Abstract 3313: The Cytosol and Mitochondria Are More Reduced in Pulmonary Artery Smooth Muscle Cells Derived From Rats With Pulmonary Hypertension When Compared to Control Rats
Introduction and hypothesis: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular obstruction leading to heart failure and death. We have previously shown that pulmonary artery smooth muscle cells (PASMCs) of Fawn-hooded rats (FHR), a strain with spontaneous PAH, display enhanced proliferation and vascular obstruction. The mitochondria in FHR PASMCs are fragmented and reactive oxygen species (ROS) are abnormally low, as measured with several techniques. Lower ROS levels could produce a reduced redox state, mimicking hypoxia, and might trigger the observed activation of HIF-1∝ and cell proliferation. Here we use compartment-specific, redox-sensitive GFP (Ro-GFP) probes to test this hypothesis.
Methods: Cytoplasmic redox GFP (Cyto-RoGFP) and a mitochondrial targeted RoGFP (Mito-RoGFP) were expressed in PASMCs isolated from FHR rats and control Sprague Dawley rats (SDR). RoGFP was excited at 400 nm (maximal excitation for the oxidized form) and 490 nm (maximal excitation for the reduced form) and the emission images were acquired at 510–560 nm. The ratio of the emission values is an indicator of the redox status with lower ratio values indicating a more reduced state. Randomly selected intracellular areas were used for the measurements.
Results: Cyto-RoGFP measurements confirmed a more reduced redox state of the cytoplasm (SDR: 0.206±0.013; FHR: 0.175±0.003; P<0.05) and the mitochondria (SDR: 0.257±0.008; FHR: 0.230±0.004; P<0.01) in FHR vs SDR PASMCs. Maximal stimulation with the oxidant H2O2 caused similar maximal increases in ratio in both cell types (SDR: 0.476±0.047; FHR: 0.493±0.038; NS).
Conclusions: FHR PASMCs have a reduced redox state when compared to SDR PASMCs, both in the mitochondria and cytosol. Since higher levels of ROS and oxidation inhibit proliferation, the reduced redox state in FHR may contribute to the hyper-proliferative state. The similar response to maximal oxidation suggests that the difference in the redox state is not related to anti-oxidant defenses, but may point to lower ROS production in FHR, consistent with prior observations in FHR. Enhancing physiological ROS production may thus be a viable strategy to limit PASMC hyper-proliferation.