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

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

Preprint posted on December 15, 2021 https://www.biorxiv.org/content/10.1101/2021.12.14.472278v1

Article now published in Frontiers in Pharmacology at http://dx.doi.org/10.3389/fphar.2022.836710

Polyacrylamide hydrogels: reproducing the physiological stiffness of the aortic wall.

Selected by Laura Alonso-Herranz

Background

Vascular smooth muscle cells (VSMCs) are the most abundant cell type in the medial layer of the arteries. They are quiescent cells that abundantly express contractile proteins (e.g. αSMA, SM22, CNN1) and are responsible for maintaining the vascular tone, with actomyosin-derived contractile forces being crucial for this purpose. However, vascular injury, inflammation, or atherogenic stimuli induce proliferation, migration, loss of contractile genes, and increased matrix synthetic activity of medial VSMCs into the intima of the arteries, in a process known as phenotypic modulation. This is common to different vascular diseases, such as atherosclerosis, acute vascular injury, aneurysms, and pulmonary artery hypertension.

A similar shift in cell phenotype is seen when VSMCs are removed from their native environment and placed in a rigid plastic plate, presumably due to the absence of the physiological signals (blood-borne factors and mechanical forces) that maintain and regulate the VSMC phenotype in the vasculature (Jensen et al., 2021). Thus, Ahmed et al. aimed to develop an in vitro contractility assay that mimics the stiffness of the aorta to study the role of microtubule stability on actomyosin activity and VSMC contraction.

Key findings

The main findings of the current study can be summarized as follows:

  1. First, the authors tested the polyacrylamide hydrogel culture system with well-known inducers of VSMC contractility. The contractile agonists angiotensin II and carbachol diminished cell area in the polyacrylamide hydrogels, an indirect measurement of cell contraction. While the reduction in cell area under angiotensin II treatment was accompanied by an increase in traction stress generation, this was not the case with carbachol treatment.
  2. To further confirm that VSMC contractility is driven by actomyosin-generated forces in their system, chemical inhibitors of actomyosin activity were used in combination with angiotensin II. This prevented the angiotensin II-induced decrease in VSMC area and caused an increase in cell volume, suggesting inhibition of VSMC contractility.
  3. Finally, the authors tested the impact of microtubule disruption on VSMC contraction. While treatment with a microtubule destabilizer (i.e. colchicine) increased angiotensin II-induced traction stress, the microtubule stabilizer paclitaxel decreased total traction stress. Altogether, these results point to a pivotal role for microtubule stability in actomyosin-driven traction stress generation and VSMC contractility.

In conclusion, Ahmed et al. developed an in vitro VSMC contractility assay that allowed them to study the role of the microtubule network in counterbalancing actomyosin-generated forces, and thus regulating the VSMC contractile phenotype. Nevertheless, complementary assays, including direct assessment of cell contraction, would help to further validate the polyacrylamide hydrogel-based system and to understand the implications of microtubule network dynamics in the VSMC contractile phenotype.

Why I chose this preprint

Most studies describing VSMC functions have been performed under standard culture conditions in which cells adhere to a rigid and static plastic plate. While these studies have contributed to the discovery of key molecular pathways regulating VSMCs, they have a significant drawback: they lack the ECM microenvironment and the mechanical forces transmitted through the matrix to VSMCs, which are sensed by VSMCs and determine their phenotype (Jensen et al., 2021). In addition, classical in vitro contraction assays (atomic force microscopy or collagen gel contraction) are either low throughput or do not reproduce the stiffness of the artery wall.

Therefore, the development of alternative contractility assays is important to gain further knowledge on the mechanisms that govern the contractile phenotype of VSMCs, how tight they are regulated, and whether transitions between de-differentiated and contractile phenotypes can be forced in vitro.

Future directions

  1. What other readouts can be used with your proposed polyacrylamide hydrogels? Is it possible to perform gene expression analysis by qPCR or protein quantification by Western blot on cells seeded on these gels?
  2. In the current study, changes in cell area are used as a parameter to assess cell contractility. However, reduction in cell area is an indirect measurement of contractility and does not always correlate with increased traction stress generation (e.g., colchicine or paclitaxel treatments in combination with angiotensin II, Figures 5 and 6). How reliable is the use of cell area as the only parameter to infer changes in cell contractility? How would you strengthen the biological relevance of the results obtained in your study?
  3. While the reduction in cell area under angiotensin II treatment correlates with an increase in traction stress generation, this is not the case for carbachol treatment. How would you explain this? Have you thought about any alternative mechanisms that could be implicated?
  4. Did you try to reproduce your results regarding microtubule stability in a different setting that mimics the mechanical forces that operate in the wall (e.g. cyclic mechanical stretch)? Which result would you predict in such a context when using microtubule stabilizing and destabilizing agents?
  5. Have you considered including a control group of untreated cells (without angiotensin II treatment) in the experiments depicted in Figures 1, 3, 4C-G, 5, and 6, in order to see whether and to what extend the chemical product added on top of angiotensin II is able to revert the cell phenotype to the baseline situation (untreated cells)?

References

Jensen, L.F., Bentzon, J.F., and Albarrán-Juárez, J. (2021). The Phenotypic Responses of Vascular Smooth Muscle Cells Exposed to Mechanical Cues. Cells 10.

Tags: actomyosin system, aortic wall, contractility assay, microtubules, phenotypic switching, stiffness, vsmc

Posted on: 29th December 2021 , updated on: 6th January 2022

doi: https://doi.org/10.1242/prelights.31218

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