Advanced Glycation End Products Induce Atherosclerosis via RAGE/TLR4 Signaling Mediated-M1 Macrophage Polarization-Dependent Vascular Smooth Muscle Cell Phenotypic Conversion

Objective. The objective of this study was to investigate the involved mechanisms of advanced glycation end product- (AGE-) exacerbated atherosclerosis (AS). Methods. Toll-like receptor 4 (TLR4) inhibitor was administrated to type 2 diabetes mellitus (T2DM) AS rats. Atherosclerotic plaque, M1 macrophage infiltration, and VSMCs phenotypes were evaluated. AGE-exposed primary macrophages were treated with specific siRNAs knocking down receptor for AGEs (RAGE) and TLR4. Phenotypes of M1 macrophage and VSMCs were identified by fluorescent stains. Contact and noncontact coculture models were established. VSMCs and macrophages were cocultured in these models. ELISA was used to detect inflammatory cytokine concentrations. Relative mRNA expression levels were determined by real-time PCR. Relative protein expression and phosphorylation levels were evaluated by Western blots assays. Results. TLR4 inhibitor treatment significantly reduced arterial stenosis, infiltration of M1 polarized macrophages, and contractile-to-synthetic phenotype conversion of VSMCs in DM AS animals. RAGE and TLR4 silencing dramatically reduced AGE-induced macrophage M1 polarization, inflammatory cytokine secretion, and RAGE/TLR4/forkhead box protein C2 (FOXC2)/signaling which inhibited delta-like ligand 4 (Dll4) expression in macrophages. AGE-treated macrophages induced VSMC phenotypic conversion via activating Notch pathway in a contact coculture model rather than a noncontact model. The VSMC phenotypic conversion induction capability of macrophages was attenuated by RAGE and TLR4 silencing. Conclusions. AGEs induced activation of RAGE/TLR4/FOXC2 signaling, which featured macrophage with Dll4 high expression during M1 polarization. These macrophages promoted contractile-synthetic phenotypic conversion of VSMCs through the Dll4/Notch pathway after direct cell-to-cell contacts.

Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch dedicated transcription factor. The Notch-dependent neoplasms require however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1, and bZIP factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally our work highlights some Notch/scrib specificities, in particular the role of the PAR domain containing bZIP transcription factor and Notch direct target Pdp1 for neoplastic growth.

Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treat-ment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provided limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory conditions and experi-mental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model of human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, including podocyte loss. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by targeting NOTCH signaling pathway. JQ1 inhibited the gene expression of the NOTCH effec-tors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.

Inhibition of RFX6 Suppresses the Invasive Ability of Tumor Cells Through the Notch Pathway and Affects Tumor Immunity in Hepatocellular Carcinoma

BackgroundThe DNA-binding protein RFX6 was overexpressed in hepatocellular carcinoma, and its expression level was correlated with the prognosis and immune cell infiltration in liver hepatocellular carcinoma. However, the mechanism of the abnormal expression and the biological effects of RFX6 in liver cancer remains unknown.MethodsTo understand the specific expression mechanism of RFX6 in liver cancer, we performed bioinformatic prediction, CHIP-qPCR assay, co-IP, and dual-luciferase assay to assess the regulating mechanism of RFX6. In the meantime, a series of biological experiments in vivo and in vitro were conducted to analyze the biological significance of RFX6 in hepatocellular carcinoma.ResultsWe demonstrated that knockdown of RFX6 in liver cancer cells significantly suppressed the proliferation, migration, and invasion of cancer cells. Moreover, inhibition of RFX6 could affect the immune response of T cells. Among a number of interacting proteins, we revealed that RFX6 directly binds to DTX2, a regulator of the Notch signaling pathway by targeting NOTCH1, and helps in its transcription stability. Furthermore, we discovered that miRNA-542-3p, the expression of which was decreased in hepatocellular carcinoma, was directly involved in the negative regulation of the expression of RFX6.ConclusionIn summary, we discovered that the miRNA-542-3p–RFX6–DTX2–NOTCH1 regulatory pathway played significant roles in the tumor progression of liver hepatocellular carcinoma.

Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treat-ment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provided limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory conditions and experi-mental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model of human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, including podocyte loss. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by targeting NOTCH signaling pathway. JQ1 inhibited the gene expression of the NOTCH effec-tors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.

Painong San, a Traditional Chinese Compound Herbal Medicine, Restores Colon Barrier Function on DSS-Induced Colitis in Mice

Objective. The intestinal barrier decreases in colitis and restores the integrity of the mucosal barriers that could be used for the treatment of colitis. Painong San (PNS), a traditional Chinese compound herbal medicine originally recorded in “Jingui Yaolve” by Zhongjing Zhang in the Later Han Dynasty, is often used in China and Japan to treat various purulent diseases including intestinal carbuncle. This study was to investigate the effect of PNS on mucosal barrier function in mice with DSS-induced colitis and its related mechanisms. Methods. BALB/C mice were given 3% DSS to induce colitis. The body weight and stool status of the mice were recorded daily, and the histopathological changes of the colon were observed after execution. The permeability of the intestinal mucosa was measured by fluorescein isothiocyanate-dextran 4000, the change of intestinal microbiota was measured by 16S rDNA, and the tight junction-related proteins and Muc-2 were investigated by immunohistochemical or immunofluorescence. The possible signaling pathways were detected by western blot. Results. Compared with the control group, the composition of the microbiota in the PNS group was close to that of the normal group, the number of goblet cells was improved, and the mucosal permeability was significantly reduced. PNS could upregulate the expression of tight junction-related proteins (ZO-1, claudin-1, and occludin) and Muc-2, and at the same time, regulate the Notch pathway. Conclusion. PNS could effectively improve the mucosal barrier function through multiple ways, including restoring the balance of intestine flora, enhancement of the mucous layer barrier, and mechanical barrier function. These protective effects may relate to inhibiting the Notch signaling pathway activated by DSS.