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

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.

Next-Generation and Single-Cell Sequencing Approaches to Study Atherosclerosis and Vascular Inflammation Pathophysiology: A Systematic Review

“…Ten articles ( 20 , 27 , 28 , 31 , 37 , 41 , 45 – 47 , 50 ) addressed the role of SMCs in atherosclerosis and vascular inflammation. Four of these articles ( 20 , 45 , 46 , 50 ) utilized single-cell sequencing exclusively, one ( 31 ) utilized next-generation RNA sequencing exclusively, one ( 47 ) utilized next-generation micro-RNA sequencing, and the remaining four ( 27 , 28 , 37 , 41 ) utilized a combination of these methodologies, including the use of ChIP-Seq ( 27 , 28 , 37 ), ATAC-Seq ( 28 ) and CITE-Seq ( 27 ).…”

“…Brandt et al ( 50 ) undertook single-cell sequencing of CD45 − cells isolated from atherosclerotic aortas of ApoE −/− mice on a normal and high cholesterol diet, with cells categorized based localization to atheroprone and atheroresistant regions (aortic arch and descending thoracic aorta, respectively). Differential regulation of genes was identified in the atheroprone cell cluster based on diet associated with apoptosis, inflammation, atherogenesis among others.…”

“…Of all cell types investigated in the included articles, SMCs featured predominantly ( n = 12), with attention paid predominantly to their phenotypic switching capacity in disease states ( 20 , 27 , 28 , 31 , 37 , 41 , 45 – 47 , 50 ). Macrophages were another prevalent focus of research ( n = 7), with specific attention paid to their phenotypic heterogeneity in plaque progression and regression ( 22 , 24 , 29 , 36 , 49 , 51 ).…”

Single-cell RNA sequencing in atherosclerosis: Mechanism and precision medicine

“…The pathogenesis of atherosclerosis is complex and involves various cells. With the development of technology in recent years, scRNA-seq has been used to construct an atherosclerosis cell atlas of mice and humans, including immune cells, such as monocytes/macrophages, dendritic cells, T cells, B cells, natural killer (NK) cells, and nonimmune cells, such as VSMCs, ECs, pericytes, and fibroblasts (Cochain et al, 2018;Kim et al, 2018;Winkels et al, 2018;Gu et al, 2019;Depuydt et al, 2020;Burger et al, 2022). Research has revealed cellular heterogeneity and provided new insights into the pathogenesis of atherosclerosis.…”

“…Li F. et al (2021) discovered a d-flow-dependent osteogenic phenotype, Spp1 hi SMC, with functions, such as osteoblast differentiation, collagen biosynthesis, and vascular remodeling, which may play a role in d-flow-induced arterial stiffness. A study by (Burger et al, 2022) showed that Lyve-1 resident-like macrophages promoted VSMC transition into osteogenic-like cells by secreting CCL24. In addition, (Kim et al, 2020) performed scRNA-seq on mouse aortic roots and showed that SMC-specific deletion of the aryl hydrocarbon receptor (AHR) resulted in increased expression of Frontiers in Pharmacology frontiersin.org chondrocyte markers (Col2a1 and Alpl) in regulated SMC chondrocytes.…”