The Role And Molecular Mechanism Of Extracellular Vesicle In Migration And Proliferation Of Vascular Smooth Muscle Cells

Hypertension is a chronic disease that seriously endangers human health,vascular remodeling is an important pathophysiological basis for the injury of heart,brain and kidney caused by hypertension.Proliferation and migration of vascular smooth muscle cells（VSMCs）are key to vascular remodeling in hypertension.Adventitia fibroblasts（AFs）plays an important role in regulating VSMCs.Extracellular vesicles（EVs）are phospholipid membrane vesicles released by cells,contain natural cargo molecules such as proteins,m RNA,mi RNA and other non-coding RNA,which play key roles in cell-cell communication.EVs often classified into exosomes and microvesicles（MVs）according to the properties of EVs such as size,density and biogenesis processes.The exosomes refer to endosome-originated membrane vesicles with a diameter of 40 to 150 nm,and the microvesicles are defined as plasma membrane shedding vesicles of 100 to 1000 nm.Since the size range of exosomes and EVs overlap,therefore,we use EVs to describe these vesicles accurately.However,the regulation and mechanism of extracellular membrane on VSMCs remain unclear.This thesis divided into three parts,respectively.（1）Effects of EVs from AFs of spontaneously hypertensive rats on VSMC migration.（2）Effects of mi R155-5p in EVs from AFs on VSMC proliferation and its molecular mechanism.（3）Effects of mi R135a-5p in EVs from AFs on VSMC proliferation and its molecular mechanism.Part 1 Extracellular vesicles-mediated transfer of ACE（angiotensin-converting enzyme）from adventitial fibroblasts of spontaneously hypertensive rats promotes vascular smooth muscle cell migration1.BackgroundMigration of VSMCs is pivotal for vascular remodeling in hypertension.Vascular adventitia close to arterial tunica media,the most abundant cell type in vascular adventitia is AFs.AFs are important in the homeostasis of vascular structure.There are complex signaling links between AFs and VSMCs involved in hypertension vascular remodeling.EVs are thought to play key roles in cell-cell communication.However,the role of AFs on VSMC migration is unclear.2.ObjectiveIn this study,we aimd to explore the effects of EVs from AFs in VSMC migration and its molecular mechanisms.3.MethodsMale Wistar-Kyoto（WKY）rats and SHR aged 8 weeks were selected,the adventitia and media were isolated from WKY and SHR thoracic aorta,and primary AFs and VSMCs were isolated by enzymatic digestion.The isolated cells were re-suspended in DMEM with 10%fetal bovine serum,100 IU/m L penicillin and 10mg/m L streptomycin maintained at 37°C in a humidified atmosphere containing 5%CO₂.EVs were isolated and purified EVs were isolated and identified from AFs medium with Pureexo（?）Exosome Isolation Kit and ultracentrifugation method.Boyden chamber assay and wound-healing assay were used to evaluate the VSMC migration.Ang II,AT₁R and ACE contents were determined with enzyme-linked immunosorbent assay（ELISA）.ACE activity was measured with a modified fluorometric assay with hippuryl-histidyl-leucine（HHL）.Western blot was used to measure protein expression in CD9,CD63,calnexin and AT₁R.The m RNA of angiotensinogen（AGT）,Ang II,AT₁R and ACE levles were anlyzed by real-time quantitative PCR.4.Results（1）EVs from AFs condition medium of SHR promoted but EVs from AFs condition medium of WKY had no significant effect on VSMC migration.Exosome inhibitor GW4869 prevents VSMC migration induced by condition medium of AFs of SHR.（2）The isolated EVs from AFs had a spherical or cup-shaped morphology,with an average size of 94 nm,contained the established exosome-associated protein markers,such as CD9 and CD63,but negative for calnexin.（3）EVs from AFs of WKY rats had no significant effects on VSMC migration,whereas EVs from AFs of SHR promotes VSMC migration in the VSMCs from both WKY and SHR（WKY-VSMCs and SHR-VSMCs）.（4）The effects of EVs from SHR on VSMC migration in VSMCs from WKY or SHR were prevented by an AT₁R（Ang II[angiotensin II]type1 receptor）antagonist losartan,or an inhibitor of ACE（angiotensin-converting enzyme）captopril.（5）There were no significant difference in Ang II and AT₁R m RNA and protein levels between EVs from SHR（SHR-EVs）and EVs from WKY（WKY-EVs）,but ACE contents and activity were much higher in EVs from SHR than those from WKY.（6）ACE m RNA and protein expressions and ACE activity were increased in AFs of SHR（SHR-AFs）but not significantly increased in VSMCs of SHR compared with those in AFs of WKY（WKY-AFs）.However,the ACE m RNA and protein expressions and ACE activity in VSMCs of SHR were lower than AFs of SHR.（7）SHR-EVs from had no significant effects on AT₁R protein and ACE m RNA levels,but increased ACE and Ang II contents in VSMCs of WKY or SHR.The increased of ACE activity and Ang II contents in VSMCs of WKY or SHR induced by the EVs from SHR were prevented by pretreatment of VSMCs with captopril.（8）ACE knockdown in AFs of SHR greatly reduced ACE contents and activity but had no significant effects on Ang II contents in EVs of SHR.（9）ACE knockdown in AFs inhibited VSMC migration induced by SHR-EVs.5.ConclusionEVs from AFs of SHR transfer ACE to VSMCs,which increased Ang II levels and activated AT₁R in VSMCs and thereby promoted VSMC migration.Part 2 Mi R155-5p in adventitial fibroblasts-derived extracellular vesicles inhibits vascular smooth muscle cell proliferation via suppressing angiotensin-converting enzyme expression1.BackgroundProliferation of VSMCs plays crucial roles in vascular remodelling and stiffening in hypertension.Our previous studies have found that AFs-derived EVs regulate VSMC migration,but the roles of EVs from AFs in VSMC proliferation and vascular remodelling in WKY and SHR still unclear.EVs are phospholipid membrane vesicles released by cells that play key roles in cell-to-cell communication,EVs contain natural cargo molecules such as mi RNA,m RNA and proteins,which are transferred to neighbouring or more distant cells to affect distinct signalling cascades.Micro RNAs（mi RNAs）are a class of small non-coding RNA molecules,which bind to the 3′-untranslated regions（3′-UTRs）of a target m RNA sequence,and induce translation repression or m RNA degradation.One recent study in our laboratory showed that the ACE content in the SHR-EVs increased compared to WKY-EVs,and the ACE in the SHR-EVs promoted the migration in VSMCs of WKY and SHR.However,the regulation and mechanism of EVs on VSMCs proliferation and vascular remodeling remain unclear.2.ObjectiveThe purpose of this study was to investigate the role and molecular mechanisms of EVs from AFs in SHR proliferation and vascular remodeling.3.Methods8 weeks old male WKY and SHR were used in the experiments,and primary AFs and VSMCs were isolated from thoracic aorta of rats with enzymatic digestion.EVs were isolated and purified with Pureexo（?）Exosome Isolation Kit and ultracentrifugation method.The isolated EVs were identified using transmission electron microscope（TEM）,Nanopartical Tracking Analysis（NTA）and western blot.VSMC proliferation was evaluated by cell counting kit-8 kits,Ed U incorporation assay and proliferation cell nuclear antigen（PCNA）protein and m RNA expression.Western blot was used to measure protein expression in PCNA,ACE and GAPDH.The m RNA of mi R155-5p,U6,ACE and GAPDH levles were anlyzed by real-time quantitative PCR.Ang II and ACE contents were determined with ELISA.Immunohistochemistry was used to detect the expression of thoracic aortic ACE.Vascular remodeling in aorta and mesenteric artery（MA）was measured with Masson’s staining.The mi R155-5p mimic and inhibitor were transfected with RNAifectin^TMtransfection reagent,observing the role of mi RNA.Dual luciferase reporter assay was used to evaluate whether mi R155-5p binds to ACE 3’UTR.Immunofluorescence was conducted to measure the fluorescence antibody intensity of ACE.4.Results（1）There was no significant difference in size and morphology between WKY-EVs and SHR-EVs.（2）WKY-EVs had no significant effect on WKY-VSMC proliferation,but attenuated SHR-VSMC proliferation.SHR-EVs promoted the proliferation of both WKY-VSMCs and SHR-VSMCs,although the SHR-VSMC proliferation was enhanced compared with that of WKY-VSMCs（3）Mi R155-5p levels in AFs and VSMCs of SHR were lower than those of WKY.Compared with WKY-EVs,mi R155-5p in SHR-EVs fell to a lower level.Mi R155-5p mimic and overexpression in VSMCs had no significant effect on WKY-VSMC proliferation,but attenuated SHR-VSMC proliferation,mi R155-5p inhibits VSMC proliferation of SHR.（4）Mi R155-5p in EVs inhibited VSMC proliferation,but the promoting effects of EVs from AFs of SHR on the proliferation of both WKY-VSMCs and SHR-VSMCs were enhanced by the mi R155-5p inhibitor.（5）ACE was a target of mi R155-5p and mi R155-5p inhibited ACE expression.WKY-EVs inhibited ACE m RNA and protein expressions in both WKY-VSMCs and SHR-VSMCs.SHR-EVs had no significant effects on ACE m RNA expression,but up-regulated the ACE protein in both WKY-VSMCs and SHR-VSMCs.（6）Captopril or losartan inhibited SHR-EVs-induced VSMC proliferation.（7）Knock down the ACE in the SHR-AFs,the VSMC proliferation of EVs was weakened,and ACE protein expression level was decreased.Mi R155-5p overexpression further attenuated VSMC proliferation and decreased ACE expression.（8）Mi R155-5p overexpression in SHR reduced vascular ACE,angiotensin II and PCNA levels,and attenuated hypertension and vascular remodelling in SHR.（9）Repetitive intravenous injection of SHR-EVs increased blood pressure and vascular ACE contents,and promoted vascular remodelling in both strains,while WKY-EVs reduced vascular ACE contents and attenuated hypertension and vascular remodelling in SHR.5.ConclusionMi R155-5p in adventitial fibroblasts-derived extracellular vesicles inhibits VSMC proliferation and vascular remodeling via suppressing ACE expression,while SHR-EVs-mediated ACE transfer promotes VSMC proliferation and vascular remodelling.Part 3 Extracellular vesicle-mediated mi R135a-5p transfer in hypertensive rat contributes to vascular smooth muscle cell proliferation via targeting FNDC51.BackgroundEVs from AFs regulate VSMC proliferation through transferring mi R155-5p and ACE,which play crucial roles in the pathogenesis of hypertension and vascular remodeling.However,it is unknown whether some other signal molecules in the EVs may play important roles in VSMC proliferation.In the course of our study,we found that mi R135a-5p contents in SHR-EVs were much higher,fibronectin type III domain containing 5（FNDC5）gene might be one of targets of mi R135a-5p,the findings aroused our great interest.FNDC5,a precursor of irisin,is a transmembrane protein encoded by the FNDC5 gene.Our previous studies have shown that FNDC5/irisin attenuates insulin resistance and hepatosteatosis,and improves glucose and lipid metabolism.Moreover,FNDC5 inhibits adipose tissue inflammation in obesity rats and in AFs of SHR.However,the role and mechanism of mi R135a-5p in VSMCs proliferation are unclear.2.ObjectiveThis study was designed to ascertain the effects of EVs-mediated transfer of mi R135a-5p in VSMC proliferation and the underlying mechanisms.3.MethodsMale WKY and SHR of 8 weeks old were selected in the experiments,and primary AFs and VSMCs were isolated with enzymatic digestion.VSMC proliferation was evaluated by cell counting kit-8 kits,Ed U incorporation assay.The mi R135a-5p mimic and inhibitor were transfected with RNAifectin^TM transfection reagent,observing the role of mi RNA.Dual luciferase reporter assay was used to evaluate whether mi R135a-5p binds to FNDC5.Western blot was used to measure protein expression in FNDC5 andβ-actin.The m RNA of mi R135a-5p,ACE,U6and GAPDH levles were anlyzed by real-time quantitative PCR.4.Results（1）Mi R135a-5p level in SHR-EVs was significantly increased.（2）Mi R135a-5p inhibitor prevented the SHR-EVs-induced VSMC proliferation.（3）FNDC5 was a target of mi R135a-5p.Mi R135a-5p mimic inhibited FNDC5m RNA and protein expressions in the VSMCs of both WKY and SHR.Mi R135a-5p inhibitor reversed the downregulation of FNDC5 m RNA and protein expressions in the VSMCs of SHR.Mi R135a-5p inhibitor prevented the SHR-EVs-induced VSMC proliferation.（4）Mi R135a-5p inhibitor not only increased FNDC5 expression,but reversed the SHR-EVs-induced FNDC5 downregulation in VSMCs of SHR.Mi R135a-5p mimic inhibited FNDC5 expression,but failed to promote the SHR-EVs-induced FNDC5downregulation in VSMCs of SHR.（5）Exogenous FNDC5 prevented the SHR-EVs-induced VSMC proliferation of both WKY and SHR.（6）Knockdown of mi R135a-5p in fibroblasts completely prevented the upregulation of mi R135a-5p in the EVs.The SHR-EVs from the mi R135a-5p knockdown-treated fibroblasts lost their roles in inhibiting FNDC5 expression and promoting proliferation in VSMCs of both WKY and SHR.5.ConclusionMi R135a-5p promoted while FNDC5 inhibited SHR-VSMCs proliferation.Increased mi R135a-5p in the SHR-EVs promoted VSMC proliferation of WKY and SHR via inhibiting FNDC5 expression.Mi R135a-5p and FNDC5 may be crucial targets for intervention of VSMC proliferation in hypertension.