Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

Abstract

The contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 µN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 µN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue.

Regulation of SMC traction forces in human aortic thoracic aneurysms

“…The traction forces are globally heterogeneous. They were also represented as histograms in the supplemental materials (Appendix B-Figs S3, S4 and S5) for each of the 24 populations, showing that they exhibit generally an exponential distribution, which is still consistent with our previous observations on a commercial cell line of aortic SMCs (Petit et al 2019a).…”

“…3, cells had developed anchorage points on the substrate, where the strain maps exhibit a traction-compression pattern. This local pattern was related to the traction force as explained in (Petit et al 2019a).…”

“…According to previous observations with gels from 0.5 to 50 kPa (Petit et al 2019a), we used 24-well plates of intermediate stiffness values: 4, 8, 12, and 25 kPa, as these stiffness values allowed reproducible quantification of bead displacements whilst lying within a range compatible with soft tissue stiffness. The stiffness values indicated by the supplier were verified using AFM indentation before performing the experiments.…”

“…Then, we applied a custom Matlab ® code for selecting an area around each cell anchorage point and we derived the traction force value. The full theory and method related to traction force measurement are explained in a previous paper (Petit et al 2019a).…”

“…To address this need, we developed an in vitro technique, based on Traction Force Microscopy (TFM), to quantify SMC basal tone and compare primary SMCs from aortic aneurysms and healthy aortas. After presenting the approach which was initially tested on a commercial cell lineage (Petit et al 2019a), we report results on three primary healthy (AoPrim) human SMC lineages and three primary aneurysmal (AnevPrim) human SMC lineages, obtained from age and gender matched donors. Eventually, we propose interpretations of the different mechanical responses of AoPrim and AnevPrim using morphological analyses and the motor-clutch model.…”