| Part 1 The regulation of VSMCs’ proliferation and apoptosis by IGF-1Objectives:Intracranial aneurysm(IA)is an abnormal structure caused by localized dilation of intracranial arterial lumen.The pathological changes are mainly characterized by rupture of vascular elastic layer,loss of middle layer and inflammatory cell infiltration.The main cause of loss of middle layer is the decrease of vascular smooth muscle cells(VSMCs).Previous studies suggested that the decrease of VSMCs was caused by apoptosis,while in vitro studies found that insulin-like growth factor-1(IGF-1)can inhibit the apoptosis of VSMCs induced by low density lipoprotein(LDL)through PI3K/Akt signaling pathway.In addition,studies on autophagy have found that the autophagy of VSMCs is inhibited after pretreatment with IGF-1.However,no studies have shown that IGF-1 can reduce the apoptosis and autophagy of VSMCs in the formation and progression of IAs.In this study,mice aortic endothelial cells(ECs)and VSMCs were co-cultured in vitro,and wall shear stress(WSS)was applied to ECs to simulate the abnormal hemodynamic environment during the formation of intracranial aneurysms.IGF-1overexpression and knockdown intervention was performed on VSMCs to explore the effects of IGF-1 on proliferation and apoptosis of VSMCs stimulated by abnormal WSS,and to explore the relationship between IGF-1 and autophagy of VSMCs.Methods:The primary ECs and VSMCs of mice aorta were extracted and subcultured in the outer and inner layers of the underside of Transwell chamber after 3-5 generations.Gradient WSS stimulation was applied to ECs of Transwell chamber by Flow chamber,and appropriate WSS and high WSS were explored as experimental conditions.VSMCs were transfected with IGF-1 overexpression(OE)and knock down(KD)adeno-associated virus(AAV),respectively,and then co-cultured with ECs.The co-culture model was divided into normal WSS group,high WSS group,high WSS+IGF-1 NC group(blank virus group),high WSS+IGF-1 OE group(overexpression group)and high WSS+IGF-1KD group(interference group).Flow cytometry was used to detect the difference of apoptosis of VSMCs between different groups.The effects of IGF-1 on VSMCs proliferation,apoptosis and autophagy were detected from m RNA and protein levels.Results:1.After WSS stimulation with different gradients was applied,the apoptosis rate of VSMCs was the lowest(14.13%)when WSS was 30 dyn/cm2,when WSS was further increased to 50 dyn/cm2 and 80 dyn/cm2,the apoptosis rate of VSMCs increased to30.94% and 43.56%,respectively.Therefore,30 dyn/cm2 was used as the normal WSS group and 50 dyn/cm2 was used as the high WSS group.The percentage of apoptotic cells in IGF-1 OE group(17.31%)was lower than that in IGF-1 NC group.The proportion of apoptotic cells in IGF-1 KD group(24.64%)was higher than that in OE group.2.The results of cell proliferation showed that the cell proliferation ability of the high WSS group was lower than that of the normal WSS group.The proliferation level of IGF-1 OE group increased(P<0.05).Moreover,the cell proliferation ability was further increased with time(P<0.05).3.Cell proliferation and apoptosis related gene and protein expression results:The expression level of Caspase3 gene in high WSS group was significantly higher than that in normal WSS group(P<0.001),the expression level of Caspase3 decreased in IGF-1OE group(P<0.001),Caspase3 expression in IGF-1 KD group was higher than that in IGF-1 OE group(P<0.001).The expression of proliferation related protein(Ki67)decreased significantly in high WSS group(P<0.001).The expression of Ki67 in IGF-1OE group was significantly increased(P<0.001),while Ki67 expression was decreased in IGF-1 KD group(P<0.001).The results of Caspase3 and Ki67 protein levels further confirmed the trend of q PCR results,and the differences were statistically significant(P<0.001).4.MRNA and protein expression of autophagy related proteins(LC3-II and Beclin-1): QPCR results showed that the expression of LC3-II and Beclin-1 was increased in the high WSS group(P<0.001),the expression of LC3-II and Beclin-1 was decreased in IGF-1 OE group(P<0.001).And the expression of LC3-II and Beclin-1 was increased in IGF-1 KD group(P<0.001).The protein expression of LC3-II and Beclin-1 also showed the same trend with statistical significance(P<0.001).Conclusion:The ECs and VSMCs co-cultured in vitro were given WSS to simulate the hemodynamic environment of IAs formation.When stimulated with high WSS,the proliferation ability of VSMCs decreased,while apoptosis and autophagy increased.IGF-1can promote the proliferation of VSMCs and inhibit their apoptosis and autophagy.Part II Effects of IGF-1 on the proliferation and apoptosis of VSMCs in mice intracranial aneurysmsObjective:In the first part of the study,we found that high WSS promoted apoptosis and autophagy of VSMCs,while inhibiting proliferation of VSMCs.IGF-1 can inhibit the apoptosis and autophagy of VSMCs induced by high WSS,and promote the proliferation of VSMCs.However,the effect of IGF-1 on VSMCs in intracranial aneurysm wall has not been verified in vivo.In this part of the study,the mouse intracranial aneurysm model was constructed and IGF-1 was overexpressed and interfered with AAV by tail vein injection to verify whether IGF-1 could affect the proliferation,apoptosis and autophagy of VSMCs in mouse intracranial aneurysm,thus affecting the progression of intracranial aneurysm.Method:Male C57BL/6 mice aged 8 weeks were randomly divided into 4 groups: Sham operation group(Sham group,n=25),aneurysm group(IA group,n=25),aneurysm +IGF-1overexpression group(OE group,n=25)and aneurysm +IGF-1 knockout group(KD group,n=25).Mice in the sham operation group were only exposed to subcutaneous tissues of neck and abdomen before suturing;In the IA group,the left common carotid artery and left kidney artery were ligated,and elastase was injected into the right anterior cross cistern of skull base.OE group and KD group were injected with IGF-1 adeno-associated virus(AAV)in caudal vein after the ligation of carotid and renal arteries,followed by intracranial stereotactic injection with elastase.All mice were fed for 4 weeks after intracranial injection of elastic protease,and then injected with bromophenol blue gelatin into the heart.The mice were sacrificed to obtain tissue samples of the Willis circle at the base of the skull.The IA formation rate of the Willis circle at the base of the skull was observed under a microscope.At the same time,the blood vessels of Willis circle at the base of the skull were collected to detect the VSMCs proliferation and the expression of apoptosis-related genes and proteins.Result:1.Intracranial aneurysm formation in mice: there was no intracranial aneurysm in the sham operation group and the tumor formation rate in the aneurysm group was 84%(21/25),the tumor formation rate in OE group was 20%(5/25),and the tumor formation rate in KD group was 92%(23/25).Significant difference was observed between groups(p<0.001).2.Compared with the sham operation group,the middle layer of the blood vessel wall of the Willis circle at the base of the skull in the aneurysm group was thickened and uneven in thickness,and the vascular remodeling phenomenon was obvious.In OE group,VSMCs proliferated,the blood vessel wall was slightly thickened and even in thickness.In KD group the blood vessel wall was uneven in thickness and the vascular remodeling was obvious.Immunohistochemical results showed that Caspase 3 expression was significantly increased in aneurysm group compared with sham group(P<0.0001),Caspase 3 expression was decreased in the OE group compared with the aneurysm group(P<0.0001),Caspase 3 expression was increased in KD group compared with OE group(P<0.0001).Tunel protein and Caspase 3 showed the same variation trend,and the difference was statistically significant(P<0.0001).Ki67 expression in aneurysm group was significantly lower than that in sham operation group(P<0.0001),while Ki67 expression was increased in OE group compared with aneurysm group(P<0.0001),ki67 expression in KD group was lower than that in aneurysm group(P<0.0001).3.Results: The expression of IGF-1 gene in aneurysm group was significantly higher than that in sham operation group(P<0.001),Ki67 gene expression increased compared with sham operation group(P<0.05),while Ki67 gene expression in OE group was higher than that in aneurysm group(P< 0.05),Ki67 expression in KD group was lower than that in OE group(P <0.05).The expression of Caspase 3 gene in aneurysm group was higher than that in sham operation group(P<0.001),and its expression in OE group was lower than that in aneurysm group(P<0.001),Caspase 3 expression in KD group was higher than that in OE group(P<0.001).Ki67 and Caspase3 protein expression showed the same trend with q PCR result,and the difference was significant.4.Results of autophagy related protein expression: The expression of autophagy related protein genes(Beclin-1 and LC3-II)in the aneurysm group was significantly higher than that in the sham group(P<0.01),Beclin-1and LC3-II gene expressions in OE group were significantly lower than those in aneurysm group(P<0.001),but the levels of both in KD group were higher than those in OE group(P< 0.001).The expression of BEClin-1 and LC3-II at protein level showed the same trend as gene expression,with statistical differences(P<0.0001).Conclusion:In mice intracranial aneurysms,VSMCs proliferation decreased,while apoptosis and autophagy increased.IGF-1 can inhibit the apoptosis and autophagy of VSMCs,promote the proliferation of VSMCs,and reduce the rate of aneurysm formation and alleviate vascular wall remodeling in mice.Part 3 Exploration of the transcriptional regulation mechanism anddownstream pathways of IGF-1 in VSMCs by FOXM1Objective:In previous studies,the effect of IGF-1 on apoptosis,autophagy inhibition and proliferation promotion of VSMCs induced by high WSS was confirmed by mouse aneurysm model and cell experiments.However,the regulatory factors and downstream signaling pathways of IGF-1 remain unclear.Previous studies in pulmonary artery models have found that FOXM1 is closely related to cell proliferation and division,and can target the expression level of IGF-1.In addition,IGF-1 also inhibits VSMCs apoptosis induced by oxidized low-density lipoprotein(ox LDL)through PI3K/Akt signaling pathway.However,it has not been concluded whether there is a similar regulatory mechanism in intracranial aneurysm.In this part of the study,we constructed an in vitro ECs-VSMCs co-culture model,applied WSS stimulation on ECs,overexpression and interference of FOXM1 on VSMCs,observed the effect of FOXM1 on proliferation and apoptosis of VSMCs,and explored the role of FOXM1 in IGF-1 transcription by immunoprecipitation technique.In addition,the expression of PI3 K and Akt was detected by overexpression and interference of IGF-1 on VSMCs,and the downstream pathways of IGF-1 regulating the proliferation and apoptosis of VSMCs in the process of aneurysm development were explored.Method:ECs-VSMCs co-culture model was carried out using Transwell,and WSS stimulation on ECs was applied by Flow Chamber.After transfection of VSMCs with FOXM1 overexpression and interference virus and co-culture with ECs,WSS stimulation was received.The co-cultured cells were divided into normal WSS group,high WSS group,high WSS+FOXM1 NC group(blank virus group),high WSS+FOXM1 OE group(FOXM1 overexpression group)and high WSS+FOXM1 KD group(FOXM1 interference group).Flow cytometry was used to detect the differences in apoptosis between VSMCs groups,RT-q PCR and WB were used to detect the effects of FOXM1 on proliferation and apoptosis of VSMCs under high WSS conditions,and the transcriptional regulation of FOXM1 on IGF-1 was analyzed by chromatin immunoprecipitation(Ch IP)technique.In addition,VSMCs transfected with IGF-1 overexpression and interference virus were co-cultured and stimulated with WSS,which were divided into normal WSS group,high WSS group,blank virus group,IGF-1 overexpression group and IGF-1 interference group.The expression of downstream IGF-1 signaling pathway was detected by RT-q PCR and WB.Result:1.Compared with the normal WSS group,the apoptosis rate of the high WSS group was higher(40.29% vs.10.99%),while the apoptosis rate of the FOXM1 overexpression group was lower than that of the blank virus group(11.66% vs.39.55%).2.Cell proliferation was weakened after WSS increased.The proliferation of VSMCs in FOXM1 overexpression group was higher than that in blank virus group(P<0.05),and cell proliferation was further enhanced with time(P<0.05).3.Results: Compared with the normal WSS group,the expression level of Caspase3 gene was significantly increased in the high WSS group(P<0.001),decreased in FOXM1 overexpression group compared with blank virus group(P<0.001),and increased in FOXM1 interference group compared with overexpression group(P<0.001).The expression of Ki67 gene in FOXM1 overexpression group was higher than that in blank virus group(P<0.001).Caspase3 and Ki67 protein expression also confirmed this result.4.Chromatin immunoprecipitation indicated that the promoter transcription level of IGF-1 increased after FOXM1 overexpression compared with that of blank virus group.After FOXM1 interference,the transcription level of IGF-1 promoter decreased.5.IGF-1 downstream pathway detection results: When WSS increased,PI3 K and AKT gene expression decreased(P<0.001);When IGF-1 was overexpressed,the expression levels of PI3 K and AKT were significantly increased(P<0.001);The expression of PI3 K and AKT in IGF-1 interference group was decreased compared with that in overexpression group(P<0.001).This result was further confirmed by PI3 K and AKT protein levels.Conclusion:FOXM1 positively regulates IGF-1,promotes the proliferation of VSMCs,and inhibits apoptosis.In addition,IGF-1 promotes proliferation and inhibits apoptosis of VSMCs through the PI3K/Akt pathway. |
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