Energetic efficiency of cell contractility is driven by the mechanics of the microenvironment

Introduction: During angiogenesis, endothelial cells expend significant energy undergoing cytoskeletal rearrangements, generating traction forces, migrating, and proliferating. Interestingly, despite these energetic costs, extracellular matrix stiffening increases cell contractility, which promotes further vascular growth and impairs vascular barrier integrity. While it is known that cellular energy is required to generate traction forces and that matrix stiffness facilitates cellular contractility, the mechanisms regulating interplay between extracellular matrix stiffness, cell contractility, and cellular energetics remains unclear. Here we investigated how matrix stiffness influences the energetic status and contractility of endothelial cells. Furthermore, we investigated how matrix stiffness, a critical mechanical property of the extracellular matrix, affects the efficiency with which cells convert energy into traction forces.

Materials and

Methods: To examine the effect of matrix stiffness on human umbilical vein endothelial cells (HUVECs), we seeded HUVEC single cells on polyacrylamide (PA) substrates coated with 0.1 mg/mL collagen. To quantify energy levels, HUVECs expressing PercevalHR, a fluorescent biomarker of intracellular ATP: ADP ratio, were seeded on PA gels with various stiffnesses and imaged with laser scanning confocal microscopy. To measure cell contractility, traction force microscopy (TFM) was utilized. Cells were treated with Y27632 or manganese to inhibit ROCK or promote integrin affinity, respectively.

Results, Conclusions, and Discussions: Results and Discussion
HUVECs seeded on PA substrates of variable stiffness demonstrated a biphasic response in energy usage and an increase in traction stress as substrate stiffness increased (Fig 1. A-C). By dividing the traction stress of an individual cell by its ATP: ADP ratio, we calculated the efficiency by which HUVECs convert energy into traction force. Utilizing this metric, we found that HUVECs seeded on the stiffest substrates had significantly higher efficiency than those on more compliant substrates, indicating that cells on stiffer substrates may exert traction stresses using less energy (Fig 1. D). Inhibition of cytoskeleton reorganization via ROCK inhibition decreased cell contractility and cellular energy efficiency (Fig. 1 E-G). When cells were treated with manganese (Mn2+) to promote integrin affinity, efficiency increased (Fig. 1H-J). Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces via forming a more stable relationship between actin cytoskeleton and focal adhesions.
Conclusion
Our study demonstrated a relationship between cell energetic levels and substrate stiffness which results in increased energy efficiency on stiff substrates. Endothelial cells exhibit increasing traction forces as substrate stiffness increases. However, energy usage displays a biphasic trend, initially increasing and then gradually decreasing as substrate stiffness increases. These findings indicated an increased cell efficiency to exert traction forces on stiffer substrates. This work suggests a previously unidentified mechanism by which matrix stiffness influences cell behavior: altering a cell’s efficiency in utilizing intracellular energy to generate traction force. As cellular mechanical sensing and cell contractility are essential for many types of cells, we expect this metric to be applicable in other cell types as well.

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