The Role And Mechanism Of GPX4-dependent Ferroptosis In Vascular Aging

Background:Vascular aging is one of the important aspects of human aging and is closely related to the overall health of the human body.Vascular aging begins early in life,and from a clinical perspective,it can be defined as a process.It is characterized by gradual changes in vascular structure and function,resulting in decreased arterial compliance,increased arteriosclerosis,and gradual loss of vascular function^[1],which is closely related to various cardiovascular diseases（CVD）^[2].Mechanisms of vascular aging include oxidative stress,genomic instability,chronic low-grade inflammation,telomere shortening,and cellular senescence^[2].Cellular senescence is a state of irreversible cell cycle arrest under various stresses,which has gradually transformed from its initial appearance as an in vitro phenomenon to a cellular hallmark of in vivo aging^[3].Vascular smooth muscle cells（VSMCs）are an important part of the vascular structure,and their senescence promotes the occurrence of atherosclerosis and plaque^[4]and is closely related to other diseases including hypertension and diabetes.Ferroptosis is a new regulated form of cell death that was formally proposed in 2012and is morphologically,biochemically and genetically distinct from other well-known cell death types such as apoptosis and necrosis^[5].It is characterized by the massive accumulation of lethal lipid reactive oxygen species（ROS）driven by ferrous iron(Fe²⁺)^[5,6].Although the precise signal transduction has not been fully elucidated,it has been demonstrated that ferroptosis is regulated by multiple layers of signaling pathways and programs,including antioxidant enzymes（especially glutathione peroxidase 4 GPX4）^[6,7],lipid Peroxidation products^[8,9],energy utilization^[10],mitochondrial function^[11],interferon-related genes^[12],etc.Ferroptosis contributes to ischemia or drug-induced cardiomyopathy^[13],acute renal failure^[14],and atherosclerosis^[15].However,little is known about the role of ferroptosis in vascular aging,and previous studies have found that aging blood vessels have iron deposition,lipid deposition,and a large amount of ROS generation,which is precisely related to ferroptosis.The main processes have a high similarity^[16].Therefore,our research group hypothesized that ferroptosis may be involved in the process of vascular aging.Methods:Clinical samples:Normal carotid tissue was obtained from middle-aged patients（<50 years old）and elderly patients（>70 years old）with clinical carotid body tumors.At the animal level:Two models of aging were used.One is to raise C57BL/6J mice to 24months of age as a natural aging model,and the other is to use angiotensin 2（Ang II,400ng/kg/min）+bleomycin（Bleo,40ng/kg/min）combined with stimulation to induce a model of vascular stress aging.The effect of ferroptosis on vascular aging was explored using the ferroptosis inhibitor Liproxstatin-1 in a stress model;GPX4 knock-in mice were constructed to evaluate the effect of ferroptosis on vascular aging.At the cellular level,primary human vascular smooth muscle cells（h VSMCs）and mouse aortic smooth muscle cells（MOVAS）were used to construct replicative aging and stress aging models for research.Results:1.Activation of ferroptosis exists in naturally aging aortae and stress-induced aging aortae.We tested ferroptosis-related markers in tissue and cellular aging models,respectively.First,the carotid arteries of middle-aged（<50 years old）and elderly（>70 years old）subjects were compared,and it was found that the ferroptosis factors ALOX15,ACSL4,and Fe²⁺were all elevated in the arteries of elderly subjects.Secondly,in the animal model,we used two vascular aging models,one is the natural aging C57BL/6J mouse（24 months old）,the other is using Angiotensin II（Ang II,400ng/kg/min）+bleomycin（Bleo,40ng/kg/min）induced vascular stress senescence.We found that ferroptosis was activated in the aortae of both aging mice compared with control C57BL/6J mice.In the cell model,we also used two cell senescence models,one was to extract primary human vascular smooth muscle cells（h VSMCs）and passaged to 35 passages to form replicative senescent cells,and the other was to use Ang II（0.1μM）+bleomycin（100n M）induced cellular stress senescence in mouse VSMCs,both of which showed activation of ferroptosis compared to controls.2.We administrated a ferroptosis activator RSL3 in mouse VSMCs and found that RSL3 at a non-cytotoxic dose（0.5μM）significantly induce VSMCs aging.At the same time,we added the culture medium of ferroptotic cells and senescent cells to normal VSMCs medium respectively,and the results showed that ferroptosis drives senescence in VSMCs,while senescent VSMCs seem to be unable to induce ferroptosis.3.We used the ferroptosis inhibitor Liproxstatin-1 in an animal stress aging model,and found that Liproxstatin-1 blocked ferroptosis,reduced vascular oxidative stress,and reduced NAD⁺loss,alleviates vascular remodeling/sclerosis,inhibits the aging phenotype and slows the vascular aging process.4.We continued to use the ferroptosis inhibitor Liproxstatin-1 on the mouse VSMCs aging model in vitro and found that Liproxstatin-1 blocked ferroptosis and reduced the unstable iron pool（LIP）in cells.It also reduces oxidative stress in cells,reduces NAD⁺loss,and slows cellular aging.5.We constructed GPX4 knock-in mice and used littermate wild-type（WT）mice as controls.Two groups of mice were reared for 24 months to a natural aging state.It was found that knock-in of GPX4 inhibited ferroptosis,slowed the vascular aging process in mice,reduced aging-related NAD⁺loss,and attenuated vascular remodeling/sclerosis in aged mice.6.Angiotensin II（Ang II,400ng/kg/min）+bleomycin（Bleo,40ng/kg/min）were used in GPX4 knock-in mice and littermate wild-type（WT）mice（8-weeks-old）respectively,combined stimulation formed stress-induced vascular senescence.It was found that knock-in of GPX4 inhibited ferroptosis,slowed the loss of NAD⁺associated with vascular stress in mice,and slowed the aging process of aortae in mice.7.In the experiment of mouse VSMCs cultured in vitro,we found that overexpression of GPX4 by transfection of plasmids in stress-induced senescent cells could alleviate oxidative stress,NAD⁺loss and degree of senescence in senescent mouse VSMCs.Conclusions:In this study,we demonstrate that ferroptosis is globally activated under conditions of vascular aging and stress.And confirmed that blocking ferroptosis pharmacologically（using the chemical inhibitor liproxstatin-1）or genetically（constructing GPX4 knock-in mice）can delay vascular aging,and attenuate aging-and stress-related NAD⁺loss and vascular stiffness in the vascular system.