Smad3 Regulates Smooth Muscle Cell Fate and Governs Adverse Remodeling and Calcification of Atherosclerotic Plaque

Background

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.

Key Findings

The authors generated an atherosclerosis murine model (in the ApoE^-/- ­background) with a mature SMC-specific Cre (Myh11^Cre-ERT2) crossed with a conditional knockout allele of Smad3 with concurrent lineage tracing (ROSA^tdTomato). Smad3 was deleted at post-natal week 8 in samples (termed Smad3^ΔSMC 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 Smad3^ΔSMC 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 Smad3^ΔSMC 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 Smad3^ΔSMC mice migrated into lesions and contributed to the plaques and fibrous caps. The SMC progenies also occupied a greater plaque volume in Smad3^ΔSMC 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 Smad3^ΔSMC 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 Smad3^ΔSMC 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 Smad3^ΔSMC 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 Smad3^ΔSMC 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 Smad3^ΔSMC 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 Smad3^ΔSMC 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.

Questions

1. 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?

References

1. 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).
2. Nikpay et al., A comprehensive 1,000 Genomes-based genome-wide association meta-analysis of coronary artery disease. Nat Genet 47, 1121-1130 (2015).
3. 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).

Tags: apoe, atherosclerosis, smad3, vascular

doi: https://doi.org/10.1242/prelights.26942

Read preprint (No Ratings Yet)