Aquaporin 1 Modulates Apoptosis Susceptibility in Pulmonary Hypertension

Abstract

Pulmonary arterial hypertension (PAH) remains a fatal diagnosis despite available therapies. PAH is characterized by extensive remodeling of the pulmonary vasculature involving the formation of vaso‐occlusive lesions and a thickened medial layer of the vascular wall, both of which contain pulmonary arterial smooth muscle cells (PASMCs). We have demonstrated that PASMCs isolated from a well‐established rat model of PAH are resistant to apoptosis under both basal and stimulated conditions. The cell membrane protein aquaporin 1 (AQP1) was initially described as a water transport channel, but more recently has been implicated in other cellular functions including migration and proliferation, and in several distinct cancer types, has been associated with apoptosis resistance. AQP1 is upregulated in PASMCs isolated from rat models of PAH, although its role in apoptosis resistance is unclear. We hypothesized that increased expression of AQP1 contributes to apoptosis resistance in PASMCs.MethodsOur animal model is the Sugen‐hypoxia (SuHx) rat model of PAH in which Sugen‐5416, a vascular endothelial growth factor receptor type 2 inhibitor is injected into rats before they are placed in chronic hypoxia (10% O2) for 3 weeks, then returned to normoxia (21% O2) for 2 weeks. Control animals receive vehicle injection and are kept in normoxic (21% O2) conditions. Cells from distal pulmonary arteries are obtained via removal of the endothelium and enzymatic digestion of the tissue to obtain a pure smooth muscle population. To deplete AQP1, we transfected control and SuHx PASMCs with small interfering RNA (siRNA) directed toward either the AQP1 protein (siAQP1) or a non‐targeted sequence (siNT). Apoptosis was measured under basal conditions by Hoechst staining and visualization of DNA condensation. In complementary experiments, control PASMCs were infected with adenoviral constructs to over‐express AQP1 (AdAQP1) or green fluorescent protein (AdGFP; infection control). Cells were treated with either PBS or the apoptotic stimulus hydrogen peroxide (250 µm for 24 h). Apoptosis was again measured by Hoechst staining and visualization of DNA condensation.ResultsIn both control and SuHx PASMCs, AQP1 knock down increased apoptosis under basal conditions. In control PASMCs with increased AQP1 expression, there was no difference in apoptosis under basal conditions. To further provoke a response in these infected cells, the apoptotic stimulus hydrogen peroxide was applied; however, again there was no difference in apoptosis levels between AdAQP1 and AdGFP infected cells.ConclusionsThese data suggest that increased AQP1 may be necessary but not sufficient for apoptosis resistance. Future studies further characterizing the role of AQP1 in apoptosis resistance are necessary to understand molecular mechanisms governing resistance to apoptosis in PASMCs and provide novel pathways for new therapeutic agents targeting vascular remodeling with the potential to reverse disease.