Related Experiment Video
Updated: Jun 6, 2026
Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments
Published on: October 4, 2021
The exploration to identify key molecules in the interaction between endothelial cells and mono-macrophages in
Boyu Li1,2, Ruiyang Zhu1,2, Na Gao1
1Center for Gene Diagnosis and Department of Clinical Laboratory Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China.
Insights
This study reveals key cell interactions in atherosclerosis, identifying the Midkine (MK) pathway involving MDK-NCL as crucial for plaque development in coronary artery disease (CAD). These findings offer potential therapeutic targets for CAD.
Area of Science:
- Cardiovascular Biology
- Immunology
- Genomics
Background:
- Coronary artery disease (CAD), driven by atherosclerosis (AS), is a major cause of cardiovascular mortality.
- Monocyte/macrophage and endothelial cell interactions are critical in atherosclerotic plaque formation and progression.
Purpose of the Study:
- To characterize single-cell profiles of carotid artery cells in atherosclerotic plaques (AC) versus adjacent healthy tissue (PA).
- To identify cell subpopulations and signaling pathways, particularly between endothelial cells (ECs) and monocyte-macrophages (MMs), altered in AS.
- To explore potential therapeutic targets for CAD.
Main Methods:
- Utilized single-cell RNA sequencing datasets from human AC and PA tissues.
- Analyzed cell-cell communication (CCC) to identify altered signaling pathways and ligand-receptor pairs.
- Performed in vitro Transwell co-culture experiments with human endothelial cells and macrophages.
Main Results:
- Identified 15 cell clusters and 6 AS-associated cell subpopulations.
- Highlighted significant alterations in MK (Midkine), GALECTIN, and SPP1 pathways in AC, with MK being most prominent.
- Found upregulation of the MDK-NCL pair in AS-associated EC and MM subpopulations, confirmed by in vitro assays.
Conclusions:
- Characterized significant cell heterogeneity in AS.
- Identified the MK pathway and MDK-NCL interaction as key players in AS pathogenesis.
- Provides potential novel targets for CAD prevention and treatment.
Abstract:
Coronary artery disease (CAD) is the leading cause of global cardiovascular deaths, and atherosclerosis (AS) is the characteristic pathological change in CAD. The interactions between monocytes/macrophages and endothelial cells play an essential role in the formation and progression of atherosclerotic plaques. In this study, we used published single-cell RNA sequencing datasets of calcified atherosclerotic core (AC) plaques and patient-matched proximal adjacent (PA) carotid artery tissue to characterize the single-cell profiles of carotid artery cells. We specifically analyzed endothelial cells (ECs) and monocyte-macrophage (MM) populations to identify cell subpopulations and signaling pathways associated with AS. By comparing cell-cell communication (CCC) between AC and PA, we identified signaling pathways and corresponding ligand-receptor pairs significantly altered in AS. For preliminary validation, Transwell co-culture experiments were conducted for 24 h with human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) co-cultured with M0, M1, or M2 macrophages. Through integrative analyses, we identified 15 cell clusters and 6 cell subpopulations closely related to AS. Among the altered pathways, MK (Midkine), GALECTIN (Galactose lectin), and SPP1 (Secreted phosphoprotein 1) showed significant changes in AC, with the MK pathway being the most prominent. Notably, the MDK-NCL pair was upregulated in the AS-associated EC and MM subpopulations. In vitro assays confirmed that MDK and NCL expression levels were significantly higher in the ECs and M1 macrophage co-culture group than in the M0 or M2 control groups. In conclusion, this work characterizes AS-related cell heterogeneity and identifies potential key signaling pathways and interactions, providing candidate targets for the prevention and treatment of CAD.
