Using Polyacrylamide Hydrogels to Model Physiological Aortic Stiffness Reveals that Microtubules Are Critical Regulators of Isolated Smooth Muscle Cell Morphology and Contractility.

Sultan Ahmed,Derek T Warren,Robert T Johnson,Reesha Solanki,Finn Wostear,Teclino Afewerki

doi:10.3389/fphar.2022.836710

Sultan Ahmed, Derek T Warren + Show 4 more

Open Access

https://doi.org/10.3389/fphar.2022.836710

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Abstract

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aortic wall and normally exist in a quiescent, contractile phenotype where actomyosin-derived contractile forces maintain vascular tone. However, VSMCs are not terminally differentiated and can dedifferentiate into a proliferative, synthetic phenotype. Actomyosin force generation is essential for the function of both phenotypes. Whilst much is already known about the mechanisms of VSMC actomyosin force generation, existing assays are either low throughput and time consuming, or qualitative and inconsistent. In this study, we use polyacrylamide hydrogels, tuned to mimic the physiological stiffness of the aortic wall, in a VSMC contractility assay. Isolated VSMC area decreases following stimulation with the contractile agonists angiotensin II or carbachol. Importantly, the angiotensin II induced reduction in cell area correlated with increased traction stress generation. Inhibition of actomyosin activity using blebbistatin or Y-27632 prevented angiotensin II mediated changes in VSMC morphology, suggesting that changes in VSMC morphology and actomyosin activity are core components of the contractile response. Furthermore, we show that microtubule stability is an essential regulator of isolated VSMC contractility. Treatment with either colchicine or paclitaxel uncoupled the morphological and/or traction stress responses of angiotensin II stimulated VSMCs. Our findings support the tensegrity model of cellular mechanics and we demonstrate that microtubules act to balance actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.

Highlights

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the arterial wall
Contractile response was measured through changes in VSMC area, which previous studies have shown correlates with isolated VSMC contractile activity (Li et al, 1999; Wang et al, 2017; Halaidych et al, 2019)
Tissue culture plastic and glass are approximately a thousand times stiffer than the aortic wall (Minaisah et al, 2016), meaning that changes in VSMC area are driven by membrane retraction rather than contraction

Summary

Introduction

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the arterial wall. Contractile VSMCs possess a greater abundance of α-smooth muscle actin (αSMA) and smooth muscle-myosin heavy chain (SM-MyHC), that enhance their ability to generate actomyosin forces and contract (Rensen et al, 2007). Both contractile and proliferative VSMCs generate actomyosin force via stimulating interactions between myosin II and filamentous actin This process is regulated in both phenotypes by blood-borne factors, such as angiotensin II, that bind to receptors on the VSMC surface and mechanical factors including matrix stiffness and circumferential tension of the aortic wall (Qiu et al, 2010; Brozovich et al, 2016; Ahmed and Warren, 2018)

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