Clock Gene Bmal1 Disruption in Vascular Smooth Muscle Cells Worsens Carotid Atherosclerotic Lesions.

BACKGROUND: Clock system disruptions are associated with cardiovascular diseases. We previously demonstrated Bmal1 (brain muscle aryl nuclear translocase like-1) expression is significantly attenuated in plaque-derived vascular smooth muscle cells (VSMCs). However, the influence of Bmal1 disruption in VSMCs and its molecular targets are still unclear. Here, we aim to define how Bmal1 disruption in VSMCs influences the atherosclerosis lesions. METHODS: The relationship among Bmal1, neurological symptoms, and plaque stability was investigated. VSMC Bmal1-/- and VSMC Bmal1+/+mice were generated and injected with adeno associated virus encoding mutant proprotein convertase subtilisin/kexin type 9 to induce atherosclerosis. Carotid artery ligation and cuff placement were performed in these mice to confirm the role of Bmal1 in atherosclerosis progression. The relevant molecular mechanisms were then explored. RESULTS: Bmal1 expression in the carotid plague was significantly lower in symptomatic patients as well as in unstable plaques. Moreover, Bmal1 reduction is an independent risk factor for neurological symptoms and plaque instability. Besides, VSMC Bmal1-/- mice exhibit aggravated atherosclerotic lesions. Further study demonstrated that Bmal1 downregulation in VSMCs increased VSMC migration, monocyte transmigration, reactive oxygen species levels, and VSMCs apoptosis. As for the mechanism, we revealed that Bmal1 suppresses VSMCs migration by inhibiting RAC1 activity in 2 ways: by activating the transcription of RhoGDIα and by interacting with RAC1. Besides, Bmal1 was shown to preserve antioxidant function in VSMCs by activating Nrf2 (nuclear factor erythroid 2-related factor 2) and Bcl-2 transcription. CONCLUSIONS: Bmal1 disruption in VSMCs worsens atherosclerosis by promoting VSMC migration and monocyte transmigration and impairing antioxidant function. Therefore, Bmal1 may be a potential therapeutic target and biomarker of atherosclerosis in the future.

miR-455-5p regulates circadian rhythms by accelerating the degradation of Clock mRNA.

Circadian rhythms are approximately 24-hr cycles generated by organisms to adapt to daily rhythms. Core circadian proteins such as CLOCK, BMAL1, PER1/2, and CRY1/2/3 form a transcription-translation feedback loop (TTFL) to maintain circadian rhythms. MicroRNAs are involved in regulating circadian rhythms; however, the detailed mechanisms remain unclear. Here, using miRNA-seq screening, we discovered that the expression level of miR-455 was controlled by CLOCK. Furthermore, miR-455-5p also binds to the 3' untranslated region (3'UTR) of Clock mRNA and regulates its stability. To further study whether such mutual regulation forms a feedback loop to regulate circadian rhythms, we recorded bioluminescence traces of Per2::Luc U2OS cells in real time and confirmed that overexpression of miR-455-5p lengthens the period and attenuates the amplitude of circadian rhythms in synchronized cells (and vice versa). We also discovered that miR-455-5p can function as a Clock modulator to induce a fine-orchestral circadian rhythm in vitro, as well as other known factors such as dexamethasone, horse serum, or temperature. In conclusion, miR-455-5p is essential for maintaining a normal circadian rhythm via regulating Clock mRNA stability. Our study reveals a new mutual regulatory mechanism between CLOCK protein, Clock mRNA, and miR-455-5p, which regulates circadian rhythms in cells.

BMAL1 induces colorectal cancer metastasis by stimulating exosome secretion.

BACKGROUND: To adapt to daily changes in the external environment, organisms have developed circadian rhythm systems with a period of approximately 24 h. Many studies have reported that both circadian rhythms and exosomes play important roles in the development and metastasis of tumors. However, whether circadian clock genes can affect the progression of tumors by regulating exosomes remains unclear. METHODS AND RESULTS: In this study, we isolated exosomes from the supernatant of human colorectal cancer (CRC) cells, including SW480, SW620, and HCT116 cells, by differential centrifugation and characterized exosomes by transmission electron microscopy, nanoparticle tracking analysis, and Western blot analysis. Then, we found that exosomes derived from SW480, SW620 and HCT116 cells could promote the migration of HCT116 and human umbilical vein endothelial cells. Exosomes derived from SW620 cells showed increased stimulating effects when we increased the expression of BMAL1, a core circadian protein. In contrast, exosomes derived from SW480 and HCT116 cells showed decreased stimulating effects when we knocked down the expression of BMAL1. Furthermore, we discovered that BMAL1 promotes the release of exosomes by HCT116 and SW620 cells. In addition, by luciferase assay, we confirmed that BMAL1 transcriptionally regulates the expression of Rab27a, a key molecule related to the secretion of exosomes. CONCLUSIONS: Our data reveal a new mechanism by which BMAL1 induces CRC metastasis by stimulating exosome secretion. This finding may help further clarify the role of circadian rhythm in the progression of CRC.

Establishment of a PRKAG2 cardiac syndrome disease model and mechanism study using human induced pluripotent stem cells.

PRKAG2 cardiac syndrome is a distinct form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular pre-excitation and progressive cardiac conduction disorder. However, it remains unclear how mutations in the PRKAG2 gene give rise to such a complicated disease. To investigate the underlying molecular mechanisms, we generated disease-specific hiPSC-derived cardiomyocytes from two brothers both carrying a heterozygous missense mutation c.905G>A (R302Q) in the PRKAG2 gene and further corrected the R302Q mutation with CRISPR-Cas9 mediated genome editing. Disease-specific hiPSC-cardiomyocytes recapitulated many phenotypes of PRKAG2 cardiac syndrome including cellular enlargement, electrophysiological irregularities and glycogen storage. In addition, we found that the PRKAG2-R302Q mutation led to increased AMPK activities, resulting in extensive glycogen deposition and cardiomyocyte hypertrophy. Finally we confirmed that disrupted phenotypes of PRKAG2 cardiac syndrome caused by the specific PRKAG2-R302Q mutation can be alleviated by small molecules inhibiting AMPK activity and be rescued with CRISPR-Cas9 mediated genome correction. Our results showed that disease-specific hiPSC-CMs and genetically-corrected hiPSC-cardiomyocytes would be a very useful platform for understanding the pathogenesis of, and testing autologous cell-based therapies for, PRKAG2 cardiac syndrome.

Clock represses preadipocytes adipogenesis via GILZ.

Adiposity is a worldwide health threat that needs to be prevented. Circadian gene Clock (circadian locomotor output cycles kaput) is closely correlated to adiposity; for example, weight gain, adipocytes size expansion, and serum lipid level rise in ClockΔ19 mice compared to C57BL/6J mice. However, the precise role of Clock during adipogenic differentiation is unknown. Herein, the circadian gene Clock is shown to regulate adipogenesis mediated by GILZ. Clock-mediated attenuation and upregulation influenced lipid synthesis and affected the levels of adipogenic transcriptional factors, C/EBP-ß, C/EBP-α, PPAR-γ, and FABP4, both in vivo and in vitro (primary adipose-derived stromal cells and 3T3-L1 cells). Chromatin immunoprecipitation (ChIP) assay, reporter gene assay, and serum shock assay found that Clock transcriptionally regulated the glucocorticoid-induced leucine zipper (GILZ). Furthermore, GILZ attenuation could relieve the inhibitory effect of Clock on lipid synthesis and GILZ overexpression also reduced the promotion role of Clock attenuation in adipogenesis suggesting that Clock inhibits adipogenic differentiation of preadipocytes via GILZ. The current results demonstrate how circadian genes are likely to regulate adiposity, affecting the adipogenic differentiation process, as well as, increasing the fat cells number. Therefore, this study may provide novel insights into the underlying mechanism explaining the correlation between Clock mutation and adiposity.

Bach1 Represses Wnt/ß-Catenin Signaling and Angiogenesis.

RATIONALE: Wnt/ß-catenin signaling has an important role in the angiogenic activity of endothelial cells (ECs). Bach1 is a transcription factor and is expressed in ECs, but whether Bach1 regulates angiogenesis is unknown. OBJECTIVE: This study evaluated the role of Bach1 in angiogenesis and Wnt/ß-catenin signaling. METHODS AND RESULTS: Hind-limb ischemia was surgically induced in Bach1(-/-) mice and their wild-type littermates and in C57BL/6J mice treated with adenoviruses coding for Bach1 or GFP. Lack of Bach1 expression was associated with significant increases in perfusion and vascular density and in the expression of proangiogenic cytokines in the ischemic hindlimb of mice, with enhancement of the angiogenic activity of ECs (eg, tube formation, migration, and proliferation). Bach1 overexpression impaired angiogenesis in mice with hind-limb ischemia and inhibited Wnt3a-stimulated angiogenic response and the expression of Wnt/ß-catenin target genes, such as interleukin-8 and vascular endothelial growth factor, in human umbilical vein ECs. Interleukin-8 and vascular endothelial growth factor were responsible for the antiangiogenic response of Bach1. Immunoprecipitation and GST pull-down assessments indicated that Bach1 binds directly to TCF4 and reduces the interaction of ß-catenin with TCF4. Bach1 overexpression reduces the interaction between p300/CBP and ß-catenin, as well as ß-catenin acetylation, and chromatin immunoprecipitation experiments confirmed that Bach1 occupies the TCF4-binding site of the interleukin-8 promoter and recruits histone deacetylase 1 to the interleukin-8 promoter in human umbilical vein ECs. CONCLUSIONS: Bach1 suppresses angiogenesis after ischemic injury and impairs Wnt/ß-catenin signaling by disrupting the interaction between ß-catenin and TCF4 and by recruiting histone deacetylase 1 to the promoter of TCF4-targeted genes.

Intracellular high cholesterol content disorders the clock genes, apoptosis-related genes and fibrinolytic-related genes rhythmic expressions in human plaque-derived vascular smooth muscle cells.

BACKGROUND: The clock genes are involved in regulating cardiovascular functions, and their expression disorders would lead to circadian rhythm disruptions of clock-controlled genes (CCGs), resulting in atherosclerotic plaque formation and rupture. Our previous study revealed the rhythmic expression of clock genes were attenuated in human plaque-derived vascular smooth muscle cells (PVSMCs), but failed to detect the downstream CCGs expressions and the underlying molecular mechanism. In this study, we examined the difference of CCGs rhythmic expression between human normal carotid VSMCs (NVSMCs) and PVSMCs. Furthermore, we compared the cholesterol and triglycerides levels between two groups and the link to clock genes and CCGs expressions. METHODS: Seven health donors' normal carotids and 19 carotid plaques yielded viable cultured NVSMCs and PVSMCs. The expression levels of target genes were measured by quantitative real-time PCR and Western-blot. The intracellular cholesterol and triglycerides levels were measured by kits. RESULT: The circadian expressions of apoptosis-related genes and fibrinolytic-related genes were disordered. Besides, the cholesterol levels were significant higher in PVSMCs. After treated with cholesterol or oxidized low density lipoprotein (ox-LDL), the expressions of clock genes were inhibited; and the rhythmic expressions of clock genes, apoptosis-related genes and fibrinolytic-related genes were disturbed in NVSMCs, which were similar to PVSMCs. CONCLUSION: The results suggested that intracellular high cholesterol content of PVSMCs would lead to the disorders of clock genes and CCGs rhythmic expressions. And further studies should be conducted to demonstrate the specific molecular mechanisms involved.

CLOCK promotes 3T3-L1 cell proliferation via Wnt signaling.

Circadian genes control most of the physiological functions including cell cycle. Cell proliferation is a critical factor in the differentiation of progenitor cells. However, the role of Clock gene in the regulation of cell cycle via wingless-type (Wnt) pathway and the relationship between Clock and adipogenesis are unclear. We found that the circadian locomotor output cycles kaput (Clock) regulated the proliferation and the adipogenesis of 3T3-L1 preadipocytes. We found that Clock attenuation inhibited the viability of 3T3-L1 preadipocytes in the cell counting kit 8. The expression of c-Myc and Cyclin D1 decreased dramatically in 3T3-L1 when Clock was silenced with short interfering RNA and was also decreased in fat tissue and adipose tissue-derived stem cells of Clock(Δ19) mice. Clock directly controls the expression of the components of Wnt signal transduction pathway, which was verified by serum shock, chromatin immunoprecipitation, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, IWR-1, a Wnt signal pathway inhibitor, inhibited the cell cycle promotion by CLOCK, which was detected by cell viability assay, flow cytometry, and qRT-PCR. Therefore, CLOCK transcription control of Wnt signaling promotes cell cycle progression in 3T3-L1 preadipocytes. Clock inhibited the adipogenesis on day 2 in 3T3-L1 cells via Oil Red O staining and qRT-PCR detection and probably related to cellular differentiation. These data provide evidence that the circadian gene Clock regulates the proliferation of preadipocytes and affects adipogenesis. © 2016 IUBMB Life, 68(7):557-568, 2016.

Clock upregulates intercellular adhesion molecule-1 expression and promotes mononuclear cells adhesion to endothelial cells.

Clock is a basic helix-loop-helix (bHLH) transcription factor that plays important role in circadian rhythms of various physiological functions. Previous study showed that the expression of intercellular adhesion molecule-1 (ICAM-1) was reduced in the liver tissues of Clock mutant mice. However, how Clock regulates ICAM-1 expression and whether Clock affects cell adhesion function remain unknown. In the present study, we found that exogenous expression of Clock upregulated the gene expressions of ICAM-1 and other adhesion-related genes including VCAM1 and CCL-2, and increased the transcriptional activity of ICAM-1 in mouse brain microvascular endothelial cell lines. In contrast, loss of Clock decreased these gene expressions and ICAM-1 transcriptional activity. Chromatin immunoprecipitation (ChIP) assay revealed that Clock binds to the E-box-like enhancer of ICAM-1 gene. ICAM-1 gene showed rhythmic expression in endothelial cells after serum shock in vitro, suggesting ICAM-1 may be a Clock-controlled gene. Clock regulates the adhesion of mononuclear cells to endothelial cells via ICAM-1. Together, our findings show that Clock is a positive regulator of ICAM-1, and promotes the adhesion of mononuclear cells to endothelial cells.

The rhythmic expression of clock genes attenuated in human plaque-derived vascular smooth muscle cells.

BACKGROUND: Acute myocardial infarction and stroke are more likely to occur in the early morning. Circadian pacemakers are considered to be involved in the process. Many peripheral tissues and cells also contain clock systems. In this study, we examined whether the primary cultured human plaque-derived vascular smooth muscle cells (VSMCs) process circadian rhythmicity; furthermore, we investigated the expression difference of clock genes between normal human carotid VSMCs and human plaque-derived VSMCs. METHODS: Fifty-six human carotid plaques provided the atherosclerotic tissue, and 21 samples yielded viable cultured primary VSMCs. The normal carotid VSMCs were cultured from donors' normal carotids. The mRNA levels of the target genes were measured by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). RESULTS: After serum shock, both types of cells showed clear circadian expressions of Bmal1, Cry1, Cry2, Per1, Per2, Per3 and Rev-erbα mRNA; meanwhile the Clock mRNA show a rhythmic expression in plaque-derived SMCs but not in normal carotid VSMCs. The expression levels of these main clock genes were significantly attenuated in human plaque-derived VSMCs compared with normal human carotid VSMCs. The rhythm of Bmal1 mRNA in plaque-derived VSMCs was changed. CONCLUSION: The present results demonstrate that the human plaque-derived VSMCs possess different circadian rhythmicity from that of normal carotid VSMCs. The rhythm changes of clock genes in plaque-derived VSMCs may be involved in the process of atherosclerosis and finally promote the rupture of plaque.

Effect of H2S on the circadian rhythm of mouse hepatocytes.

BACKGROUND: Dysregulation of circadian rhythms can contribute to diseases of lipid metabolism. NAD-dependent deacetylase sirtuin-1(SIRT1) is an important hub which links lipid metabolism with circadian clock by its deacetylation activity depends on intracellular NAD+/NADH content ratio. Hydrogen sulfide (H2S) is an endogenous reductant which can affect the intracellular redox state. Therefore, we hypothesized that exogenous H2S can affect the expression of circadian clock genes mediated by sirt1 thereby affecting body's lipid metabolism. And also because the liver is a typical peripheral circadian clock oscillator that is intimately linked to lipid metabolism. Thus the effect of H2S were observed on 24-hour dynamic expression of 4 central circadian clock genes and sirt1gene in primary cultured hepatocytes. RESULTS: We established a hepatocyte model that showed a circadian rhythm by serum shock method. And detected that the expression level and the peak of circadian clock genes decreased gradually and H2S could maintain the expression and amplitude of circadian clock genes such as Clock, Per2, Bmal1 and Rev-erbαwithin a certain period time. Accordingly the expression level of sirt1 in H2S group was significantly higher than that in the control group. CONCLUSION: Exogenous reductant H2S maintain the circadian rhythm of clock gene in isolated liver cells. We speculated that H2S has changed NAD+/NADH content ratio in hepatocytes and enhanced the activity of SIRT1 protein directly or indirectly, so as to maintain the rhythm of expression of circadian clock genes, they play a role in the prevention and treatment of lipid metabolism-related disease caused by the biological clock disorders.

The circadian clock regulates metabolic responses to physical exercise.

It has been proposed for years that physical exercise ameliorates metabolic diseases. Optimal exercise timing in humans and mammals has indicated that circadian clocks play a vital role in exercise and body metabolism. Skeletal muscle metabolism exhibits a robust circadian rhythm under the control of the suprachiasmatic nucleus of the hypothalamus. Clock genes also control the development, differentiation, and function of skeletal muscles. In this review, we aimed to clarify the relationship between exercise, skeletal muscles, and the circadian clock. Health benefits can be attained by the scheduling of exercise at the best circadian time. Exercise therapy for metabolic diseases and cardiovascular health is a key adjuvant method. This review highlights the importance of exercise timing in maintaining healthy metabolism and circadian clocks.

Genome-wide CRISPR Screening Reveals Pyrimidine Metabolic Reprogramming in 5-FU Chronochemotherapy of Colorectal Cancer.

Objective: Disruption of the circadian rhythm is associated with cancer occurrence, response to chemotherapy, and poor prognosis. Thus, using internal clock-based chronotherapy to optimize the administration time may improve the therapeutic effects of anticancer drugs while reducing the side effects. Chronotherapy with 5-fluorouracil (5-FU) has been observed in colorectal cancer (CRC) for a long time, but its effect is under controversial and the mechanism remains unclear. Methods: Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening and RNA-sequencing were combined to identify the potential genes or pathways involved in 5-FU chronochemotherapy. Genetic deletion or overexpression of pyrimidine metabolic pathway genes were conducted to examine cellular viability with or without 5-FU via flow cytometry. Western blotting, qPCR, chromatin immunoprecipitation, gain-of-function and loss-of-function assays of several CRC cell lines in vitro and in vivo were used to elaborate and validate the mechanism of 5-FU chronotherapeutic effects. Results: Chronochemotherapeutic effects of 5-FU on CRC in vivo were verified. Furthermore, 5-FU chronochemotherapy related genes such as UPP2, UCK2 and UMPS in the pyrimidine metabolic pathway were identified. Disturbance in these genes, especially UMPS, perturbs 5-FU treatment outcomes in CRC cells. Mechanistically, the core circadian gene, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1), extensively regulate gene expression in pyrimidine metabolic pathway by binding to E-box element in the promoter region of key genes such as UMPS and perturb their enzymatic activities, thereby maintain diurnal efficacy of 5-FU in CRC cells. Conclusion: This study uncovered a new mechanism by which a core circadian gene BMAL1 increases the effectiveness of 5-FU by enhancing the expression and enzymatic activities of key genes in the pyrimidine metabolic pathway in CRC cells. The findings suggest a novel strategy for CRC chemotherapy by targeting chrono-modulated genes of the 5-FU metabolic pathway.

CLOCK regulates Drp1 mRNA stability and mitochondrial homeostasis by interacting with PUF60.

Circadian genes such as Clock, Bmal1, Cryptochrome1/2, and Period1/2/3 constitute the precise circadian system. ClockΔ19 is a commonly used mouse model harboring a circadian clock gene mutation, which lacks the EXON-19-encoded 51 amino acids. Previous reports have shown that ClockΔ19 mice have severe metabolic abnormalities. Here, we report that the mitochondria of ClockΔ19 mice exhibit excessive fission and dysfunction. We also demonstrate that CLOCK binds to the RNA-binding protein PUF60 through its EXON 19. Further, we find that PUF60 directly maintains mitochondrial homeostasis through regulating Drp1 mRNA stability, while the association with CLOCK can competitively inhibit this function. In ClockΔ19 mice, CLOCKΔ19 releases PUF60, leading to enhanced Drp1 mRNA stability and persistent mitochondrial fission. Our results reveal a direct post-transcriptional role of CLOCK in regulating mitochondrial homeostasis via Drp1 mRNA stability and that the loss of EXON 19 of CLOCK in ClockΔ19 mice leads to severe mitochondrial homeostasis disorders.

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.

E2A ablation enhances proportion of nodal-like cardiomyocytes in cardiac-specific differentiation of human embryonic stem cells.

BACKGROUND: Human sinoatrial cardiomyocytes are essential building blocks for cell therapies of conduction system disorders. However, current differentiation protocols for deriving nodal cardiomyocytes from human pluripotent stem cells (hPSCs) are very inefficient. METHODS: By employing the hPSCs to cardiomyocyte (CM) in vitro differentiation system and generating E2A-knockout hESCs using CRISPR/Cas9 gene editing technology, we analyze the functions of E2A in CM differentiation. FINDINGS: We found that knockout of the transcription factor E2A substantially increased the proportion of nodal-like cells in hESC-derived CMs. The E2A ablated CMs displayed smaller cell size, increased beating rates, weaker contractile force, and other functional characteristics similar to sinoatrial node (SAN) cells. Transcriptomic analyses indicated that ion channel-encoding genes were up-regulated in E2A ablated CMs. E2A directly bounded to the promoters of genes key to SAN development via conserved E-box motif, and promoted their expression. Unexpect enhanced activity of NOTCH pathway after E2A ablation could also facilate to induct ventricle workingtype CMs reprogramming into SAN-like cells. INTERPRETATION: Our study revealed a new role for E2A during directed cardiac differentiation of hESCs and may provide new clues for enhancing induction efficiency of SAN-like cardiomyocytes from hPSCs in the future. FUNDING: This work was supported by the NSFC (No.82070391, N.S.; No.81870175 and 81922006, P.L.), the National Key R&D Program of China (2018YFC2000202, N.S.; 2017YFA0103700, P.L.), the Haiju program of National Children's Medical Center EK1125180102, and Innovative research team of high-level local universities in Shanghai and a key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005).

Postnatal ontogenesis of clock genes in mouse suprachiasmatic nucleus and heart.

BACKGROUND: The master clock within the hypothalamic suprachiasmatic nucleus (SCN) synchronizing clocks in peripheral tissues is entrained by the environmental condition, such as the light-dark (LD) cycle. The mechanisms of circadian clockwork are similar in both SCN and peripheral tissues. The aim of the present work was to observe the profiles of clock genes expression in mouse central and peripheral tissues within postnatal day 5 (P5). The daily expression of four clock genes mRNA (Bmal1, Per2, Cry1 and Rev-erb alpha) in mouse SCN and heart was measured at P1, P3 and P5 by real-time PCR. RESULTS: All the studied mice clock genes began to express in a circadian rhythms manner in heart and SCN at P3 and P5 respectively. Interestingly, the daily rhythmic phase of some clock genes shifted during the postnatal days. Moreover, the expressions of clock genes in heart were not synchronized with those in SCN until at P5. CONCLUSION: The data showed the gradual development of clock genes in SCN and a peripheral tissue, and suggested that development of clock genes differed between in the SCN and the heart. Judging from the mRNA expression, it was possible that the central clock synchronized the peripheral clock as early as P5.

BMAL1 regulates mitochondrial fission and mitophagy through mitochondrial protein BNIP3 and is critical in the development of dilated cardiomyopathy.

Dysregulation of circadian rhythms associates with cardiovascular disorders. It is known that deletion of the core circadian gene Bmal1 in mice causes dilated cardiomyopathy. However, the biological rhythm regulation system in mouse is very different from that of humans. Whether BMAL1 plays a role in regulating human heart function remains unclear. Here we generated a BMAL1 knockout human embryonic stem cell (hESC) model and further derived human BMAL1 deficient cardiomyocytes. We show that BMAL1 deficient hESC-derived cardiomyocytes exhibited typical phenotypes of dilated cardiomyopathy including attenuated contractility, calcium dysregulation, and disorganized myofilaments. In addition, mitochondrial fission and mitophagy were suppressed in BMAL1 deficient hESC-cardiomyocytes, which resulted in significantly attenuated mitochondrial oxidative phosphorylation and compromised cardiomyocyte function. We also found that BMAL1 binds to the E-box element in the promoter region of BNIP3 gene and specifically controls BNIP3 protein expression. BMAL1 knockout directly reduced BNIP3 protein level, causing compromised mitophagy and mitochondria dysfunction and thereby leading to compromised cardiomyocyte function. Our data indicated that the core circadian gene BMAL1 is critical for normal mitochondria activities and cardiac function. Circadian rhythm disruption may directly link to compromised heart function and dilated cardiomyopathy in humans.

CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition.

Carotid artery stenosis is a leading cause of ischemic stroke, but the underlying mechanism remains unclear. We aimed to determine the molecular mechanisms of carotid plaque progression. We analyzed the molecular and morphometric characteristics of carotid plaque samples obtained from 30 patients who underwent carotid endarterectomy. Additionally, we established a mouse model of carotid atherosclerosis by partially ligating the left common carotid arteries of male ClockΔ19/Δ19 (Clk) and wild-type (WT) C57BL/6J mice fed a high-fat diet. Clk and WT primary mouse aortic endothelial cells (pMAECs) were exposed to disturbed flow (DF) or undisturbed flow (UF) with or without treatment with the IRE-1α inhibitor STF-083010 or the PERK inhibitor GSK2606414. In human carotid artery plaques, CLOCK expression was lower in the lipid-rich necrotic core than in transitional regions, especially in the endothelium. Decreased CLOCK mRNA levels were associated with more extensive stenosis, intraplaque hemorrhage, and complex plaque in human carotid plaques. In mice, the ClockΔ19/Δ19 mutation significantly increased neointima formation and neovascularization but decreased collagen content and lumen area in partially ligated carotid arteries. In addition, ClockΔ19/Δ19 mutants exhibited significantly decreased Cdh5 expression and increased expression of endothelial-mesenchymal transition (EndMT) and endoplasmic reticulum (ER) stress markers in mice with partially ligated carotid arteries and pMAECs exposed to DF. Notably, inhibition of the IRE1α-XBP1 axis abrogated the increased EndMT caused by ClockΔ19/Δ19 mutation and DF in pMAECs. In conclusion, the disruption of CLOCK function aggravates EndMT via the IRE1α-XBP1 axis, contributing to carotid artery stenosis.