The Role And Mechanism Of Ferritinophagy Inhibition In Hypoxia-Induced Proliferation Of Pulmonary Artery Smooth Muscle Cells

BackgroundHypoxic pulmonary hypertension（HPH）is a complex cardiopulmonary disease caused by hypoxia.Hypoxia,which can lead to pulmonary hypertension,can be caused by high altitude environments,respiratory pathologies,etc.The dominant clinical feature of HPH is a progressive increase in pulmonary artery pressure,often accompanied by fatigue,dyspnea,chest tightness and limited mobility.Early hypoxia can trigger pulmonary vasoconstriction,and long-term hypoxia can lead to pulmonary vascular remodeling,which is the main pathological characteristics of HPH.The pathological process of pulmonary vascular remodeling involves several kinds of vascular cells,including pulmonary vascular endothelial cell dysfunction,fibroblast activation,and smooth muscle cell overproliferation.Overproliferation of pulmonary artery smooth muscle cells（PASMCs）is the critical mechanism of pulmonary vascular remodeling.Therefore,an in-depth study on the mechanism of excessive proliferation of PASMCs induced by hypoxia has significant implications for the prevention and treatment of HPH.It has been shown that iron homeostasis is commonly present in patients with pulmonary hypertension and that iron homeostasis is also involved in the regulation of hypoxia-induced pulmonary vascular remodeling.The body’s oxygen demand increases during hypoxia and body’s iron homeostasis is altered to accommodate the high iron demand for hemoglobin synthesis and erythropoiesis.As an essential microelement for intracellular protein and enzyme activities,iron is participating in cellular respiration,oxygen sensing,oxygen transport,oxygen metabolism,energy metabolism,DNA synthesis and repair,and signal transmission.The reduction of intracellular iron ions can influence the normal function of cells;while too much iron ions may also create an increase in intracellular reactive oxygen species,leading to cellular oxidative stress and even Ferroptosis.Thus,the intracellular iron ion content needs to be precisely regulated to better adapt to the hypoxic environment.Most of the intracellular iron is stored in ferritin（FT）,which cannot be directly utilized by cells,and the minor portion of iron that can be directly available to cells exists as free iron in the cytoplasm,also known as the labile iron pool（LIP）.FT can be degraded in the lysosome via the Ferritinophagy pathway,releasing ferric ions to the LIP.Iron regulatory proteins（IRPs）sense changes in intracellular iron content and maintain intracellular iron homeostasis by regulating iron uptake,export,storage and release pathways.Is there an imbalance in iron homeostasis in pulmonary artery smooth muscle cells under hypoxic conditions?What is the mechanism?Does the imbalance of iron homeostasis modulation affect smooth muscle cell proliferation?It is still unclear.Consequently,this study intends to investigate the hypoxia-induced changes in iron homeostasis of PASMCs and its underlying mechanisms,and further clarify its role in pulmonary artery smooth muscle cell proliferation and pulmonary vascular remodeling by regulating iron homeostasis,so as to provide new ideas and effective targets for the prevention and treatment of HPH.Methods1.Primary mouse PASMCs were placed in a hypoxic（1%O₂）environment for 48h to construct a hypoxia-induced proliferation model of PASMCs.The cell proliferation were detected by CCK-8 and EdU.The free iron content in PASMCs were detected by Ferro Orange.The protein expression of IRPs in PASMCs were detected by WB.Free iron content in PASMCs were increased by exogenous addition of Ferric ammonium citrate（FAC）,and cell proliferation were detected by CCK-8 method and EdU method;free iron content in PASMCs were detected by Ferro Orange method.2.The mice were randomly divided into 4 groups:normoxic group,normoxic high-iron diet group,hypoxic group and hypoxic high-iron diet group.The normoxic and hypoxic groups were fed a normal diet（iron content about 50 ppm）,while the normoxic group on a high iron diet and the hypoxic group on a high iron diet were fed a high iron diet（iron content about 500 ppm）.The hypoxic groups were placed in a low-pressure oxygen chamber,and the conditions were set to simulate an altitude of 5000 m.The mice were kept under hypoxic conditions for 4 weeks to develop HPH.The right ventricular systolic function were measured by cardiac ultrasound.The right ventricular systolic pressure were measured by catheterization.The pulmonary vascular alterations were detected by HE staining.Determination of free iron ions in isolated pulmonary artery smooth muscle cells is measured by Ferro Orange.3.The mRNA and protein expression of iron homeostasis genes（TFR,FPN and FTH）in PASMCs were detected by RT-qPCR and WB.The expression of NCOA4 and autophagy molecule LC3 in PASMCs were detected by WB.The ferritin in lysosomes in PASMCs were detected by immunofluorescence labeling of ferritin and lysosomes,and the free iron in lysosomes in PASMCs were detected by immunofluorescence labeling of free iron and lysosomes.Rapamycin were used to interfere with ferritinophagy,and cell proliferation were detected by CCK-8 and EdU assays;free iron content in PASMCs were detected by Ferro Orange assay;protein expression of iron homeostasis genes（TFR and FTH）in PASMCs after ferritinophagy intervention were detected by WB assay.Results1.The hypoxia-induced cell proliferation model was successfully replicated,and compared with the normoxia group,hypoxia significantly enhanced the proliferation activity of PASMCs（P<0.01）,significantly increased the EdU positivity rate（P<0.01）,and significantly reduced the free iron content in PASMCs（P<0.01）.The HPH mouse model was successfully replicated and hypoxia significantly reduced free iron content within isolated PASMCs of HPH mouse（P<0.05）.Hypoxia significantly upregulated the protein expression of iron regulatory proteins（IRP1 and IRP2）in PASMCs（P<0.05）.FAC significantly increased free iron ion content in PASMCs（P<0.01）,significantly inhibited hypoxia-induced cell proliferation activity（P<0.01）,and significantly reduced EdU positivity（P<0.05）.2.High-iron diet significantly increased free iron content in PASMCs of HPH mice（P<0.01）,significantly improved right heart function（P<0.01）,significantly decreased right ventricular systolic pressure（P<0.05）,significantly reduced right heart hypertrophy（P<0.01）,and significantly inhibited pulmonary vascular thickening（P<0.01）.3.Hypoxia significantly increased mRNA expression of TFR in PASMCs（P<0.05）and significantly inhibited mRNA expression of FPN（P<0.01）and FTH（P<0.05）;TFR protein levels were significantly increased in PASMCs（P<0.05）and intracellular FTH protein levels were significantly increased after hypoxia（P<0.01）;after hypoxia,ferritin in lysosome were significantly increased,lysosomal acidity were significantly decreased,and free iron in cytoplasm and lysosomes were significantly decreased.Rapamycin enhanced ferritinophagy in PASMCs,significantly increased intracellular free iron content（P<0.01）,significantly inhibited hypoxia-induced cell proliferation activity（P<0.05）,significantly decreased EdU positivity（P<0.01）,and significantly decreased TFR（P<0.01）and FTH protein levels（P<0.05）.Conclusion1.Hypoxia induces proliferation and free iron reduction in PASMCs,and iron supplementation significantly inhibits hypoxia-induced cell proliferation.2.High-iron diet significantly increased free iron content in PASMCs of HPH mouse.High iron diet significantly improves right heart function,reduces right ventricular pressure,and inhibits right ventricular hypertrophy and pulmonary vascular remodeling.3.Hypoxia promotes the proliferation of PASMCs by inhibiting the lysosome-dependent ferritinophagy pathway.Activation of ferritinophagy significantly increased free iron content in PASMCs and significantly inhibited hypoxia-induced cell proliferation Hypoxia regulates the proliferation of MPASMCs by inhibiting the lysosome-dependent ferritinophagy.4.Inhibition of ferritinophagy in pulmonary vascular smooth muscle cells plays an important role in the formation of HPH and targeting ferritinophagy is promised to be a new strategy to prevent and treat HPH.