Role Of Piezo1 Ion Channel In The Formation Of Intracranial Aneurysms Induced By Hemodynamics

Part 1:Effect of Piezo1 ion channel of mouse endothelial cells on the phenotype of vascular smooth muscle cellsObjective:To prove the response of the mechanically-sensitive ion channel Piezo1of endothelial cells（ECs）to wall shear stress（WSS）.And to investigate the effect of EC Piezo1 on the phenotype of co-cultured vascular smooth muscle cells（VSMCs）in vitro.Methods:Mice primary ECs and VSMCs were subcultured for 3-5 generations.For co-culture experiments,ECs were seeded in the bottom layer of transwell and co-cultured with VSMCs seeded in the inner layer.Using a parallel plate Flow chamber,the WSS stimulation was applied on the ECs layer.The intracellular free Ca²⁺fluorescence intensity was measured by flow cytometry to reflect the sensing of Piezo1 channel to Yoda1（agonist）,WSS stimulation and Gs MTx4（inhibitor）in the standard medium and medium with Ca Cl_2,respectively.By comparing the Ca²⁺influx in ECs treated with Gs MTx4,the inhibition of Piezo1-Cas9 lentivirus transfection on Piezo1 was verified.The expression of VSMC phenotype-related protein（α-SMA,SM22α,OPN and MMP2）were detected under normal WSS and high WSS.The effect of EC Piezo1 inhibition on VSMC phenotype was explored under the above conditions.Moreover,it was further detected under static conditions using Piezo1 agonist Yoda1.Finally,the expression of Piezo1 was explored under both normal WSS and high WSS.Results:1.The intracellular Ca²⁺fluorescence intensity was increased under Yoda1（P<0.0001）and WSS（15dyn/cm2）stimulation（P<0.01）compared to the static state.However,the intracellular Ca²⁺fluorescence intensity was not significantly changed when WSS increased from 15dyn/cm2to 30dyn/cm2（P=0.1662）.On this basis,the fluorescence intensity decreased when Gs MTx4 was applied（P<0.01）.In contrast,the intracellular fluorescence intensity of Ca²⁺increased in each group when Ca Cl₂was added to the medium.Under this condition,the intracellular Ca²⁺fluorescence intensity significantly increased when WSS increased from 15dyn/cm2to 30dyn/cm2（P<0.01）.The fluorescence intensity of Ca²⁺decreased significantly when EC Piezo1 was knocked down under the stimulation of30dyn/cm2WSS（P<0.0001）.And the fluorescence intensity of Ca²⁺after treatment with Gs MTx4（P<0.001）was lower than that of Piezo1 knockdown.2.The m RNA level of VSMC contraction marker geneα-SMA（P<0.05）and SM22α（P<0.0001）were upregulated under normal WSS stimulation for 24h compared to the static state.And the expression of proteins also increased（P<0.01）correspondingly.While synthetic marker gene OPN was upregulated both in m RNA（P<0.0001）and protein（P<0.01）levels.When EC Piezo1 was inhibited,the m RNA level ofα-SMA（P<0.05）and SM22α（P<0.001）was downregulated,and the expression ofα-SMA and SM22αprotein（P<0.05）was also reduced.In contrast,the m RNA expression（P<0.001）and protein expression of OPN were increased（P<0.01）.3.Compared with normal WSS,high WSS stimulation led to the downregulation of m RNA ofα-SMA（P<0.05）and SM22α（P<0.0001）and also the expression of the proteins（P<0.05）.When EC Piezo1 was inhibited under high WSS stimulation,the m RNA expression ofα-SMA（P<0.001）and SM22α（P<0.0001）was downregulated.Similarly,the protein expression ofα-SMA（P<0.01）and SM22α（P<0.05）was decreased.4.Under the static condition,the m RNA expression ofα-SMA（P<0.0001）and SM22α（P<0.0001）was upregulated when EC was stimulated with Yoda1.Meanwhile,the protein expression ofα-SMA（P<0.0001）and SM22α（P<0.0001）was increased.On this basis,knocking down EC Piezo1 inhibited the m RNA expression ofα-SMA（P<0.0001）and SM22α（P<0.0001）while promoting the m RNA expression of MMP2（P<0.05）.In this process,the protein expression ofα-SMA（P<0.001）and SM22α（P<0.001）was also increased.5.Compared with normal WSS,high WSS stimulation inhibited both the m RNA（P<0.05）and protein expression（P<0.05）of EC Piezo1.Conclusion:EC Piezo1 channels open by sensing WSS stimulation,leading to the extracellular Ca²⁺influx.The degree of Ca²⁺influx positively correlates with WSS stimulation intensity.Normal WSS stimulation maintains the VSMC phenotype,but high WSS leads to VSMC phenotypic transformation.In this process,the expression of EC Piezo1is decreased,showing negative feedback to the rise of WSS.Inhibition of EC Piezo1 channel promotes VSMC phenotypic transformation.Part 2:Effects of endothelial cell Piezo1 knockout on the mouse intracranial aneurysms and the vascular smooth muscle cells phenotypic modulationObjective:To study the effect of EC Piezo1 on VSMC phenotype,vascular remodeling and the development of intracranial aneurysm（IA）by the IA mouse model.Methods:The EC Piezo1-specific knockout mice were obtained by injecting AAV2/BR1-Tie2-Cre into the tail vein of 6-8-week-old male C57BL/6 Piezo1^flox/flox mice as KO group（n=24）.Then 36 male C57BL/6 wild-type mice aged 6-8 weeks were randomly divided into the sham group（n=18）and IA group（n=18）.All mice were raised for 4 weeks until immunofluorescence verified the EC Piezo1-specific knockout in KO group mice.After exposing the common carotid artery and renal artery in the sham group,the skin incision was sutured.In contrast,the IA and KO group mice were generated by ligating the left common carotid artery and renal artery combined with the stereotactic elastase injection in the anterior cruciate pool and high saline diet.Three weeks after the operation,the cerebral vessels of mice in each group were perfused with bromophenol blue gelatin.The vascular morphology of the Willis circle at the skull base and the aneurysm formation rate of mice in each group were observed.The mice brain tissue was obtained for HE and immunohistochemical staining to observe vascular remodeling and the expression of VSMC phenotype-related proteins（α-SMA,SM22α,OPN and MMP2）.In addition,the expression of the above proteins was further detected by rt-q PCR and WB in the Willis circle vessels.Results:1.The IA formation rate was 0（0/18）in the sham group,44.45%（8/18）in the IA and 79.17%（19/24）in the KO group,respectively.And the IA formation rate of the KO group was higher than that of the IA group（P<0.05）.HE staining showed that the inner elastic layer of the Willis circle of the IA group was broken and disappeared,and the structure of the middle layer was disordered compared to the sham group.However,the Willis circle of the KO group showed even more disordered in the middle layer,uneven blood vessel thickness,and irregular lumen with local protrusion.Immunohistochemical staining showed that the expression of contractive marker proteinα-SMA（P<0.01）and SM22α（P<0.05）of the Willis circle was higher in the IA group than that in the KO group.2.Compared with the sham group,the m RNA level ofα-SMA（P<0.0001）and SM22α（P<0.01）in the Willis circle was decreased in the IA group,and the protein level ofα-SMA（P<0.01）and SM22α（P<0.0001）was also decreased.The expression ofα-SMA（P<0.001）and SM22α（P<0.05）in the m RNA level of the KO group was lower than that in the IA group.In addition,the protein expression of SM22αin the KO group was lower than in the IA group（P<0.05）.However,the protein expression of OPN（P<0.001）and MMP2（P<0.01）was higher in the KO group than that in the IA group.Conclusion:Knocking out of Piezo1 in EC promotes the VSMC phenotypic transformation in mouse IA models,which also aggravates vascular remodeling and promotes the formation of IA.Part 3:Differential gene expression profiles and intercellular communication changes after inhibition of Piezo1 in ECObjective:Our previous research has confirmed that inhibition of EC Piezo1promotes VSMC phenotypic transformation in vitro and in vivo.However,the specific mechanism of VSMC phenotypic regulation is still unclear.In this study,we explored the differential gene expression profiles and intercellular communication changes through RNA-seq,aiming to lay a foundation for subsequent research.Methods:Piezo1-inhibited ECs and control ECs were co-cultured with VSMCs in the transwell chamber,respectively.The Flow chamber device applied high WSS stimulation to the EC layer for 12h.After collecting samples of ECs and VSMCs,the full-length single-cell RNA-seq library was prepared using Smart-seq2 protocol,and then the samples were sequenced through Illumina Nova Seq 6000 sequencing platform.Next,the quality control and comparison of sequencing data were conducted,and the expression of differential genes was analyzed in ECs and VSMCs.Then,annotation and functional enrichment analysis of differential expression genes were conducted by GO and KEGG analysis.In the final,Cellphone DB v2.0 was used to analyze intercellular communication between ECs and VSMCs.Results:1.After EC Piezo1 was inhibited,4690 genes were significantly differential expressed in ECs,3517 of which were upregulated and 1173 were downregulated.Notably,the expression of PDGFB was significantly upregulated（FC=8.65,P=0.0189）in ECs.In addition,901 genes were significantly differential expressed in VSMC,756 of which were upregulated and 145 were downregulated.2.GO enrichment analysis showed that EC biological processes,such as ATP metabolism,oxidative phosphorylation,positive regulation of ion transport,cell adhesion,and Wnt signal pathway of ECs,were affected by the inhibition of EC Piezo1.At the same time,the VSMC biological processes,such as ion channel activity,glucose metabolism,smooth muscle cell differentiation,migration,and actin cytoskeleton reorganization and regulation of VSMCs,were affected by the inhibition of EC Piezo1.KEGG enrichment analysis showed that the differentially expressed genes of ECs were mainly enriched in oxidative phosphorylation,adhesive plaque,glycolysis,fluid shear WSS and atherosclerosis and ECM receptor interaction after inhibition of EC Piezo1.While the differentially expressed genes of VSMCs were mainly concentrated in gap junction,hypertrophic cardiomyopathy,calcium signaling pathway,insulin regulation and amino acid metabolism.3.Intercellular communication analysis showed that receptor/ligand pairs between ECs and VSMCs increased from 235 to 618 after EC Piezo1 was inhibited.Seventy-seven receptor/ligand pairs were significantly differential expressed,70 of which were upregulated and 7 of which were downregulated.Among the 77 receptor/ligand pairs,platelet-derived growth factor（PDGF）,transforming growth factorβ（TGFβ）,the Notch pathway and the Wnt pathway were related to VSMC phenotypic regulation.Conclusion:The intercellular communication between ECs and VSMCs is enhanced,and some receptor/ligand pairs related to VSMC phenotypic regulation are upregulated when EC Piezo1 is inhibited.Among the receptor/ligand pairs,PDGFB＿PDGFRβprobably promotes VSMC phenotypic transformation in this process.Part 4:EC Piezo1 regulates VSMCs phenotype by affecting PDGFBB expressionObjective:To Verify whether the PDGFB＿PDGFRβreceptor/ligand pair regulates VSMC phenotype mediated by EC Piezo1 in vitro study.And preliminarily explore the regulatory mechanism of Piezo1 on PDGFBB.Methods:Piezo1-inhibited ECs and control ECs were co-cultured with VSMCs in the transwell chamber,respectively.High WSS stimulation was applied to the EC layer for12h by the Flow chamber device.The concentration of PDGFBB in the medium of ECs’and VSMCs’was detected by ELISA.The exogenous PDGFBB was used to stimulate mono-cultured VSMC in static states,and the expression ofα-SMA,SM22α,OPN and MMP2 was detected by rt-q PCR and WB.Then,imatinib mesylate was used to antagonize the PDGFRβof VSMCs based on the co-cultured and high WSS stimulation system,followed by the detection of VSMC phenotype-related proteins expression.Finally,after EC Piezo1 was inhibited under high WSS stimulation,the free Ca²⁺concentration of ECs was altered by adding Ca Cl₂（Ca Cl₂ group）and calcium chelator BAPTA-AM（BAPTA-AM group）into the ECs medium.Both concentrations of PDGFBB and expression of VSMC phenotype-related proteins were detected.Results:1.Compared with the control group,the concentration of PDGFBB in the EC medium and VSMC medium increased significantly（P<0.05）after the EC Piezo1channel was inhibited.2.Stimulation of mono-cultured VSMC with exogenous PDGFBB significantly increased the m RNA level ofα-SMA（P<0.05）and SM22α（P<0.01）,but decreased the m RNA level of OPN（P<0.001）and MMP2（P<0.01）.Similarly,the exogenous PDGFBB promoted the expression ofα-SMA and SM22αprotein（P<0.05）but inhibited the expression of OPN and MMP2 protein（P<0.05）.3.After pretreatment VSMCs with imatinib mesylate based on the EC Piezo1 inhibition,the m RNA expression ofα-SMA（P<0.01）and SM22α（P<0.05）was upregulated,while OPN（P<0.05）and MMP2（P<0.001）was downregulated.Meanwhile,the protein level ofα-SMA（P<0.05）and SM22α（P<0.05）was increased,while OPN（P<0.01）and MMP2（P<0.05）were decreased.4.Compared with the control group,the concentration of PDGFBB in the EC medium（P<0.05）and VSMC medium（P<0.05）was increased in the BAPTA-AM group.However,the concentration of PDGFBB in the EC medium（P<0.01）and VSMC medium（P<0.01）was decreased in the Ca Cl₂ group compared with those in the BAPTA-AM group.5.Compared with the control group,the m RNA expression ofα-SMA（P<0.05）and SM22α（P<0.01）was downregulated,but OPN（P<0.05）and MMP2（P<0.05）were upregulated in the BAPTA-AM group.Meanwhile,the protein expression ofα-SMA（P<0.01）was inhibited,but the OPN（P<0.01）and MMP2（P<0.05）were increased.As to the Ca Cl₂ group,the m RNA expression ofα-SMA（P<0.001）and SM22α（P<0.01）was upregulated,while OPN（P<0.001）and MMP2（P<0.001）were downregulated compared to the BAPTA-AM group.Similarly,the protein expression ofα-SMA（P<0.01）and SM22α（P<0.05）was increased,while OPN（P<0.01）and MMP2（P<0.001）were decreased.Conclusion:Inhibiting the EC Piezo1 channel increases PDGFBB expression,leading to the VSMC phenotypic transformation by interacting with PDGFRβof the VSMCs.Mechanically,the regulation of PDGFBB expression by EC Piezo1 is caused by the intracellular free Ca²⁺concentration.