Endothelium Derived Extracellular Vesicles In Hypertension Associated Arterial Stiffening:Function And The Underlying Mechanism

ObjectiveHypertension is a major risk factor for cardiovascular disease and has become the leading cause of disease burden worldwide.Hypertension features as arterial remodeling at the early stage,resulting in organ damage and cardiovascular events.Arterial remodeling is characterized by endothelial cell(EC)dysfunction and smooth muscle cell(SMC)phenotype switch,which in turn damages vascular structure and function,further accelerates the deteriorative progression of blood pressure and generates a vicious cycle ultimately.It is of great significance to study the underlying mechanism of abnormal stiffness caused by vascular remodeling and develop effective measures for the prevention and treatment of hypertension.The disturbed communication between endothelial cells(EC)and subendothelial smooth muscle cells(SMC)plays a crucial role in the arterial remodeling.Extracellular vesicles(EV)are emerging as an important mediator for regulating intercellular communication and diagnostic marker for many diseases by typically containing proteins,nucleic acids,and lipids to reflect their cell origins and mediating intercellular communication under normal and pathological conditions.Besides the intensively studied hormone and cytokines in the process,EV should be also involved,though the details are largely unknown.In the present study,we first expounded the function and underlying mechanism of endothelium derived EV in hypertension associated arterial stiffening with the technical advantages of vascular ultrasound imaging and developed a delivery strategy of engineered EV for prevention and treatment of vascular remodeling.Our study has not only provided novel insights on the mechanism of hypertension associated blood vessel wall remodeling,but also shed light on therapeutic intervention of arterial stiffening.Methods and resultsPart One.Characterization of endothelium derived extracellular vesicles and changes under application of mechanical force.In order to explore whether EV involved in the intercellular communication between EC and subendothelial SMC,the ultrastructure of the artery was analyzed by transmission electron microscopy.Amounts of vesicles were in the basolateral side of the endothelium,while some of the vesicles were undergoing secretion.The vesicles were in the size range of 80-100 nm in diameter and displayed a typical ball-shaped morphology.To further confirm EV distribution in mice,EVs were harvest from the culture medium of HUVEC by ultracentrifugation.The particle size for EV was about 100 nm in diameter.In addition,EVs were confirmed by exosomal inclusive markers and exclusive marker GM130.Besides the main distribution in liver,EVs were also found in other organs.Notably,there were also EVs in the blood vessels,especially the branched area.To mimicking the blood pressure force pushing the blood vessel wall,HUVECs were subjected to cyclic weight overloading.The EVs derived from control or 2 g weight loading cells were denoted as EVctrl and EV2g respectively.Weight loading didn’t change the EV size,morphology and marker expression,while the yield under 2 g weight was moderately increased.Smooth muscle cells(MOVAS)were incubated with DiI labeled EVctrl and EV2g.Both EVctrl and EV2g could be efficiently taken up by MOVAS,while the uptake efficiency of EV2g was markedly higher than that of EVctrl.We found that EVs could circulate into the subendothelial SMC in vivo by tracing DiI labeled EVs after tail vein injection,and there were more EV2g colocalized with the SMC in the blood vessel.To further confirm the finding,EVctrl and EV2g were loaded with cel-miR-54,which has no homolog in mouse,and cel-miR-54 expression in the aorta was analyzed by qPCR.Consistent with the fluorescence data,there were more cel-miR-54 delivered into the aorta in the EV2g group.Besides efficient uptake by SMC,EC derived EVs could be also taken up by endothelium both in vitro and in vivo.One-way ANOVA was used for group comparisons,and results with significant differences are further compared by post hoc analysis with Bonferroni corrected t-test.For all analyses,P<0.05 was considered statistically significant.Part Two.Endothelium derived extracellular vesicles contributed to the phenotype switch of vascular smooth muscle cell.1.EVs from the injured endothelium recapitulate the phenotype in hypertension.We explored whether the EVs from the injured endothelium conferred the functional information in vivo.EVs were injected into mice via tail vein once a week for 4 weeks,and the arterial stiffness was then assessed by PWV.The aorta PWV in mice of EV2g treatment showed a significant higher PWV in comparison with the control.Moreover,HE staining showed that the vascular wall thickness was markedly increased in EV2g group mice.And smooth muscle fibers were sparse and disordered with prominent gaps in the media.The lumen diameter had no significant change,while the wall and lumen ratio increased in the EV2g treatment group.Consistently,there was increased collagen deposition in vascular wall in EV2g mice group as revealed by Masson’s trichrome staining.Moreover,the mRNA expression of Col3a1,Colla1 and Spp1,which were the synthetic phenotype marker of SMC,was found significantly increased in EV2g treatment group,suggesting that EV2g treatment might promote SMC phenotype switch.Accordingly,EV2g treatment also promoted the synthetic phenotype switch of MOVAS,as shown by up-regulated expression of Col3a1,Col1a1 and Spp1.2.Significant miRNA changes in the EVs derived from dysfunctional endothelium under hypertension.The difference of miRNA profiles in plasma EVs between normal people and hypertension patients were profiled.The particle size for plasma-EV was 150-200 nm in diameter expressing typical EV markers.Then EVs were sequenced by HiSeq 2500.Totally,1228 EV-miRNAs were identified in samples of control and hypertension and there were 19 miRNAs with high abundance in either group showed significant difference between two groups,including 10 up-regulated and 9 down-regulated in hypertension group.Among these miRNAs,we were specifically interested in miR-320d and miR-423-5p for their most obvious fold change.To further explore the correlation between miR-320d/miR-423-5p abundance and the blood pressure as well as PWV,11 more volunteers were recruited.Interestingly,we found a significant positive correlation between mean blood pressure and the abundance of the two EV-miRNAs(EV-miR-320d:R2=0.3845,P=0.0418;EV-miR-423-5p:R2=0.3854,P=0.0415),and a significant positive correlation between PWV and EV-miRNA expression(EV-miR-320d:R2=0.703,P=0.0013;EV-miR-423-5p:R2=0.552,P=0.0088).3.EV-miR-320d/423-5p from the dysfunctional endothelium contribute to the arterial remodeling.In view of the above data,we assumed that EVs derived from dysfunctional endothelial cells might participate in the arterial remodeling via transferring miRNA-320d/423-5p.As expected,EV2g treatment significantly increased expression of miRNA-320d/423-5p in the aorta.To verify the effect of EV-miRNA-320d/423-5p,miR-320d-mimics and miR-423-5p-mimics were thus encapsulated into EV derived from control endothelial cells respectively by electroporation.qPCR analysis showed that miRNA-mimics were efficiently loaded into the EVs.Then,the EVs were tail vein injected once a week for 4 weeks,the arterial stiffness was measured by PWV.As expected,both of the EVmiR-320d-mimics group and EVmiR-423-5p-mimics group had significant higher PWV,compared with the negative control group.Moreover,the thickness of vascular wall and the wall/lumen ratio increased in the EVmiR-320d-mimics group and EVmiR-423-5p-mimics group.Histology analysis showed increased collagen deposition and disordered arrangement of SMC in the groups treated with the EVmiR-320d-mimics and EVmiR-423-5p-mimics.Accordingly,mRNA expression of Col3a1,Col1a1 and Spp1,was found increased in the two groups.Part Three,Therapeutic delivery of engineered extracellular vesicles improved arterial stiffness in hypertension.In the following experiments,we explored whether intervention of the intercellular communication would be therapeutic.The PWV of SHR was much higher compared with the control Wistar-Kyoto(WKY)rats.Similarly,the thickness of vascular wall and wall/lumen ratio of SHR were much larger.Moreover,more collagen deposition and prominent gaps increased as Masson’s trichrome staining showed in SHR compared with WKY.qPCR revealed higher mRNA expression of Col3al,Col1a1 and Spp1 in SHR rats.All of these suggested that high blood pressure aggravated the vascular stiffness and dysfunction in the SHR model,resembling the phenotype in hypertension patients.Furthermore,miR-320d-inhibitor and miR-423-5p-inhibitor were thus encapsulated into EVs by electroporation respectively and were injected via tail vein once a week for one month.Compared with control group,EVmiR-320d-inhibitor and EV miR-423-5p-inhibitor treatment significantly decreased the aorta PWV in SHR rats.Consistent with the functional change,the thickness of vascular wall and the wall/lumen ratio was decreased after EVmiR-320d-inhibitor and EVmiR-423-5p-inhibitor treatment.In addition,EVmiR-320d-inhibitor and EVmiR-423-5p-inhibitor treatment reduced collagen deposition and down-regulated the mRNA expression of Col3a1,Collal and Spp1 in the blood vessels of SHR.We next explored the downstream target of miR-320d and miR-423-5p.Inhibition of miR-423-5p with the miRNA inhibitor significantly blocked the effects of EV2g on the synthetic phenotype switch of vascular SMC in vitro,however the inhibition of miR-320d had no effect.The paradoxical roles of miR-320 in EV between in vivo animal experiment and the in vitro cell experiments suggested that miR-320 might regulate other cell types rather than directly on the SMC.In order to further clarify the direct target of miR-423-5p,the mRNA level of Mybl2 in MOVAS treated with miR-423-5p-mimic transfection was analyzed by qPCR,which is a putative regulator of synthetic phenotype switch of vascular SMC.As expected,miR-423-5p significantly reduced the expression of Mybl2.Accordingly,EV mediated delivery of miR-423-5p significantly reduced Mybl2 expression in the aorta.All the data suggested that miR-423-5p in EV2g or EV from hypertension might regulate SMC synthetic phenotype switch at least via Mybl2.Moreover,ROS in the HUVEC with 2 g weight treatment were much higher than that in the control group.ROS scavenger N-acetylcysteine(NAC)treatment significantly reduced the ROS,together with the abundance of miR-320d and miR-423-5p in donor cells and EVs.The expression of miR-320d and miR-423-5p was significantly up-regulated in both the weight overload cells and the derived EV2g,suggesting that the increased abundance in EVs might be due to the enhanced expression of the two miRNAs in the donor cells.Together,these findings suggested that hypertension related mechanical force increased miR-320d and miR-423-5p abundance in EVs in an ROS dependent manner.Conclusions1.EVs derived from the endothelial cells could be taken up by the subendothelial smooth muscle cells in the artery.2.EVs from the ROS-injured endothelial cells could remodel the vessel wall and increase arterial stiffness at least partially via switching the smooth muscle cell phenotype by targeting Mybl2 with miRNAs encapsulated.3.Therapeutic delivery of miR-320d/423-5p inhibitors via engineered EVs could alleviate the phenotype of vascular remodeling caused by hypertension and improve arterial stiffness.