Biology subjects

Shanahan, C.

Publications and source records attributed to Shanahan, C..

3 recordsLinked to original sources

bioRxiv2026

An ncBAF-ETS2 Chromatin-Remodelling Axis Drives Vascular Smooth Muscle Cell Osteogenic Reprogramming in Vascular Calcification

Introduction: Vascular calcification is a detrimental ageing-related pathology that is markedly accelerated in metabolic disorders. It is driven by osteogenic differentiation of vascular smooth muscle cells (VSMCs), however epigenetic regulatory pathways activated early in this transition remain poorly defined. Methods: An in vitro calcification model was developed using primary human aortic VSMCs cultured with or without mineral stress. Epigenetic changes were assessed using targeted PCR arrays and CUT&RUN sequencing. Key findings were validated in vivo using single-cell sequencing datasets from human large arteries and spatial transcriptomic analysis in atherosclerotic carotid plaques. Transcriptomic and CUT&RUN analyses identified gene targets altered by epigenetic remodelling, and molecular tools were applied to study effects on metabolism, inflammation, apoptosis, and calcification. Results: During early calcification in response to mineral stress, SWI/SNF chromatin remodelling complexes shift toward ncBAF enrichment in pre-osteogenic VSMCs. ncBAF complexes activated transcriptional programs involved in inflammation, apoptosis, and glycolysis-all hallmarks of calcifying VSMCs. The transcription factor ETS2 was identified as a novel component of ncBAF complexes. Disruption of ncBAF or ETS2 impaired osteogenic differentiation and calcification. Notably, ETS2 expression was regulated by ncBAF, forming a positive feedback loop that reinforced VSMC phenotypic switching. Co-activation of ETS2 and ncBAF and the resulting transcriptional shifts were confirmed in human arterial single-cell datasets, with osteogenic/inflammatory clusters showing NFkB and RUNX2 activation. Spatial transcriptomics further suggested that a macrophage-rich microenvironment may promote the differentiation of smooth muscle cells toward an overt osteogenic/inflammatory phenotype. Immunohistochemistry showed that ETS2 levels correlated with calcification severity in human vessels supporting the potential clinical relevance of ETS2. Conclusions: Our findings identify a novel epigenetic mechanism in vascular calcification, where ncBAF and ETS2 cooperate to drive VSMC phenotypic switching. This ncBAF-ETS2 axis represents a potential therapeutic target to modulate VSMC plasticity and intervene early in the progression of cardiovascular calcification.

cell biology↗

bioRxiv2023

Sox9 Regulates Extracellular Matrix Stiffness during Vascular Smooth Muscle Cell ageing by increased secretion of LH3 via extracellular vesicles.

RationaleVascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a master regulator of chondrogenesis, also expressed in the vasculature, that has been implicated in vascular smooth muscle cell (VSMC) osteo-chondrogenic conversion. ObjectiveHere, we investigated the relationship between vascular ageing, calcification and Sox9-driven ECM regulation in VSMCs. Methods and ResultsImmunohistochemistry in human aortic samples showed that Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed it was mechanosensitive with increased expression and nuclear translocation in senescent cells and on stiff matrices. Manipulation of Sox9 via overexpression and depletion, combined with atomic force microscopy (AFM) and proteomics, revealed that Sox9 regulates ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs whereby senescent cells plated onto ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a Sox9 target, and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles (EVs) and Sox9 promoted EV secretion, leading to increased LH3 deposition within the ECM. ConclusionsThese findings identify cellular senescence and Sox9 as a key regulators of ECM stiffness during VSMC ageing and highlight a crucial role for ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle whereby cellular senescence and increased ECM stiffening promote Sox9 expression which drives further ECM modifications that act to accelerate vascular stiffening and cellular senescence.

molecular biology↗

bioRxiv2020

Matrix stiffness and blood pressure together regulate vascular smooth muscle cell phenotype switching and cofilin dependent podosome formation

Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. In pre-atherosclerotic lesions, VSMCs switch from a contractile to a synthetic phenotype and subsequently remodel the microenvironment, leading to further disease progression. Ageing and associated mechanical changes of the extracellular matrix as well as hypertension are major risk of atherosclerosis. Consequently, we sought here to systematically study the impact of mechanical stimulations on VSMC phenotypic switching, by modulating both stiffness and hydrodynamic pressure. Thereby we find that hemodynamic pressure and matrix stiffness individually affect the VSMC phenotype. However, only the combination of hypertensive pressure and matrix compliance, and as such mechanical stimuli that are prevalent during atherosclerosis, lead to a full phenotypic switch including the formation of matrix degrading podosomes. We further analyse the molecular mechanism in stiffness and pressure sensing and identify a regulation through different, but overlapping pathways, culminating in the regulation of the actin cytoskeleton through cofilin. Altogether, our data shows how different pathological mechanical signals combined, but through distinct pathways accelerate a phenotypic switch that will ultimately contribute to atherosclerotic disease progression.

cell biology↗