Abstract 16915: Potential Role of Endothelial Cell WWOX in the Pulmonary Vascular Response to Hypoxia

Abstract

Introduction: Pulmonary arterial hypertension (PAH) is characterized by abnormal proliferation and dysfunction of pulmonary artery smooth muscle and endothelial cells (PASMC, PAEC). The WWOX/ Wwox gene encodes a tumor suppressor WW domain-containing oxidoreductase, known as WWOX. WWOX has been linked to several protein networks, highlighting its functions in cell homeostasis, survival and proliferation. Loss of WWOX gene expression is observed in human cancer cells, as lung cancer. We hypothesize that during hypoxia, WWOX expression is modulated in PAECs, regulating cell proliferation and selecting apoptosis-resistant cells, thus causing vascular remodeling. Methods and Results: Endothelial cell specific Wwox knockout mice were developed by a conditional ablation of Wwox gene expression using the Cre -Lox site-specific recombination system. We crossed Wwox flox/flox mice with CDH5 Cre+ mice, generating Wwox floxed, CDH-Cre+ specific mice that are tamoxifen-inducible (EC- WWOX KO). EC-WWOX KO mice were treated daily with tamoxifen or vehicle for 5 days and exposed to 10% hypoxia. After four weeks of hypoxia exposure, EC-WWOX KO mice showed increased right ventricular systolic pressure (RVSP) (P=0.0003) and Fulton index (RV (LV+S)) ( P=0.049), when compared to controls, suggesting PH development. In vitro , PAECs exposure to 3% hypoxia for 6 hours showed an increase of WWOX mRNA (P=0.0154) and protein (P<0.05) expression levels, decreased of cell proliferation detected by CyQUANT assay and PCNA protein expression (P=0.0024). These effects did not extend to cells exposed to 3% hypoxia for 24h. Immunoblots showed activation of apoptotic markers, such as cleaved caspase 3, PARP and Bax, suggesting that in hypoxia there is a potential controlled balance between increased WWOX expression, decreased cell proliferation and stimulation of apoptosis in these cells. Conclusion: Loss of EC WWOX expression plays a significant role in the development of PH in mice exposed to chronic hypoxia. We speculate that increased WWOX expression with acute hypoxia exposure could contribute to the selection of proliferative and pro-apoptotic PAECs, characteristic of PAH. Studies seeking to understand the mechanisms surrounding WWOX expression in PAECs are ongoing.