Background and Aims

Autophagy is a cell survival mechanism, which has been implicated in atherogenesis in mouse models by studying core autophagy machinery proteins using knock-out models. MAP1LC3A and MAP1LC3B play a key role in autophagy activity and have been implicated as prognostic factors in several human cancers. However, data on the involvement of autophagy in human atherosclerotic disease and plaque vulnerability are still sparse and completely lacking with regards to the involvement of MAP1LC3.

Aim: To characterize MAPLC3 involvement during plaque progression and plaque vulnerability.

Methods

Mouse models of atherosclerosis and plaque rupture, human tissues from two different carotid plaque biobanks as well as primary human carotid smooth muscle cells were analysed by different histological, immunofluorescent, cell and molecular approaches.

Results

In in vivo models, we show that Map1lc3a mRNA is downregulated but no change in Map1lc3b expression during the progression of atherosclerosis in hypercholesterolemic mice. Using two independent biobanks of human carotid atherosclerotic plaques, we observe that MAP1LC3A mRNA and protein levels are decreased in plaques from patients with symptomatic disease compared to asymptomatic. Notably, MAP1LC3A mRNA levels strongly correlate with vascular smooth muscle cell (VSMC) markers, while MAP1LC3B does not. Upon vascular hyperplasia, induced by balloon-injury, Map1lc3a is downregulated. Preliminary in vitro studies show that silencing of MAP1LC3A affects VSMC phenotypic switch. Further analyses of migration, proliferation and cell death parameters are ongoing.

Conclusions

These results demonstrate that reduced MAP1LC3A expression is a relevant marker of vulnerable plaque phenotype, suggesting an impact on VSMC biology in the context of atherogenesis.

Background and Aims

Melanocortin receptor-1 (MC1-R) subtype is expressed in leukocytes, where it mediates anti-inflammatory actions. We have previously observed that global deficiency of MC1-R signaling perturbed cholesterol homeostasis and increased arterial leukocyte accumulation in ApoE^-/- mice. Therefore, we aimed at investigating the contribution of leukocyte MC1-R to the development of atherosclerosis in ApoE^-/- mice.

Methods

ApoE^-/- mice were irradiated and received bone marrow (BM) cells from either ApoE^-/- or MC1-R deficient ApoE^-/- MC1-R^e/e mice. After 6 weeks of recovery, mice were maintained on chow diet or placed on western-type (HFD) diet for 10 weeks and analyzed for leukocyte counts in the blood, spleen and aorta by flow cytometry. Plaque size and composition in the aortic root were determined. Tissue expression of inflammatory markers was also assessed by qPCR and Western blotting.

Results

ApoE^-/-MC1-R^e/e engrafted mice showed significantly elevated leukocyte counts in the blood and spleen in both chow- and HFD-fed groups. This was primarily due to increased lymphocyte count, specifically in CD4⁺ T cells. Aortic lymphocyte count was also increased in chow-fed mice, while no differences were observed after HFD. Unexpectedly, ApoE^-/-MC1-R^e/e mice showed down-regulation of pro-inflammatory markers in the spleen and aorta, an affect that was more pronounced in the HFD group. Despite robust leukocytosis, plaque size and composition were unaffected by leukocyte MC1-R deficiency.

Conclusions

Our data demonstrate that leukocyte MC1-R deficiency in ApoE^-/- mice enhances leukocyte counts, particularly CD4⁺ T cells, without affecting the development of atherosclerotic plaques. Nonetheless, MC1-R appears to significantly regulate leukocyte production and behavior.

Background and Aims

Bifurcations are known as one of the most atheroprone regions in the coronary vasculature. In order to one day predict disease progression in this region, the development of a biomechanical computer model based on patient-specific in vivo data is essential. This investigation aims to develop such a biomechanical model from clinically indicated angiography of the left main bifurcation region, including surrounding coronary branches, capable of accurately analysing parameters, such as shear stress, that lead to atherosclerosis progression.

Methods

A fluid-structure interaction model of the left main bifurcation region, including the left main, left circumflex, left obtuse marginal, left anterior descending and the first diagonal branch from clinically indicated angiography planes is proposed. The Finite Element Method (FEM) was used to analyse the contraction of the heart muscle, pulsatile blood flow, change in artery wall thickness (intima, media and adventitia) and the active contraction of the media layer. Material properties for artery layers, blood and plaques were used from clinical data in the literature.

Results

The influence of heart muscle contraction (including increasing heart rate with exercise) and changes in artery wall thickness, evidenced in vascular remodelling, is discussed in relation to shear and artery stress distribution with links to atherosclerosis progression made.

Conclusions

Many parameters influence the shear and artery stress distributions in bifurcation regions and, hence, alter the risk of atherosclerosis progression. The developed biomechanical computer model shows promise for predicting both these distribution changes as well as atherosclerosis initiation and progression.

Background and Aims

Different cell populations of arteries such as vascular smooth muscle cells, endothelial cells and immune cells undergo specific gene expression changes during the process of atherosclerosis development. Recently, application of single cell RNA sequencing (scRNA-seq) on leukocytes has identified 11-13 distinct immune cell populations and their transcriptional landscape in atherosclerosis prone mice. Since, diabetes accelerates atherosclerosis development, we have extended this approach by applying scRNA-seq to all aortic cells with the specific aim to identify cell specific gene expression signature of the diabetic aorta.

Methods

Diabetes was induced with streptozotocin and mice were followed for 10 weeks. Cells from digested aortae were FACS-sorted for viable and metabolically active cells. These cells were loaded on the Chromium Single Cell Controller (10X Genomics) to generate single cell and gel bead emulsion. ScRNA-seq libraries were prepared with Single Cell 3’ Solution V2 kit (10X Genomics) and sequenced with Illumina Nova-seq 6000.

Results

We have applied scRNA-seq to unbiasedly uncover cell populations and their transcriptional landscape in the aortae of diabetic and control Apoe^-/- mice. Based on SEURAT algorithm, scRNA transcriptomic analysis have identified aortic cell populations and subpopulations with their distinct gene expression signature validating the immune cell heterogeneity in atherosclerotic aorta. Cell specific gene set enrichment analysis has shown several dysregulated pathways including oxidative stress, lipid metabolism, cytokine secretion and inflammation.

Conclusions

This is the first scRNA transcriptomic landscape of the whole aorta in diabetes. These exciting novel findings uncover the transcriptomic landscape of diabetes at the single cell resolution in the atherosclerotic aorta.