Abstract 12953: Targeting NEDD9 and SMAD3 in the Pathogenesis of Chronic Thromboembolic Pulmonary Hypertension

Abstract

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by unresolved pulmonary thrombi due, in part, to impaired fibrinolysis. We showed previously that oxidation of the Cas protein NEDD9 at cysteine-18 (Cys 18 ) inhibits complex formation between NEDD9 and the anti-fibrinolytic transcription factor SMAD3 in human pulmonary artery endothelial cells (HPAECs). However, the effect of NEDD9-Cys 18 oxidation on SMAD3 bioactivity in HPAECs is not known. Hypothesis: NEDD9-Cys 18 oxidation increases bioavailable SMAD3, which upregulates SERPINE1 that encodes plasminogen activator inhibitor (PAI)-1 to inhibit fibrinolysis pathways in HPAECs. Methods/Results: To assess the effect of SMAD3 on SERPINE1 transcription, HPAECs were transfected with cDNA-SMAD3 (1 μg/μl) for 72 hr to induce SMAD3 overexpression. Compared to vehicle (V) control, cDNA-SMAD3 increased PAI-1 and SERPINE1 expression by 35% (P=0.004) by immunoblot and 70% (P=0.003) by real-time PCR, respectively (N=3). To determine the effect of NEDD9 inhibition on SMAD3 activation, we analyzed the ratio of P-SMAD-Ser423/425:SMAD3 by immunoblot. Compared to scrambled RNA (scRNA), transfection with siRNA against NEDD9 (siNEDD9) for 48 hr increased the relative quantity of activated SMAD3 by 1.4-fold (P=0.006) and upregulated PAI-1 (P=0.004) expression by 1.7-fold in HPAECs (N=4). Next, we monitored the effect of oxidant stress on SMAD3 bioactivity. Compared to V-control, the expression of NEDD9, total SMAD3, and P-SMAD3 was increased by 66% (P=0.04), 81% (P=0.03), and 29% (P=0.04), respectively, in HPAECs treated with hydrogen peroxide (H 2 O 2 ) (500 μM) for 20 min. Next, the effect of NEDD9 silencing on H 2 O 2 accumulation in HPAECs was measured by Amplex Red assay. HPAECs were transfected with scRNA or siNEDD9 for 48 hr followed by measurement of H 2 O 2 production. Compared to scRNA, NEDD9 silencing significantly decreased H 2 O 2 concentration by 56% (N=4, P=0.02). Conclusion: These data demonstrate that oxidant stress increases SMAD3 activity and SMAD3-dependent upregulation of PAI-1 through a mechanism involving redox regulation of NEDD9. Identifying prothrombotic control of SMAD3 by NEDD9 may have important therapeutic implications for CTEPH.