Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile ...lncRNAs derived specifically from the aortic intima of
mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 (
) is highly expressed in the vascular endothelium and decreases during lesion progression.
knockdown accelerated atherosclerotic lesion formation by 2.4-fold in
mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of
protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that
interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of
reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD
, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by
knockdown.
expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.
Abstract
Aims
Recent evidence suggests that ‘vulnerable plaques’, which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture ...and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage.
Methods and results
We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity.
Conclusions
NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.
Graphical Abstract
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.
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-β1 (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 600–1197 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.
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.
Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as ...biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown.
Disease stage-specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells.
Disease progression drove significant convergence (
<0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold (
<0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules
,
, and
in human carotid artery smooth muscle cells and
,
, and
in human aortic valvular interstitial cells significantly modulated calcification.
The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.
prostaglandin E(2), by ligation of its receptor EP4, suppresses the production of inflammatory cytokines and chemokines in macrophages in vitro. Thus, activation of EP4 may constitute an endogenous ...anti-inflammatory pathway. This study investigated the role of EP4 in atherosclerosis in vivo, and particularly its impact on inflammation.
Ldlr(-/-) mice transplanted with EP4(+/+) or EP4(-/-) bone marrow consumed a high-fat diet for 5 or 10 weeks. Allogenic bone marrow transplantation promoted exacerbation of atherosclerosis irrespective of EP4 genotype, compatible with prior observations of exacerbated atherogenesis by allogenicity. EP4 deficiency had little effect on plaque size or morphology in early atherosclerosis, but at the later time point, mice deficient in EP4 displayed enhanced inflammation in their atherosclerotic plaques. Expression of monocyte chemoattractant protein-1 and interferon-γ inducible protein 10 increased, and there was a corresponding increase in macrophage and T-cell infiltration. These plaques also contained fewer smooth muscle cells. Despite these changes, mice deficient in EP4 in bone marrow-derived cells at an advanced stage had similar lesion size (in both aorta and aortic root) as mice with EP4.
this study shows that in advanced atherosclerosis, EP4 deficiency did not alter atherosclerotic lesion size, but yielded plaques with exacerbated inflammation and altered lesion composition.
Objectives This study investigated the regulation of glucose uptake in cells that participate in atherogenesis by stimuli relevant to this process, to gain mechanistic insight into the origin of ...the18fluorine-labeled 2-deoxy-D-glucose (FdG) uptake signals observed clinically. Background Patient studies suggest that positron emission tomography (PET) using FdG can detect "active" atherosclerotic plaques, yet the mechanism giving rise to FdG signals remains unknown. Methods We exposed cells to conditions thought to operate in atheroma and determined rates of glucose uptake. Results Hypoxia, but not pro-inflammatory cytokines, potently stimulated glucose uptake in human macrophages and foam cells. Statins attenuated this process in vitro, suggesting that these agents have a direct effect on human macrophages. Immunohistochemical study of human plaques revealed abundant expression of proteins regulating glucose utilization, predominantly in macrophage-rich regions of the plaques--regions previously proved hypoxic. Smooth-muscle cells and endothelial cells markedly increased rates of glucose uptake when exposed to pro-inflammatory cytokines. Conclusions Glucose uptake and, probably, FdG uptake signals in atheroma may reflect hypoxia-stimulated macrophages rather than mere inflammatory burden. Cytokine-activated smooth-muscle cells also may contribute to the FdG signal.
This study investigated the regulation of glucose uptake in cells that participate in atherogenesis by stimuli relevant to this process, to gain mechanistic insight into the origin of the ...(18)fluorine-labeled 2-deoxy-D-glucose (FdG) uptake signals observed clinically.
Patient studies suggest that positron emission tomography (PET) using FdG can detect "active" atherosclerotic plaques, yet the mechanism giving rise to FdG signals remains unknown.
We exposed cells to conditions thought to operate in atheroma and determined rates of glucose uptake.
Hypoxia, but not pro-inflammatory cytokines, potently stimulated glucose uptake in human macrophages and foam cells. Statins attenuated this process in vitro, suggesting that these agents have a direct effect on human macrophages. Immunohistochemical study of human plaques revealed abundant expression of proteins regulating glucose utilization, predominantly in macrophage-rich regions of the plaques-regions previously proved hypoxic. Smooth-muscle cells and endothelial cells markedly increased rates of glucose uptake when exposed to pro-inflammatory cytokines.
Glucose uptake and, probably, FdG uptake signals in atheroma may reflect hypoxia-stimulated macrophages rather than mere inflammatory burden. Cytokine-activated smooth-muscle cells also may contribute to the FdG signal.
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