A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-ß1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600­1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

Nicotine Modulates CTSS (Cathepsin S) Synthesis and Secretion Through Regulating the Autophagy-Lysosomal Machinery in Atherosclerosis.

OBJECTIVE: Increased CTSS (cathepsin S) has been reported to play a critical role in atherosclerosis progression. Both CTSS synthesis and secretion are essential for exerting its functions. However, the underlying mechanisms contributing to CTSS synthesis and secretion in atherosclerosis remain unclear. Approach and Results: In this study, we showed that nicotine activated autophagy and upregulated CTSS expression in vascular smooth muscle cells and in atherosclerotic plaques. Western blotting and immunofluorescent staining showed that nicotine inhibited the mTORC1 (mammalian target of rapamycin complex 1) activity, promoted the nuclear translocation of TFEB (transcription factor EB), and upregulated the expression of CTSS. Chromatin immunoprecipitation-qualificative polymerase chain reaction, electrophoretic mobility shift assay, and luciferase reporter assay further demonstrated that TFEB directly bound to the CTSS promoter. mTORC1 inhibition by nicotine or rapamycin promoted lysosomal exocytosis and CTSS secretion. Live cell assays and IP-MS (immunoprecipitation-mass spectrometry) identified that the interactions involving Rab10 (Rab10, member RAS oncogene family) and mTORC1 control CTSS secretion. Nicotine promoted vascular smooth muscle cell migration by upregulating CTSS, and CTSS inhibition suppressed nicotine-induced atherosclerosis in vivo. CONCLUSIONS: We concluded that nicotine mediates CTSS synthesis and secretion through regulating the autophagy-lysosomal machinery, which offers a potential therapeutic target for atherosclerosis treatment.

Reactive Oxygen Species-Responsive Nanoparticle Delivery of Small Interfering Ribonucleic Acid Targeting Olfactory Receptor 2 for Atherosclerosis Theranostics.

Atherosclerosis (AS) is a chronic inflammatory disorder characterized by arterial intimal lipid plaques. Small interfering ribonucleic acid (siRNA)-based therapies, with their ability to suppress specific genes with high targeting precision and minimal side effects, have shown great potential for AS treatment. However, targets of siRNA therapies based on macrophages for AS treatment are still limited. Olfactory receptor 2 (Olfr2), a potential target for plaque formation, was discovered recently. Herein, anti-Olfr2 siRNA (si-Olfr2) targeting macrophages was designed, and the theranostic platform encapsulating si-Olfr2 to target macrophages within atherosclerotic lesions was also developed, with the aim of downregulating Olfr2, as well as diagnosing AS through photoacoustic imaging (PAI) in the second near-infrared (NIR-II) window with high resolution. By utilization of a reactive oxygen species (ROS)-responsive nanocarrier system, the expression of Olfr2 on macrophages within atherosclerotic plaques was effectively downregulated, leading to the inhibition of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and interleukin-1 ß (IL-1ß) secretion, thereby reducing the formation of atherosclerotic plaques. As manifested by decreased Olfr2 expression, the lesions exhibited a significantly alleviated inflammatory response that led to reduced lipid deposition, macrophage apoptosis, and a noticeable decrease in the necrotic areas. This study provides a proof of concept for evaluating the theranostic nanoplatform to specifically deliver si-Olfr2 to lesional macrophages for AS diagnosis and treatment.

Targeting HDAC6 attenuates nicotine-induced macrophage pyroptosis via NF-κB/NLRP3 pathway.

BACKGROUND AND AIMS: During the development of atherosclerosis, nicotine activates macrophage inflammation. However, whether nicotine induces macrophage pyroptosis and the underlying mechanisms remain unclear. This study aimed to investigate the role of histone deacetylase 6 (HDAC6) in nicotine-induced macrophage pyroptosis. METHODS: For the in vivo study, nicotine was administered to 8-week-old ApoE-/- mice fed a high-fat diet (HFD) for 12 weeks. TUNEL/CD68 and Caspase-1/CD68 staining was used to assess macrophage pyroptosis in plaque. For the in vitro study, Western blotting, lactic dehydrogenase activity (LDH), coimmunoprecipitation, chromatin immunoprecipitation and immunofluorescence were used to evaluate pyroptosis and related signaling pathway in RAW264.7 cells. RESULTS: A high-fat diet and nicotine upregulated macrophage pyroptosis in atherosclerotic lesions. Nicotine promoted pyroptosis in RAW264.7 cells, as evidenced by increased expression of cleaved Caspase1, NLRP3, IL-1ß, IL-18, and elevated LDH release. Inhibition of HDAC6 suppressed nicotine-induced pyroptosis, which is partly mediated by p65 acetylation and NLRP3 transcription. Silencing p65 or NLRP3 resulted in decreased pyroptosis in RAW264.7 cells. CONCLUSIONS: Nicotine induces macrophage pyroptosis in atherosclerosis through HDAC6/NF-κB/NLRP3 signaling pathway.

Epigenetic Control of circHNRNPH1 in Postischemic Myocardial Fibrosis through Targeting of TGF-ß Receptor Type I.

Postischemic myocardial fibrosis is a factor for the development of cardiac dysfunction and malignant cardiac arrhythmias, and no effective therapy is currently available. Circular RNAs are emerging as important epigenetic players in various biological functions; however, their roles in cardiac fibrosis are unknown. With the use of a rat model of postischemic myocardial fibrosis, we identified an increase in circHNRNPH1 in the ischemic myocardium after myocardial infarction, particularly in cardiac fibroblasts. In cardiac fibroblasts, circHNRNPH1 was responsive to transforming growth factor ß1 (TGF-ß1), the principal profibrotic factor. The downregulation of circHNRNPH1, in contrast to its overexpression, promoted myofibroblast migration and α-smooth muscle actin and collagen I expression and inhibited myofibroblast apoptosis. The recombinant adeno-associated virus 9 (rAAV9)-mediated, cardiac-specific knockdown of circHNNRPH1 accordingly facilitated cardiac fibrosis and aggravated cardiac dysfunction. Mechanistically, circHNRNPH1 colocalized with and sponged microRNA (miR)-216-5p in the cytoplasm of cardiac fibroblasts to induce SMAD7 (protein family of signal transduction component of the canonical transforming growth factor-ß signaling pathway) expression, accelerating the degradation of TGF-ß receptor I. Thus, our results indicated that circHNRNPH1 negatively regulates the fibrogenesis of cardiac fibroblasts and may provide a new therapeutic strategy for postischemic myocardial fibrosis.

The interaction between STAT3 and nAChRα1 interferes with nicotine-induced atherosclerosis via Akt/mTOR signaling cascade.

During atherosclerosis development, nicotine and its α1 nicotinic acetylcholine receptors (nAChRα1) activate atherogenic inflammation. However, the effect of signal transducer and activator of transcription 3 (STAT3)-related inflammatory pathways in nicotine-induced atherosclerosis has been poorly studied. This study investigated the transcriptional mechanism of STAT3 in nicotine/nAChRα1-induced atherosclerosis. In vivo, ApoE-/- mice were used to establish an atherosclerotic model. Plaque area and composition were assessed by oil red O staining and immunohistochemistry. In vitro, vascular smooth muscle cells and macrophages were used to investigate cell migration, proliferation, inflammation and related signaling pathways by Transwell migration assay, EdU assay, immunofluorescence, western blotting, coimmunoprecipitation and chromatin immunoprecipitation. nAChRα1 knockdown significantly decreases the nicotine-induced upregulation of p-STAT3, p-Akt and p-mTOR in vitro, while nAChRα1 overexpression has the opposite effects. The inhibition of STAT3 attenuated nicotine-induced atherosclerosis, by reducing the proliferation and migration of vascular smooth muscle cells and inflammation in macrophages. Moreover, there is a direct interaction between STAT3 and nAChRα1 that modulates STAT3 nuclear translocation and its binding to the Akt promoter region upon nicotine exposure. Taken together, STAT3 and nAChRα1 blockade attenuates nicotine-induced atherosclerosis by reducing the migration and proliferation of vascular smooth muscle cells and inflammation in macrophages via the Akt/mTOR pathway.

Exosomes from nicotine-stimulated macrophages accelerate atherosclerosis through miR-21-3p/PTEN-mediated VSMC migration and proliferation.

Rationale: During the development of atherosclerosis, macrophages secrete exosomes that regulate vascular smooth muscle cells (VSMCs); however, whether nicotine, a major constituent of cigarettes, can modulate this communication in the context of atherogenesis remains to be further studied. In this study, we hypothesized that nicotine induces macrophages to secrete atherogenic exosomes containing microRNAs (miRNAs) to mediate cell-to-cell crosstalk and encourage proatherogenic phenotypes of VSMCs. Methods: In an in vivo study, nicotine was administered subcutaneously to 8-week-old male ApoE-/- mice fed a high-fat diet (HFD) for 12 weeks. Oil red O and hematoxylin and eosin (HE) were used to stain atherosclerotic lesions. Lesion macrophages, VSMCs and exosomes were stained for CD68, α-smooth muscle actin (α-SMA) and CD9, and plaque exosomes were observed by transmission electron microscopy (TEM). Exosomes derived from control macrophages (M-Exos) and from nicotine-treated macrophages (NM-Exos) were isolated by ultracentrifugation, purified by sucrose density gradient centrifugation and characterized based on specific morphology and surface markers. The IVIS® Spectrum in vivo imaging system showed the biodistribution of NM-Exos and M-Exos in circulation. Chitosan hydrogel-incorporated exosomes were applied to simulate exosome secretion in situ. Scratch wound assay, transwell assay and EdU staining were conducted to assess the effects of NM-Exos on the migration and proliferation of mouse VSMCs. RNA-seq was performed to determine the miRNA profiles of M-Exos and NM-Exos. Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of miRNAs and mRNAs. The roles of the candidate miRNA and its target gene were assessed using specific RNA inhibitors, siRNAs and miRNA mimics. Western blotting was used to detect candidate protein expression levels. A dual-luciferase reporting system was utilized to confirm the binding of a specific miRNA to its target gene. Results: Nicotine induced atherosclerotic lesion progression and resulted in plaque exosome retention in vivo. The biodistribution of NM-Exos showed that plaque-resident exosomes might be secreted in situ. VSMCs cocultured in vitro with nicotine-stimulated macrophages presented an increased capacity for migration and proliferation, which was exosome-dependent. In addition, isolated NM-Exos helped promote VSMC migration and proliferation. miRNA profiling showed that miR-21-3p was enriched in NM-Exos, and this miRNA was shown to play a key role in regulating NM-Exos-induced effects by directly targeting phosphatase and tension homologue (PTEN). Conclusion: Exosomal miR-21-3p from nicotine-treated macrophages may accelerate the development of atherosclerosis by increasing VSMC migration and proliferation through its target PTEN.

Inhibition of circHIPK3 prevents angiotensin II-induced cardiac fibrosis by sponging miR-29b-3p.

BACKGROUND: Circular RNAs (circRNAs) are emerging as powerful regulators of cardiac development and disease. Nevertheless, detailed studies describing circRNA-mediated regulation of cardiac fibroblasts (CFs) biology and their role in cardiac fibrosis remain limited. METHODS: PCR and Sanger sequencing were performed to identify the expression of circHIPK3 in CFs. Edu corporation assays, Transwell migration assays, and immunofluorescence staining assays were conducted to detect the function of circHIPK3 in CFs in vitro. Bioinformatics analysis, dual luciferase activity assays, RNA immunoprecipitation, and fluorescent in situ hybridization experiments were conducted to investigate the mechanism of circHIPK3-mediated cardiac fibrosis. Echocardiographic analysis, Sirius Red staining and immunofluorescence staining were performed to investigate the function of circHIPK3 in angiotensin II (Ang II) induced cardiac fibrosis in vivo. RESULTS: circHIPK3 expression markedly increased in CFs and heart tissues after the treatment of Ang II. circHIPK3 silencing attenuates CFs proliferation, migration and the upregulation of a-SMA expression levels induced by Ang II in vitro. circHIPK3 acted as a miR-29b-3p sponge and overexpression of circHIPK3 effectively reverses miR-29b-3p-induced inhibition of CFs proliferation and migration and alters the expression levels of miR-29b-3p targeting genes (a-SMA, COL1A1, COL3A1) in vitro. Combination of circHIPK3 silencing and miR-29b-3p overexpression had a stronger effect on cardiac fibrosis suppression in vivo than did circHIPK3 silencing or miR-29b-3p overexpression alone. CONCLUSIONS: Our data suggest that circHIPK3 serves as a miR-29b-3p sponge to regulate CF proliferation, migration and development of cardiac fibrosis, revealing a potential new target for the prevention of Ang II-induced cardiac fibrosis.

Combination of doxorubicin liposomes with left atrial appendage radiofrequency catheter ablation to reduce post-ablation recovery of electrical conduction.

AIMS: To determine whether use of radiofrequency catheter ablation (RFCA) combined with intravenously administered liposomal doxorubicin (L-DOX) facilitates a reduction in the recovery of post-ablation electrical conduction. METHODS: Circumferential ablation was performed on the epicardial surface of the left atrial appendage (LAA) in New Zealand White rabbits, and L-DOX was then administered intravenously. Fluorescence spectrophotometry was used to assess reagent bio-distribution, while Western blots and immunohistochemistry were used to assess the localization of the apoptotic markers Bcl-2, Bax, and cleaved CASP3 in the LAA. Liver, kidney, and cardiac functions were also measured to evaluate the safety of this approach. RESULTS: At 1 week and 1 month after RFCA, a pacing electrocardiogram could not be detected in most of the rabbits that had received the combined RFCA and L-DOX therapy. L-DOX began to target the LAA on the second day after RFCA. L-DOX treatment increased the apoptosis of cardiomyocytes in the regions peripheral to the necrotic area induced by RFCA. Doxorubicin had some effect on liver and kidney function, but these effects were reversible and did not affect survival. CONCLUSION: The present results provide evidence that L-DOX treatment can reduce the recovery of electrical conduction after RFCA therapy owing to L-DOX-induced apoptosis of cardiomyocytes in the ablated area and the proximal transition zone of the LAA.

circHIPK3 regulates lung fibroblast-to-myofibroblast transition by functioning as a competing endogenous RNA.

Myofibroblasts predominantly emerging through fibroblast-to-myofibroblast transition (FMT) are considered to be the key collagen-producing cells in pulmonary fibrosis. Circular RNAs (circRNAs) are important players involved in many biological processes. circHIPK3 has been identified as the one of the most abundant circRNAs in human lung. In this study, we characterized the role of circHIPK3 in pulmonary fibrosis. We revealed that circHIPK3 is upregulated in bleomycin-induced pulmonary fibrosis mice model, FMT-derived myofibroblasts. circHIPK3 silencing can ameliorate FMT and suppress fibroblast proliferation in vivo and vitro. Fundamentally, circHIPK3 regulates FMT by functioning as an endogenous miR-338-3p sponge and inhibit miR-338-3p activity, thereby leading to increased SOX4 and COL1A1 expression. Moreover, dysregulated circHIPK3 expression was detected in the clinical samples of patients with idiopathic pulmonary fibrosis. Intervention of circHIPK3 may represent a promising therapy for pulmonary fibrosis.

Rabbit model to simulate the residual conduction gaps after radiofrequency ablation on the anterior wall of left atrial appendage.

PURPOSE: Radiofrequency ablation (RFA) is widely used to treat patients with atrial fibrillation (AF), but its recurrence rate is still high mainly due to pulmonary vein reconnection and residual conduction gaps. We aim to establish a rabbit model to simulate the residual conduction gaps after ablation. METHODS: Sixty-nine adult New Zealand white rabbits were randomly assigned to six groups. RFA on the anterior wall of left atrial appendage (LAA) were performed with the ablation power from 6 to 21 W. The electrophysiological pacing and mapping technology was used to evaluate the bidirectional conduction of LAA. Histological study and fluorescence techniques were used to evaluate the effect of RFA and the accumulation of drug-loaded liposome on the loop ablation lesions of LAA. RESULTS: Typical loop ablation lesions of LAA could be observed in vivo and vitro of rabbit models. Histological evaluation revealed coagulative necrosis on the loop ablation lesions. Electrical conduction between inside and outside loop lesions recovered after 1 or 2 weeks after initial unidirectional conduction block. The recurrence rates were significantly different among six groups with varying ablation powers (p < 0.05). Compared with exit conduction block, entrance conduction block was significantly different at 5 min after ablation (p = 0.02). IR-775-loaded liposomes were accumulated on the loop ablation lesions at 48 h after RFA. CONCLUSIONS: RFA associated with electrophysiological pacing and mapping technology successfully established a novel rabbit model to simulate the residual conduction gaps after RFA.