Roles And Mechanisms Of Histone Acetylation Homeostasis In Ferroptosis Of Vascular Smooth Muscle Cells

Objective: Ferroptosis characterized with iron overload,accumulation of reactive oxygen species and lipid peroxidation was reported as a novel cell death.The ferroptosis of vascular smooth muscle cells(VSMCs)is closely related to the loss of VSMCs in aortic dissection,and was reported a new pathological mechanism of aortic dissection.This study focused on the ferroptosis of VSMCs,and explored the role of histone acetyltransferase and histone deacetylase in ferroptosis of VSMCs.We expect to find new therapeutic targets for preventing and treating ferroptosis of VSMCs.Methods: 1.Ferroptosis models of VSMCs were established and histone acetylation levels were examined under the ferroptosis models: Human aortic smooth muscle cells were extracted and used in the experiments.The cystine deprivation and imidazole keton erastin(IKE)were selected to induce the ferroptosis of VSMCs,and the ferroptosis models were evaluated by lipid peroxidation level examination.Then,the expression of histone acetylation was examined by western blot.2.Effect of histone acetyltransferase E1A-associated 300-k Da protein(P300)deficiency on ferroptosis of VSMCs were evaluated: Firstly,the expression pattern of histone acetyltransferase P300 was examined by western blot in the VSMCs under the ferroptosis models.The effects of P300 knockdown or inhibition on cell viability and lipid peroxidation level of VSMCs under the ferroptosis models were evaluated.Further,the pathway of P300 regulating ferroptosis of VSMCs was identified through the examination of the protein expression of the key molecules of ferroptosis.Then,the coimmunoprecipitation was performed to examine the interaction between P300 and HIF-1α,and the interaction between HIF-1α and P53 to clarify the mechanism of P300 regulating the ferroptosis of VSMCs.3.Effect of histone deacetylase 6(HDAC6)on ferroptosis of VSMCs were evaluated: The expression of HDAC6 in VSMCs under ferroptosis models were examined by western blot.Then,the cell viability and lipid peroxidation level of VSMCs with treatment of HDAC6 knockdown or inhibition under the ferroptosis models were examined to verify the effect of HDAC6 on VSMCs ferroptosis.The association between HDAC6 and ferroptosis of VSMCs and the mechanism of HDAC6 regulating ferroptosis of VSMCs were further identified by bioinformatics analysis.Results: 1.The ferroptosis model was successfully established after cystine deprivation and 2.5 μM IKE stimulating VSMCs for 16 hours.Under the ferroptosis models of VSMCs,the expression of H3K23 ac,H4K8ac and H4K12 ac were significantly decreased.2.Under the VSMCs ferroptosis models,the level of P300 protein was significantly decreased.P300 knockdown or P300 inhibition by A-485 significantly promoted VSMCs ferroptosis.P300 deficiency induced VSMCs ferroptosis by activating the HIF-1α/HMOX1 axis,and the aggravated effects of P300 knockdown on VSMCs ferroptosis were largely nullified by HIF-1α knockdown or HIF-1α inhibitor BAY87-2243.3.The protein level of HDAC6 was significantly increase in VSMCs after treatment with cystine deprivation and IKE.HDAC6 knockdown or HDAC6 inhibition by TBSA significantly protected VSMCs from ferroptosis.Knockdown of HDAC6 significantly inhibited cell autophagy to protect VMSCs from ferroptosis.The protection effects of HDAC6 knockdown on VSMCs ferroptosis were largely nullified by Fe SO4.Conclusions: Histone acetylation homeostasis is of great importance for the ferroptosis of VSMCs.Histone acetyltransferase P300 deficiency promotes the ferroptosis of VSMCs through regulating HIF-1α/HMOX1 axis,while histone deacetylase HDAC6 downregulation protects VSMCs from ferroptosis by inhibiting autophagy.Histone acetylation is expected to become new therapeutic strategy for the treatment of vascular diseases characterized by ferroptosis of VSMCs.