The Effect Of Volume-Regulated Anion Channel Protein LRRC8A On Vascular Remodeling In Hypertension

The incidence of cardiovascular disease（CVD）is rising continuously due to the rapid development of China’s economy and the accelerating ageing of the population,which result in the incidence of cardiovascular disease has been the leading cause of total death of urban and rural residents in China.And most importantly,hypertension is the top incidence disease of all cardiovascular disease.Hypertension is a clinical syndrome characterized by increased systemic arterial blood pressure（systolic or diastolic blood pressure）,and it is also a major risk factor for cardiovascular and cerebrovascular diseases.Hypertension can cause various complications such as stroke,heart failure,atherosclerosis,dissecting aneurysm and renal failure,and vascular remodeling（VR）is an important pathological basis for the pathogenesis of these organ injury.Badly,vascular remodeling and hypertension are mutually causal,which aggravating the damage of tissues and organs and finally lead to the deterioration of the disease.Therefore,while actively controlling hypertension and further explore the pathological mechanism of the occurrence and development of vascular remodeling will provide a theoretical basis for finding new targets for intervention in vascular remodeling.And this is of important significance for the prevention and treatment of hypertensive complications.Vascular remodeling often refers to pathological vascular remodeling which is caused by abnormal vascular wall cell proliferation,apoptosis/necrosis,migration,and synthesis or degradation of extracellular matrix（ECM）.The normal structure and function of blood vessels are losing.This is a complex and dynamic process.This change is a very important common pathological process of atherosclerosis,aneurysm,restenosis after angioplasty,especially high blood pressure and other clinical cardiovascular diseases.Therefore,the prevention and treatment strategies for vascular remodeling have important clinical value.The vascular smooth muscle cell layer is in the media of the arterial blood vessels,it is the main functional structure of the vascular wall.The vascular smooth muscle cell layer is responsible for the regulation of vasoconstriction and relaxation and is the structural basis for the elasticity and integrity of blood vessels.Vascular smooth muscle cell has a high degree of plasticity.The significant dedifferentiation of vascular smooth muscle cell can occur,including proliferation,migration,and the transformation from a contractile phenotype to a synthetic secretory phenotype once under the stimulation of damaging factors such as hypertensive pressure.And a large amount of extracellular matrix is secreted by vascular smooth muscle cell to promote vessel wall structure undergoes disorderly changes,with centripetal thickening and narrowing.Therefore,it is possible to inhibit vascular remodeling by interfering the dedifferentiation of vascular smooth muscle cell.Hence,exploring the new mechanism of vascular smooth muscle cell dedifferentiation and finding new targets to reverse or delay the dedifferentiation of vascular smooth muscle cell is one of the hot issues that need to be resolved in the cardiovascular field.Ion channels have always been a hot spot in life sciences and drug research and development.Channels are the key"gateway"of cells,they can affect many important life processes and are reported closely relate to many human diseases.Among them,the volume regulated anion channel is widely distributed in the tissue cells of mammals and it has a variety of pathophysiological effects such as regulating cell volume,cell proliferation,differentiation,and migration.In 2014,researches in Science and Cell magazine discovered that the protein LRRC8A encoded by the lrrc8a gene is a key component of cell VRAC for the first time,which provides an important foundation for in-depth research.The research on LRRC8A protein and vascular remodeling is still in the preliminary exploration stage at present.Whether LRRC8A can regulate VSMC dedifferentiation,including proliferation,migration,especially contraction-synthetic phenotype transformation,is not yet clear.This is the purpose and innovation of this study.The aim（1）To observe the changes of vascular remodeling,vascular smooth muscle dedifferentiation and LRRC8A protein expression in the constructed animal model of hypertension.（2）To explore whether the intervention of LRRC8A regulates vascular remodeling and vascular smooth muscle dedifferentiation.（3）To explore the molecular regulation mechanism of LRRC8A on the dedifferentiation of vascular smooth muscle.The methods1.Preparation of knockout mice:LRRC8A^floxp+/+and Tagln-Cre⁺c57 mice were crossed to breed LRRC8A vascular smooth muscle conditional knockout mice that were identified for their genotypes.2.The experimental animals were divided into 4 groups:Control group（WT+NS）,model group（WT+AngⅡ）,LRRC8A conditional knockout group（LRRC8A c KO+AngⅡ）,LRRC8A conditional knockout normal control group（LRRC8A c KO+NS）.3.AngⅡadministration and blood pressure measurement:The osmotic micro-pumps were implanted under the skin of the experimental mice,and AngⅡwas continuously pumped at a dose of 1.5 mg/kg/d for 4 weeks.Animal blood pressure was measured by instrument dynamically.4.Observation of vascular pathology:HE staining to observe the pathological changes of mouse aortic blood vessels,and observation of the pathological changes of blood vessels under a microscope.5.Observation of vascular collagen fibers:Masson’s three-color staining method to observe and analyze the deposition of collagen fibers in the aorta of mice microscopically.6.Observation of vascular elastic fibers:EVG staining method to observe the changes of collagen elastic fibers in mouse aorta microscopically.7.Immunohistochemical stain:Observe the mouse aortic proliferation markers PCNA and Ki67,migration markers MMP2 and MMP9,contractile phenotype marker proteins calponin,a-SMA,SM22,SMMHC,and secretion phenotype markers vimentin and OPN expression.8.Real-time quantitative PCR（q PCR）:Quantitative detection of mouse aortic proliferation markers PCNA and Ki67,migration markers MMP2 and MMP9,contractile phenotype marker proteins calponin,a-SMA,SM22,SMMHC and secreted phenotype markers Expression of vimentin,OPN,collagenⅠ,collagenⅧm RNA.9.Immunofluorescence staining method:Smooth muscle cells were labeled with proliferation markers Ki67 and PCNA to observe their expression.Label calponin,a-SMA,SM22 markers to observe the migration of vascular smooth muscle in mouse abdominal aorta tissue.10.Western-Blot:detect the protein expression of arterial contraction phenotype markers calponin,a-SMA,SM22,SMMHC and secretion phenotype markers vimentin and OPN.11.Isolation and culture of primary vascular smooth muscle cells:Isolate rat aortic media routinely,cut into pieces,digest,collect cells,harvest vascular smooth muscle cells and culture them for use.12.Lentiviral packaging si RNA:Silencing the expression of LRRC8A in rat vascular smooth muscle cells.13.PDGF stimulates rat vascular smooth muscle cells:Construction of an in vitro vascular smooth muscle dedifferentiation model.14.Cell Counting Kit-8 was used to test vascular smooth muscle cell proliferation rate.15.Flow cytometry:Detection of smooth muscle cell proliferation cycle.16.Edu assay:to detect the number of S phase cells in the smooth muscle cell cycle.17.Scratch test and Transwell migration test:cell migration ability detection.18.Transcriptome sequence screening:Look for the differential expression genes in vascular smooth muscle cells which were knocked LRRC8A down and stimulated by PDGF,finally get enrich pathways.The results1.We successfully bred LRRC8A-specific knockout mice for vascular smooth muscle cells.2.AngⅡ-hypertensive mice and vascular remodeling models were successfully constructed.Comparing with control group,model group can find:（1）.Mice systolic blood pressure SBP,diastolic blood pressure DBP and mean arterial pressure increased significantly（P<0.05,n=6）;（2）HE staining showed vascular smooth muscle hyperplasia and hypertrophy,smooth muscle arrangement disorder,and vessel wall thickening;（3）The q PCR test showed that the expression of Ki-67 and PCNA m RNA in the aorta was significantly increased（P<0.05,n=6）,and immunohistochemical staining showed that the positive staining of Ki-67 and PCNA protein increased,and the number of positive cells for nuclear localization also increased;（4）The m RNA and protein levels of aortic migration-related proteins MMP2 and MMP9were significantly increased by q PCR（P<0.05,n=6）and Western-Blot assay;（5）The m RNA expressions of calponin,SM22a,SMMHC,and a-SMA m RNA of vascular smooth muscle contraction phenotype markers were significantly decreased（P<0.05,n=6）and the synthetic phenotypic markers vimentin,OPN,collagen I and collagenⅧm RNA were significantly increased（P<0.05,n=6）;（6）Western-Blot detection showed that the expression of the core markers of aortic smooth muscle contraction phenotype calponin,SM22a,and SMMHC protein decreased significantly（P<0.05,n=3）;the synthetic phenotypic proteins vimentin and OPN increased significantly（P<0.05,n=3）;3.AngⅡpromoted the high expression of LRRC8A in the vascular tissues of mice.Compared with the control group,the aortic LRRC8A m RNA expression was significantly increased（P<0.05,n=6）and the LRRC8A protein was positive deeper stained by immunohistochemistry in the AngⅡmodel group.4.The effect of knocking out LRRC8A on vascular remodeling in AngⅡhypertensive mice:（1）Compared with control group,the systolic blood pressure,diastolic blood pressure and mean arterial pressure continued to increase at 0,1,2,3,and 4 weeks in the AngⅡmodel group（P<0.05,n=6）;There was a significant drop in blood pressure in LRRC8A knocking out mice（P<0.05,n=6）;（2）Ultrasound of the abdominal aorta showed that the diameter of the aorta in the AngⅡmodel group was significantly larger than that in control group（P<0.05,n=6）;Knockout of LRRC8A reduced the diameter of the artery（P<0.05,n=3）;（3）HE staining showed that vascular smooth muscles are hypertrophy in the thoracic aorta of mice in the AngⅡmodel group were arranged disorderly,vessel wall was thickened and the dissected abdominal aortic aneurysm formed.Knocking out LRRC8A relieved the hypertrophy and disorder of the media and keep it intact and inhibit the formation of abdominal aortic dissection;（4）Masson staining showed that the deposition of collagen fibers in the media and vessel wall of the aorta was significantly increased in AngⅡmodel group.Knockout of LRRC8A inhibited the further deposition of collagen;（5）EVG staining showed that the elastic fibers of aorta in AngⅡgroup mice were thickened and disordered and the elastic fibers of the abdominal aorta were broken,forming a dissecting aneurysm.Knockout of LRRC8A inhibited elastic fiber breakage and maintained the integrity of the vessel media;（6）Immunohistochemistry and immunofluorescence staining showed that in media of the aorta,especially the abdominal aortic aneurysm,the proliferation antigen Ki67 and PCNA expression were increased,and the number of positive cell nuclei increased significantly in AngⅡgroup mice.The Ki67 and PCNA m RNA in the blood vessels in AngⅡgroup mice were significantly increased（P<0.05,n=6）,knocking out LRRC8A inhibited the protein expression of Ki67 and PCNA,and reduced the number of positive nucleus,simultaneously,down-regulated Ki67 and PCNA m RNA（P<0.05,n=6）.（7）Immunohistochemical staining showed that the expression of migration-related proteins MMP2 and MMP9 increased in AngⅡgroup aortic,q PCR found that its m RNA expression was up-regulated（P<0.05,n=6）;Immunofluorescence staining with specific markers of smooth muscle showed that the smooth muscle was breaking through the media to migrates to outside in a large amount in abdominal aortic aneurysm.Knockout of LRRC8A not only inhibited the up-regulation of MMP2 and MMP9 m RNA（P<0.05,n=6）,inhibited its protein expression,but also inhibited the migration of vascular smooth muscle;（8）The q PCR results showed that the m RNA expression of smooth muscle contraction phenotype markers calponin,SM22a,SMMHC,and a-SMA in aortic in AngⅡgroup was significantly decreased（P<0.05）;The synthetic phenotypic markers vimentin,OPN,collagen I,and collagenⅧm RNA expression were significantly increase（P<0.05,n=6）;Knockout of LRRC8A up-regulated the systolic phenotype marker m RNA and down-regulated synthetic phenotypic markers vimentin,OPN,collagen I,and collagenⅧm RNA（P<0.05,n=6）;Similarly Western-Blot detected that the expression of aortic proteins calponin,SM22a,and SMMHC in WT+AngⅡgroup were decreased（P<0.05,n=3）,and the expression of vimentin and OPN were increased（P<0.05,n=3）.Knocking out LRRC8A inhibited the decrease and increase of the expression of these marker proteins,respectively.5.Construction of vascular smooth muscle dedifferentiation model in vitro:（1）The effect of AngⅡon vascular smooth muscle cells:After AngⅡstimulated vascular smooth muscle cells,no cell proliferation changes were found,and no changes in expression of smooth muscle contraction phenotype molecules m RNA（P>0.05,n=6）;only the migration molecule MMP2 m RNA（P<0.05,n=6）but the MMP9 m RNA was up-regulated.Scratch experiment showed that vascular smooth muscle migrated.It suggested that AngⅡdid not induce an ideal vascular smooth muscle cell dedifferentiation model in vitro（2）The effect of PDGF on vascular smooth muscle cells:CCK-8 and Edu staining found that PDGF stimulation in vitro cause significant proliferation of vascular smooth muscle cells;Transwell migration assay and scratch experiment found that PDGF stimulation promotes the migration of vascular smooth muscle;q PCR found that PDGF stimulation can significantly decrease the contractile phenotypic marker m RNA of vascular smooth muscle（P<0.05,n=6）,and up-regulate the synthetic phenotypic marker collagenⅧm RNA（P<0.05,n=6）but for the expression of vimentin,OPN and collagenⅠm RNA.It is suggested that PDGF is an ideal inducer for the dedifferentiation model of vascular smooth muscle cells in vitro.6.The effect of lentiviral knockdown of LRRC8A on the dedifferentiation of vascular smooth muscle:（1）CCK-8 and flow cytometry showed that knocking down LRRC8A significantly inhibited PDGF induced cell proliferation（P<0.05,n=6）;（2）q PCR,scratch experiment and Transwell migration experiment found that knocking down LRRC8A inhibits the migration of vascular smooth muscle cells and inhibits the expression of MMP9 m RNA（P<0.05,n=6）;（3）q PCR found that knockdown of LRRC8A in vitro can significantly inhibit the PDGF-induced vascular smooth muscle down-regulation of the four phenotype markers m RNA and the up-regulation of the synthetic phenotype marker collagenⅧm RNA（P<0.05,n=6）;For the non-elevated synthetic phenotype marker vimentin,OPN and collagenⅠm RNA,knockdown of LRRC8A also has a down-regulation effect（P<0.05,n=6）;（4）Western-Blot detection found that knocking down LRRC8A not only inhibited the down-regulation of contractile phenotype proteins calponin,SM22a,and SMMHC（P<0.05,n=3）induced by PDGF,but also inhibited the normal expression of vimentin and OPN（P<0.05,n=3）.7.Screened the signal proteins related to vascular smooth muscle dedifferentiation:After transcriptome sequencing,the differential genes were screened out and enriched to get the top 30 signal pathway genes with obvious changes.The first two pathways RHO GTPases activate PAKs and IRAK2 mediated activation of TAK1 complex that related to vascular smooth muscle were selected.As the literature reported that they had a high correlation with vascular smooth muscle,which laying the foundation for the next in-depth study.The conclusion1.In the successful construction of hypertensive mouse model,we find that the expression of LRRC8A in remodeled blood vessels is increased.2.It is confirmed that hypertension induce obvious dedifferentiation changes in mouse vascular smooth muscle cells,including active proliferation,migration and the transition from contractile phenotype to secretory phenotype.3.For the first time,we find that the volume-regulating anion channel protein LRRC8A is involved in the regulation of hypertensive vascular remodeling.Knockout of vascular smooth muscle LRRC8A significantly reduce the vascular remodeling and blood pressure of hypertensive mice and significantly inhibit the process of hypertensive vascular remodeling.More importantly,the transition of vascular smooth muscle from contraction phenotype to synthetic secretion phenotype in the process of vascular remodeling in hypertension,as well as proliferation,migration and dedifferentiation process all are inhibited.This suggests that LRRC8A can be used as an intervention target for the treatment of hypertensive vascular remodeling.4.Screening out two pathways related to LRRC8A regulating the dedifferentiation of vascular smooth muscle and their differential genes,which providing clues for in-depth research.