Matrix Stiffness Regulates Endothelial Function Through TRPV4/miR-6740/ET-1 Mechanically Activated Pathway

Cardiovascular diseases(CVDs)are the primary cause of death in human beings and seriously endanger human health.Cardiovascular diseases are systemic diseases with complex and diverse pathogenesis.The occurrence and development of CVDs are generally accompanied by vascular sclerosis.Stiffening is mechanically manifested as increased stiffness of the blood vessel,which increases the capacity of the blood to resist elastic deformation.Specifically,a blood vessel is less likely to deform after being subjected to blood flow and thus becomes inelastic,increasing the risk of congestion and rupture.Clinical evidence shows that an abnormal increase in arterial stiffness leads to an increased incidence of cardiovascular disease and mortality.This suggests that exploring the role and mechanism of vascular stiffness changes in the occurrence and development of cardiovascular diseases is expected to be an important entry point to explain the pathogenesis of cardiovascular diseases and improve the prognosis of diseases.The main reasons for vascular stiffening are the increase of collagen,the degradation of elastin,calcification in the vascular wall,and the proliferation and migration of smooth muscle cells in the vascular wall to the intima.This series of abnormal changes in vascular structure and composition lead to changes in the mechanical properties of the vascular wall,especially the gradual increase in the stiffness of ECM in Endothelial Cells(ECs).It has been reported that the elastic modulus of the endothelial extracellular matrix is about 2.5-8 kPa under normal physiological conditions and can be increased to over 30kPa under cardiovascular diseases.Vascular endothelial cells are monolayers closely arranged on the inner side of blood vessels.They can synthesize and secrete a variety of vasoactive substances to maintain vascular homeostasis.Endothelial cells maintain vascular homeostasis by producing Nitric Oxide(NO)and endothelin-1(ET-1),which are regarded as representative indicators of vascular endothelial function.NO is catalyzed by Endothelial Nitric Oxide Synthase(eNOS)and is the most important vascular relaxant.Et-1 is a long-acting endogenous peptide produced by endothelial cells that promotes vasoconstriction.Upregulation of ET-1 inhibits eNOS expression and reduces the production of NO,leading to homeostasis imbalance of vasoactive substances and endothelial dysfunction,which is a key factor in cardiovascular disease.Therefore,the regulation effect of extracellular matrix stiffness on endothelial cells'production of vasoactive factors and maintenance of vascular homeostasis is the entry point to study cardiovascular diseases caused by increased vascular stiffness.At present,the mechanical environment of the extracellular matrix was simulated by the cultured substrate in vitro,and it was found that the change of substrate stiffness could affect the spreading,adhesion,proliferation,and migration of vascular endothelial cells.Previous studies in our laboratory also found that changes in the stiffness of the substrate increase the instability of traditional reference gene expression in endothelial cells.However,the effect of changes in stiffness on the important function of endothelial cells in maintaining vascular homeostasis remains unclear.We hypothesized that increased stiffness is detrimental to endothelial cells'ability to maintain vascular homeostasis and plays an important role in the occurrence and development of cardiovascular diseases.In this study,we through different stiffness(4,25 and 50 kPa)polyacrylamide hydrogel basal simulation of physical mechanics of Endothelial Cells under the environment,to cultivate the original generation of Umbilical Vein Endothelial Cells(Human Umbilical Vein Endothelial Cells,HUVECs),found that the basal stiffness increased inhibition of the expression of Endothelial cell eNOS,promote the expression and secretion of ET-1,prompt abnormal vascular stiffness increased to induce Endothelial cell function obstacle,conduction and mechanics of concrete mechanism also need to be further explored.In recent years,microRNA(miRNA)is among the most concerned molecules in investigating the mechanisms of diseases.We focused on miRNA in order to further discover how increased stiffness induces endothelial dysfunction.Mi RNA is a kind of endogenous non-coding single-strand RNA,which specifically binds to the 3'end untranslated region(3'-UTR)of the target mRNA inhibiting the expression of the target gene at the post-transcriptional level.ECs cultured on 4kPa and 50kPa hydrogel basement were used as samples to screen the differentially expressed miRNA genes using miRNA microarray analysis.There were 22 up-regulated miRNA genes and 8down-regulated miRNA genes in ECs cultured on 50kPa,compared with 4kPa.Among them,the bioinformatics prediction analysis found that ET-1 is the potential target of hsa-miR-6740-5p(miR-6740),which has been validated by qPCR and dual-luciferase report gene assay.ET-1 expression was detected after transfecting miR-6740 mimics and inhibitors into ECs.We found that the ET-1 is the target gene of miR-6740,stiffening significantly downregulates miR-6740 and increases ET-1expression and secretion through the above experiment.The key problem to investigate the mechanism of stiffening-induced endothelial dysfunction is how endothelial cells feel changes in stiffness and accordingly regulate miR-6740 expression and downstream ET-1.Matrix stiffness,as a part of the cellular microenvironment,stimulates cells mechanically.The mechanical receptors on the cell surface are the key elements to transform the extracellular microenvironmental mechanical signals into intracellular biochemical signals.Transient receptor potential vanilloid 4(TRPV4)is one of the cellular mechanical receptors widely distributed in multiple organs and tissues.As a calcium ion channel,TRPV4 in ECs and myocardial cells change its permeability to promote calcium(Ca²⁺)influx regulating vascular tension and myocardial excitability in response to shear force.Also,TRPV4 is the major cellular second messenger that affects the activity of nuclear transcription factors through a couple of signaling pathways participating in the regulation of gene expression.In our study,ECs have been treated with TRPV4 specific activator GSK1016790A(GSK101)and inhibitor GSK2193847(GSK219).Changes in the activity of TRPV4 ion channels were measured by calcium ion imaging and patch-clamp experiment.Expressions of TRPV4 in ECs on hydrogel basement with three Young's modulus were detected by qPCR and cell immunofluorescence.The results showed that the increase in basement stiffness inhibited the expression of TRPV4 and its ion channel activity in ECs,inhibited the intracellular Ca²⁺influx,and activation of TRPV4 promoted the expression of miR-6740 and inhibited the expression of ET-1 in ECs.In addition,this study answered whether miR-6740 has the application potential for clinical biomarkers.It is essential to determine whether ECs secret miR-6740 outside of ECs.As a small single-stranded RNA,miRNA is extremely unstable outside the cell and usually exists in exosomes.Exosome extraction,identification,and miRNA qPCR detection have been performed.The results showed that ECs secreted extracellular miR-6740 through exosomes.The content of miR-6740 in the exosomes from ECs cultured on the 4kPa basement was significantly higher than that on the50kPa basement,suggesting that organisms contain miR-6740 in blood.Then,we collected plasma samples from 15 healthy people and 21 patients with arteriosclerosis.It was found that the plasma miR-6740 content in healthy people was significantly higher than that in patients with atherosclerosis,and plasma miR-6740 was correlated with atherosclerosis.Above all,this study clarified that the increase of basement stiffness promotes the adhesion and cytoskeleton generation of endothelial cells,promotes the production of diastolic factor NO,and inhibits the expression and secretion of vasoconstrictor factor ET-1.This study found a potential basal stiffness pathway that regulates endothelial cell function:the TRPV4/miR-6740/ET-1 pathway.It was first proposed that the TRPV4 calcium ion channel is the stiffness sensor of endothelial cells,and it was the first time to reveal the biological role of miR-6740 in inhibiting ET-1 expression and the correlation between miR-6740 in plasma and the occurrence of atherosclerosis.This study is expected to provide a research basis for the diagnosis and prognosis of cardiovascular disease biomarkers and provide a theoretical reference for the development of new vascular biomaterials and the optimization of mechanical properties.