Increased AQP1 abundance contributes to reduced apoptosis susceptibility in human lung microvascular endothelial cells (hLMVECs) exposed to shear stress

Background: The pulmonary vascular endothelium is in contact with laminar blood flow, resulting in physiologic shear stress exerted on endothelial cells (ECs), the effect of which may be underappreciated in traditional static in vitro conditions. We noted AQP1 protein is not detected in vitro in hLMVECs cultured under static conditions despite abundant presence in vivo; however, after exposure to physiologic shear stress, AQP1 protein is readily detected in vitro. AQP1 confers apoptosis resistance in pulmonary vascular smooth muscle cells, though its regulation and role in EC survival is less described. Hypothesis: hLMVECs exposed to physiologic shear stress will exhibit less apoptosis susceptibility due to increased AQP1 protein levels. Methods: hLMVECs were plated in six-well plates, grown to confluence in EC growth media (Lonza) with 5% FBS and placed in static conditions or exposed to physiologic shear stress (12 dyn/cm 2 ) on an orbital shaker. After 24 h, plates were treated with vehicle or the apoptotic stimulus staurosporine (20 nM) for an additional 24 h, after which apoptosis was measured by Hoescht staining. Depletion of endogenous AQP1 was achieved using 10 nM of siRNA targeting AQP1 (siAQP1) for 6 h in serum- and antibiotic-free media. siRNA with non-targeting siRNA (siNT) used as control. After 6 h, serum was added for a total concentration of 5% FBS. siRNA transfection was performed 24 h prior to exposure to shear stress and adequate protein knockdown was verified by immunoblot. After knockdown and exposure to static conditions or shear stress, apoptosis in response to staurosporine was measured by Hoescht staining. Results: Compared to static conditions, AQP1 protein abundance increased 8-fold after exposure to 24 h of physiologic shear stress (shear-adapted). Under non-stimulated conditions, apoptosis rates were <1% in both static and shear-adapted culture. After staurosporine treatment, apoptosis in hLMVECs under static culture was 15%, compared to apoptosis of only 2% in shear-adapted conditions. siAQP1 transfection decreased AQP1 protein abundance to 60% of levels in cells transfected with siNT. In static cells, levels of staurosporine-induced apoptosis after AQP1 protein depletion (16%) were similar to those in siNT treated cells. In shear-adapted cells, however, AQP1 protein depletion led to increased apoptosis susceptibility to 7% compared to 2% in cells treated with siNT. Conclusions: Our data indicate dynamic regulation of AQP1 protein in response to shear stress, with increased AQP1 abundance rendering hLMVECs resistant to apoptosis compared to static hLMVECs. These results suggest AQP1 plays a vital role in EC survival in response to extracellular stress. Further investigation of the exact pathways involved in regulation of AQP1 protein in response to shear stress and the downstream mechanisms by which AQP1 confers apoptosis resistance is warranted. NIH T32 HL007534; NIH F32 HL176197; Johns Hopkins School of Medicine Bauernschmidt Fellowship Award; NIH R01s HL159906 and HL126514 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.