Physiologic shear stress regulates cell shape and apoptosis susceptibility through calcium-dependent regulation of aquaporin 1 in human lung microvascular endothelial cells (hLMVECs)

Rationale: The pulmonary endothelium is in contact with blood flow, resulting in physiologic shear stress, the effect of which may be underappreciated in static in vitro conditions. We noted morphologic differences between static-cultured hLMVECs and those adapted to shear stress, and similarly that aquaporin 1 (AQP1) protein is not detected in static-cultured hLMVECs despite abundance in vivo. AQP1 expression is calcium-dependent and confers apoptosis resistance in pulmonary smooth muscle cells, however its regulation and role in hLMVECs are not described. TRPV4 is a calcium-permeable, mechanosensitive channel that may be activated by shear stress in hLMVECs. Hypothesis: We hypothesize that shear stress regulates hLMVEC shape and apoptosis by increasing [Ca 2+ ]i through TRPV4, leading to increased AQP1 protein. Methods: [Ca 2+ ]i was measured under shear stress (12 dyn/cm2) in parallel plates using Fura-2. Changes in [Ca 2+ ]i were compared between static and shear conditions with or without the TRPV4 antagonist HC-067047. hLMVECs were also exposed to 12 dyn/cm2 in 6-well plates using an orbital shaker. AQP1 protein was measured after 24 h (static or shear conditions) with or without HC-067047, and under static conditions after 24 h treatment with the TRPV4 agonist GSK-1016790A. Morphologic assessment was done at 24 h by calculating the ratio of the longest to shorted dimension of the cells. Apoptosis susceptibility to staurosporine (24 h) was measured by Hoescht staining and caspase-3 activity. Under shear conditions, TRPV4 inhibition (HC-067047) and AQP1 protein knockdown (siAQP1) were used. Under static conditions, the effect of TRPV4 activation (GSK-1016790A) and AQP1 viral overexpres-sion (AdAQP1) on cell shape and apoptosis was measured. Results: Compared to static conditions, [Ca 2+ ]i rose acutely after exposure to shear stress and AQP1 protein abundance increased. TRPV4 inhibition resulted in attenuation of both find-ings. AQP1 levels increased in static conditions with TRPV4 activation, though not to the level seen in shear. Shear stress exposure led to cell elongation and alignment with flow as well as reduced apoptosis susceptibility. Both findings were attenuated after loss of function of TRPV4 and AQP1. Under static conditions, activation of TRPV4 and forced AQP1 expression led to decreased apoptosis susceptibility, but did not cause any changes in cell shape. Conclusions: Our data indicate that physiologic shear stress activates TRPV4 on hLMVECs, leading to [Ca 2+ ]i increase. We observed dynamic regulation AQP1 protein that is in part cal-cium-dependent through TRPV4. This increase in AQP1 protein is necessary and sufficient to confer changes in apoptosis susceptibility, suggesting AQP1 plays a vital role in EC survival. Interestingly, TRPV4 activation and AQP1 protein appear to be necessary for shear-induced cell morphology changes but are not sufficient to induce changes in the absence of shear stress, warranting further investigation. Funding: NIH T32 HL007534; NIH F32 HL176197; Johns Hopkins School of Medicine Bau-ernschmidt Fellowship Award; NIH R01s HL159906 and HL126514 This abstract was presented at the American Physiology Summit 2026 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.