The Effect And Mechanism Of Kv7 Channels On Placental Chorionic Arteries Vessels From Preeclampsia Complicating Pregnancy

Objective: The aim of this study was to extend prevsious work on Kv7 channels of CPAs by establishing the expression profiles of KCNQ subunits on CPAs taken from women with preeclampsia(PE),as well as normotensive controls(NP),and examining the functional relevance of the data on an altered expression profiles.To study the effect and involved mechanisms of Kv7 channels to the vasomotor of placental chorionic arteries from Preeclampsia complicating pregnancy.Methods:(1).Measurement of vascular tension in vitro:(1)The effect of XE991(Kv7 channels blocker;10-7-10-4M)was assessed on basal artetial tone of CPAs from NP group(n=10)and PE group(n=10),and analysis the action of basal tension changes of XE991(10-6,10-5,5x10-5 mol/L)on CPAs vascular ring of NP and PE group;(2)the effect of thromboxane A2 analogue U46619(10-10-3X10-6mol/L)on CPAs vascular tension of NP group(n=10)and PE group(n=10)in presence or absence of XE991(10-5 mol/L);and analysis the different effect of XE991(10-5 mol/L)on the contraction of U46619 on the CPAs vascular tension of NP group and PE group;(3)the effect of different types Kv7 channels openers[Retigabine ? BMS-204352 ? ICA-27243 ? ML277(10-7-10-4 mol/L)]to U46619-precontraction CPAs vascular rings tension from NP group(n=10)and PE group(n=10);(2).Quantitative RT-PCR:15 patients from preeclampsiacomplicating pregnancy as PE group,and 15 normotensive patients as NP group;then extract total RNA of CPAs vascular smooth muscle,synthesize cDNA and carry out SYBR Green I real-time fluorescence quantitative PCR reaction,get the corresponding Ct value,calculate?Ct(=the target gene Ct subtract the Ct value of reference gene),the first sample of NP group as reference factors to calculate??Ct(=?Ct value of target gene subtract ?Ct value of the first sample gene of NP group),carry out the relative expression after processing of 2-??Ct;(3).Western blotting:the research object is derived from the QT-PCR experiments;First,total protein of CPAs vascular smooth muscle was extracted and determinate the protein concentration,separate the protein samples through SDS-PAGE electrophoresis;then transfer to the PVDF film,and followed with antibody incubation;last,imaging with Bio-Rad gel system,measurement the gray value with Quantity-One software,the ratio of target protein and ?-Actin protein grey value as the relative expression of target protein.The result of all experiments are expressed in meanstandard deviation(),the SPSS 17.0 and GraphPad Prism 5.0 software were used for statistical analysis.Results:(1).the results of measurement of vascular tension in vitro:(1).KCNQ channels blockers XE991(10-8-10-4 mol/L)induced concentr ation-dependent vasoconstrictions of CPAs artetial rings from NP group(n=10)compared with DMSO control group(n=10,?P>0.05,**P<0.01,***P<0.001,two-way ANOVA);the most significant change was achieved at5×10-5M/L,the mean contraction at this concentration had an amplitude44.67±9.52% of that induce by 80 mM K+;When upon addition of 10-4M/L,Vessels displayed relaxtion with tension amplitude returning around29.61±6.47% towards the baseline.Contrast to the obvious contraction effects of the channels broad spectrum blocker XE991 in vascular basal tone of normotensive CPAs,the ability of XE991(10-6,10-5,5x10-5M/L)to contract CPAs of preeclampsia was considerably attenuated(n=10,normotensive vs.preeclampsia by two-way ANOVA,*P<0.01,***P<0.001).(2).A significantly increased U46619(10-10-3X10-6 mol/L)-induced contraction of CPAs was observed in the presence of 10-5 M/L XE991(n=10;each concentration showed significance,*P>0.05,**P<0.01 vs.XE991-absent control;).The potent contraction effect of XE991(10-5M/L)on U46619(10-8,10-7,10-6M/L)-induced contraction was almost abrogated in CPAs of preeclampsia women.(n=10,normotensive vs.preeclampsia by two-way ANOVA,?P>0.05).(3)BMS-204352(n=10)and retigabine(n=10)produced a concentration-dependent relaxation in the U46619(10-7M)precontracted CPAs,two-way ANOVA test comparing the Kv7 activators and DMSO at each concentration show significance,**P<0.01?***P<0.001.But ICA-27243 and ML277 relatively had no effect in tension of the precontracted CPAs(?P>0.05,?P>0.05,two-way ANOVA).The ability of the Kv7 channel activators to relax agonist-induced CPAs vessels from preeclampsia women was significantly altered.The effect of BMS-204352(n=10)and retigabine(n=10)to the relaxation of U46619(10-7M/L)-precontracted CPAs vessels from preeclampsia women wasalso obviously down-regulated(?P>0.05,*P<0.05,**P<0.01,***P<0.001,two-way ANOVA)and interestingly the relaxation ability of ICA-27243 to precontracted CPAs vessels of preeclampsia women(n=10,*P<0.05,two-way ANOVA)was enhanced in 10-5M/L and 10-4M/L Application of BMS-204352(10-4M)and retigabine(10-4M)only relaxed 9.99±15.67% and32.39±5.60% of the vessel contraction in the preeclampsia CPAs.The relaxation of ICA-27243(10-4M)to precontracted CPAs from preeclampsia women(n=10)increased to 47.20±13.04% compared with a 23.91±8.83%relaxation in the normotensive CPAs.?.The relative expression quantity of QT-PCR experiments for KCNQ1-5 gene in CPAs vessels from NP group(n=15)and PE group(n=15)was respectively as follows: 1.1148±0.0351 ?1.0172±0.0247 ? 1.0337±0.0224 ? 1.0223±0.0473 ? 1.0280±0.0510 ?1.1017±0.0547 ? 0.9993±0.0589 ? 1.7448±0.0678 ? 0.1123±0.0117 ?0.1133±0.0129 in the PE group.There was no signifinant differences between the two groups for KCNQ1 and KCNQ2(?P>0.05,?P>0.05),the KCNQ3 gene mRNA levels of PE group were significantly increased(***P<0.001),the KCNQ4 ? KCNQ5 gene mRNA levels of PE group were obviously lower(***P<0.001,***P<0.01).?.The relative protein expression level of KCNQ3?KCNQ4?KCNQ5 gene in the NP group(n=15)and PE group(n=15)was respectively as follows: 0.1845±0.032?0.6147±0.0426?0.5738±0.0192 in the NP group and 0.5083±0.0377?0.2657±0.0461?0.2594±0.0587 in the PE group,the relative protein expression level of KCNQ3 gene in the PE groupwere significantly increased(***P<0.01),on the contrary,the level of KCNQ4?5 gene were obviously lower(***P<0.01 ? ***P<0.01).Conclude: 1.Kv7 channels can effectively regulate CPAs vascular smooth muscle excitability and the vascular tension of CPAs vessels from normotensive pregnancy,blocking these channels can caused vasoconstriction response;Compared with CPAs vessels from normal pregnancy,the function impact of Kv7 channels to the tension of CPAs vessels from preeclampsia was significantly diminished;2.The expression of mRNA for KCNQ3 was specifically up-regulated,whereas that for KCNQ4 and KCNQ5 was down-regulated in CPAs of preeclampsia women compared with normotensive controls.Same observations were found in subsequent probing for protein levels of KCNQ 3-5 in the CPAs vessels from preeclampsia women.3.KCNQ4 and KCNQ5 channel uniforms play a predominant role in regulating the the vascular tension of CPAs vessels.Significant decreased function effects of Kv7 channels to the tension of CPAs vessels from preeclampsia might be predominantly mediated by considerable lower expression of KCNQ4 and KCNQ5 found in CPAs of preeclampsia women in our study.4.the up-regulation of KCNQ3 relevant to preeclampsia may contribute to the vasodilation of CPAs vessels from preeclampsia.The up-regulation of KCNQ3 isoforms could be viewed as a compensation mechanism for the reduced placental perfusion of preeclampsia women caused by considerable down-regulation of KCNQ4 and KCNQ5.