Abstract 14360: Gene Transfer of Sarco/Endoplasmic reticulum Ca2+-ATPase 2a Restores Pulmonary Artery Endothelial Catechol-O-Methyltransferase Activity to Decrease Norepinephrine Levels in Pulmonary Hypertension

Abstract

Elevated levels of norepinephrine (NE) may occur in pulmonary hypertension (PH), in part, when activity of catechol- O -methyltransferase (COMT) is decreased. COMT is expressed by pulmonary artery endothelial cells (PAEC), degrades NE, and is negatively regulated by aldosterone (ALDO) and cytosolic Ca 2+ . Expression of the cardiac isoform of the sarco/endoplasmic Ca 2+ -ATPase (SERCA2a) modulates ALDO and cytosolic Ca 2+ and is downregulated in pulmonary arteries in PH. We hypothesized that gene transfer of SERCA2a to pulmonary arteries would prevent the decrease in COMT activity and lower NE levels. Accordingly, human PAEC were infected with an adenovirus encoding SERCA2a to increase expression 4.2-fold (p<0.01) or control virus and exposed to ALDO (10 -7 mol/L), which decreases COMT activity by 59.2 ± 6.2% (p<0.01). Compared to controls, SERCA2a overexpression in PAEC had no effect on COMT mRNA or protein levels but increased COMT activity by 148.8 ± 14.5% (p<0.01) by decreasing cytosolic Ca 2+ levels by 38.2 ± 6.7% (p<0.05). This resulted in a decrease in NE levels in the medium (229.4 ± 20.6 vs. 139.9 ± 14.8 vs. pg/mg protein, p<0.01) and collagen I expression. To examine the in vivo relevance of SERCA2a gene transfer on COMT activity, the rat monocrotaline model of PH was treated with inhaled adeno-associated virus 1 carrying SERCA2a (AAV1.SERCA2a). There was no difference in COMT expression in AAVI.SERCA2a-treated or saline control PH-rats; however, lung COMT activity was increased 139.8 ± 8.2% (p<0.01) while lung levels of NE (366.7 ± 38.6 vs. 504 ± 30.2 pg/ml/mg, p<0.01), serum ALDO, and pulmonary artery collagen 1 were decreased by AAV1.SERCA2a. In the pig pulmonary vein banding model of PH, inhaled AAV1.SERCA2a gene therapy decreased mean pulmonary artery pressure (54 ± 20 vs. 29 ± 5 mmHg, p<0.03); increased COMT activity by 121.1 ± 6.2% (p<0.05); and decreased levels of ALDO by 43.3 ± 4.6% (p<0.03), NE by 43.3 ± 4.6% (p<0.03), and collagen I in remodeled vessels. Taken together, these data indicate that gene therapy with inhaled AAV1.SERCA2a increases COMT activity by decreasing intracellular Ca 2+ , thereby decreasing NE levels. This extends the actions of SERCA2a beyond pulmonary vascular remodeling in PH to modulating levels of circulating catecholamines.