Expression Of L-type Calcium Channel In Human Vascular Endothelial Cells And Its Relationship With Inflammation

Objectives:Preliminary clinical experiments have shown that L-type calcium channels（LTCCs）are related to hypertension and endothelial cell dysfunction.Therefore,the purpose of this study was to observe the expression of LTCCS protein subunits in human umbilical vein endothelial cells（（HUVECs））,and to explore the effect of its channel agonist and its main functional subunit Cav1.3 LTCCsα1 coding gene CACNA1D on the inflammatory response of HUVECs.Methods:1.The expression of LTCCs protein subunits on HUVECs was studied by q RT-PCR,Nucleic Acid Electrophoresis,and Immunofluorescence staining.2.The changes of intracellular calc ium(Ca²⁺)concentration in HUVECs treated with Bay-k8644 were dynamically observed by laser confocal fluorescence microscope.3.q RT-PCR and Western Blot were us ed to detect the expression of HUVECs adhesion molecules ICAM-1 and VCAM-1 and the changes of related signal molecules induced by Bay-k 8644.4.The differences of molecular expression,disease correlation and signal pathway of HUVECs after overexpression of Cav1.3 coding gene（CACNA1D）were analyzed by gene chip technology,and the role of Cav1.3 in endothelial cell inflammation and other physiological functions was discussed.Results:1.The expression of LTCCs in HUVECs and its ion channel function:Ⅰ.The results of q RT-PCR showed that the expression of LTCCsα1 subunit m RNA of Cav1.2 and Cav1.3 in HUVECs was higher than that of Cav1.1 and Cav1.4.Ⅱ.The positive expression of LTCCSβ2 subunits of Cacnb1,Cacnb2,Cacnb3and Cacnb4,and LTCCsα2δsubunit m RNAs of Cacna2d1,Cacna2d2 and Cacna2d3 were also detected.Ⅲ.The calcium fluorescence experiment confirmed that the fluorescence intensity of Ca²⁺in HUVECs cells increased at first and then decreased after the addition of agonists.2.LTCCs participates in the up-regulation of HUVECs adhesion molecules ICAM-1,VCAM-1 and its mechanism:Ⅰ.Through the q RT-PC experiment,it was found that when the concentration of LTCCS agonist was 20μM,the ICAM-1（DMSO VS 20μM P=0.02）,VCAM-1（DMSO VS 20μM,P=0.003）were significantly up-regulated.Ⅱ.The specific mechanism was studied by setting different stimulation time gradients with 20μM as the best stimulation concentration,and the Western Blot expression showed that ICAM-1,VCAM-1 and NF-κB and the phosphorylation level of PKC,Erk and IκB increased with time（P<0.05）.3.Study on the difference of HUVECs molecular expression after overexpression of CACNA1D gene.Ⅰ.Through the analysis of gene chip technology:compared with the NC control group,after CACNA1D overexpression,the number of up-regulated genes was 213,and the number of down-regulated genes was 260.Ⅱ.The results of classical pathway analysis based on IPA showed that leukocyte exudation-related signal pathways were significantly activated after CACNA1D overexpression.Ⅲ.The results of disease correlation analysis showed that CACNA1D was closely related to inflammatory diseases and cardiovascular diseases.Conclusions:1.LTCCs is expressed in endothelial cells and may play an important role in mediating the influx of fine Ca²⁺in vascular endothelium.2.Preliminary verification that the up-regulation of ICAM-1 and VCAM-1expression in endothelial cells after LTCCs activation may be related to the activation of Ca²⁺/PKC/Erk/NF-κB signal axis.3.The expression profile of HUVECs molecules changed significantly after CACNA1D overexpression,in which the expression of many adhesion molecules and leukocyte exudation-related signal pathways showed a tendency of up-regulation and activation.It indicates that Cav1.3 may be involved in the inflammatory response process of endothelial cells after overexpression...