RNA sequencing of blood in coronary artery disease: involvement of regulatory T cell imbalance

Abstract

Background Cardiovascular disease had a global prevalence of 523 million cases and 18.6 million deaths in 2019. The current standard for diagnosing coronary artery disease (CAD) is coronary angiography. Surprisingly, despite well-established clinical indications, up to 40% of the one million invasive cardiac catheterizations return a result of ‘no blockage’. The present studies employed RNA sequencing of whole blood to identify an RNA signature in patients with angiographically confirmed CAD. Methods Whole blood RNA was depleted of ribosomal RNA (rRNA) and analyzed by single-molecule sequencing of RNA (RNAseq) to identify transcripts associated with CAD (TRACs) in a discovery group of 96 patients presenting for elective coronary catheterization. The resulting transcript counts were compared between groups to identify differentially expressed genes (DEGs). Results Surprisingly, 98% of DEGs/TRACs were down-regulated ~ 1.7-fold in patients with mild to severe CAD (> 20% stenosis). The TRACs were independent of comorbid risk factors for CAD, such as sex, hypertension, and smoking. Bioinformatic analysis identified an enrichment in transcripts such as FoxP1, ICOSLG, IKZF4/Eos, SMYD3, TRIM28, and TCF3/E2A that are likely markers of regulatory T cells (Treg), consistent with known reductions in Tregs in CAD. A validation cohort of 80 patients confirmed the overall pattern (92% down-regulation) and supported many of the Treg-related changes. TRACs were enriched for transcripts associated with stress granules, which sequester RNAs, and ciliary and synaptic transcripts, possibly consistent with changes in the immune synapse of developing T cells. Conclusions These studies identify a novel mRNA signature of a Treg-like defect in CAD patients and provides a blueprint for a diagnostic test for CAD. The pattern of changes is consistent with stress-related changes in the maturation of T and Treg cells, possibly due to changes in the immune synapse.

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“…To determine whether this cell-specific effect was due to an interpretation bias, the transcriptomes were compared to cell-type-specific transcriptomes from the Human Blood Atlas. The Blood Atlas’ data has been previously used to demonstrate enrichment in specific blood cell types in RNAseq samples [ 14 ], and was able to detect changes in T-cell abundance in coronary artery disease [ 16 ]. Average levels of the cell-type-specific transcripts are shown for control/incidental and critical patients ( Fig 1 , Panel C).…”

Whole-Blood Transcriptional Profiles Enable Early Prediction of the Presence of Coronary Atherosclerosis and High-Risk Plaque Features at Coronary CT Angiography

“…However, profiling was performed using microarrays, which are limited in dynamic range. Only recently have a few studies used RNA-Seq to look for transcriptional markers of the degree of coronary stenosis or related conditions, although in all of these the vessels were being investigated using invasive angiography in symptomatic patients [ 12 , 13 , 14 , 15 ].…”

“…The CAD phenotype was associated with transcriptional profiles of T lymphocytes (Tregs, T-CD4 + , T-CD8 + , and follicular T cells) and B lymphocytes (naive and memory cells), whereas the noCAD group was enriched with cell types associated with the innate immune system, such as granulocytes, monocytes, and mast cells. Although atherosclerosis is considered a chronic inflammatory disorder whose progression is driven by the innate immune response, with the principal involvement of myeloid cells, the hypothesis that both innate and adaptive immunity are involved in the development of this disease has emerged from recent studies [ 38 ], and involvement of regulatory T cell (Tregs) imbalance has been described [ 14 ]. The presence of B cells and T cells at the level of the lesion suggests that atherosclerotic pathology may also be characterized by the presence of an important autoimmune component [ 28 , 38 ].…”

“…Preliminary studies have identified gene expression profiles that would distinguish women with acute myocardial infarction (MI) with nonobstructive CAD from those with MI-CAD or controls [ 12 ], and subjects with coronary stenosis ≥50% from those with stenosis <50% [ 13 ]. Two larger studies documented transcriptional expression signatures that discriminated a low degree of stenosis (≤20%) from an intermediate degree (>20% but <70% in any vessel) in patients presenting for elective coronary catheterization [ 14 ], and subjects with early MI from those with subclinical coronary atherosclerosis (detected as a high degree of coronary artery calcification (CAC)) and from controls without a history of MI or high CAC [ 15 ]. In all these studies, the presence or absence of coronary atherosclerosis had been documented by invasive coronary angiography.…”

Identification of molecular subtypes of coronary artery disease based on ferroptosis- and necroptosis-related genes

“…Coronary artery disease (CAD) is a common cardiac disease and the primary cause of cardiovascular disease-related death ( Agha et al, 2019 ; McCaffrey et al, 2021 ). CAD is caused by atherosclerosis or atherosclerotic occlusions of the coronary arteries ( Thomas et al, 2018 ).…”