Role of mitochondrial reactive oxygen species (ROS) and TRPV4 activation in microvascular endothelial cell dysfunction in PAH.
Authors: Suresh Karthik , Servinsky Laura , Jiang Haiyang , Bigham Zahna , Zaldumbide Joel , Huetsch John , Kliment Corrine , Damarla Mahendra , Shimoda Larissa 
Published: June 2021

Rationale

In pulmonary arterial hypertension (PAH), endothelial cells (ECs) exhibit increased migration/proliferation leading to formation of vaso‐occlusive lesions. The mechanisms underlying this pro‐migratory/proliferative state remain unclear. Increased ROS and intracellular Ca2+ concentration ([Ca2+]i) are known to promote migration and proliferation in other vascular beds, and there is evidence for increased oxidant stress and basal [Ca2+]i in ECs from humans with PAH. Although the exact source of increased EC oxidant stress in PAH ECs is not fully known, mitochondria are a significant source of ROS in ECs, and mitochondrial dysfunction is present in human PAH ECs. However, the links between ROS production, elevated [Ca2+]i and enhanced migration and proliferation in PAH ECs are incompletely understood.

We previously showed that exogenous ROS increased [Ca2+]i in lung microvascular endothelial cells (MVECs) via the transient receptor potential vanilloid‐4 (TRPV4) calcium channel. To study endogenous ROS production and the possible role of ROS‐induced Ca2+ influx in promoting MVEC migration and proliferation in PAH, we isolated MVECs from the lungs of rats subjected to normoxia (N‐MVEC) or SU5416/hypoxia (SuHx‐MVEC), a robust model of PAH, and examined ROS production, [Ca2+]i, mitochondrial morphology/function and cellular migration and proliferation.

Methods

Ca2+ was measured using Fura‐2AM‐loaded MLMVEC in a flow chamber perfused with Krebs buffer. Mitochondrial morphology measurements were made using validated algorithms on confocal images of mitochondria in N‐ and SuHx‐MVEC transfected with MitoRFP. ROS measurements were made using the ratiometric ROS sensor roGFP. Migration and proliferation were measured using transwell assay and BrDU incorporation, respectively.

Results

ROS levels were increased in SuHx‐MVEC and attenuated following treatment with MitoQ (MQ) a mitochondrion‐specific antioxidant. In SuHx‐MVECs, mitochondrial number, network fragmentation and activation of the fission‐inducing protein dynamin‐related protein 1 were increased, while oxidative phosphorylation and mitochondrial membrane potential were both decreased. Basal [Ca2+]i, migration and proliferation were all increased in SuHx‐MVEC, but similarly attenuated following treatment with: 1) MQ; 2) a global ROS scavenger (TEMPOL); or 3) HC‐067047, a specific TRPV4 inhibitor. While total TRPV4 expression was unchanged between N‐ and SuHx‐MVEC, activation of membrane bound TRPV4 (using the specific agonist GSK1016790A ‐ GSK) produced greater [Ca2+]i influx in SuHx‐MVEC. Biotinylation of TRPV4 revealed greater membrane‐bound expression of TRPV4 in SuHx‐MVEC. Interestingly, quenching mitochondrial ROS attenuated GSK‐induced [Ca2+]i influx in SuHx‐MVEC.

Conclusion

These data suggest that, in SuHx‐MVEC, mitochondrial ROS promote membrane translocation of TRPV4, leading to increased basal [Ca2+]i and enhanced migration and proliferation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.