Identification of ADORA1-PDE10A Complex Formation and Its Bidirectional Regulation of Intracellular Cyclic AMP Levels as a Novel Therapeutic Target for Treating Pulmonary Hypertension

Abstract

Despite substantial advancements in the treatment of pulmonary arterial hypertension (PAH), obstacles still remain in achieving the optimal outcomes. Various treatments have been developed to target signaling pathways mostly leading to increased intracellular cAMP. The existence of intracellular cAMP microenvironment and various cAMP regulators and effectors as signalosome, adds a different level of complexity to the cAMP signaling. In vitro studies were performed majorly in donor and idiopathic PAH (IPAH) human pulmonary artery smooth muscle cells (PASMCs). To study the cAMP complex components, I used co-immunoprecipitation, immunofluorescence staining, and proximity ligation assay. Functional studies include assessment of proliferation and apoptosis in the presence or absence of ADORA1 and/or PDE10A siRNAs and inhibitors. cAMP levels were measured using ELISA kits. In vivo studies involve treatment of MCT-PAH and SU5416+Hypoxia-PAH rat models with dual ADORA1/PDE10A inhibitor for 14 days after establishment of PAH. After the treatment protocol, rats are subjected to cardiac MRI, right heart catheterization, and isolated lungs were taken for morphometric analysis. From the expression studies, (Figure 4.19 A) I observed that ADORA1 was highly expressed under the disease condition. Screening for cAMP inhibiting PDEs that can be colocalized with ADORA1, I observed a close proximity of ADORA1 with PDE10A compared to other cAMP targeting PDEs exclusively under PAH setting. PDE10A was also upregulated under the disease condition. From the functional studies, I demonstrate that genetic and pharmacological inhibition of ADORA1 and PDE10A induces pro-apoptotic and anti-proliferative effects in PASMCs isolated from IPAH patient lungs via increased cAMP levels. Impressively, in in vivo studies, the dual inhibitor treatment improved survival in the MCT-PAH rats compared to placebo. The hemodynamics and MRI data indicated that dual inhibitor treatment of MCT- and SuHx- rat models of PAH, significantly lowered pulmonary vascular resistance, reduced right ventricular hypertrophy, and improved cardiac performance. The morphometric analysis revealed that the MCT- and SuHx- PAH rats treated with dual inhibitors reversed pulmonary vascular remodeling i.e. reduced medial wall thickness and muscularization compared to the placebo group. In conclusion, these results show that targeting ADORA1/PDE10A signalosome regulated cAMP microenvironment is a promising step towards the development of novel and potent therapeutic strategy in the field of PAH.