The Mechanism Of Heme Inducing Osteogenic Phenotypic Transformation Of Aortic Valve Interstitial Cells Through Endoplasmic Reticulum Stress

BackgroundDegenerative aortic valve stenosis(DAVS)is the predominate type of aortic valve stenosis(AS).Its pathological features mainly include thickening,fibrosis and calcification remodeling of arterial valve.The pathogenesis of degenerative aortic valve sclerosis is mediated by inflammation at the initial stage.Stimulation through related molecular mechanism changes the local valvular environment and triggers aortic valve endothelial cell(AVECs)and aortic valvular interstitial cell(AVICs)to ossify and calcify,resulting in collagen deposition and progressive calcification.The conversion of AVICs to osteogenesis phenotype is a basic step of valvular calcification.In recent years,more and more studies have shown that there is a large scale of neovascularization and intravalvular bleeding in the calcified aortic valve tissue,and the intravalvular bleeding proportion is an independent factor positively related to the rate of calcification progress of the degenerative aortic valves.Heme,an iron porphyrin compound,is the product decomposed from red blood cells and hemoglobin.Free heme is insoluble in water but has good affinity to organic substance.It is toxic to cells through lipid and protein oxidation,DNA damage,and inducing pro-inflammatory response.Heme can be degraded into Fe2+,leading to the increase of intracellular labile iron pool(LIP),which on the other hand can also promote the production of intracellular ROS and lipid peroxidation.In atherosclerosis,heme induces endoplasmic reticulum stress in vascular smooth muscle cells and promotes the progression of atherosclerosis.Endoplasmic reticulum stress happens when a wide range of cell dysfunction caused by external stimuli results in endoplasmic reticulum dysfunction and unfold proteins accumulation.In order to repair abnormal state,the activation of a series of pathways called unfold protein response(UPR)is regarded as endoplasmic reticulum stress.Some studies have shown that endoplasmic reticulum stress is involved in promoting aortic valve calcification in hypercholesterolemic mice.There are few studies revealing the mechanism of intravalvular hemorrhage promoting aortic valve calcification.The previous studies only explore the effect of erythrocytes on valvular interstitial cells.Researches on erythrocyte metabolites such as iron and heme in DAVS have not been reported.ObjectiveThe objective of this study is to explore the relationship between intravalvular hemorrhage,heme,endoplasmic reticulum stress and osteogenic phenotype of aortic valve interstitial cells.Methods1.The aortic valve tissue was sectioned,stained and observed.2.Heme assay kit was used to detect heme content in aortic valve tissue.3.Aortic valve interstitial cells(AVIC)was isolated from valves and cultured in vitro.4.Fluorescence probes were used to detect the levels of Fe2+,ROS,mitochondrial oxidation and lipid peroxidation in AVICs treated with heme,DFO and Fer1 in vitro.5.Western blot and q-PCR were applied to detect the expression of endoplasmic reticulum stress-related protein,osteogenesis-related protein and mRNA in AVICs treated with heme,DFO and Fer-1.6.After applying siRNA kit to reduce the expression of ATF4,western blot and q-PCR were used to detect heme and endoplasmic reticulum stress-related protein and osteogenic-related protein in AVICs under the treatment of DFO and Fer-1.Results1.Neovascularization,red blood cell infiltration and hemosiderin deposition are observed in the tissue sections of the aortic valve of human degenerative aortic valve.The heme in aortic valve tissue of human degenerative aortic valve is more than that in normal aortic valve tissue.2.Heme induces the increase of Fe2+,ROS production,mitochondrial oxidation and lipid peroxidation in porcine AVICs in vitro,which can be antagonized by iron chelator DFO and ferroptosis specific antioxidant Fer-1.3.Heme induces osteogenic phenotype transition and endoplasmic reticulum stress of AVICs,which can be inhibited by DFO and Fer-1.4.Silencing the endoplasmic reticulum stress-related protein ATF4 can inhibit the phenotypic transition of porcine AVICs osteogenesis induced by heme.Conclusion1.There is a large scale of neovascularization and erythrocyte infiltration discovered in the degenerative atherosclerotic aortic valve tissue,where the content of heme is higher than that in the normal aortic valve.2.Heme induces oxidative stress in porcine AVICs and induces osteogenic phenotypic transformation of AVICs.3.Heme induces endoplasmic reticulum stress in porcine AVICs by oxidative stress.4.Silencing the endoplasmic reticulum stress-related protein ATF4 can inhibit the osteogenic phenotypic transformation of AVICs induced by heme.