Effects Of RNA Interference Targeting Angiotensin Type 1 Receptor And Angiotensin-converting Enzyme On Spontaneous Hypertensive Rats

ObjectiveHypertension is the importantly risk factor of congestive heart failure,stroke and renal failure.According to the statistics of World Health Organization,15-37%of all world adults are hypertensive patients.The incidence of hypertension even comes up to 50%in a group of over 60-year-olds.It is estimated that the number of hypertensive patients across the world will go up to 1,560,000,000 in 2025.However regrettably, only one third of patients has been treated successfully.The main cause is that hypertension is a kind of lifelong disease,but all drugs decreasing blood pressure have short half-life period which is no longer than 24 hours.Hence,patients must take medicine every day,which is not easy to insist on.Moreover,the nonspecific effects and side effects of drugs are also not easy to accept for patients.Therefore,pursuing a kind of lasting and effective antihypertensive gene therapy by regulating gene expression is always scientists' aim and dream.RNA interference(RNAi)is a new genetic interference technology,which specially degrades the corresponding sequence mRNA by the mediate of double-stranded RNA(dsRNA)which are diced up 21-23 nucleotide-long small interfering RNA(siRNA)by the enzyme Dicer to induce the post-transcriptional gene silencing.Short hairpin RNA(shRNA)is a kind of siRNA.The shRNA is more effective than the siRNA.In this study,we designed and constructed the expression vector of shRNA targeting ACE and AT₁R of RAS closely associated with hypertensive occurance and development in order to degrade ACE and AT₁R mRNA and decrease corresponding protein expression in vivo and vitro experiment with RNAi technology.By this way,we investigated the application prospective of RNAi in antihypertensive treatment.Methods1.Construction of eukaryotic expression vector for shRNA targeting ACE and AT₁R geneFirstly,four RNAi sites targeting rat ACE and AT₁R gene in GenBank were selected according to the guidelines for the selection of highly effective siRNA sequences and then BLAST test was performed.We selected two siRNA sequences with stronger effects of gene silence aiming at rat ACE and AT1R by a lot of previous experiments.Secondly,using molecule cloning technique,two pairs of oligonuleotides fragments were synthesized and annealed to double strands.Thirdly,the double strands were cloned into plasmid vectors PEGFP6-1 and Pgenesil-hH1.Fourthly,The recombinant plasmids were identified by restriction enzyme digestion and assayed sequence of them.The two kinds of recombinant plasmids were named pACE-shRNA and pAT₁R-shRNA.Finally,The target gene fragments of ACE and AT1R were identified by restriction enzyme digestion and assayed sequence of them.The recombinant plasmid carrying the promoter of hU6 and hH1 was named pACE-AT₁R-shRNA.2.Optimization of the transfection condition oftranfecting pACE-AT₁R-shRNA to rat aortic endothelial cells(1)The aorta was removed from rat in bacteria free condition.The vessel was opened longitudinally and cut into lmmxlmm pieces.These pieces were pasted inside culture bottle. Aortic endothelial cells were cultured in Dulbecco's modified Eagle's medium(DMEM) containing 10%or 20%heat-inactivated fetal bovine serum(FBS).Cells were identified by morphological(growth characteristic)and immunological(cellular markers)criteria.(2)Rat aortic endothelial cells was transfected with complex of different amount of pACE-AT₁R -shRNA and different concentration of METAFECTENE 2000(polycationic liposome transfection reagent).Transfection efficiency was determined by using fluorescence microscope and cells viability was calculated by using MTT assay after 48h of transfection to select the optimal ratio of pACE-AT₁R-shRNA to METAFECTENE 2000.3.Study pACE-AT₁R-shRNA on the inhibition of ACE and AT₁R mRNA and protein expression in rat aortic endothelial cellsRat aortic endothelial cells were transfected with complex of the optimal ratio of pACE-shRNA,pAT₁R-shRNA and pACE-AT₁R-shRNA to METAFECTENE 2000.Total RNA and total protein were extracted from aortic endothelial cells before transfection and after 24h, 48h,72h of transfection.The expression level of ACE and AT1R mRNA was evaluated by real-time quantitative PCR and the expression level of ACE and AT1R protein was evaluated by Western blot.Rat aortic endothelial cells were divided into five groups:(1)blank control group, (2)plasmid control group,(3)pACE-shRNA group,(4)pAT₁R-shRNA group,(5) pACE-AT₁R-shRNA group.4.Construction of recombinant adenoviral vectors expressing shRNA targeting rat ACE and AT₁R geneThe rat ACE-AT₁R-shRNA segments were obtained from plasmids PACE-AT₁R-shRNA which were constructed at an earlier date by restriction endonucleases digestion and then cloned into the shuttle plasmids pDC316 to form recombinant plasmids pDC316-hH1-AT₁RB-U6-ACEB-EGFP.The recombinant plasmids were cotransfected with genomic plasmids pBHGlox_E1,3Cre into 293 cells to package recombinant adenovirus which were named Ad5-hH1-AT₁RB-hU6-ACEB-EGFE Then the recombinant adenovirus were propagated,purified and identified.5.Study on the therapeutic effects of shRNA targeting ACE and AT₁R gene by adenovirus-mediated in spontaneously hypertensive rats(SHR)SHR were randomly divided into five groups:(1)blank control group(injected intravenously with normal saline),(2)adenovirus control group(injected intravenously with control adenovirus),(3)Ads-EGFP-ACE-shRNA treat group(injected intravenously with Ads-EGFP-ACE-shRNA),(4)Ad5-EGFP-AT₁R-shRNA treat group(injected intravenously with Ads-EGFP-AT₁R-shRNA),(5)Ad5-EGFP-ACE-AT₁R-shRNA treat group(injected intravenously with Ad5-EGFP-ACE-AT₁R-shRNA).At the same time,Wistar-Kyoto rats(WKY)were selected as normal control group which were injected intravenously with normal saline.All rats were injected two times at the first day and the seventeenth day in the experiment.The following items were measured during experiment.(1)Systolic blood pressure(SBP)of the caudal artery and heart rate were measured before and after injection.(2)Aorta,myocardium,cerebral,lung and kidney of rats in treat group were observed by using fluorescence microscope to identify the sites of Ads-EGFP-ACE-shRNA,Ad5-EGFP-AT₁R-shRNA and Ad5-EGFP-ACE-AT₁R-shRNA expression at the third day after first injection.(3)At the third day after first injection,serum concentration of ACE and Angâ…¡were measured by ELISA.The expression levels of ACE and AT₁R mRNA in aorta,myocardium,cerebral and kidney were evaluated by real-time quantitative PCR and the expression levels of ACE and AT₁R protein in aorta,myocardium,cerebral and kidney were evaluated by Western blot.(4)When the experiment was finished,the ratio of total heart weight(HW)to body weight(BW)and left cardiac ventricle weight(LU)to body weight(BW)were calculated.Myocardial hydroxyproline and collagen levels were measured. Aorta,myocardium,cerebral and kidney pathological changes were observed by light microscope,myocardial and kidney ultrastructures were observed by transmission electron microscope.At the same time,the functions of liver and kidney were measured.6.Study on the therapeutic mechanism of shRNA targeting ACE and AT₁R gene by adenovirus-mediated in spontaneously hypertensive rats(SHR)SHR group and therapy were same as above.The following items were measured during experiment.(1)Systolic blood pressure(SBP)of the caudal artery and heart rate were measured before and after injection.(2)At the third day after first injection,tissue content of ACE and Angâ…¡was measured by ELISA in myocardium,cerebral and kidney.The expression levels of ACE2 and AT₂R mRNA in aorta, myoeardium,cerebral and kidney were evaluated by real-time quantitative PCR and the expression levels of ACE2 and AT₂R protein in aorta,myocardium,cerebral and kidney were evaluated by Western blot.(3)At the third day after first injection,serum concentration of NO was measured, tissue content of NO was measured in myocardium,cerebral and kidney.Results1.Restriction enzyme digestion analysis showed that eukaryotic expression vectors of shRNA targeting ACE and AT₁R gene had been constructed successfully.Sequencing results revealed that the sequences of the vectors were all right.2.(1)After three days of explant culture,endothelial cells migrating out of the aortic intima could be seen at the edge by a inverted phase-contrast microscope and formed confluent monolayers which showed a cobblestone shape 10 days later.Passaged cells grew faster than primary cells and could form confluent monolayers 4-5 days later.After passaged four generations,cells became deformed,detached from the culture bottle.Characteristic green fluorescence was observed by fluorescence microscope after immunofluorescence labeling for von Willebrand factor.(2)The optimal ratio of pACE-AT₁R-shRNA to METAFECTENE 2000 was"1ug:4ul"according to transfection efficiency and cells viability.At this transfection condition,transfection efficiency was 57.7%and cells viability was 91.6%.3.Compared with control groups,the expression level of ACE mRNA and protein of pACE-shRNA group and pACE-AT₁R-shRNA group were significantly suppressed after 48h of transfection(P＜0.05),the expression level of AT₁R mRNA and protein of pAT₁R-shRNA group and pACE-AT₁R-shRNA group were significantly suppressed after 48h of transfection (P＜0.05)and both were nearly undetectable 72h later(P＜0.01).The suppression effect of pACE-AT₁R-shRNA group was stronger,the result was caused by cooperative effects of double genes combined silencing.There were no significant difference about expression level of ACE and AT₁R mRNA and protein in blank control group and plasmid control group before and after transfection.4.Recombinant adenoviral vectors Ad5-EGFP-ACE-AT₁R-shRNA were constructed successfully,which was confirmed by restriction enzyme digestion and PCR and EGFP expression.5.(1)There was no significant difference in the SBP of the caudal artery among SHR groups before intervention(P＞0.05),but SBP of the caudal artery of all SHR groups was much higher than that of normal control group(P＜0.01).After two days of single injection,SBP of the caudal artery of treat group was effectively reduced by(25.2±5.3)mmHg,(23.5±4.8)mmHg and (27.1±6.4)mmHg respectively.The biggest range reached 24mmHg,22mmHg and 26 mmHg. This antihypertensive effect could last up to 15 days.After two days of the second injection,SBP of the caudal artery of treat group was effectively reduced by(22±6)mmHg,(20±5)mmHg and (23±7)mmHg.The biggest range reached 23mmHg,22mmHg and 23mmHg.This antihypertensive effect could last up to 20 days.However,rats of blank control group and adenovirus control group produced a continued increase in blood pressure and without significant difference between these two groups.(2)Under the fluorescence microscope,marked expression could be observed in heart,cerebral,lung,aorta and kidney.(3)Serum concentration of ACE in each treat group was significantly lower than that of blank control group and adenovirus control group respectively(P＜0.05),similar to that of normal control group(P＞0.05). Serum concentration of Angâ…¡in Ad5-EGFP-ACE-shRNA group was significantly lower than that of blank control group and adenovirus control group(P＜0.05).Serum concentration of Angâ…¡in Ad5-EGFP-AT₁R-shRNA group was significantly higher than that of blank control group and adenovirus control group(P＜0.05).Serum concentration of Angâ…¡in AdS-EGFP-ACE-AT₁R-shRNA was between above two treatment groups.The expression levels of ACE and(or)AT1R mRNA and protein in heart,cerebral,aorta and kidney of each treat group were significantly decreased compared with blank control group and adenovirus control group(all P＜0.05),The expression levels of ACE mRNA and protein in AdS-EGFP-AT₁R-shRNA group were similar to that of blank control group and adenovirus control group(P＞0.05).(4)Compared to blank control group and adenovirus control group,myocardial,cerebral,aorta and kidney hypertrophy and ultrastrueture of myocardial and kidney in each treat group were significantly improved.The LV/BW and the level of myocardial collagen of treat group were all significantly lower than those of blank control group(both P＜0.05)and adenovirus control group(both P＜0.05),hut still higher than those of normal control group (P＞0.05).(5)The heart rate and the function of kidney and liver were not affected during the experiment.6.(1)Serum concentration of ACE and Angâ…¡in myocardial,cerebral and kidney of each treat group was significantly lower than that of blank control group and adenovirus control group respectively(P＜0.05),similar to that of normal control group(P＞0.05).(2)Serum and tissue of myocardial,cerebral and kidney concentration of NO in each treat group was significantly higher than that of blank control group and adenovirus control group(P＜0.05),similar to that of normal control group(P＞0.05).(3)The expression levels of ACE2 mRNA and protein in heart, cerebral,aorta and kidney of each treat group were significantly higher compared with blank control group and adenovirus control group(all P＜0.05),similar to that of normal control group(P＞0.05).The expression levels of AT₂R mRNA and protein in heart,cerebral,aorta and kidney of blank control group,adenovirus control group and Ad5-EGFP-AT₁R-shRNA group were significantly higher compared with normal control group,Ad5-EGFP-ACE-shRNA group, Ad5-EGFP-ACE-AT₁R-shRNA group(all P＜0.05),especially Ad5-EGFP-AT₁R-shRNA group was higher.Conclusions1.Construct successfully eukaryotic expression vector pACE-AT₁R-shRNA. 2.By explant technique,we successfully accomplished the isolation,primary culture and passaged culture of rat aorta vascular endothelial cells.The cells purity was above 90%.By optimizing the parameters,METAFECTENE 2000 could transfect pACE-AT₁R-shRNA to rat aorta vascular endothelial cells with high effect,at the same time,with high cells viability.3.Transfection of pACE-AT₁R-shRNA to rat aorta vascular endothelial cells could initiate sequence-specific post-transcriptional gene silencing and inhibit the expression of ACE and AT₁R mRNA,subsequently inhibit the protein expression.4.Recombinant adenovirus vectors expressing ACE-AT₁R-shRNA were constructed successfully,which laid the foundation of application ofgene therapy to antihypertension.5.Introduction of shRNA targeting ACE and AT₁R gene by adenovirus-mediated to SHR led to successful inhibition of ACE and(or)AT₁R mRNA and protein,which subsequently produced significance and lasting decrease in blood pressure.Moreover,myocardial remodeling was significantly improved.Myocardial,cerebral,aorta and kidney hypertrophy and ultrastructure of Myocardial and kidney in each treat group were significantly improved.At the same time,there were no obvious side-effects.Combined gene silence group was superior to single gene group(P＞0.05),probably by the cooperative effects of double genes combined silencing.The RNAi technology may become a new strategy of gene therapy for hypertension.The experiment may offer clinical application prospect for combined silencing therapy to antihypertension with low dosage of Ad5-ACE-AT₁R-shRNA.6.Introduction of shRNA targeting ACE and AT₁R gene by adenovirus-mediated to SHR led to successful expression of AT₂RmRNA/AT₁RmRNA and ACE2 mRNA and protein,block the imbalance expression of two types carboxypeptidase,which subsequently produced significance and lasting decrease in blood pressure and avoided damage of target organs in hypertension;meanwhile,ACE and Angâ…¡in myocardial,cerebral and kidney of each treat group was significantly lower,NO in each treat group was significantly higher than that of blank control group and adenovirus control group(P＜0.05).