AMP-Activated Protein Kinase Alpha 2 Deletion Induces VSMC Phenotypic Switching and Reduces Features of Atherosclerotic Plaque Stability.

AMP-activated protein kinase (AMPK) has been reported to play a protective role in atherosclerosis. However, whether AMPKα2 controls atherosclerotic plaque stability remains unknown. The aim of this study was to evaluate the impact of AMPKα2 deletion on atherosclerotic plaque stability in advanced atherosclerosis at the brachiocephalic arteries and to elucidate the underlying mechanisms. Features of atherosclerotic plaque stability and the markers for contractile or synthetic vascular smooth muscle cell (VSMC) phenotypes were monitored in the brachiocephalic arteries from Apoe(-/-)AMPKα2(-/-) mice or VSMC-specific AMPKα2(-/-) mice in an Apoe(-/-) background (Apoe(-/-)AMPKα2(sm-/-)) fed Western diet for 10 weeks. We identified that Apoe(-/-)AMPKα2(-/-) mice and Apoe(-/-)AMPKα2(sm-/-) mice exhibited similar unstable plaque features, aggravated VSMC phenotypic switching, and significant upregulation of Kruppel-like factor 4 (KLF4) in the plaques located in the brachiocephalic arteries compared with those found in Apoe(-/-) and Apoe(-/-)AMPKα2(sm+/+) control mice. Pravastatin, an AMPK activator, suppressed VSMC phenotypic switching and alleviated features of atherosclerotic plaque instability in Apoe(-/-)AMPKα2(sm+/+) mice, but not in Apoe(-/-)AMPKα2(sm-/-) mice. VSMC isolated from AMPKα2(-/-) mice displayed a significant reduction of contractile proteins(smooth muscle actin-α, calponin, and SM-MHC [smooth muscle-mysion heavy chain]) in parallel with increased detection of synthetic proteins (vimentin and osteopontin) and KLF4, as observed in vivo. KLF4-specific siRNA abolished AMPKα2 deletion-induced VSMC phenotypic switching. Furthermore, pharmacological or genetic inhibition of nuclear factor-κB significantly decreased KLF4 upregulation in VSMC from AMPKα2(-/-) mice. Finally, we found that AMPKα2 deletion markedly promoted the binding of nuclear factor-κBp65 to KLF4 promoter. This study demonstrated that AMPKα2 deletion induces VSMC phenotypic switching and promotes features of atherosclerotic plaque instability in nuclear factor-κB-KLF4-dependent manner.

Ding Y ，Zhang M ，Zhang W ，Lu Q ，Cai Z ，Song P ，Okon IS ，Xiao L ，Zou MH ... -  《-》

Protein deglycase DJ-1 deficiency induces phenotypic switching in vascular smooth muscle cells and exacerbates atherosclerotic plaque instability.

Protein deglycase DJ-1 (DJ-1) is a multifunctional protein involved in various biological processes. However, it is unclear whether DJ-1 influences atherosclerosis development and plaque stability. Accordingly, we evaluated the influence of DJ-1 deletion on the progression of atherosclerosis and elucidate the underlying mechanisms. We examine the expression of DJ-1 in atherosclerotic plaques of human and mouse models which showed that DJ-1 expression was significantly decreased in human plaques compared with that in healthy vessels. Consistent with this, the DJ-1 levels were persistently reduced in atherosclerotic lesions of ApoE-/- mice with the increasing time fed by western diet. Furthermore, exposure of vascular smooth muscle cells (VSMCs) to oxidized low-density lipoprotein down-regulated DJ-1 in vitro. The canonical markers of plaque stability and VSMC phenotypes were evaluated in vivo and in vitro. DJ-1 deficiency in Apoe-/- mice promoted the progression of atherosclerosis and exaggerated plaque instability. Moreover, isolated VSMCs from Apoe-/- DJ-1-/- mice showed lower expression of contractile markers (α-smooth muscle actin and calponin) and higher expression of synthetic indicators (osteopontin, vimentin and tropoelastin) and Kruppel-like factor 4 (KLF4) by comparison with Apoe-/- DJ-1+/+ mice. Furthermore, genetic inhibition of KLF4 counteracted the adverse effects of DJ-1 deletion. Therefore, our results showed that DJ-1 deletion caused phenotype switching of VSMCs and exacerbated atherosclerotic plaque instability in a KLF4-dependent manner.

Wang ZY ，Cheng J ，Liu B ，Xie F ，Li CL ，Qiao W ，Lu QH ，Wang Y ，Zhang MX ... -  《-》

microRNA let-7g suppresses PDGF-induced conversion of vascular smooth muscle cell into the synthetic phenotype.

Platelet-derived growth factor (PDGF) can promote vascular smooth muscle cells (VSMCs) to switch from the quiescent contractile phenotype to synthetic phenotype, which contributes to atherosclerosis. We aimed to investigate the role of microRNA let-7g in phenotypic switching. Bioinformatics prediction was used to find let-7g target genes in the PDGF/mitogen-activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal-regulated kinase (ERK)/Krüppel-like factor-4 (KLF4) signalling pathway that affects VSMC phenotypic switching. The luciferase reporter assay and let-7g transfection were used to confirm let-7g target genes. Two contractile proteins alpha-smooth muscle actin (α-SMA) and calponin were VSMC-specific genes and were measured as the indicators for VSMC phenotype. Lentivirus carrying the let-7g gene was injected to apolipoprotein E knockout (apoE-/- ) mice to confirm let-7g's effect on preventing atherosclerosis. Through the PDGF/MEKK1/ERK/KLF4 signalling pathway, PDGF-BB can inhibit α-SMA and calponin. The PDGFB and MEKK1 genes were predicted to harbour let-7g binding sites, which were confirmed by our reporter assays. Transfection of let-7g to VSMC also reduced PDGFB and MEKK1 levels. Moreover, we showed that let-7g decreased phosphorylated-ERK1/2 while had no effect on total ERK1/2. KLF4 can reduce VSMC-specific gene expression by preventing myocardin-serum response factor (SRF) complex from associating with these gene promoters. The immunoprecipitation assay showed that let-7g decreased the interaction between KLF4 and SRF. Further experiments demonstrated that let-7g can increase α-SMA and calponin levels to maintain VSMC in the contractile status. Injection of lentivirus carrying let-7g gene increased let-7g's levels in aorta and significantly decreased atherosclerotic plaques in the apoE-/- mice. We demonstrated that let-7g reduces the PDGF/MEKK1/ERK/KLF4 signalling to maintain VSMC in the contractile status, which further reduce VSMC atherosclerotic change.

Wang TM ，Chen KC ，Hsu PY ，Lin HF ，Wang YS ，Chen CY ，Liao YC ，Juo SH ... -  《-》

Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo.

Cai Z ，Ding Y ，Zhang M ，Lu Q ，Wu S ，Zhu H ，Song P ，Zou MH ... -  《-》

Melatonin Ameliorates Atherosclerotic Plaque Vulnerability by Regulating PPARδ-Associated Smooth Muscle Cell Phenotypic Switching.

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE-/-) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE-/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis.

Chen SJ ，Chien HC ，Tsai SH ，Jheng YS ，Chen Y ，Hsieh PS ，Tsui PF ，Chien S ，Tsai MC ... -  《-》