Iron chelation inhibits the development of pulmonary vascular remodeling

Abstract

Reactive oxygen species (ROS) have been implicated in the pathogenesis of pulmonary hypertension. Because iron is an important regulator of ROS biology, this study examined the effects of iron chelation on the development of pulmonary vascular remodeling. The administration of an iron chelator, deferoxamine, to rats prevented chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling. Various iron chelators inhibited the growth of cultured pulmonary artery smooth muscle cells. Protein carbonylation, an important iron-dependent biological event, was promoted in association with pulmonary vascular remodeling and cell growth. A proteomic approach identified that Rho GDP-dissociation inhibitor (a negative regulator of RhoA) is carbonylated. In human plasma, the protein carbonyl content was significantly higher in patients with idiopathic pulmonary arterial hypertension than in healthy controls. These results suggest that iron plays an important role in the ROS-dependent mechanism underlying the development of pulmonary hypertension.

Fig. 1. Effects of deferoxamine (DFO) on chronic hypoxia-induced pulmonary hypertension in intact rats

Rats were intraperitoneally injected daily with saline (vehicle control) or 20 mg/kg body weight of DFO during 2-week exposure to normoxia or hypoxia (10% O₂). (A) Right ventricular systolic pressure (RVSP) was measured using a Millar catheter (n = 3). (B) RV/(LV+S) values (n = 3 – 6). (C) H & E staining showing small pulmonary arteries (PA) and arterioles with the diameter ranging 53–113 μm. Scale bars, 50 μm. The bar graph represents means ± SEM of % wall thickness (n = 3). *, P<0.05 vs. normoxia; a, P<0.05 vs. hypoxia.

Fig. 2. Effects of metal chelators on pulmonary artery SMC growth

Growth-arrested bovine pulmonary artery SMCs were pre-treated for 30 min with (A, B, and C) deferoxamine (DFO), (D) hinokitiol (HIN), and (E) HBED. Cells were then treated with (A) 30 nM ET-1 for 6 days, (B) 10 ng/ml PDGF for 6 days, or (C, D & E) 10% FBS for 3 days. Cell growth was monitored by XTT assay. Bar graphs represent means ± SEM (n = 7 – 24). *, P<0.05 vs. untreated; a, P<0.05 vs. ET-1, PDGF or FBS.

Fig. 3. Effects of chronic hypoxia on protein carbonylation in an in vivo model of pulmonary hypertension

Rats were subjected to chronic hypoxia (10% O₂) for indicated durations. After treatments, pulmonary arteries were isolated and homogenized. Proteins were derivatized with DNPH and subjected to Western blotting, with rabbit polyclonal IgG for DNP used to monitor carbonylated proteins. Total protein levels were monitored by Coomassie Blue staining. The bar graph represents means ± SEM (n = 3 – 4). *, P<0.05 vs. normoxia (0 day hypoxia).

Fig. 4. Effects of hydralazine (HDZ) on pulmonary artery SMC growth

(A) Cell lysates from FBS-stimulated pulmonary artery SMCs (control) were treated in vitro with HDZ before derivatization with DNPH and subsequent immunoblotting to demonstrate that HDZ interacts with protein carbonyl groups and competes with DNPH. The bar graph represents means ± SEM (n = 3 – 4). *, P<0.05 vs. untreated control. (B) Growth-arrested bovine pulmonary artery SMCs were pre-treated for 30 min with HDZ and then treated with 10% FBS for 3 days. Cell growth was monitored by XTT assay. The bar graph represents means ± SEM (n = 8).*, P<0.05 vs. untreated control; a, P<0.05 vs. FBS.

Fig. 5. Chronic hypoxia promotes RhoGDI-α carbonylation and activates RhoA signaling in an iron-dependent fashion in the pulmonary arteries of intact rats

(A) Rats were intraperitoneally injected with saline or 20 mg/kg body weight of deferoxamine (DFO) and were then subjected to chronic hypoxia (10% O₂) for 1 day. Isolated pulmonary arteries were homogenized. Proteins were derivatized with DNPH and subjected to immunoprecipitation with anti-DNP IgG and to Western blotting with anti-RhoGDI-α IgG in order to monitor carbonylated RhoGDI-α. (B) Western blotting to monitor total RhoGDI-α expression. (C) Rats were injected with saline or DFO and were then subjected to chronic hypoxia for 4 days. Western blotting was performed on pulmonary arterial homogenates to show phosphorylation of MYPT1 at Serine 507 (p-MYPT1) and total MYPT1 expression. Bar graphs represent means ± SEM (n = 3 – 4). *, P<0.05 vs. normoxia; a, P<0.05 vs. hypoxia. ns: not significantly different from each other.

Fig. 6. Total protein carbonyl contents in IPAH patients and control subjects

Plasma samples from IPAH patients and age- and gender-matched control subjects were derivatized with DNPH and subjected to (A) 1-dimensional SDS-PAGE or (B) 2-dimensional gel electrophoresis consisting of isoelectric focusing and SDS-PAGE. Samples were then immunoblotted with DNP antibody. The graph represents means ± SEM (n = 6 – 7) of total carbonyl content in arbitrary units (a.u.). The symbol (*) denotes that the two groups are significantly different from each other at P < 0.05. No signals were detected without DNPH derivatization (data not shown).