Ferroptosis Of Vascular Smooth Muscle Cells Facilitates Vascular Calcification In Chronic Kidney Disease

Background:Vascular calcification is a common pathologic condition in patients with chronic kidney disease(CKD),highly associated with mortality of cardiovascular disease in patients with CKD.Vascular calcification is a process mediated by vascular smooth muscle cells(VSMCs),which is similar to bone formation.Pathological factors such as calcium and phosphate metabolism disorder,oxidative stress and programmed cell death play critical roles in regulating vascular calcification.However,there is no effective method to prevent and treat vascular calcification due to lack of fully understanding the mechanisms underlying vascular calcification.Ferroptosis is a form of cell death resulting from excessive iron-dependent reactive oxygen species(ROS)and lipid peroxidation.Cells eliminate accumulated lipid peroxide and resist ferroptosis mainly through glutathione peroxidase 4(GPX4)using glutathione(GSH)as substrate.GSH synthesis requires cystine/glutamate reverse transporter(system xc-),mediated by SLC7A11,to transport raw materials.SLC7A11/GSH/GPX4 axis is also considered to be a key pathway to regulate ferroptosis.Inhibition of ferroptosis attenuates atherosclerosis.However,it is unclear whether ferroptosis of vascular smooth muscle cells(VSMCs)regulates vascular calcification in CKD via SLC7A11/GSH/GPX4 pathway.Objective:To investigate the role of ferroptosis in vascular calcification and the molecular regulatory mechanism.Methods:1.Calcification of VSMCs and arterial rings was induced by high calcium and phosphate,and ferroptosis inhibitor Ferroptosis-1 was used to treat VSMCs and arterial rings.The extent of ferroptosis was determined by assessing cytoplasmic ROS,lipid peroxidation,ptgs2 mRNA expression,GSH contents and cell viability in VSMCs.Calcification was detected by alizarin red staining and quantitative analysis.Calcium concentration was measured.Expression of osteogenic markers Runx2 and BMP2 was analyzed by western blot.2.VSMCs were treated with inhibitors targeting SLC7A11/GSH/GPX4 signaling pathway and siRNA to determine the role of SLC7A11/GSH/GPX4 signaling pathway in VSMC calcification.3.CKD rats and vitamin D3-overload mice were used to determine the role of ferroptosis in vascular calcification in vivo,indicated by alizarin red staining.Results:We found that high calcium and phosphate treatment increased the levels of ROS and lipid peroxidation,leading to ferroptosis.Alizarin red staining and calcium contents showed that inhibiting ferroptosis reduced calcium deposition in VSMCs under osteogenic conditions.Gene expression analysis revealed that inhibition of ferroptosis repressed osteogenic differentiation of rat VSMCs.Similarly,in vitro experiments showed that inhibition of ferroptosis ameliorated calcification of rat and human arterial rings.Furthermore,in vivo experiments showed that inhibition of ferroptosis attenuated aortic calcification in rats or mice.Mechanistically,high calcium and phosphate reduced the contents of GSH in VSMCs by down-regulating expression of SLC7A11.GSH depletion induced by Erastin,system xc-inhibitor,significantly promoted calcification of VSMCs under osteogenic conditions,whereas GSH supplement by biosynthetic precursor reduced calcification of VSMCs.Consistently,knockdown of SLC7A11 by siRNA markedly promoted VSMC calcification.In addition,high calcium and phosphate treatment also down-regulated expression of GPX4 and reduced the activity of glutathione peroxidase.Both GPX4 inhibitor RSL3 and knockdown of GPX4 promoted VSMC calcification.Conclusions:1.High calcium and phosphate induces ferroptosis in rat VSMCs.2.Inhibition of ferroptosis represses calcium/phosphate-induced rat VSMC calcification.3.Inhibition of ferroptosis attenuates calcification of rat and human arterial rings.4.Inhibition of ferroptosis attenuates aortic calcification in rats with chronic kidney disease or vitamin D3-overloaded mice.5.Repression of SLC7A11/GSH/GPX4 axis is required for ferroptosis-mediated vascular calcification.