Cook, S. L. | Search bioRxiv

bioRxiv2025

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

BackgroundEndothelial 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. MethodsTwenty-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. ResultsPairwise 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. ConclusionsCCBE1 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 ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/670989v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@4e609aorg.highwire.dtl.DTLVardef@1a85810org.highwire.dtl.DTLVardef@12d97cdorg.highwire.dtl.DTLVardef@494ab3_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LIThe endothelium plays a critical role in vascular health and susceptibility to atherosclerosis. C_LIO_LIMitochondrial dysfunction has been implicated in atherosclerosis, but its role and mechanism in individual susceptibility to CAD in humans is not known. C_LI What New Information Does This Article Contribute?O_LINovel approach integrating CT imaging with ECFC functional data to link vascular structure with endothelial biology ex vivo. C_LIO_LIPatients with CT-defined CAD had 3.6-fold higher CCBE1 expression compared to non-CAD within the Validation Cohort. C_LIO_LICis-eQTL-association analysis revealed increased odds of CAD. C_LIO_LICCBE1 knockdown improved mitochondrial function in human endothelial cells. C_LIO_LITogether, these 4 lines of evidence point to a novel and causal role for CCBE1 in human CAD. C_LI

cell biology↗

bioRxiv2024

IL-7 mediated upregulation of VLA-4 increases accumulation of adoptively transferred T lymphocytes in murine glioma.

The efficacy of T cell-activating therapies against glioma is limited by an immunosuppressive tumor microenvironment and tumor-induced T cell sequestration. We investigated whether peripherally infused non-antigen specific autologous lymphocytes (ALT) could accumulate in intracranial tumors. We observed that non-specific autologous CD8+ ALT cells can indeed accumulate in this context, despite endogenous T cell sequestration in bone marrow. Rates of intratumoral accumulation were markedly increased when expanding lymphocytes with IL-7 compared to IL-2. Pre-treatment with IL-7 ALT also enhanced the efficacy of multiple tumor-specific and non-tumor-specific T cell-dependent immunotherapies against orthotopic murine and human xenograft gliomas. Mechanistically, we detected increased VLA-4 on mouse and human CD8+ T cells following IL-7 expansion, with increased transcription of genes associated with migratory integrin expression (CD9). We also observed that IL-7 increases S1PR1 transcription in human CD8+ T cells, which we have shown to be protective against tumor-induced T cell sequestration. These observations demonstrate that expansion with IL-7 enhances the capacity of ALT to accumulate within intracranial tumors, and that pre-treatment with IL-7 ALT can boost the efficacy of subsequent T cell-activating therapies against glioma. Our findings will inform the development of future clinical trials where ALT pre-treatment can be combined with T cell-activating therapies. Brief SummaryT cell immunotherapies are limited by few T cells in glioma. Adoptively transferred lymphocytes expanded with IL-7 exhibit increased VLA-4 expression and accumulate in tumors.

cancer biology↗