Role of Endothelial Anoctamin-1 in Pulmonary Arterial Hypertension

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ABSTRACT Pulmonary arterial hypertension (PAH) is associated with significant morbidity and mortality. PAH pathology includes vasoconstriction, medial and adventitial remodeling, and microvascular endothelial cell (EC) proliferation leading to vaso-occlusive plexiform lesions. EC in PAH are both apoptosis resistant and hyperproliferative. There are no effective treatments for the severe vascular remodeling observed with PAH, and therapeutic strategies that target the dysfunctional EC and plexiform lesions are urgently needed. We have discovered that calcium activated chloride channel, Ano1, is localized to both the EC plasma membrane (pl-Ano1) and mitochondria (mito-Ano1). Ano1 expression is upregulated in EC in settings of PAH and associated with a hyperproliferative and apoptosis resistant phenotype. However, unregulated activation of this channel results in apoptosis of hyperproliferative apoptosis-resistant ECs. Our overall objective in this proposal is to delineate the mechanisms underlying these seemingly conflicting observations to identify therapeutic opportunities to decrease EC proliferation and target hyperproliferative ECs for apoptosis, thereby improving pulmonary vascular remodeling in PAH. We hypothesize that epigenetically regulated increased expression of Ano1 results in EC proliferation and apoptosis resistance, yet unregulated opening of Ano1 in the context of overexpression is detrimental and can potentially be exploited to promote apoptosis of hyperproliferative ECs in PAH. In Aim 1, we will determine the mechanism of increased Ano1 expression in PAH EC with a focus on DNA methylation. We will use complementary in vitro and in vivo approaches to design novel constructs to selectively alter the methylation pattern of Ano1 regulatory elements in lung EC and test if this strategy prevents or treats PAH. In Aim 2, we will determine the effect of increased pl- and mito- Ano1 expression on EC proliferation and apoptosis resistance and elucidate the underlying mechanisms. We will investigate the signaling pathways in vitro and then use selective inhibition of Ano1 expression in lung EC in vivo to determine its effect on preventing or treating PAH. In Aim 3, we will determine the mechanism of the effect of pl- and mito-Ano-1 opening on EC apoptosis in vitro and test if opening of Ano1 by a small molecule delivered by inhalation attenuates PAH. As a result of these studies we expect to delineate the mechanism of Ano1-mediated EC proliferation and apoptosis resistance in settings of PAH as well as develop novel therapeutic strategy targeting Ano1 to improve pulmonary vascular remodeling and PAH.