The Function And Mechanism Of TRPV4 Coupling Complex In Vascular Function

The incidence of cardiovascular diseases is increasing year by year,and the diagnosis and treatment of cardiovascular diseases is a medical problem that needs to be solved urgently.The bottlenecks in the diagnosis and treatment of cardiovascular disease include the lack of understanding of vascular lesions and the lack of precise pharmacological interventions.Endothelial cells are a layer of cells that make up the endothelium and are in direct contact with the blood,feeling the impact of blood flow and metabolic fluctuations,and their function is highly susceptible to damage,thus inducing the occurrence of permeability,diastolic,inflammation,plaque and other lesions,resulting in the temporal and spatial diversity of vascular lesions in terms of the disease process and the location of the lesions,which may also be the entry point for analysing the complexity of vascular lesions.Previous studies have identified transient receptor potential（TRP）ion channels as an important class of non-selective cationic transmembrane proteins in endothelial cells,and their activation is usually accompanied by the coupling of other cellular components to form TRP complexes involved in the regulation of vascular function.In the present study,we carried out research on both the role and mechanism of TRPV4-coupled complex in vascular function.The main results are as follows:1.By constructing a hypertensive mouse model,single-cell transcriptome sequencing of four segments of the aorta（ascending aorta/aortic arch/thoracic aorta/abdominal aorta）in normal and hypertensive mice revealed a subpopulation of endothelial cells with CD106⁺TRPV4^highNOS3^high.It was also found to be highly expressed in the thoracic aortic segment by immunofluorescence and flow sorting.Subsequently,at the cellular level,it was demonstrated that TRPV4 function was not altered in CD106⁺TRPV4^highNOS3^high endothelial cells of aortic four segments in normal and hypertensive mice by detecting the inward flow of calcium ions(Ca²⁺)as well as the release of nitric oxide（NO）,although the release of NO was significantly reduced in the hypertensive mice was significantly reduced and was most pronounced in the thoracic aortic segment.In addition,TRPV4 and endothelial nitric oxide synthase（eNOS）expression were not significantly different in normal and hypertensive mice.Next,immunoprecipitation and immunofluorescence staining revealed that TRPV4 was physically and functionally coupled to eNOS in CD106⁺TRPV4^highNOS3^high endothelial cells,and this coupling was significantly reduced during hypertension.The possible binding regions of TRPV4 and eNOS were screened by molecular docking,and then these regions were deletion mutated by using the targeted mutagenesis technique.The mutated TRPV4 plasmid and eNOS plasmid were transfected into HEK293 cells,and their coupling was detected by using the fluorescence resonance energy transfer（FRET）method,and it was found that the deletion of the AR2 region（aa190-236）of TRPV4 and the A region（aa491-500）of eNOS significantly reduced the TRPV4-eNOS interaction.These results suggest that impaired TRPV4-eNOS interaction is critical for the progression of hypertension.2.The eNOS adeno-associated virus was constructed,and FRET and vascular tone assays revealed that enhancing TRPV4-eNOS interaction increased the vasodilatory ability of normal mice mediated by acetylcholine and GSK1016790A,and decreased blood pressure in mice.On the contrary,weakening TRPV4-eNOS interaction decreased the vasodilatory ability of normal mice mediated by acetylcholine（ACh）and GSK1016790A,and increased blood pressure in mice.However,enhancing or weakening TRPV4-eNOS interaction had no effect on the vasodilation ability and blood pressure of endothelium-specific TRPV4 knockout(TRPV4_EC^-/-)mice.Therefore,based on the TRPV4-eNOS interaction site,a series of drug small molecules were synthesised by molecular docking technique,in which the JNc-463molecule was predicted to link to the AR2 region of TRPV4 and the A region of eNOS.In addition,FRET experiments also confirmed that JNc-463 could enhance TRPV4-eNOS interactions,enhance the vasodilatory capacity mediated by ACh and GSK1016790A in hypertensive mice,and reduce blood pressure in mice,but had no effect on blood pressure in TRPV4_EC^-/-mice.The above results suggest that JNc-463 improves impaired TRPV4-eNOS interactions in hypertension and reduces blood pressure in mice.3.By constructing a high-fat diet-induced obese mouse model,single-cell transcriptome sequencing of three branches of the coronary artery（right coronary artery/left coronary artery anterior descending branch/left coronary artery gyratory branch）in mice on normal diet and mice on high-fat diet revealed a subpopulation of CD133⁺TRPV4^high endothelial cells,which were found in mice on normal diet and mice on high-fat diet by immunofluorescence,flow sorting,and immunoblotting.TRPV4 activity in CD133⁺TRPV4^high endothelial cells was subsequently demonstrated by membrane clamp detection of TRPV4 opening frequencies as well as currents at the single-cell level.In addition,fluid shear-induced Ca²⁺inward currents were significantly enhanced in CD133⁺TRPV4^high endothelial cells from normal diet and TRPV4 endothelium-specific TRPV4 knockin(TRPV4_EC^+/+)mice compared with normal diet and TRPV4_EC^-/-mice,indicating that TRPV4 has a physiological function in CD133⁺TRPV4^high endothelial cells.Next,coronary artery branches were found to respond to fluid shear-induced vasodilatation by vascular tone experiments,and the degree of vasodilatation of coronary artery branches was significantly increased in TRPV4_EC^+/+mice compared with normal diet mice,whereas the degree of vasodilatation of coronary artery branches was significantly decreased in TRPV4_EC^-/-mice.Finally,the use of Doppler ultrasound revealed a decrease in blood flow in all branches of coronary arteries in TRPV4_EC^+/+mice and an elevation in blood flow in all branches of coronary arteries in TRPV4_EC^-/-mice compared with normal diet mice.These results suggest that TRPV4 is essential for coronary blood flow regulation.4.In high-fat diet-induced obese mice,Ca²⁺fluorescence imaging revealed that TRPV4function was not altered in CD133⁺TRPV4^high endothelial cells.Using membrane clamp techniques,immunoprecipitation and immunofluorescence staining,TRPV4 was found to be physically coupled and functionally coupled to the medium conductance calcium-activated potassium channel（KCa3.1）in CD133⁺TRPV4^high endothelial cells,and this coupling was significantly reduced in high-fat diet mice.Next,the possible binding regions of TRPV4 and KCa3.1 were screened by molecular docking,followed by deletion mutation of these regions.The mutated TRPV4 plasmid and KCa3.1 plasmid was transfected into HEK293 cells,and FRET revealed that deletion of the AR1 region（aa283-286）of TRPV4 and AR3 region（aa223-232）of KCa3.1 significantly reduced the TRPV4 binding.By comparing the serum metabolomic profiles of normal and high-fat diet mice,it was found that1-heptadecyl-2-hydroxy-sn-glycero-3-phosphorylcholine（LPC）was significantly elevated in high-fat diet mice and that LPC could competitively bind to the interaction site of TRPV4 and KCa3.1,decreasing the TRPV4-KCa3.1 interaction and reducing the GSK1016790A-mediated vasodilatory capacity and increased blood flow in mice.Using KCa3.1 adeno-associated virus,enhancing TRPV4-KCa3.1 interaction increased the ability of normal mice to GSK1016790A-mediated vasodilation,while weakening TRPV4-KCa3.1interaction decreased the ability of normal mice to GSK1016790A-mediated vasodilation,but enhancing or weakening TRPV4-KCa3.1 interaction had no effect on the vasodilatory ability in TRPV4_EC^-/-mice.Therefore,based on the TRPV4-KCa3.1 interaction site,folic acid was found to have a strong affinity for TRPV4 and KCa3.1 by molecular docking technique.In addition,FRET experiments revealed that folic acid enhanced TRPV4-KCa3.1 interaction in high-fat diet mice,improve the vasodilation ability of high-fat diet mice mediated by GSK1016790A and fluid shear force,reduce the coronary artery blood flow of mice,and do not change the expression of TRPV4 and KCa3.1.These results indicate that the TRPV4-KCa3.1 interaction is involved in the regulation of coronary blood flow,and folic acid can improve the damaged TRPV4-KCa3.1 interaction in the coronary arteries of obese mice and reduce blood flow.In conclusion,the TRPV4 coupling complex plays an important role in vascular tone regulation of the aorta and coronary arteries,and drugs designed to target the coupling complex provide a new strategy for the treatment of cardiovascular diseases.