Extracellular superoxide dismutase overexpression can reverse the course of hypoxia-induced pulmonary hypertension

Abstract

Hypoxia leads to free radical production, which has a pivotal role in the pathophysiology of pulmonary hypertension (PH). We hypothesized that treatment with extracellular superoxide dismutase (EC-SOD) could ameliorate the development of PH induced by hypoxia. In vitro studies using pulmonary microvascular endothelial cells showed that cells transfected with EC-SOD had significantly less accumulation of xanthine oxidase and reactive oxygen species than nontransfected cells after hypoxia exposure for 24 h. To study the prophylactic role of EC-SOD, adult male wild-type (WT) and transgenic (TG) mice, with lung-specific overexpression of human EC-SOD (hEC-SOD), were exposed to fraction of inspired oxygen (FiO(2)) 10% for 10 d. After exposure, right ventricular systolic pressure (RVSP), right ventricular mass (RV/S + LV), pulmonary vascular wall thickness (PVWT) and pulmonary artery contraction/relaxation were assessed. TG mice were protected against PH compared with WT mice with significantly lower RVSP (23.9 ± 1.24 versus 47.2 ± 3.4), RV/S + LV (0.287 ± 0.015 versus 0.335 ± 0.022) and vascular remodeling, indicated by PVWT (14.324 ± 1.107 versus 18.885 ± 1.529). Functional studies using pulmonary arteries isolated from mice indicated that EC-SOD prevents hypoxia-mediated attenuation of nitric oxide-induced relaxation. Therapeutic potential was assessed by exposing WT mice to FiO(2) 10% for 10 d. Half of the group was transfected with plasmid containing cDNA encoding human EC-SOD. The remaining animals were transfected with empty vector. Both groups were exposed to FiO(2) 10% for a further 10 d. Transfected mice had significantly reduced RVSP (18.97 ± 1.12 versus 41.3 ± 1.5), RV/S + LV (0.293 ± 0.012 versus 0.372 ± 0.014) and PVWT (12.51 ± 0.72 versus 18.98 ± 1.24). On the basis of these findings, we concluded that overexpression of EC-SOD prevents the development of PH and ameliorates established PH.

Figure 1

HPMECs nontransfected (N) and transfected (T) with EC-SOD after exposure to hypoxia for 24 h compared with a cell line incubated with FiO₂ 21% for 24 h. (A) Western blot for hEC-SOD protein and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. (B) XO concentration. (C) Intracellular reactive species and hydrogen peroxide measuring using DCF fluorescence over time. *Transfected cell line versus nontransfected cell line, P < 0.05. —◆—, RA; —■—, hypoxic nontransfected; —▲—, hypoxic transfected.

Figure 2

Evaluation of ROS concentration after exposure to hypoxia (FiO₂ 10% × 10 d) in WT and TG adult mice compared with normoxic groups. (A) XO concentration. (B) Total ROS concentration. Data are the mean of six animals per group ± SEM. *TG hypoxic versus WT hypoxic group, P < 0.05.

Figure 3

Evaluation of PH and cardiac hypertrophy after exposure to hypoxia (FiO₂ 10% × 10 d) in WT and TG adult mice compared with normoxic groups. (A) RV/S + LV ratio. (B) RVSP. (C) Images of BV stained for α-SMA and measurement showing the method of measurement of wall thickness. (D) Wall thickness of medium-sized pulmonary arteries assessed by immunostaining for anti–α-SMA. Data are the mean of six animals per each group ± SEM. *TG hypoxic versus WT hypoxic group, P < 0.05.

Figure 4

White blood cell counts and PMNs after exposure to hypoxia (FiO₂ 10% × 10 d) in WT and TG adult mice compared with normoxic groups. Data are the mean of six animals per group ± SEM. *TG hypoxic versus WT hypoxic group, P < 0.05. BAL, bronchoalveolar lavage; , BAL, WBCs count × 1,000; , BAL, PMNs count × 1,000.

Figure 5

Pulmonary artery function studies were performed on ~180 μm pulmonary arteries isolated from adult WT and TG animals exposed to hypoxia of FiO₂ 10% for 10 d compared with normoxic groups. (A) Contraction (grams) induced by PE (1–100 nmol/L = PE⁻⁹ to PE⁻⁷). (B) Relaxation (grams) induced by Ach (10 nmol/L to 10 μmol/L = Ach⁻⁹ to Ach⁻⁵). (C) Contraction induced by 5-HT (10 nmol/L to 10 μmol/L = 5-HT⁻⁸ to 5-HT⁻⁵). (D) Relaxation induced by spermine NONOate (NO, 1 nmol/L to 10 μmol/L, NO⁻⁹ to NO⁻⁵). Data are expressed as mean of four animals per each group. RA, room air; —◆—, WT RA; —■—, TG RA; —▲—, WT hypoxia; —×—TG hypoxia.

Figure 6

RV/S + LV ratio after exposure to hypoxia (FiO₂ 10% × 20 d) in both WT adult mice groups transfected with plasmid and EC-SOD or plasmid only (after 10 d of the exposure), compared with WT normoxic groups that were treated the same way. (A) Western blot for hEC-SOD protein. (B) RV/S + LV ratio. (C) RVSP. (D) Wall thickness of medium-sized pulmonary arteries assessed by immunostaining for anti–α-SMA. Data are the mean of six animals per group ± SEM. *Hypoxic transfected with plasmid + hEC-SOD versus hypoxic transfected with plasmid alone, P < 0.05.