Mechanism Research Of Polycystin-1 On Phenotype Switch Of Vascular Smooth Muscle Cells

Polycystin-1(PC-1),a transmembrane protein,belongs to Polycystin family,which is mainly expressed within epithelial cell and also found within other kinds of cells from different tissues including vascular smooth muscle,myocardium,skeletal muscle as well as endocrine cell.It has been shown that PC-1 involves various physiological activities such as interaction with lots of proteins,adherence among cells or between cells and matrix and participation in different signal pathways.PC-1 plays an important role during the development of normal kidney.However,closely association between ADPKD and PC-1 has been also established since almost 85% of ADPKD patients are suffered due to mutation of PKD-1 leading to abnormal expression and function of PC-1.Other extrarenal complications of ADPKD are also found and current study aims to explore the influence on cardiovascular system,of which,aortic dissection(AD)is a key element.AD is fatal disease whose mechanism is still unknown.Currently,VSMC of aortic wall is associated with dissection closely and reduction as well as altered phenotype of VSMC are observed among patients with AD.Altered phenotype of VSMC is considered as an important molecule pathogenesis of AD.Similar damage within aortic wall of early stage AD including intramural hematoma has been found in mouse model with knock out of PC-1 gene in previous study.It has been demonstrated that the expression of PC-1 within aortic wall decreased among patients with AD in our study.Therefore,based on closely association between altered phenotype of VSMC and AD,the expression levels of PC-1 in both damaged wall with AD and normal wall are collected and meanwhile,the relationship between reduced PC-1 and altered phenotype of VSMC as well as mechanism and target of PC-1 are also explored through cell experiment.Part I The Expression of PC-1 Among Patients with ADObjective: To observe the expression levels of PC-1 in both damaged wall with AD and normal wall.Methods: A total of 4 aortic walls from patients with AD were taken as AD group and equally,4 aortic walls from patients with CAD were also taken as control group without any differences in age and gender.Taken walls were fixed and stained by HE.Morphology of walls was recorded and the difference of collagens within walls was observed by Masson stain.The levels of PC-1 were tested by immunochemistry and Western Blot.Finally,the m RNAs of PC-1 were tested by RT-PCR.Results: All three layers of aortic wall were not clear.Isolated tunica media was relatively thinner while tunica adventitia was thicker.The chaos or even absence of smooth muscle cells within tunica media.Cystic necrosis was found(Arrow).Compared with control group,chaotic collagens increased and smooth muscle cells decreased significantly in AD group.Large intercellular deposited collagens were also found.PC-1 was shown as brown through immunochemistry.Brown positive particles were mainly distributed within cytoplasm of VSMC of tunica media.Compared with control group,the number of positive cells in AD decreased significantly.The m RNA of PC-1 decreased significantly in AD group(p<0.05)by RT-PCR.The expression of PC-1decreased significantly in AD group(p<0.05).Conclusion: The expression and m RNA of PC-1 within aortic wall were lower significantly among patients with AD.Part II Downregulation of PC-1 for Phenotype Switch of VSMCObjective: to study the effect of low PC1 expression on the phenotype of VSMC cells in vitro.Methods: Cultured human VSMC line was transfected by RNAi(LV-PC-kd group)and the effect was tested through RT-PCR and Western Blot.Later proliferation of cells in both two groups was tested by CCK-8.Changes of cell cycle in both two groups were analyzed through flow cytometry.Migration was tested by Transwell.Finally,contractibility biomarkers of VSMC as well as m RNA in both two groups were tested by Western Blot and RT-PCR.Results: Compared with control group,the expression as well as m RNA of PC-1 in LV-PC-kd group were decreased significantly based on both RT-PCR and Western Blot.After 72 h,compared with control group,the proliferation of LV-PC-kd group was higher significantly by CCK-8.The number of cells during G0/G1 stage was lower while that during G2/M stage was higher significantly(p<0.05)through flow cytometry.Compared with control group,the migration of VSMC and the number of migrated cells in LV-PC-kd group were higher significantly(p<0.05)by transwell(Fig 2-6).Contractibility biomarkers of VSMC including SM22α,ACTA2 and CNN1 in LV-PC-kd group were lower significantly(p<0.05)through Western Blot and RT-PCR.Conclusion: the expression of pc-1 can lead to the transformation of phenotype of VSMC.Conclusion: Downregulation of PC-1 promotes alteration of phenotype of human VSMC into synthesis type.Part three: The Signal Pathway of PC-1 on Altered Phenotype of VSMCObjective: To explore the signal pathway involving alteration of phenotype of VSMC by PC-1.Methods: Cultured human VSMC line was divided into three groups as control,LV-PC1-kd+DMSO and LV-PC1-kd+Comp.No any intervention was taken in the control group and VSMC cells were transfected by RNAi in the remaining two groups.The effect was tested by both RT-PCR and Western Blot.Also,Western Blot was used to analyze phosphorylation of proteins involving pathway including p38,JNK,PI3 K,ERK and MEK.Part of cells in LV-PC-kd group were handled by inhibitor of SCH772984.Proliferation of cells in all three groups was tested by CCK-8.Changes of cell cycle in all three groups were analyzed through flow cytometry.Finally,contractibility biomarkers of VSMC were tested by Western Blot.Results: Compared with control group,phosphorylation of p38,JNK and PI3 K was not changeable significantly,while,that of ERK and MEK was higher significantly(p<0.05).Both Myc and Cyclin D1 are downstream proteins of MEK/ERK and compared with control group,the expression levels of Myc and Cyclin D1 were higher significantly(p<0.05).After 72 h,compared with LV-PC1-kd+Comp group,the proliferation of LV-PC1-kd+DMSO group was higher significantly by CCK-8(p<0.05).The number of cells during G2/M stage was higher significantly(p<0.05)through flow cytometry in LV-PC1-kd+DMSO group.Contractibility biomarkers of VSMC including SM22α and ACTA2 in both LV-PC1-kd+DMSO and LV-PC1-kd+Comp groups were not significant(p>0.05)through Western Blot.Conclusion: MEK/ERK pathway is involved into the alteration of phenotype of human VSMC into synthesis type by downregulated PC-1.Part four: The Research of PC-1 Key Function SiteObjective: To confirm the key sites of PC-1 inducing alteration of phenotype of VSMC.Methods: Cultured human VSMC line was divided into four groups.No any intervention was taken in the control group.Lentivirus was used to induce high expression of intracellular part of PC-1 within VSMC in another group and VSMC cells were transfected by RNAi in the remaining two groups,one of which,S4166 of PC-1was mutated into alanine by site-directed mutagenesis.The expression of p-ERK,ERK,SM22,ACTA2,CNN1,cyclin D and myc in four groups was tested by Western Blot.Results: After site-directed mutagenesis,compared with PC-1 knock out group,the expression of p-ERK,ERK,SM22,ACTA2,CNN1,cyclin D and myc in mutation group was not significant(p>0.05),while,compared with control,that of SM22,ACTA2 and CNN1 was lower significantly(p<0.05),however,that of p-ERK,cyclin D and myc was higher significantly(p<0.05).Conclusion: The intracellular part of PC-1 plays an important role in inducing alteration of phenotype of VSMC by PC-1 and S4166 may be a critical functional target.The mutation of S4166 of PC-1 within VSMC activates ERK pathway leading to alteration of phenotype.