Endothelial Colony-Forming Cell Transcriptomic Profiling in CT-defined Coronary Artery Disease from the BioHEART-CT Study Implicate CCBE1 in Mitochondrial Dysfunction-associated Atherosclerosis

Authors:
  • SCSarah L. Cook
    Sarah L. Cook
  • GPGareth S.D. Purvis
    Gareth S.D. Purvis
  • KSKirsten Schnitzler
    Kirsten Schnitzler
  • EGElijah Genetzakis
    Elijah Genetzakis
  • MBMarie Besnier
    Marie Besnier
  • MCMadhuvanthi Chandrakanthan
    Madhuvanthi Chandrakanthan
  • ATAndy Tran
    Andy Tran
  • DSDario Strbenac
    Dario Strbenac
  • CMCarmen Mifsud
    Carmen Mifsud
  • KBKatharine Bate
    Katharine Bate
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Abstract
Background: Endothelial dysfunction is an early contributor to atherosclerosis. This study combined CT imaging of coronary artery disease (CAD) and patient-dervied endothelial colony-forming cells (ECFCs) transcriptional profiling to investigate potential mechanisms underlying endothelial dysfunction in atherosclerosis. Methods: Twenty-six individuals with CT-defined CAD and eighteen non-CAD controls were included in the Discovery Cohort for bulk RNA sequencing and transcriptomic analysis of ECFCs. Differential gene expression analysis was performed, and candidate genes were selected based on logFC and p-value. Candidate genes were carried forward for gene expression validation using quantitative real-time PCR (qRT-PCR) in a Validation Cohort. Mitochondrial reactive oxygen species (mROS) production and mitochondrial mass were assessed using multi-colour flow cytometry. Functional validation of the top candidate was conducted in using human umbilical vein endothelial cells (HUVECs) using loss-of-function genetic approach. Expression Quantitative Trait Loci (eQTL)-association analysis was conducted using genotype data from the BioHEART-CT cohort. Results: Pairwise analysis identified six differentially expressed protein-coding genes in CAD ECFCs: CCBE1 (Collagen and Calcium Binding EGF Domain-Containing 1), SPINT2, CRISPLD1, PIEZO2, EPB41L3 , and AC005943.1 . qRT-PCR in the Validation Cohort confirmed significantly higher CCBE1 expression in CAD patients. Individuals with relative CCBE1 fold change expression>10 had a 2.8-fold increase in the log-odds ratio of CT-defined CAD. CAD ECFCs displayed elevated mROS and mitochondrial mass. CCBE1 knockdown in HUVECs reduced mROS and mitochondrial mass without affecting proliferation or permeability, but shifted cells into a metabolically elevated state, marked by increased ATP production, respiration and glycolysis. CCBE1 cis-eQTLs were associated with increased odds of CAD in the BioHEART-CT cohort. Conclusions: CCBE1 expression in ECFCs was higher in patients with CT-defined CAD versus non-CAD. Quantitative assessment of transcript levels supported a causal relationship between greater CCBE1 expression and CAD burden and risk, and functional experiments on CCBE1 knockdown demonstrated improved mitochondrial function in human endothelial cells. GRAPHICAL Novelty and Significance: What is Known?: The endothelium plays a critical role in vascular health and susceptibility to atherosclerosis. Mitochondrial dysfunction has been implicated in atherosclerosis, but its role and mechanism in individual susceptibility to CAD in humans is not known. What New Information Does This Article Contribute?: Novel approach integrating CT imaging with ECFC functional data to link vascular structure with endothelial biology ex vivo . Patients with CT-defined CAD had 3.6-fold higher CCBE1 expression compared to non-CAD within the Validation Cohort. Cis -eQTL-association analysis revealed increased odds of CAD. CCBE1 knockdown improved mitochondrial function in human endothelial cells. Together, these 4 lines of evidence point to a novel and causal role for CCBE1 in human CAD.

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