Abstract Rationale The pulmonary endothelium is in contact with laminar blood flow, resulting in physiologic shear stress, the effect of which may be underappreciated in static in vitro conditions. Changes in shear stress accompany several pulmonary vascular disorders, including pulmonary embolism and pulmonary hypertension, the effects of which are not well defined. We noted aquaporin 1 (AQP1) protein is not detected in static-cultured hLMVECs despite abundant presence in vivo. AQP1 protein 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. Methods Intracellular calcium concentration (Ca2+i) was measured under shear stress (12 dyn/cm2) in Ibidi parallel plates using Fura-2. Changes in Ca2+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. 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 with GSK-1016790A and AQP1 viral overexpression (AdAQP1, MOI 50) on apoptosis was measured. Statistics were performed by two-way ANOVA. Results Compared to static conditions, shear stress increased Ca2+i and AQP1 protein abundance, both of which were significantly attenuated by TRPV4 inhibition. Shear stress exposure also reduced apoptosis susceptibility compared to static conditions, which was partially restored after loss of function of TRPV4 and AQP1. Under static conditions, TRPV4 activation led to acute Ca2+i as well as an increase in AQP1 protein, though not to the level seen in shear. Additionally, activation of TRPV4 and forced AQP1 expression led to decreased apoptosis susceptibility. Conclusions Our data indicate physiologic shear stress activates TRPV4 on hLMVECs, increasing Ca2+i. We observed dynamic regulation AQP1 protein due in part to TRPV4-dependent calcium influx. The increase in AQP1 protein is necessary and sufficient to confer changes in apoptosis susceptibility, suggesting AQP1 plays a vital role in EC survival in response to extracellular stress. Further investigation of this pathway, including additional contributing channels and downstream mechanisms of AQP1-induced apoptosis resistance, is warranted. This abstract is funded by: NIH-F32-HL176197
Croglio et al. (Fri,) studied this question.