Using polyacrylamide hydrogels to model physiological aortic stiffness reveals that microtubules are critical regulators of isolated smooth muscle cell morphology and contractility

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 and we demonstrate that microtubules act to balance the actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.

A microtubule stability switch alters isolated vascular smooth muscle calcium flux in response to matrix rigidity

“…Actomyosin generated tension drives VSMC contraction and microtubules maintain VSMC morphology, protecting against strain induced cellular damage (Stamenović, 2005). The tensegrity model predicts that disruption of the microtubule cytoskeleton alters the equilibrium of these stresses and results in increased VSMC traction stress generation, a hypothesis previously confirmed by both wire myography and traction force microscopy (Ahmed et al, 2021; Zhang et al, 2000). We show that in rigid environments, the VSMC tensegrity equilibrium becomes unbalanced.…”

“…Human adult aortic VSMCs (passages 3-10) were purchased from Cell Applications Inc. (354-05a). Standard VSMC culture was performed as previously described (Ahmed et al, 2021; Johnson et al, 2024). VSMCs were seeded onto polyacrylamide hydrogels in basal media (Cell Applications Inc, Cat# 310-500), 18 hours prior to the beginning of the experiment to induce quiescence.…”

“…Briefly, bead displacement was measured using the first and last image of the movie sequence. The cell region was determined by overlaying the traction map with the phase image, selecting the cell traction region with an ROI and extracting the traction forces in each pixel using the XY coordinate function in FIJI (Ahmed et al, 2021; Porter et al, 2020).…”

“…The tensegrity model predicts the interplay between actomyosin and microtubules in VSMCs. Previous studies have shown that microtubule stabilisation decreases VSMC actomyosin activity, whereas microtubule destabilisation increases VSMC actomyosin activity (Ahmed et al, 2021; Zhang et al, 2000). Other models have been proposed that describe VSMC mechano-adaption responses, yet we lack an in-depth mechanistic understanding of pathways linking changes in VSMC behaviour to the proposed models of mechanotransduction (Steucke et al, 2017).…”