Acrolein-induced apoptosis of smooth muscle cells through NEAT1-Bmal1/Clock pathway and a protection from asparagus extract.

Apoptosis of vascular smooth muscle cells (VSMCs) accelerates manifestation of plaque vulnerability in atherosclerosis. Long noncoding RNA NEAT1 participates in the proliferation and apoptosis of cells. In addition, circadian clock genes play a significant role in cell apoptosis. However, whether acrolein, an environmental pollutant, affects the apoptosis of VSMCs by regulating NEAT1 and clock genes is still elusive. We established VSMCs as an atherosclerotic cell model in vitro. Acrolein exposure reduced survival rate of VSMCs, and raised apoptosis percentage through upregulating the expression of Bax, Cytochrome c and Cleaved caspase-3 and downregulating Bcl-2. Asparagus extract (AE), as a dietary supplementation, was able to protect VSMCs against acrolein-induced apoptosis. Expression of NEAT1, Bmal1 and Clock was decreased by acrolein, while was ameliorated by AE. Knockdown of NEAT1, Bmal1 or Clock promoted VSMCs apoptosis by regulating Bax, Bcl-2, Cytochrome c and Caspase-3 levels. Correspondingly, overexpression of NEAT1 inhibited the apoptosis. We also observed that silence of NEAT1 repressed the expression of Bmal1/Clock and vice versa. In this study, we demonstrated that VSMCs apoptosis induced by acrolein was associated with downregulation of NEAT1 and Bmal1/Clock. AE alleviated the effects of proapoptotic response and circadian disorders caused by acrolein, which shed a new light on cardiovascular protection.

Chen L ，Wu X ，Zeb F ，Huang Y ，An J ，Jiang P ，Chen A ，Xu C ，Feng Q ... -  《-》

Regulation of circadian rhythms by NEAT1 mediated TMAO-induced endothelial proliferation: A protective role of asparagus extract.

Trimethylamine N-oxide (TMAO) promotes atherosclerosis in association with the functions of endothelial cells. Clock and Bmal1, as two main components of molecular circadian clock, play important regulatory roles during progression of atherogenesis. However, whether Clock and Bmal1 are involved in the regulation of endothelial proliferation disturbed by TMAO are unclear. We observed that cell proliferation of human umbilical vein endothelial cells (HUVECs) was inhibited after exposed to TMAO for 24 h. Besides, TMAO caused increased expression of lncRNA-NEAT1, Clock and Bmal1, and inhibited MAPK pathways. While MAPK pathways were blocked, the expression of Clock and Bmal1 was elevated. NEAT1 showed a circadian rhythmic expression in HUVECs, and its overexpression reduced cell proliferation. Knockdown or overexpression of NEAT1 might decrease or increase the expression of Clock and Bmal1 respectively, while raised or suppressed the expression of MAPK pathways correspondingly. Asparagus extract (AE) was found to improve the TMAO-reduced HUVECs proliferation. Moreover, it ameliorated the disorders of NEAT1, Clock, Bmal1, and MAPK signaling pathways induced by TMAO. Therefore, our findings indicated that NEAT1 regulating Clock-Bmal1 via MAPK pathways was involved in TMAO-repressed HUVECs proliferation, and AE improved endothelial proliferation by TMAO, proposing a novel mechanism for cardiovascular disease prevention.

Wu X ，Chen L ，Zeb F ，Huang Y ，An J ，Ren J ，Yang F ，Feng Q ... -  《-》

Clock-Bmal1 mediates MMP9 induction in acrolein-promoted atherosclerosis associated with gut microbiota regulation.

Circadian rhythm is believed to play important roles in atherosclerosis. The gut microbiota is found to be closely related to atherogenesis, and shows compositional and functional circadian oscillation. However, it's still unclarified whether circadian clock and intestinal microbiota are involved in the progression of atherosclerosis induced by environmental pollutant acrolein. Herein, patients with atherosclerosis showed higher MMP9, a promising biomarker for atherosclerosis, and lower Bmal1 and Clock expression in the plasma. Interestingly, acrolein exposure contributed to the increased MMP9, decreased Clock and Bmal1, and activated MAPK pathways in human umbilical vein endothelial cells (HUVECs). We found that knockdown of Clock or Bmal1 lead to upregulation of MMP9 in HUVECs, and that Clock and Bmal1 expression was elevated while MAPK pathways were blocked. Atherosclerotic apolipoproteinE-deficient mice consumed a high-fat diet were used and treated with acrolein (3 mg/kg/day) in the drinking water for 12 weeks. Upregulation of MMP9, and downregulation of Clock and Bmal1 were also observed in plasma of the mice. Besides, acrolein feeding altered gut microbiota composition at a phylum level especially for an increased Firmicutes and a decreased Bacteroidetes. Additionally, gut microbiota showed correlation with atherosclerotic plaque, MMP9 and Bmal1 levels. Therefore, our findings indicated that acrolein increased the expression of MMP9 through MAPK regulating circadian clock, which was associated with gut microbiota regulation in atherosclerosis. Circadian rhythms and gut microbiota might be promising targets in the prevention of cardiovascular disease caused by environmental pollutants.

Wu X ，Chen L ，Zeb F ，Li C ，Jiang P ，Chen A ，Xu C ，Haq IU ，Feng Q ... -  《-》

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

Lin C ，Tang X ，Zhu Z ，Liao X ，Zhao R ，Fu W ，Chen B ，Jiang J ，Qian R ，Guo D ... -  《Lipids in Health and Disease》

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

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. 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. 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-β-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. Our results highlight the importance of BMAL1 as a major regulator in VSMCs phenotypic switch towards fibroblast-like cells which stabilize an atherosclerotic plaque.

Shen Y ，Xu LR ，Yan D ，Zhou M ，Han TL ，Lu C ，Tang X ，Lin CP ，Qian RZ ，Guo DQ ... -  《-》