BMAL1 modulates smooth muscle cells phenotypic switch towards fibroblast-like cells and stabilizes atherosclerotic plaques by upregulating YAP1.

BACKGROUND: Ischemic heart diseases and ischemic stroke are closely related to circadian clock and unstable atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) can stabilize or destabilize an atherosclerotic lesion through phenotypic switch. BMAL1 is not only an indispensable core component in circadian clock but also an important regulator in atherosclerosis and VSMCs proliferation. However, little is known about the modulation mechanisms of BMAL1 in VSMCs phenotypic switch and atherosclerotic plaque stability. METHODS: We integrated histological analysis of human plaques, in vivo experiments of VSMC-specific Bmal1-/- mice, in vitro experiments, and gene set enrichment analysis (GSEA) of public datasets of human plaques to explore the function of BMAL1 in VSMCs phonotypic switch and plaque stability. FINDINGS: Comparing to human unstable plaques, BMAL1 was higher in stable plaques, accompanied by elevated YAP1 and fibroblast maker FSP1 which were positively correlated with BMAL1. In response to Methyl-ß-cyclodextrin-cholesterol, oxidized-low-density-lipoprotein and platelet-derived-growth-factor-BB, VSMCs embarked on phenotypic switch and upregulated BMAL, YAP1 and FSP1. Besides, BMAL1 overexpression promoted VSMCs phonotypic switch towards fibroblast-like cells by transcriptionally upregulating the expression of YAP1. BMAL1 or YAP1 knock-down inhibited VSMCs phonotypic switch and downregulated FSP1. Furthermore, VSMC-specific Bmal1-/- mice exhibited VSMCs with lower YAP1 and FSP1 levels, and more vulnerable plaques with less collagen content. In addition, BMAL1 suppressed the migration of VSMCs. The GSEA results of public datasets were consistent with our laboratory findings. INTERPRETATION: Our results highlight the importance of BMAL1 as a major regulator in VSMCs phenotypic switch towards fibroblast-like cells which stabilize an atherosclerotic plaque.

Identification of potential therapeutic targets for atherosclerosis by analysing the gene signature related to different immune cells and immune regulators in atheromatous plaques.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease that affects multiple arteries. Numerous studies have shown the inherent immune diversity in atheromatous plaques and suggest that the dysfunction of different immune cells plays an important role in atherosclerosis. However, few comprehensive bioinformatics analyses have investigated the potential coordinators that might orchestrate different immune cells to exacerbate atherosclerosis. METHODS: Immune infiltration of 69 atheromatous plaques from different arterial beds in GSE100927 were explored by single-sample-gene-set enrichment analysis (presented as ssGSEA scores), ESTIMATE algorithm (presented as immune scores) and CIBERSORT algorithm (presented as relative fractions of 22 types of immune cells) to divide these plaques into ImmuneScoreL cluster (of low immune infiltration) and ImmuneScoreH cluster (of high immune infiltration). Subsequently, comprehensive bioinformatics analyses including differentially-expressed-genes (DEGs) analysis, protein-protein interaction networks analysis, hub genes analysis, Gene-Ontology-terms and KEGG pathway enrichment analysis, gene set enrichment analysis, analysis of expression profiles of immune-related genes, correlation analysis between DEGs and hub genes and immune cells were conducted. GSE28829 was analysed to cross-validate the results in GSE100927. RESULTS: Immune-related pathways, including interferon-related pathways and PD-1 signalling, were highly enriched in the ImmuneScoreH cluster. HLA-related (except for HLA-DRB6) and immune checkpoint genes (IDO1, PDCD-1, CD274(PD-L1), CD47), RORC, IFNGR1, STAT1 and JAK2 were upregulated in the ImmuneScoreH cluster, whereas FTO, CRY1, RORB, and PER1 were downregulated. Atheromatous plaques in the ImmuneScoreH cluster had higher proportions of M0 macrophages and gamma delta T cells but lower proportions of plasma cells and monocytes (p < 0.05). CAPG, CECR1, IL18, IGSF6, FBP1, HLA-DPA1 and MMP7 were commonly related to these immune cells. In addition, the advanced-stage carotid plaques in GSE28829 exhibited higher immune infiltration than early-stage carotid plaques. CONCLUSIONS: Atheromatous plaques with higher immune scores were likely at a more clinically advanced stage. The progression of atherosclerosis might be related to CAPG, IGSF6, IL18, CECR1, FBP1, MMP7, FTO, CRY1, RORB, RORC, PER1, HLA-DPA1 and immune-related pathways (IFN-γ pathway and PD-1 signalling pathway). These genes and pathways might play important roles in regulating immune cells such as M0 macrophages, gamma delta T cells, plasma cells and monocytes and might serve as potential therapeutic targets for atherosclerosis.

Effects of RNA interference-induced tryptase down-regulation in P815 cells on IL-6 and TNF-alpha release of endothelial cells.

OBJECTIVE: To explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) of vascular endothelial cells. METHODS: Tryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-alpha in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: IL-6 and TNF-alpha mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-alpha protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group. CONCLUSION: Reduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-alpha in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.

Roles of cyclooxygenase-2 in microvascular endothelial cell proliferation induced by basic fibroblast growth factor.

BACKGROUND: The level of basic fibroblast growth factor (bFGF) increases rapidly after cerebral ischemia. However, the molecular mechanisms for the effects of bFGF on cerebral microvascular endothelial cells (cMVECs) have not yet been fully elucidated. In this study, a murine cMVEC line, bEnd.3, was employed to study the effects of bFGF on cyclooxygenase (COX) expression and its downstream effects in cMVECs. METHODS: After treatment with bFGF, RT-PCR and Western blotting analyses were carried out to evaluate the changes in COX-2 mRNA and protein expression, respectively. MTT assays were performed to measure cell proliferation. The prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) concentrations in the culture medium were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: COX-2 mRNA and protein expressions in bEnd.3 cells were induced by bFGF in time- and dose-dependent manners. The bFGF-induced COX-2 upregulation led to enhanced PGE2 production by bEnd.3 cells, and this effect was abolished by the selective COX-2 inhibitor NS-398. bFGF also increased VEGF production by bEnd.3 cells, and this effect was blocked by NS-398 and the EP1/2 (PGE2 receptors) antagonist AH6809. Furthermore, exogenous PGE2 increased VEGF production in bEnd.3 cells, and AH6809 blocked this effect. CONCLUSION: bFGF increases VEGF production in an autocrine manner by increasing COX-2-generated PGE2 in cMVECs and subsequently stimulates MVEC proliferation and angiogenesis.

[Dynamic changes in focal adhesion kinase during cell migration induced by bFGF and the significance].

Cell migration plays an important role in repair of injury, angiogenesis, cancer metastasis and so on. In this paper the effects of basic fibroblast growth factor (bFGF) of different concentrations on ECV-304 cell migration, and the dynamic changes in focal adhesion kinase (FAK) were observed. The relationship between FAK and cell migration induced by bFGF was studied. A ECV-304 cell scratch wound model was established and the images of cell migration were quantitatively measured using a computer-assisted videomicroscopic system. The dynamic changes in FAK content (Western blot), FAK activity (immunoprecipitation plus Western blot) and FAK mRNA (RT-PCR) were measured in vitro. The expression of integrin alpha3 was investigated using immunocytochemical staining (ABC method). The results showed that bFGF produced a dual-phase regulatory effect on ECV-304 migration when the cell confluent areas reached 90%-95% in culture. It was found that compared with the control group (0 ng/ml bFGF), the cell migration was stimulated (P<0.05), inhibited (P<0.05) and unchanged when the cultured cells were treated with bFGF at the concentrations of 5, 10 and 15 ng/ml, respectively. The content and activity of FAK protein were markedly up-regulated in 5 ng/ml bFGF group and down-regulated in 15 ng/ml bFGF group, respectively. FAK mRNA expression came to the peak in 5 ng/ml bFGF group after 6 h culture and there was a significant difference compared with the control group. In various experimental groups there were no significant differences in the expression of integrin alpha3 compared with the control group according to the immunocytochemical staining. The results mentioned above suggest that different concentrations of bFGF have a dual-phase effect on the migration of cultured ECV-304 cells, which correlates positively with FAK content, activity and mRNA in cultured ECV-304 cell scratch wound model. The FAK plays an important role in the signal transduction pathway of cell migration induced by bFGF, while bFGF can regulate the content of FAK in ECV-304 cells at gene transcription level.