| PART I Activation of hypoxia signal pathway is involved in the occurrence and development of aortic dissection Objective: By comparing the clinical aortic tissue and plasma samples of patients with aortic dissection(AD),patients with valvular disease and organ donors.To explore whether there is activation of hypoxia signal pathway in the occurrence and development of AD.Method: From March 2018 to June 2019,50 patients with AD were enrolled in the Department of Cardiovascular surgery,Renmin Hospital of Wuhan University.These patients were diagnosed as Stanford type An AD,on admission and underwent emergency aortic replacement within 48 hours of admission.The patients’ plasma and dissected aortic tissue were collected during the operation.As a control,20 patients with valvular disease and 10 heart transplant donors were included in the control group.Plasma samples were taken from patients with valvular disease when they were admitted to hospital as a normal control.Normal aortic tissue was taken from the donor heart during heart transplantation,and the basic clinical information of the patient was included in the comparative analysis.The above tissues were stained with Masson,Elastic Stain,TUNEL and reactive oxygen species(ROS),the expression of related proteins and hypoxia-related signal pathways were detected,and the level of mi R-199 b in plasma samples was detected.Results: Based on the analysis of the basic clinical information of the patients included in the study,it was found that the age of Stanford type An AD patients was higher than that of organ donors,and Stanford type An AD had obvious gender orientation,with a male proportion of 80.0%.The body mass index of Stanford type An aortic dissection patients was significantly higher than that of organ donors and valvular disease patients.Moreover,the proportion of Stanford type An AD patients complicated with hypertension was significantly higher than that of organ donors and valvular disease patients.Through the comparison of histological staining of aortic tissue between AD patients and organ donors,it was found that compared with organ donors,Masson staining analysis showed that the elastic fibers in the middle layer of aorta of AD patients were more broken and disarranged,and Elastic Stain staining analysis showed that the deposition of collagen fibers in the middle layer of aorta of AD patients increased.TUNEL staining analysis showed that the apoptosis of aortic middle tissue in patients with AD was significantly more than that in organ donors,and ROS staining analysis showed that the level of ROS in aortic middle tissue in AD patients was increased.Western Blot detection of proteins in aortic tissues of patients with AD and organ donors showed that compared with organ donors,the expression of matrix metalloproteinase(MMPs),osteopontin(OPN),BAX and HIF-1 α in aortic tissue of AD patients increased,while the expression of α-smooth muscle actin(α-SMA)and Bcl-2 decreased.At the same time,the plasma q RT-PCR analysis of patients with AD and valvular disease showed that the level of circulating mi R-199 b related to hypoxia in patients with AD was lower than that in patients with valvular disease.Conclusion: AD aortic tissue showed middle aortic degeneration,including increased degradation of extracellular matrix,increased apoptosis of aortic smooth muscle cells,increased transformation of aortic smooth muscle cells to synthetic phenotype,and activation of hypoxia-related signals in aortic tissue.PART II miR-199 b participates in hypoxia-induced aortic medial degeneration by targeted regulation of HIF-1αObjective: On the basis of the first part,the in vitro experiment was conducted to explore whether hypoxia can induce the aortic media degeneration(AMD).And whether mi R-199 b can participate in hypoxia-induced AMD through targeted regulation of HIF-1α.Method: In vitro,human aortic smooth muscle cells(HASMC),were cultured in threegas incubator to simulate hypoxia.HASMC were cultured in hypoxia for 0,4,8,12 and24 hours,respectively.The effects of hypoxia on HASMC were observed,and the expression of MMPs,phenotypic markers,proliferation and apoptosis were mainly detected.The lentivirus infection HASMC model with overexpression of mi R-199 b was constructed to verify whether overexpression of mi R-199 b could inhibit the expression of HIF-1 α,and then double luciferase assay was used to verify whether mi R-199 b targeted inhibition of HIF-1 α by amplifying the predicted target sequence.Results: Hypoxia stimulation of HASMC can increase the expression of HIF-1 α and increase the level of ROS,which is positively correlated with the time of hypoxia,not only down-regulating the level of mi R-199 b,but also negatively related to the time of hypoxia.No obvious change in stress state of HASMC was observed at 4 hours and 8hours after hypoxia stimulation of HASMC.Western Blot detection showed that there were no significant changes in the expression of matrix metalloproteinase-2(MMP-2),MMP-9,BAX,Bcl-2 and phenotypic markers.When the hypoxia time was prolonged to 12 hours and 24 hours,Western Blot detection showed that the expression of MMP-2,MMP-9,BAX and OPN increased significantly,while the expression of Bcl-2 and α-SMA decreased significantly.Flow cytometry showed that hypoxia increased the apoptosis of HASMC.With the prolongation of hypoxia time,the apoptosis of HASMC increased significantly,and hypoxia for 4 hours had no obvious inhibition on the proliferation of HASMC.When the hypoxia time reached 8,12 and 24 hours,hypoxia significantly inhibited the proliferation of HASMC.With the extension of hypoxia time,the inhibition of HASMC proliferation was more obvious.The construction of the model of HASMC infected by mi R-199 b overexpression lentivirus showed that overexpression of mi R-199 b could inhibit the expression of HIF-1 α protein,but did not significantly reduce the m RNA level of HIF-1α.At the same time,double luciferase assay also confirmed that mi R-199 b could inhibit the expression of HIF-1α by targeting m RNA binding to HIF-1α.Conclusion: Hypoxia stimulation has no significant effect on the state of HASMC,but the later stage of hypoxia stimulation can induce AMD changes in HASMC.Mi R-199 b can participate in hypoxia-induced AMD by targeting regulation of HIF-1α expression.PART III Effect of HIF-1α functional deficiency on hypoxia-induced aortic media degeneration Objective: On the basis of the first two parts,by specifically interfering with the expression of HIF-1α,to explore the role of HIF-1α in the response of HASMC to hypoxia stimulation,and the role of HIF-1α in 3-aminopropionitrile fumarate(BAPN)-induced AD.Method: RNAi lentivirus was used to infect HASMC to specifically interfere with the expression of HIF-1α.HASMC with specific HIF-1α interference was stimulated in hypoxia for 4 hours and 24 hours,respectively.The changes of MMPs expression,phenotypic markers,proliferation and apoptosis were observed and analyzed under the stimulation of different hypoxia time.The mouse model of aortic specific interference with HIF-1α was established by injecting RNAi lentivirus into the aorta of mice via tail vein,and then the mouse model of AD was induced by feeding mice with purified feed containing 0.25%BAPN to observe the effect of aortic specific interference HIF-1α on the occurrence and development of AD.Results: RNAi lentivirus infection could specifically reduce the protein expression level and m RNA level of HIF-1α in HASMC,but had no significant effect on ROS level.Four hours after hypoxia stimulation of HASMC,Western Blot assay showed that the expression of MMP-2,MMP-9,BAX and OPN in HIF-1α interfered cells was higher than that in normal cells,while the expression of α-SMA and Bcl-2 decreased.Flow cytometry showed that compared with hypoxia-stimulated normal cells for 4hours,hypoxia-stimulated HIF-1α-interfered cells increased apoptosis and inhibited proliferation.However,at 24 hours after hypoxia stimulation of HASMC,Western Blot detection showed that the expression of MMP-2,MMP-9,BAX and OPN in HIF-1α-interfered cells was lower than that in normal cells,while the expression of α-SMA and Bcl-2 was increased.Flow cytometry showed that compared with hypoxia-stimulated normal cells for 24 hours,hypoxia-stimulated HIF-1α-interfered cells reduced apoptosis and alleviated the inhibition of proliferation.In the in vivo experiment,it was found that 0.25%BAPN feeding could significantly induce the occurrence of AD in mice,and there was no significant difference in the incidence of AD between the mice with aortic specific HIF-1α interference and normal mice,but the incidence of AD rupture was significantly decreased,and aortic specific HIF-1 α interference could prolong the survival time of mice.Conclusion: After hypoxia stimulation for 4 hours,HIF-1α protects HASMC from hypoxia injury,and HIF-1α interference aggravates hypoxia injury.24 hours after hypoxia,overexpression of HIF-1α aggravates HASMC injury induced by hypoxia,while HIF-1α interference partially reverses hypoxia injury.Aortic specific interference with HIF-1 α can alleviate the pathological changes of AD induced by BAPN. |
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