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The role of necroptosis in atherogenesis and plaque stability
Smooth muscle cells plasticity in plaque stability
GASDERMIN D DEFICIENCY DELAYS ATHEROGENESIS BUT STIMULATES PLAQUE APOPTOSIS IN APOE-/- MICE
Abstract
Background and Aims
Gasdermin D (Gsdmd) is a key executioner of NLRP3 inflammasome- and caspase 1-dependent pyroptotic cell death. Recently, expression of Gsdmd and caspase 1-mediated cleavage of Gsdmd was described in atherosclerotic plaques. Plaque rupture is associated with strong immunoreactivity for caspase 1, suggesting that Gsdmd-mediated pyroptosis and inflammation are involved in plaque destabilization. Therefore, we aimed to study the effects of Gsdmd deficiency on advanced plaques in ApoE-/- mice.
Methods
ApoE-/- Gsdmd-/- (n=16) and ApoE-/- Gsdmd+/+ (n=18) mice were fed a Western-type diet (WD) for 16 weeks to induce plaque formation. BMDMs were isolated from Gsdmd-/- and Gsdmd+/+ mice and treated with LPS followed by nigericin or ATP to characterize Gsdmd-dependent cell death and inflammation in vitro.
Results
Atherosclerotic plaques in the brachiocephalic artery of WD-fed ApoE-/- Gsdmd-/-mice were significantly smaller as compared to controls (114±18 vs. 186±16 103 µm2, P=0.006). Moreover, plaques of ApoE-/- Gsdmd-/- mice showed features of increased plaque stability, such as a decrease in necrotic core area (19±4 vs. 37±7 103 µm2, P=0.03) and an increase in the vascular smooth muscle cell/macrophage ratio (αSMA/Mac3 ratio: 1.6±0.3 vs. 0.7±0.1, P=0.01). Interestingly, a significant increase of TUNEL positive cells was observed in plaques of ApoE-/- Gsdmd-/- mice as compared to controls (141±25 vs. 62±8 cells/mm2, P=0.005), suggesting a switch to apoptosis. This switch from pyroptotic to apoptotic cell death was also confirmed in vitro in Gsdmd-/- BMDM after LPS/nigericin and LPS/ATP treatment.
Conclusions
Gsdmd deficiency delays atherogenesis but induces a switch to apoptosis in ApoE-/- mice.
LAMIN A/C EXPRESSION IN HEMATOPOIETIC CELLS REGULATES MOUSE ATHEROSCLEROSIS: A POTENTIAL MECHANISM CONTRIBUTING TO AGE-DEPENDENT ATHEROSCLEROSIS
Abstract
Background and Aims
Nuclear lamin A/C play key structural and functional roles in many cell types. We have shown that immune cell function is regulated by lamin A/C, which undergoes age-dependent downregulation in these cells. Since aging is the main cardiovascular risk factor, we aimed to investigate whether atherosclerosis is affected by changes in Lamin A/C expression in immune cells, major regulators of atherosclerotic plaque development.
Methods
We generated by CRISPR-Cas9 a new transgenic mouse model with Cre-LoxP driven inducible lamin A overexpression (LAO). For atherosclerosis studies, we reconstituted lethally-irradiated Ldlr-KO mice with control, lamin A/C-KO (LKO) or LAO bone marrow (BM). After recovery, transplanted mice were challenged with a high-fat diet for 6 weeks and aortas were processed for characterization of plaque size and composition, and for sc-RNAseq. We also performed intravital microscopy to assess leukocyte/endothelium interactions in vivo.
Results
Circulating blood cell populations, body weight, and lipid profile were undistinguishable between experimental groups; however, atherosclerosis burden was increased and reduced in Ldlr-KO mice transplanted with LKO and LAO BM, respectively. These results were associated with changes in the expression of genes involved in leukocyte migration (sc-RNAseq studies), and with increased and reduced number of extravasated leukocytes in mice receiving LKO and LAO BM, respectively (intravital microscopy).
Conclusions
These findings highlight an important role of lamin A/C in atherosclerosis development, mediated at least in part through regulation of leukocyte extravasation. We propose age-dependent downregulation of lamin A/C in immune cells as a new mechanism contributing to atherosclerosis development during aging.
SMOOTH MUSCLE CELL-SPECIFIC DELETION OF S100A4 REROUTES SMOOTH MUSCLE CELL FATE AND MODIFIES THE INFLAMMATORY STATUS OF MURINE ATHEROSCLEROTIC LESIONS
Abstract
Background and Aims
S100A4, a small calcium binding protein, plays an important role in vascular smooth muscle cell (SMC) phenotypic switch but its implication in atherosclerotic plaque development and particularly in SMC phenotypic plasticity is not clear yet. By neutralizing S100A4 using a monoclonal antibody, we have previously shown a reduction in overall atherosclerotic burden. However, this strategy did not distinguish the different contribution between SMCs or other S100A4-expressing cells (e.g. macrophages).
Methods
Herein, we used a lineage tracing mouse model in which we induced a SMC specific deletion of S100A4 (SMC-S100A4Δ/Δ) in an ApoE-/- background. High cholesterol diet was maintained for 12 weeks, after which, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed and aortas processed for staining and single cell RNA sequencing (scRNA-seq).
Results
We showed that S100A4 deletion modulated plaque composition rather than plaque size. ScRNA-seq analysis from SMC-S100A4Δ/Δ, compared with SMC-S100A4wt/wt, showed a reduction in macrophages (28% vs 58%) and dendritic cells (9% vs 1%), and an increase in total SMCs (16% vs 42%), likely participating in fibrous cap stabilization. SMC populations showed a decrease in macrophage-like inflammatory phenotype and an increase in fibroblast-like repair phenotype, as well as retention of specific SMC markers, namely Acta2 and Myh11. Gene expression analysis revealed a reduction in the expression of inflammation markers, fatty acid metabolism, and extracellular matrix-related genes, and an increase in classical contractile SMC markers.
Conclusions
We report S100A4 as a regulator of SMC fate within the plaque, and a global impact on the inflammatory status of the atherosclerotic plaque.
THE EPIGENETIC ENZYME DOT1L ORCHESTRATES VSMC–MONOCYTE CROSSTALK TO PROTECT AGAINST ATHEROSCLEROSIS VIA THE NF-KB PATHWAY
Abstract
Background and Aims
Cardiovascular diseases are the principal cause of death and disability worldwide, and resulting in an estimated total economic burden of €210 billion. H3K79 dimethylation is a key epigenetic mark uniquely induced by methyltransferase disruptor of telomeric silencing 1-like (DOT1L). We aimed to determine whether DOT1L modulates vascular smooth muscle cell (VSMC) phenotype and how it might affect atherosclerosis in vitro and in vivo.
Methods
Gene expression screening of VSMCs, upon platelet-derived growth factor isoform BB (PDGF-BB) treatment, led us to identify DOT1L as an early upregulated epigenetic factor. Mouse and human atherosclerotic lesions were assessed for Dot1l expression, which resulted specifically localized in the VSMC compartment. The relevance of DOT1L to atherosclerosis pathogenesis was assessed through deletion of its gene in the VSMCs via an inducible, tissue-specific-knockout mouse model crossed with the ApoE‑/- high-fat diet model of atherosclerosis.
Results
We found that inactivation of DOT1L significantly reduced atherosclerosis progression. By combining RNA sequencing and H3K79me2-chromatin immunoprecipitation, we then found that DOT1L and its induced H3K79me2 mark directly regulate the transcription of NF-κB-1 and -2, master modulators of inflammation, which in turn induces the expression of CCL5 and CXCL10, both fundamentally involved in atherosclerosis development. Finally, a correlation between coronary artery disease and genetic variations in the DOT1L gene was found, since specific polymorphisms are associated with increased mRNA expression.
Conclusions
DOT1L plays a key role in the epigenetic control of VSMC gene expression, leading to atherosclerosis development. Results identify DOT1L as a potential therapeutic target for vascular diseases.
UNDERSTANDING 3D CULTURE IN SMOOTH MUSCLE CELL BIOLOGY AND THE MODELLING OF ATHEROSCLEROSIS
Abstract
Background and Aims
Even though the advantages of 3D culture in mimicking cellular environment and drug discovery is widely reported in cancer biology, there are limited studies on 3D culture in vascular biology and atherosclerosis modelling. Our study aims to investigate the significance of 3D spheroid culture on cellular physiology and in atherosclerosis modelling using vascular smooth muscle cells(VSMC). We also explore 3D vessel modelling via co-culturing of endothelial cells(EC) and VSMCs.
Methods
Commercial SMC and patient-derived SMCs(CD45-/CD31-/CD90-), which were isolated using enzymatic dissociation and magnetic microbeads sorting from primary arterial tissue, were used.
Results
Transcript evaluation identified that SMCs in their adherent 2D culture and 3D floating culture (spheroid) have differentially expressed genes related to cellular phenotype and their response to oxLDL stimuli. 3D SMCs have significantly higher expression of genes encoding for foam cell-like characteristics(CD36 and LOX-1) and extracellular matrix-related proteins(MMP2, Fibronectin, and VCAM-1) compared to 2D VSMCs. Moreover, upon introducing ECs to VSMC 3D culture, we observed specific movement and self-organization with EC localization within the core region of spheroids, regardless of the time of EC introduction and cell ratio.
Conclusions
Thus, we conclude that transcripts differences of the 2D and 3D culture vascular could be utilized to create a patient-specific model, allowing precise therapy planning and drug testing ex vivo. The 3D vessel model could generate patient-specific disease model and provide greater understanding of VSMCs characteristic in the cellular microenvironment mimicking vessel wall and atherosclerosis. Moreover, the proposed 3D model would provide insights into the ECs and SMCs interaction on single-cell resolution.