Single-Cell Analysis Uncovers Osteoblast Factor Growth Differentiation Factor 10 as Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation Associated with Plaque Rupture in Human Carotid Artery Disease.

Karim J Brandt,Rafaela Fernandes Da Silva,Fabienne Burger,Francois Mach,Fabrizio Montecucco,Aline Roth,Kapka Miteva,Daniela Baptista

doi:10.3390/ijms23031796

Abstract

(1) Background: Vascular smooth muscle cells (VSMCs) undergo a complex phenotypic switch in response to atherosclerosis environmental triggers, contributing to atherosclerosis disease progression. However, the complex heterogeneity of VSMCs and how VSMC dedifferentiation affects human carotid artery disease (CAD) risk has not been clearly established. (2) Method: A single-cell RNA sequencing analysis of CD45− cells derived from the atherosclerotic aorta of Apolipoprotein E-deficient (Apoe−/−) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the site-specific predisposition to atherosclerosis was performed. Growth Differentiation Factor 10 (GDF10) role in VSMCs phenotypic switch was investigated via flow cytometry, immunofluorescence in human atherosclerotic plaques. (3) Results: scRNAseq analysis revealed the transcriptomic profile of seven clusters, five of which showed disease-relevant gene signature of VSMC macrophagic calcific phenotype, VSMC mesenchymal chondrogenic phenotype, VSMC inflammatory and fibro-phenotype and VSMC inflammatory phenotype. Osteoblast factor GDF10 involved in ossification and osteoblast differentiation emerged as a hallmark of VSMCs undergoing phenotypic switch. Under hypercholesteremia, GDF10 triggered VSMC osteogenic switch in vitro. The abundance of GDF10 expressing osteogenic-like VSMCs cells was linked to the occurrence of carotid artery disease (CAD) events. (4) Conclusions: Taken together, these results provide evidence about GDF10-mediated VSMC osteogenic switch, with a likely detrimental role in atherosclerotic plaque stability.

Highlights

Cardiovascular diseases are the leading cause of death in developed countries [1,2], with atherosclerosis being the predominant underlying cause
The adventitia was carefully excised by sharp surgical dissection in a clearly defined plane to leave a naked media composed of vascular smooth muscle cells (VSMCs) and endothelial cells (CD45− cells)
Aorta cells isolated from the arch and root (AA&R) and DT aorta of Apoe−/− mice fed either an normal cholesterol diet (NCD) for 16 weeks or high cholesterol diet (HCD) for 11 weeks (Figure 1a)

Summary

Introduction

Cardiovascular diseases are the leading cause of death in developed countries [1,2], with atherosclerosis being the predominant underlying cause. The disruption of the atheroprotective layer of vascular smooth muscle cells (VSMCs), which forms the fibrous cap that covers the atherosclerotic plaque [3], induces acute thrombo-occlusive events, such as myocardial infarction and stroke [4] Evidence has demonstrated that the different embryonic ascending and descending aortic domains exhibit distinct phenotypes [5] which impact disease progression [6]. Site-specific development of atherosclerotic lesions is observed in both murine models of atherosclerosis and in humans [7]. The observed spatial dissemination of vascular diseases could be explained by differences in hemodynamic and vessel structure [8,9]. Respecting the site-specific predisposition of atherosclerosis, we undertook a precise approach to reveal disease-associated cell populations, genes and molecular determinants by performing single-cell RNA-sequencing (scRNAseq) of atherosclerosis-prone sites separately from atherosclerosis-resistant sites

Methods

Results

Conclusion