Using polyacrylamide hydrogels to model physiological aortic stiffness reveals that microtubules are critical regulators of isolated smooth muscle cell morphology and contractility
Posted on: 29 December 2021 , updated on: 6 January 2022
Preprint posted on 15 December 2021
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
Categories: cell biology, molecular biology
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:
- 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.
- 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.
- 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
- 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?
- 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?
- 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?
- 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?
- 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.
doi: https://doi.org/10.1242/prelights.31218
Read preprint
(No Ratings Yet)Sign up to customise the site to your preferences and to receive alerts
Register hereAlso in the cell biology category:
Motor Clustering Enhances Kinesin-driven Vesicle Transport
Sharvari Pitke
Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis
Ankita Walvekar
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
Also in the molecular biology category:
Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor
Jonathan Townson
HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos
Anchel De Jaime Soguero
The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites
Felipe Del Valle Batalla
preLists in the cell biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
| List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
| List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
| List by | Dey Lab, Amanda Haage |
Also in the molecular biology category:
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
| List by | Nathalie Krauth |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |