulating module of DEGs and hub genes were screened through MCODE and cytoHubba in Cytoscape. Three most significant hub genes were further analyzed by GSEA analysis. Moreover, we verified the predicted hub genes by performing RT qPCR analysis in animals and human samples. Besides, the relative fraction of 22 immune cell types in adipose tissue was detected by using the deconvolution algorithm of CIBERSORT (Cell Type Identification by Estimating Relative Subsets of RNA Transcripts). Furthermore, based on the significantly changed types of immune cells, we performed correlation analysis between hub genes and immune cells. And, we performed immunohistochemistry and immunofluorescence analysis to verify that the hub genes were associated with adipose tissue macrophages (ATM). RESULTS: Thirty DEGs were commonly expressed across three datasets, most of which were upregulated. DEGs mainly participated in the process of multiple immune cells' infiltration. In protein-protein interaction network, we identified CSF1R, C1QC, and TYROBP as hub genes. GSEA analysis suggested high expression of the three hub genes was correlated with immune cells functional pathway's activation. Immune cell infiltration and correlation analysis revealed that there were significant positive correlations between TYROBP and M0 macrophages, CSF1R and M0 macrophages, Plasma cells, and CD8 T cells. Finally, hub genes were associated with ATMs infiltration by experimental verification. CONCLUSIONS: This article revealed that CSF1R, C1QC, and TYROBP were potential hub genes associated with immune cells' infiltration and the function of proinflammation, especially adipose tissue macrophages, in the progression of obesity-induced diabetes or insulin-resistance.

The subcellular redistribution of NLRC5 promotes angiogenesis via interacting with STAT3 in endothelial cells.

Angiogenesis is a critical step in repair of tissue injury. The pattern recognition receptors (PRRs) recognize pathogen and damage associated molecular patterns (DAMPs) during injury and achieve host defense directly. However, the role of NLR family CARD domain containing 5 (NLRC5), an important member of PPRs, beyond host defense in angiogenesis during tissue repair remains unknown. Methods:In vitro, western blot and real-time PCR (RT-PCR) were used to detect the expression of NLRC5 in endothelial cells (ECs). Immunofluorescence microscopy was used to reveal the subcellular location of NLRC5 in ECs. Cell proliferation, wound healing, tube formation assays of ECs were performed to study the role of NLRC5 in angiogenesis. By using Tie2Cre-NLRC5flox/flox mice and bone marrow transplantation studies, we defined an EC-specific role for NLRC5 in angiogenesis. Mechanistically, co-immunoprecipitation studies and RNA sequencing indicated that signal transducer and activator of transcription 3 (STAT3) was the target of NLRC5 in the nucleus. And Co-IP was used to verify the specific domain of NLRC5 binding with STAT3. ChIP assay determined the genes regulated by interaction of STAT3 and NLRC5. Results: Knockdown of NLRC5 in vitro or in vivo inhibited pathological angiogenesis, but had no effect on physiological angiogenesis. NLRC5 was also identified to bind to STAT3 in the nucleus required the integrated death-domain and nucleotide-binding domain (DD+NACHT domain) of NLRC5. And the interaction of STAT3 and NLRC5 could enhance the transcription of angiopoietin-2 (Ang2) and cyclin D1 (CCND1) to participate in angiogenesis. Conclusions: In the ischemic microenvironment, NLRC5 protein accumulates in the nucleus of ECs and enhances STAT3 transcriptional activity for angiogenesis. These findings establish NLRC5 as a novel modulator of VEGFA signaling, providing a new target for angiogenic therapy to foster tissue regeneration.

Decreased plasma musclin levels are associated with potential atrial fibrillation in non-diabetic patients.

BACKGROUND: Musclin is involved in the regulation of natriuretic peptide (NP) clearance and may affect the concentration of atrial natriuretic peptide (ANP). It has also been found to play an important role in several diseases, such as diabetes mellitus and hypertension. Both abnormalities in ANP and associated medical history are involved in the pathogenesis of atrial fibrillation (AF). However, plasma concentration of musclin as a biomarker for risk stratification in patients with AF has not been fully investigated. METHODS: Plasma musclin levels were measured in 290 patients with AF (including 199 paroxysmal AF patients and 91 persistent AF patients) and 120 control subjects. The association between plasma musclin levels and AF onset, as well as its predictive effects on clinical outcomes after cryoballoon ablation were analyzed. RESULTS: AF patients were found to have a lower concentration of plasma musclin than healthy controls. Moreover, in the non-diabetic group and normal N-terminal pro-brain natriuretic peptide (NT-proBNP) level group, the association between lower plasma concentration of musclin and AF remained significant. According to receiver operating characteristic (ROC) curve analysis, the optimal cut-off point of musclin for predicting AF onset was 54.94 ng/mL, which had a sensitivity of 81.67% and a specificity of 31.47% [area under the ROC curve (AUC) =60.71%]. In follow-up studies, both diabetes and left atrial size were independent predictors of AF recurrence after ablation, while musclin showed only a modest relationship with the outcome of cryoballoon ablation. CONCLUSIONS: Our data indicated that decreased musclin was associated with the onset of AF. Moreover, lower plasma levels of musclin were an independent risk factor of AF in non-diabetic patients. Our studies suggest that musclin could be a predictive factor for the onset of AF.

miR­124­3p regulates angiogenesis in peripheral arterial disease by targeting STAT3.

Peripheral arterial disease (PAD) is the third leading cause of cardiovascular morbidity worldwide, after coronary artery disease and stroke. As endogenous regulators of gene expression, microRNAs (miRs) are implicated in the development and progression of various diseases, including types of cancer, autoimmune diseases and heart diseases. In the present study, the role of miR­124­3p in PAD was investigated. The reverse transcription­quantitative PCR results indicated that the expression levels of miR­124­3p were significantly increased in the ischemic tissue of the hindlimb ischemia (HLI) model and in hypoxic human umbilical vein endothelial cells compared with the corresponding control groups. Proliferation, wound healing and tube formation assays demonstrated the inhibition of miR­124­3p on angiogenesis in vitro and the HLI model indicated the same function of miR­124­3p in vivo. A dual­luciferase reporter revealed STAT3 as the target of miR­124­3p. The expression levels of miR­124­3p in human blood were negatively correlated with ankle­brachial index, which is an index for the evaluation of the severity of PAD. Collectively, the present study indicated that miR­124­3p was a critical regulator of angiogenesis in PAD, and a potential diagnostic, prognostic and therapeutic target for PAD.

Plasma vaspin is an effective biomarker for evaluation of future cardiovascular events in patients with chest pain: a 5-year retrospective observational study.

BACKGROUND: Our previous study showed that visceral adipose tissue-derived serpin (vaspin) was an independent predictor of coronary artery disease (CAD). Further, plasma vaspin levels in patients with unstable angina pectoris were lower than those in patients with stable angina pectoris. In this study, we investigated the prognostic relevance of plasma vaspin levels in patients with CAD and non-CAD. METHODS: It was a retrospective observational study. A total of 197 patients with chest pain were enrolled, of which 88 patients with CAD and 109 patients with non-CAD were confirmed by angiography. Plasma vaspin levels and clinical parameters were measured at baseline. Incidence of major adverse cardiac event (MACE) was determined on follow-up. RESULTS: One hundred eighty-nine patients were successfully followed up for 5 years, of which 63 patients experienced MACEs. Patients with low vaspin levels (<0.385 ng/mL) experienced a higher incidence of MACE as compared to patients with high vaspin levels (>0.385 ng/mL) (42.55% vs. 24.21%, respectively; P=0.007). In both CAD and non-CAD groups, patients with high vaspin levels showed improvement in left ventricular ejection fraction. Kaplan Meier survival curves showed that patients with low vaspin levels had an obviously higher timing of incidence of MACE in the whole population (P=0.006) and in the non-CAD subgroup (P=0.009); however, the trend was not significant in the CAD subgroup. On multivariate analyses, plasma vaspin level was found to be an independent predictor of MACE, particularly in the non-CAD group. CONCLUSIONS: Plasma vaspin may be a useful biomarker for prediction of MACE in patients with chest pain.

NLRC5 inhibits neointima formation following vascular injury and directly interacts with PPARγ.

NLR Family CARD Domain Containing 5 (NLRC5), an important immune regulator in innate immunity, is involved in regulating inflammation and antigen presentation. However, the role of NLRC5 in vascular remodeling remains unknown. Here we report the role of NLRC5 on vascular remodeling and provide a better understanding of its underlying mechanism. Nlrc5 knockout (Nlrc5-/-) mice exhibit more severe intimal hyperplasia compared with wild-type mice after carotid ligation. Ex vivo data shows that NLRC5 deficiency leads to increased proliferation and migration of human aortic smooth muscle cells (HASMCs). NLRC5 binds to PPARγ and inhibits HASMC dedifferentiation. NACHT domain of NLRC5 is essential for the interaction with PPARγ and stimulation of PPARγ activity. Pioglitazone significantly rescues excessive intimal hyperplasia in Nlrc5-/- mice and attenuates the increased proliferation and dedifferentiation in NLRC5-deficient HASMCs. Our study demonstrates that NLRC5 regulates vascular remodeling by directly inhibiting SMC dysfunction via its interaction with PPARγ.