Caspase-6 Is a Non-Apoptotic Effector of Shear-Induced Morphological Adaptation in Pulmonary Artery Endothelial Cells In Vitro

Caspases are known for their roles in cell death and inflammation. However, emerging evidence suggests they also mediate non-lethal processes, governed by a finely tuned balance of localization, activity, kinetics, and substrate availability. Given that many caspase substrates are implicated in mechanoadaptive processes, we investigated if caspases contribute to morphological adaptation of human pulmonary artery endothelial cells to fluid shear stress and other morphology-altering stimuli in vitro. Using selective inhibitors, we screened all major caspases for a role in endothelial cell adaptation to unidirectional laminar shear stress (15 dyn/cm², 72 h). Selective inhibition of caspase-6, but not other caspases, impaired morphological shear adaptation. Only 5.5% of caspase-6-inhibited cells shear-adapted vs. 75.2% of vector controls. Live-cell FRET imaging revealed progressive caspase-6 activation starting at 18 h of shear stress, coinciding with the onset of morphological remodeling. The active caspase-6 localized predominantly perinuclearly, while caspase-3 remained inactive throughout shear exposure. Caspase-6 inhibition did not affect elongation in response to alternative biomechanical or biochemical stimuli, including uniaxial cyclic stretch (5%, 1 Hz), spatial confinement on narrow micropatterned RGD-lines, or TNF-α stimulation, nor did it impair cell adhesion, directed migration, wound healing, or barrier recovery after wounding. Our study uncovers a previously unidentified role of caspase-6 as a non-apoptotic, mechanosensitive effector specifically required for shear-induced morphological adaptation of pulmonary artery endothelial cells, highlighting a novel regulatory axis in vascular mechanoadaptation.