Ferritinophagy inhibition regulates hypoxia-induced proliferation of pulmonary artery smooth muscle cells

Objective To investigate the mechanism of hypoxia-induced free iron reduction in mouse pulmonary artery smooth muscle cells (PASMCs) and its role in the proliferation of PASMCs and pulmonary vascular remodeling. Methods ① Primary mouse PASMCs were cultured and divided into normoxic group(C), normoxic ferric ammonium citrate group (C+FAC), normoxic rapamycin group (C+R), hypoxic group (H), hypoxic ferric ammonium citrate group (H+FAC) and hypoxic rapamycin group (H+R). The C, C+FAC and C+R groups were incubated in normoxic (21% O2) enviroment for 48 h. The H, H+FAC and H+R groups were incubated in hypoxic (1% O2) environment for 48 h. The C+FAC and H+FAC groups were treated with ferrous ammonium citrate (FAC), and the C+R and H+R groups were treated with rapamycin (R). Cell proliferation was detected with CCK-8 assay and EdU proliferation assay. The content of intracellular free iron was measured by FerroOrange staining. The protein levels of iron regulatory proteins (IRPs), transferrin receptor (TFR), ferroportin (FPN) and ferritin heavy chain (FTH) were detected with Western blotting. ②Twenty-four C57/BL6N mice (male, 6 weeks old, weighting 18~22 g) were divided into normoxic group (C-4W), normoxic high-iron diet group (C+HID-4W), hypoxic group (H-4W) and hypoxic high-iron diet group (H+HID-4W) (n=6). The H-4W and H+HID-4W groups were placed in a low-pressure hypoxic environment at a simulated altitude of 5 000 m for 4 weeks. Right heart function, right ventricular systolic pressure, and pulmonary vascular remodeling in each group were observed and measured. Results Compared with C group, significantly enhanced proliferation (P < 0.01), reduced intracellular free iron content (P < 0.01), and up-regulated protein level of FTH were observed in the PASMCs of H group (P < 0.01). Compared with H group, the free iron content was obviously increased (P < 0.01) and cell proliferation was decreased in H+FAC group (P < 0.01). The H+R group had notably increased free iron content (P < 0.01), decreased cell proliferation (P < 0.05), and reduced protein level of FTH when compared with H group (P < 0.05). Compared with H-4W group, the mice in the H+HID-4W group showed significantly improved right heart function (P < 0.01), lower right ventricular systolic pressure (P < 0.05), reduced right heart hypertrophy (P < 0.01), and inhibited pulmonary artery thickening (P < 0.01). Conclusion Hypoxia mediates the reduction of free iron by inhibiting the ferritinophagy pathway, and thus promotes the proliferation of PASMCs and pulmonary artery remodeling.