View Abstract

A3758 - Endothelial Mitochondrial Dysfunction and Reactive Oxygen Species Production in the Sugen/Hypoxia Model of Pulmonary Arterial Hypertension
Author Block: K. Suresh, L. Servinsky, H. Jiang, J. Zaldumbide, Z. Bigham, J. Huetsch, C. Kliment, L. A. Shimoda; Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore, MD, United States.
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 reactive oxygen species (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. Though the exact source of increased EC oxidant stress in PAH ECs is not fully known, mitochondria are 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 ECs (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. Migration and proliferation were measured using transwell assay and BrDU incorporation, respectively.
Results
SuHx-MVEC appeared morphologically abnormal, with larger size, acquisition of smooth muscle cell markers and loss of “cobblestone” morphology. 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 either 1) MQ, 2) a global ROS scavenger (TEMPOL) or 3) HC-067047, a specific TRPV4 inhibitor. These data suggest that, in SuHx-MVEC, mitochondrial ROS activate TRPV4 leading to increased basal [Ca2+]i and enhanced migration and proliferation.