The Role Of Calcium-activated Chloride Channel ANO1 In Phenotypic Transformation Of Vascular Smooth Muscle Cells In Spontaneously Hypertensive Rats

ObjectiveThe role of calcium activated chloride channel ANO1 in phenotypic transformation of vascular smooth muscle cells（VSMCs）in spontaneously hypertensive rats,which was observed by using spontaneously hypertensive rats as a animal model and primary cultured vascular smooth muscle cells（VSMC）as a cell model.By observing the relationship between calcium activated chloride channel（ANO1）and phenotypic transformation of VSMCs（Vascular Smooth Muscle Cells）,and make a further study about the role of ANO1in phenotypic transformation of VSMCs in spontaneously hypertensive rats and the possible mechanism involved,which can provide a new idea and target for the prevention and treatment of clinical essential hypertension.Method1.Animal experimentSpontaneous hypertensive rats（SHRs）of male at 6-8 weeks with relatively stable blood pressure after blood pressure measurement were selected as experimental group and Wistar-Kyoto（WKY）rats as control group.The expression of ANO1,SM-22,SM-α-actin,PCNA and IL-1βin thoracic aorta was detected by Western blot.2.Cell experimentThe primary aortic smooth muscle cells（VSMCs）were cultured by tissue patch method.Cells of passage which were used in the experiment of 5-8th generation primary aortic smooth muscle cells.The morphological differences between VSMCs derived SHRs and VSMCs derived WKY were observed by inverted microscope.（1）Immunofluorescence:The expression of SM-α-actin protein in vascular smooth muscle cells of WKY and SHR was observed through immunofluorescence by using the fifth generation of vascular smooth muscle cells.（2）Primary vascular smooth muscle cells（VSMCs）were cultured by the method of thoracic aortic tissue patch.The fifth generation cells were selected to detect ANO1,SM-22,SM-α-actin,PCNA,IL-1β,Trk B and migration by Western blot,which reflected the phenotypic transformation of vascular smooth muscle cells（VSMCs）.（3）The experiments was divided into four groups:WKY,WKY+A01,SHR,SHR+A01.After inhibiting the activity of ANO1（T16Ainh-A01）,ANO1,SM-22,SM-α-actin,PCNA and IL-1βwere detected by Western blot reflecting the index of phenotypic transformation of vascular smooth muscle cells（VSMCs）to observe the effect of calcium activated chloride channel ANO1 on phenotypic transformation and migration of vascular smooth muscle cells.（4）The experiments was divided into six groups:WKY,WKY+Si-NC,WKY+Si-ANO1,SHR,SHR+Si-NC,SHR+Si-ANO1.The changes of ANO1,SM-22,SM-α-actin,PCNA,IL-1βreflecting phenotypic transformation of vascular smooth muscle cells（VSMCs）and their effects on cell migration were observed.（5）The experiments was divided into four groups:WKY,WKY+A01,SHR,SHR+A01.After inhibiting the activity of ANO1（T16Ainh-A01）,ERK1/2,P-ERK1/2 were used to study the possible mechanism of calcium-activated chloride channel ANO1 on the phenotypic transformation of VSMCs.Result1.Morphological differences between SHR derived vascular smooth muscle cells and Wistar-Kyoto（WKY）derived vascular smooth muscle cells.Primary vascular smooth muscle cells（VSMCs）were cultured from thoracic aorta of WKY rats and SHR rats by tissue patch method.Compared with Wistar-Kyoto（WKY）-derived VSMCs,SHR-derived VSMCs were hypertrophic.The quantity of SM-α-actin was higher than that of VSMCs derived WKY,and the fluorescence intensity of SM-α-actin in cytoplasm was significantly decreased.And VSMCs derived WKY was fusiform.2.Changes of protein expression level in aorta.Compared with Wistar-Kyoto（WKY）rats,the expression of SM-22,SM-α-actin protein in（SHRs）aorta of spontaneously hypertensive rats was significantly decreased（P<0.05）.The expression of Trk B protein increased significantly（P<0.05）.3.Changes of phenotypic transformation of primary cultured vascular smooth muscle cells.Compared with the vascular smooth muscle cells of Wistar-Kyoto（WKY）rats,the expression of SM-22,SM-α-actin protein in（SHRs）vascular smooth muscle cells of spontaneously hypertensive rats was significantly decreased（P<0.05）.The expression of Trk B protein was significantly increased（P<0.05）,which was consistent with the change of aortic tissue.4.Effect of calcium-activated chloride channel ANO1 on phenotypic transformation of VSMCs.T16A_inh-A01,an inhibitor of calcium activated chloride channel（ANO1）,could significantly increase the expression of SM-22,SM-α-actin protein and inhibit the expression of ANO1,PCNA and IL-1βprotein in vascular smooth muscle cells of spontaneously hypertensive rats compared with vascular smooth muscle cells of Wistar-Kyoto（WKY）rats（P<0.05）.5.The possible mechanism of calcium-activated chloride channel ANO1 affecting phenotypic transformation of VSMCs was investigated by detecting ERK1/2.Calcium-activated chloride channel ANO1 promotes phenotypic transformation of vascular smooth muscle cells in spontaneously hypertensive rats,which may be mediated by ERK1/2 pathway.Conclusion1.Calcium-activated chloride channel ANO1 may promote the transformation of vascular smooth muscle cells from contractility to proliferation in spontaneously hypertensive rats.2.The effect of calcium-activated chloride channel ANO1 on phenotypic transformation of vascular smooth muscle cells in spontaneously hypertensive rats may be mediated by ERK1/2 pathway.