G566(P) Acetazolamide partially restores pulmonary artery smooth muscle cell de-differentiation in experimental pulmonary hypertension

Aims Pulmonary hypertension (PH) is a serious disease with limited therapeutic options. Inflammation has been shown to be causally linked to pulmonary arterial hypertension pathogenesis along with vascular smooth muscle cell (VSMC) phenotypic switching. We hypothesized that Acetazolamide (ACTZ), vis-a-vis carbonic anhydrase inhibition and induction of extracellular acidosis, restores rat pulmonary artery smooth muscle cell (RPASMC) phenotypic switching in experimental PH. Methods Adult male Sprague-Dawley rats were injected with Sugen (20 mg/kg) or vehicle and were exposed to hypoxia (9% O2) or normoxia for 3 weeks followed by 2 days of normoxia to induce pulmonary hypertension. Lungs were harvested subsequent to induction. In vitro studies were conducted as follows: 70,000 primary RPASMCs from healthy rats were seeded in 6-well culture dishes and serum-deprived (0.5% fetal bovine serum) for 48 hours prior to stimulation with 20% FBS. In some experiments, cells were treated with TNF-α (10 ng/ml) and exposed to ACTZ (500 mM) or media buffered to pH 6.8. In other experiments, baseline differences between PASMC proliferation from 3 experimental groups (control, Sugen/Hypoxia or Sugen/Hypoxia+ACTZ animals) were compared. Cell numbers were evaluated with a haemochromocytometer using trypan blue cell exclusion 1, 2 and 3 after stimulation in triplicates. RT-qPCR was conducted to determine the expression of proliferative, contractile and de-differentiation markers. Results RPASMCs exposed to TNF-α for 24 hours showed significant downregulation of markers of a contractile phenotype and upregulation of proliferative and de-differentiation markers. Exposure to TNF-α also resulted in increased proliferation, which was significantly reduced by exposure to ACTZ or acidosis. ACTZ or acidosis treatment also restored mRNA levels of contractile markers. Additionally, PASMCs isolated from control, SU/Hx and ACTZ-treated animals retained their respective phenotypes in vitro across a panel of markers. Subsequent to stimulation with 20% FBS, PASMCs of SU/Hx rats underwent significantly higher rates of proliferation, while cells from ACTZ-treated rats proliferated similarly to controls. Conclusion Acetazolamide partially restores RPASMC phenotypic switching and it may do so through induction of extracellular acidosis.