Relationship Between TRPV4 And Pulmonary Microvascular Endothelial Permeability In Rats With Pulmonary Hypertension Induced By Monocrotaline

Pulmonary vascular endothelium is the first barrier of blood flow impact.Endothelial cells（EC）damage are closely related to the pathogenesis of pulmonary hypertension（PH）.In recent years,the functional heterogeneity of vascular endothelial cells has received more and more attention.One of the most prominent features is that pulmonary microvascular endothelial cells（PMVECs）have a unique permeability barrier function.Tight Junction（TJ）is an important component affecting the function of the endothelial barrier.Studies have shown that Ca²⁺,which enters cells through the cation channel of the transient receptor potential vanilloid 4 channel（TRPV4）,plays a key regulatory role in endothelial permeability and barrier integrity.However,the expression of TRPV4 on PMVECs in PH and its effect on endothelial barrier function are rarely reported in the literature.Objective:To investigate whether TRPV4 channel function and endothelial permeability of PMVECs induced by monocrotaline（MCT）in rats with pulmonary hypertension have changed,and its effect on endothelial permeability.Methods:（1）Clean grade rat（sprague dawley rat,SD）,male,randomly divided into 2groups:CON group and MCT group.The model was identified by right ventricular systolic pressure（RVSP）,right ventricular mass index（RVMI）and HE staining.（2）After the primary PMVECs were cultured by tissue-blocking method,high-purity PMVECs were identified by immunofluorescence,CD31,vWF and FITC-BSI binding assays;（3）Transmission electron microscopy:detection of changes in TJ structure in PMVECs in CON and MCT groups;（4）IHC:Observing the expression and distribution of TJ-related proteins Claudin-5,Occludin and ZO-1 in pulmonary vascular endothelium;（5）Transendothelial electrical resistance（TER）assay:After constructing a single-layer model of PMVECs using Transwell,an inhibitor or agonist of TRPV4 was added to the PMVECs of CON group and divided into CON,CON+HC-067（TRPV4 inhibitor）.,CON+GSK101（TRPV4 agonist）three groups;Similarly,MCT is also divided into three groups:MCT,MCT+HC-067,MCT+GSK101,measuring resistance value;Fluorescein isothiocyanate（FITC）labeled Portuguese Glycan method:grouping the same as above,measuring the permeability of PMVECs;（6）Real-time PCR and Western Blot assay were used to determine the expression levels of related genes mRNA and protein in PMVECs.Results:（1）The RVSP and RVMI of the MCT group were significantly higher than those of the CON group.HE section showed that the MCT group was thicker than the con-vascular wall,the lumen was smaller,and the endometrium was broken.It suggested that the MCT group was successfully prepared.（2）Tissue block adherence method can successfully culture high-purity primary rat PMVECs.（3）The resistance of the CON group was significantly higher than that of the MCT group,indicating that the permeability of PMVECs in the MCT group was significantly increased.The results of transmission electron microscopy showed that the TJ structure of the MCT group was destroyed and the intercellular space was widened.Both of them suggested that the increased permeability of PMVECs in the MCT group was related to the structural damage of TJ.（4）In the CON group and the MCT group,the resistance values of PMVECs and GSK101 were significantly decreased after activation of TRPV4 channel;The resistance value of HC-067 was significantly increased after inhibiting TRPV4 channel.The results of FITC-labeled dextran method showed that the cell-to-cell permeability was significantly increased after stimulating TRPV4channel,and the permeability was significantly decreased after inhibiting TRPV4channel.The above results suggest that the permeability of PMVECs in PH rats is closely related to TRPV4 activity.（5）The histochemical method indicated that the TJ-related proteins Occludin,Claudin-5 and ZO-1 were distributed on the microvascular intima,and the expression of MCT group was significantly decreased.（6）Real-time PCR results showed that the expression of TPPV4 mRNA in PMVECs of MCT group was significantly higher than that of CON group,while the TJ protein Occludin and ZO-1 were significantly lower than that of CON group,suggesting that high expression of TPPV4 in PMVECs mediates increased pulmonary vascular permeability,and the increase of pulmonary vascular endothelial permeability was closely related to the significant decrease of TJ protein expression.Western Blot results showed that TRPV4 protein expression was significantly increased in GSK101-expressing TRPV4 channels in both CON group and MCT group,while Occludin,Claudin-5 and ZO-1 protein expression was significantly decreased.After TRPV4 channel was inhibited,the expression of Occludin,Claudin-5 and ZO-1 protein was significantly increased except for the decrease of TRPV4 protein expression.The expression of TRPV4 protein in MCT group was significantly higher than that in CON group,and the expression of TJ protein was significantly lower than that in CON group,suggesting that the enhancement of TRPV4 channel function at the time of PH increased pulmonary vascular permeability by inhibiting TJ protein expression.（7）The expression of p38MAPK mRNA in PMVECs of MCT-PH rats was significantly up-regulated compared with CON group,while PKC-βmRNA was decreased.PKC-αand PKC-δwere not statistically significant compared with CON group,suggesting that PKC-βand p38MAPK may be involved in the changes of PMVECs injury at the time of PH and affect its permeability.Conclusion:This work has initially proved that the permeability of PMVECs induced by MCT is increased.At the occurrence of PH,the function of TRPV4 channel is enhanced.By reducing the expression of TJ proteins Occludin,Claudin-5 and ZO-1,the TJ structure is destroyed and the pulmonary vascular endothelial barrier is damaged.The signal molecules p38MAPK and PKC-βmay participate in this process.