Smad3 Regulates Smooth Muscle Cell Fate and Governs Adverse Remodeling and Calcification of Atherosclerotic Plaque
Preprint posted on 14 November 2020 https://www.biorxiv.org/content/10.1101/2020.09.15.299131v2
Coronary artery disease (CAD) is still a leading cause of death globally. To find novel therapeutic strategies, genome-wide association studies (GWAS) have been implemented to discover CAD risk factors. In particular, genes which regulate smooth muscle cell (SMC) behaviour are known to contribute to CAD1. Notably, an allelic variation at 15q22.33 which is associated with SMAD3 expression in SMCs has been identified by several GWAS2.
TGF-β signaling is central to SMC biology in development and disease. Canonical TGF-β signaling is mainly mediated by the SMAD family proteins, including SMAD3. Since the mechanisms by which SMAD3 contribute to plaque development are poorly understood, Cheng et al. established a murine atherosclerosis model with an SMC-specific deletion of Smad3 to investigate these processes. Their findings centered around key phenotypes, namely, plaque volume, vascular calcification, outward remodeling of vessels, and inflammatory cell recruitment.
The authors generated an atherosclerosis murine model (in the ApoE-/- background) with a mature SMC-specific Cre (Myh11Cre-ERT2) crossed with a conditional knockout allele of Smad3 with concurrent lineage tracing (ROSAtdTomato). Smad3 was deleted at post-natal week 8 in samples (termed Smad3ΔSMC henceforth) but not in controls. The mice were subjected to Western high-fat diet from post-natal week 8–24.
Changes in SMC progeny phenotypes and composition contribute to Smad3ΔSMC vascular features
Given the key role of Smad3 in SMC cell fate decisions, the authors hypothesized that SMC progenies transitioned into distinct cellular phenotypes following Smad3 deletion. Single-cell transcriptomic profiling demonstrated that a greater portion of lineage-traced cells no longer expressed mature SMC markers in Smad3ΔSMC mice compared to controls. Five clusters of SMC progenies were identified: mature SMCs, pericytes, fibromyocytes, pro-calcific chondromyocytes, and a novel disease-associated population, termed remodeling-SMCs (R-SMCs) henceforth.
Histological studies showed that the SMCs in Smad3ΔSMC mice migrated into lesions and contributed to the plaques and fibrous caps. The SMC progenies also occupied a greater plaque volume in Smad3ΔSMC mice. In agreement with this, GO category analyses revealed that the proliferation of SMC progenies increased with the loss of Smad3.
There was also more vascular calcification in Smad3ΔSMC mice. This may be explained by increased chondromyocyte proliferation. There was also a greater extent of SMC transition into chondromyocytes and R-SMCs at the expense of the fibromyocyte population numbers.
Furthermore, in Smad3ΔSMC mice, while the lumina sizes remained unchanged, the areas encapsulated by the elastic lamina enlarged. This means that outward or ‘positive’ remodeling of the vessels occurred. R-SMCs highly expressed genes involved in extracellular matrix (ECM) remodeling, including matrix metalloproteinase-3 (Mmp3). Since MMP3 is required for outward remodeling3 and was most prominent in the disrupted elastic lamina, MMP3 may contribute to R-SMC invasion through elastic lamina to drive positive remodeling.
There was also an increase in inflammatory cell recruitment in Smad3ΔSMC mice, as indicated by larger lesion areas populated with monocytes and macrophages. R-SMCs may have a role in recruiting inflammatory cells, because PANTHER analysis showed that biological processes related to the regulation of inflammation were enriched in R-SMCs. R-SMCs also expressed higher levels of chemoattractants whose receptors are mainly restricted to macrophages or monocytes.
The roles of TGF-β signaling and co-regulatory factors in modulating genes in Smad3ΔSMC progenies
To understand the molecular mechanisms linked to the vascular features observed, the authors identified 83 differentially expressed genes (DEGs) in chondromyocytes, R-SMCs, and fibromyocytes between Smad3ΔSMC and control mice. Consistent with the role of Smad3 in mediating TGF-β signaling, this pathway was significantly enriched among these DEGs. When the authors compared these DEGs with upregulated genes in disease-related murine de-differentiated SMCs, they identified Lox and Mfap5 as significantly down-regulated genes in Smad3ΔSMC mice.
In human coronary artery SMCs (HCASMCs), Smad3 knockdown negated TGF-β-mediated regulation of Mmp3 and Lox expression, which suggested that their transcription was directly affected by TGF-β via SMAD3. However, a subset of genes, including Mfap5, were not TGF-β responsive, but sensitive to the Smad3 knockdown. This indicated that additional co-regulatory factors may influence their transcription.
Hox and Sox transcription factor motifs were enriched among the 83 DEGs. The authors investigated the role of Sox9, which regulates calcification, and HoxB2, the most highly expressed Hox gene in human coronary SMCs. Similar to Smad3, the authors showed that both Sox9 and HoxB2 also regulate the transcription of Mmp3 and MFAP5. Co-IP experiments demonstrated that SMAD3 was directly bound to either His-HOXB2 or Flag-SOX9. Furthermore, SMAD3 was essential for the SOX9- and HOXB2-mediated activation of a reporter gene containing an enhancer element found near the human MFAP5 gene. This suggested that these factors interact together to regulate MFAP5 expression.
Why I think this work is important
This work unraveled the potential mechanisms of how the risk variant encoding Smad3 contributes to atherosclerosis pathogenesis. It highlights vascular calcification, ECM remodeling, and recruitment of inflammatory cells as features of atherosclerosis which can be studied further for developing novel therapies. This work also presents a starting point for determining whether R-SMCs are present in other vascular disease models, and the role they play in the pathogenesis and pathophysiology. The authors also demonstrate that SMAD3 is the point of convergence for the crosstalk of TGF-B signalling with other molecular factors in a context-dependent manner, which will inform future studies aiming to modulate downstream substrates of TGF-β signaling for pharmacological treatments.
- What would be the next step to move towards the end goal of translating these findings to accelerate treatments for CAD, especially in terms of targeting the phenotypes you highlighted in your paper?
- Liu et al., Genetic Regulatory Mechanisms of Smooth Muscle Cells Map to Coronary Artery Disease Risk Loci. Am J Hum Genet 103, 377-388 (2018).
- Nikpay et al., A comprehensive 1,000 Genomes-based genome-wide association meta-analysis of coronary artery disease. Nat Genet 47, 1121-1130 (2015).
- R. Alexander et al., Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice. J Clin Invest 122, 70-79 (2012).
Posted on: 12 January 2021Read preprint
Also in the cell biology category:
mTORC1 is required for differentiation of germline stem cells in the Drosophila melanogaster testis
|Selected by||Diego Sainz de la Maza|
Expansion microscopy at one nanometer resolution
|Selected by||Nadja Hümpfer|
Actin-driven protrusions generate rapid long-range membrane tension propagation in cells
|Selected by||Nicolaes Hyun-Kee Min|
preListscell biology category:in the
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
|List by||Osvaldo Contreras|
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
|List by||Alex Eve|
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
|List by||Ana Dorrego-Rivas|
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
|List by||Ana Dorrego-Rivas|
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
|List by||Dey Lab, Samantha Seah|
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
|List by||Yamini Ravichandran|
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
|List by||Maiko Kitaoka et al.|
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
|List by||Paul Gerald L. Sanchez and Stefano Vianello|
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
|List by||Hiral Shah|
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
|List by||Madhuja Samaddar et al.|
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
|List by||Dey Lab|
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
|List by||Sandra Malmgren Hill|
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
|List by||Rob Hynds|
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
|List by||Pablo Ranea Robles|
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
|List by||Dey Lab|
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
|List by||Sandra Franco Iborra|
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
|List by||Joseph Jose Thottacherry|
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
|List by||Dey Lab, Amanda Haage|