Role Of Wnt1/beta-catenin Signaling Pathway Regulating Smooth Muscle Cell Phenotype Transformation In The Development Of Intracranial Aneurysms

Intracranial aneurysms are cerebrovascular diseases that seriously threaten human health.The incidence of intracranial aneurysms is between 0.4% and 6% reported all over the world.An imaging examination based on hospital population in China found that the incidence of intracranial aneurysms is as high as 8.8%.Subarachnoid hemorrhage(SAH)caused by intracranial aneurysms accounts for 85% of all spontaneous SAH events.Even with the rapid development of medical treatment and the improvement of neurological intensive care,the mortality rate of SAH events caused by ruptured aneurysms reach to45%,and about 30% of the remaining survivors still exist serious disabilities.In clinic,the treatment strategy of intracranial aneurysms includes clipping and vascular intervention.However,no matter what treatment method is adopted,there are many problems,such as high cost,high risk,high recurrence rate,postoperative complications and poor prognosis.Therefore,exploring the mechanism of the occurrence and development of intracranial aneurysms is a key process to intervene the natural history of aneurysms and to find non-invasive therapeutic targets for aneurysms.The middle layer in cerebral artery is mainly composed of vascular smooth muscle cells(VSMCs).The steady state of the number,structure and function of VSMCs in physiological state is an important basis for the normal function of vascular wall.When VSMCs are stimulated by external stress or inflammatory factors,VSMCs transformed from mature and differentiated contractile cells to dedifferentiated and proinflammatory cells,also known as phenotypic transformation of VSMCs.At present,many studies have confirmed that phenotypic transformation of VSMCs is an important mechanism in the occurrence of intracranial aneurysms.Wnt1/beta-catenin signaling pathway plays an important role in the differentiation,migration and proliferation of VSMCs.The purpose of this study is to discuss the role of Wnt1/beta-catenin signaling pathway in regulating smooth muscle cell phenotype transformation during the development of intracranial aneurysms in vivo and in vitro.Part Ⅰ: Wnt1/beta-catenin signaling pathway regulates phenotypic transformation of VSMCs in intracranial aneurysms of SD ratsOBJECTIVE: To verify the phenotypic transformation of VSMCs and the activation of Wnt1/beta-catenin signaling pathway in SD rat intracranial aneurysm model.Methods: SD rats were divided into sham group,aneurysm model group and inhibitor intervention group.Rat aneurysm models were successfully constructed by ligating the common carotid artery in one side,the contralateral external carotid artery and pterygomandibular artery in SD rats.The sham group was not treated,while the inhibition group was injected with specific inhibitors of signal pathway.Rats were raised for 3 months.Aneurysm formation in anterior intracranial communicating complex was observed by electron microscopy,remodeling of vascular wall was observed by HE section,and expression of phenotypic transforming protein was observed by immunohistochemistry.Realtime-PCR and Western Blot were used to detect the expression of Wnt1 gene and its downstream WISP-1 gene,as well as the expression of matrix metalloproteinases(MMP-9).Results: 1.Scanning electron microscopy showed that compared with sham group,aneurysm formation rate in aneurysm model group increased significantly,aneurysm formation rate of signal pathway inhibitor group decreased significantly(p < 0.05);2.HE results showed that vascular remodeling phenomenon of model group was more significant than sham group,and vascular remodeling phenomenon of inhibitor group was less observant(p < 0.001);3.Immunohistochemistry showed VSMCs phenotype transformation in aneurysm model group.The expression of a-smooth muscle actin(a-SMA)decreased and the expression of a-SMA increased in the inhibitor group.4.RT-PCR and WB detection showed that the expression of Wnt1 gene(p < 0.01)and WISP-1 gene(p < 0.001)increased in the aneurysm model group.5.WB detection showed that the expression of MMP-9 protein increased in the aneurysm model group(p< 0.001).CONCLUSION: Intracranial aneurysm model in SD rats was successfully constructed.The phenotypic transformation of VSMCs was verified in the aneurysm model.Wnt1/beta-catenin signaling pathway was activated during the phenotypic transformation of VSMCs.The phenotypic transformation of VSMCs and aneurysm formation decreased after the expression of specific inhibitory signaling pathway.Part Ⅱ: Wnt1/beta-catenin signaling pathway regulates phenotypic transformation of VSMCs in human cerebrovascular smooth muscle cellsOBJECTIVE: To confirm the role of Wnt1/beta-catenin signaling pathway inphenotypic transformation of human brain VSMCs cultured in vitro.METHODS: Human brain VSMCs were cultured in vitro and stimulated by tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),platelet derived growth factor(PDGF)to induce phenotypic transformation of VSMCs.The cells were divided into control group,inflammatory factor stimulation group and inhibitor group.The expression of Wnt1 gene and its downstream WISP-1 gene and matrix metalloproteinases MMP-9were detected by rt-PCR and WB.The results were as follows: 1.TNF-α,a phenotypic transforming inflammatory stimulator,was successfully induced human brain VSMCs phenotypic transformion in vitro.Compared with the control group,rt-PCR and WB showed that the expression of VSMCs phenotypic transforming marker gene in TNF-α group was significantly reduced(p < 0.01),while that in inhibitor group was up-regulated(p < 0.05).2.RT-PCR detected expression of Wnt1 gene(p < 0.01)and WISP-1 gene(p < 0.001)in inflammatory stimulation group were increased.The expression of WISP-1 gene was increased in the inflammatory stimulation group,and decreased in the inhibitor group(p < 0.05),the same results were observed in WB.3.The expression of MMP-9 was increased in the inflammatory stimulation group(p < 0.01)and decreased in the inhibitor group(p < 0.05),which is also evidenced by WB.CONCLUSION: TNF-alpha can induce phenotypic transformation of VSMCs in human brain.Wnt1/beta-catenin signaling pathway is activated during phenotypic transformation,and the expression of inflammatory factors is increased.After inhibiting the expression of signal pathway,the phenotypic transformation of VSMCs decreased and the expression of inflammatory factors decreased accordingly.Summary: Phenotypic transformation of VSMCs in intracranial aneurysms has been confirmed by in vivo and in vitro experiments.This change may be mediated by Wnt1/beta-catenin signaling pathway.Inhibition of phenotypic transformation of VSMCs may bring new directions for the research and treatment of intracranial aneurysms.