Effect And Mechanism Of EETs On Thoracic Aortic Aneurysm In Marfan Syndrome Mice

Background :The mortality rate of thoracic aortic aneurysms in patients with Marfan syndrome(MFS)is extremely high.At present,open thoracic vascular replacement or endovascular repair is the main treatment of MFS thoracic aortic aneurysm,there is still a lack of early drug intervention.Studies have shown that mitochondrial dysfunction caused by mitochondrial biogenesis in vascular smooth muscle cells(VSMC)can cause phenotypic transformation of VSMC cells,promote the secretion of matrix metalloproteinases(MMP)by VSMC,and then promote the occurrence and development of MFS thoracic aortic aneurysm.Mitochondrial transcription factor A(TFAM)is a protein related to mitochondrial biogenesis and plays an important role in maintaining the normal function of mitochondria.Therefore,targeted treatment of VSMC mitochondrial dysfunction by changing the expression of TFAM is considered to be a potentially effective therapeutic strategy.It has been found that arachidonic acid metabolism is involved in the occurrence of thoracic aortic aneurysm in patients with MFS.Epoxyeicosatrienoic acids(EETs),as a downstream metabolite of arachidonic acid,has a strong cardiovascular protective effect.In addition,EETs can protect mitochondria by reducing the production of reactive oxygen species,reducing mitochondrial apoptosis and inhibiting mitochondrial dysfunction.More importantly,the level of hypoxia inducible factor-1α(HIF-1α)is significantly increased in patients with aortic aneurysm.HIF-1α can be translocated to the nucleus and downregulated the expression of mitochondrial metabolism-related target genes in the nucleus,while proline hydroxylase domain protein(PHD)can promote the degradation of HIF-1α,and EETs can regulate the activity of PHD.To sum up,we speculate that EETs in MFS thoracic aortic aneurysm may inhibit the transfer of HIF-1α from cytoplasm to nucleus,prevent the binding of HIF-1α to HRE on Tfam promoter in nucleus,reverse the inhibitory effect of HIF-1α on Tfam expression,and then restore mitochondrial biogenesis and function,and play a protective role in MFS thoracic aortic aneurysm.Objective:1.To clarify the involvement of reduced levels of EETs in the development of thoracic aortic aneurysms in MFS;2.To elucidate the mechanisms by which EETs improve thoracic aortic aneurysms in MFS;3.To explore the specific mechanisms by which EETs increase the expression of TFAM.Methods:1.The metabonomic differences between MFS thoracic aortic aneurysm individuals and healthy controls were compared:(1)The sera of patients with MFS thoracic aortic aneurysm and healthy controls were taken for metabonomic detection,and the contents of downstream metabolites were analyzed and compared;(2)MFS thoracic aortic aneurysm mice were constructed and phenotypic verification: Thoracic aortic diameter of MFS thoracic aortic aneurysm mice aged 8,12,14,16 and 20 weeks were detected by ultrasound.EVG staining was used to detect the disintegration of elastic fibers in the media of thoracic aorta of mice;(3)The sera of MFS and WT mice were taken for metabolites detection,and the contents of downstream metabolites were analyzed and compared;(4)QPCR and Western blot were used to detect the expression of proteins related to differential metabolites in the media of thoracic aorta of MFS and WT mice.2.To change the level of EETs in MFS thoracic aortic aneurysm mice and observe the effect of EETs on MFS thoracic aortic aneurysm.The specific methods are as follows:(1)To construct two models:(1)Liter the concentration of EETs in vivo: 8-week-old MFS thoracic aortic aneurysm mice were intragastrically administered with 10mg/kg/d TPPU.After 12 weeks of intervention,samples were taken at 20 weeks old.(2)Model of reducing the concentration of EETs in vivo: adenoassociated virus AAV9-s EH,which overexpressed s EH,was injected into the tail vein of 4-week-old MFS mice with thoracic aortic aneurysm to reduce the concentration of EETs in vivo.After the completion of the modeling,the following tests were performed:(2)The concentration of EETs in each group was detected by liquid chromatography-mass spectrometry,and the expression level of enzyme s EH that degraded EETs was detected by qPCR and Western blot.(3)The phenotype of mice in each group was detected by ultrasound:(1)The diameter of thoracic aorta was detected by ultrasound;(2)The vascular morphology was observed by HE staining;(3)The collagen deposition of thoracic aorta was detected by Masson staining;(4)The disintegration of elastic fibers in intima of thoracic aorta was detected by EVG staining.(5)QPCR and Western blot were used to detect the expression level of genes related to VSMC phenotypic transformation in each group;(6)Elisa method was used to detect the expression level of MMP2 and MMP9 in plasma;(7)Tail cuff method was used to detect blood pressure in each group;(4)RNA sequencing preliminarily explored the mechanism of EETs improving MFS thoracic aortic aneurysm:(1)RNA sequencing detected the gene expression level of MFS thoracic aortic tumor mice and increased EETs concentration group,and analyzed the enrichment of different genes;(2)The morphology and quantity of mitochondria in media VSMC of mouse thoracic aorta were observed by transmission electron microscope;(3)The differential genes related to mitochondrial metabolism were screened by qPCR;(4)The corresponding protein levels were verified by Western blot,immunofluorescence and immunohistochemistry.3.Cell experiments explore the specific mechanism of EETs increasing TFAM expression:(1)Sh Fbn1 lentivirus knocks down the Fbn1 gene of mouse aortic VSMC to construct a cell model;(2)CCK8 method to determine the optimal conditions for TPPU intervention in cells;(3)TPPU interferes with Fbn1 knockout mouse aortic VSMC to increase cellular EETs level,and the following detection:(1)QPCR and Western blot to detect the expression level of VSMC phenotypic transformation related genes in each group;(2)QPCR and Western blot were used to detect the expression of TFAM and HIF-1α;(3)Immunofluorescence to detect the location of HIF-1α expression in cells;(4)Bioinformatics to predict mitochondrial production;(5)The expression level of PHD,an enzyme that degrades HIF-1α,was detected.Results:1.Compared with healthy controls,the metabolic pathway of arachidonic acid in patients with MFS thoracic aortic aneurysm was disordered,and the level of EETs,a downstream metabolite of arachidonic acid,was most significantly down-regulated(P < 0.05).The model of MFS thoracic aortic aneurysm was successfully established: compared with WT mice of the same age,the thoracic aorta of MFS mice dilated mainly in the root of thoracic aorta and ascending aorta.The diameter of thoracic aorta dilated progressively with the increase of age,and there was significant rupture and disintegration of elastic fibers in the media of thoracic aorta in MFS mice compared with WT mice(all P <0.05).Compared with WT mice,the metabolic pathway of arachidonic acid was significantly disordered in MFS mice,especially in EETs.In addition,the expression level of s EH enzyme in thoracic aorta of MFS mice was significantly higher than that of WT mice(all P < 0.05).2.EETs improved the biogenesis of mitochondria and improved MFS thoracic aortic aneurysm:(1)Compared with the same-week-old WT mice,20-week-old MFS thoracic aortic aneurysm mice significantly decreased the level of EETs,significantly dilated thoracic aorta,increased collagen deposition in media tissue,significantly broken elastic fibers,VSMC phenotype changed from contractile type to synthetic type,and the expression of MMP2 and MMP9 increased.After increasing the level of EETs in MFS mice with thoracic aortic aneurysm,the diameter of thoracic aorta decreased,the collagen deposition and elastic fiber rupture in media tissue improved,the phenotypic transformation of VSMC was reversed,and the expression levels of MMP2 and MMP9 decreased(all P < 0.05).(2)The RNA sequencing suggested that the genes related to mitochondrial function in medial tissue of thoracic aorta were changed after increasing EETs level in mice with MFS thoracic aortic aneurysm.Transmission electron microscope images showed that compared with WT mice,the number of mitochondria in VSMC of thoracic aorta media tissue of MFS mice was swollen and decreased,and the morphological number of mitochondria in mice was improved after increasing the level of EETs(all P < 0.05).The results of qPCR and Western blot showed that compared with WT mice,the expression of TFAM was down-regulated and the expression of HIF-1α was up-regulated in the media of thoracic aorta of MFS mice,while the expression of TFAM was up-regulated and the expression of HIF-1αwas down-regulated after increasing EETs level(all P < 0.05).3.The mechanism of EETs increasing TFAM expression by promoting HIF-1α degradation:(1)The mouse aortic VSMC Fbn1 gene knockout model was successfully constructed;(2)The optimal TPPU concentration of interfering cells was determined by CCK8 method;(3)The results of qPCR and Western blot showed that the expression of VSMC synthesis phenotypic markers in Fbn1 knockdown cells was higher than that in Ctrl group.After increasing the level of EETs,VSMC changed from synthetic type to contractile type(all P < 0.05).(4)The results of qPCR and Western blot showed that compared with Ctrl group,the TFAM level of knockdown Fbn1 gene cells decreased,the expression of HIF-1α increased,and the level of TFAM increased and the expression of HIF-1α decreased after increasing EETs level(all P < 0.05).(5)Immunofluorescence detection of HIF-1α showed that the expression level of HIF-1α in knockdown Fbn1 gene cells was higher than that in Ctrl group,and partly located in the nucleus,while the expression level of HIF-1α decreased after increasing the level of EETs,and the part located in the nucleus decreased(all P < 0.05).(6)After biological prediction of HIF-1α and Tfam promoter,it was found that HIF-1 α could bind to Tfam promoter.(7)The enzyme PHD2 related to HIF-1α degradation was detected by qPCR.The results showed that the PHD2 level of knockdown Fbn1 genomic cells was lower than that of Ctrl group,and the expression of PHD2 was up-regulated after increasing the level of EETs.(all P < 0.05).Conclusion:1.The decrease of EETs level is involved in the occurrence and development of thoracic aortic aneurysm in MFS.2.EETs can improve MFS thoracic aortic aneurysm by increasing the level of TFAM and decreasing the level of HIF-1α to enhance the mitochondrial biogenesis of VSMC.3.The mechanism of EETs increasing TFAM expression is to increase PHD2 activity,inhibit HIF-1α nuclear translocation and promote TFAM expression.Figures:45;tables:12;references:112.