Knockdown Of Angiotensin II Receptor Subtype 1a (AT_1aR) By RNA Interference And The Effects Of Which On Blood Pressure And Ventricular Hypertrophy Of Rats With Renovascular Hypertension

Background & ObjectiveHypertension is a clinical syndrome caused by complex factors of genetics and environment. It can result in progressive damage of many target organs such as brain, heart, kidney, eye and aorta and is the chief criminal of the cardiovascular and cerebrovascular diseases.Rennin-angiotensin-aldosterone system (RAAS) plays important role in the pathophysiologic process of hypertension and the related target organ damages, and in which , the angiotensin II is the key effector molecule, as a circulatory or autocrine/paracrine hormone, mainly through the angiotensin II receptor type1(AT1) pathway, leading to a series of biological effect, such as vasoconstriction, aldosterone and vasopressin release, salt and water retention, sympathetic activation. In addition, Ang II, via AT1 receptor, directly causes cell growth, proliferation and migration, regulates the gene expression of various bioactive substances, and activates multiple intracellular signaling cascades. These actions are supposed to participate in the pathophysiology of cardiac hypertrophy and remodeling, heart failure, vascular thickening, atherosclerosis, and glomerulosclerosis. AT1 receptor has become an important intervention target of current medication on cardiovascular disease, many selective antagonists such as losartan, candesartan, valsartan etc have been developed and in use clinically.Despite several drugs for the treatment of hypertension, there are many patients with poorly controlled high blood pressure. This is partly due to that all of the available drugs are short-lasting (<24 hours), have side effects, and are not highly specific. Gene therapy offers a possibility of producing longer-lasting effects with precise specificity based on the genetic design. Previous studies on gene therapy for hypertension have taken 2 approaches. One is to increase vasodilator proteins such as kallikrein, atrial natriuretic peptide, and endothelin NO synthase , et al. The other is anti-sense approach, including anti-sense oligodeoxynucleotides and anti-sense of the full-length DNA to decrease the vasoconstrictors or their receptors such as angiotensinogen and the angiotensin type 1 (AT1) receptor. All the above studies show a decrease in blood pressure lasting several days to weeks or months.RNAi, an ancient and ubiquitous phenomenon, had been cognized not long ago as a kind of post-transcriptional gene silencing mechanism induced by double strand RNA (dsRNA). 21- to 23-nucleotide, double-stranded RNAs can act as small interfering RNAs (siRNAs) to elicit gene specific inhibition in mammalian cells has made RNAi possible in mammalian systems as a brand-new anti-sense technology for gene knockdown. Recent exciting advances allow delivery of siRNAs into mammalian cells by a plasmid or viral DNA vector.In this study, we choose the angiotensin II receptor subtype 1a (AT1aR) of the rat as a target and choose a plasmid vector to deliver specific siRNAs. We intended to selectively knockdown the expression of AT1a receptor on rat vascular smooth muscle cells ( VSMCs ) and neonatal rat ventricular myocytes (NRVMs) by RNA interference, and test the effect on cellular viability and proliferation of VSMCs in vitro. In addition, we attempted to test the effect of RNA interference (RNAi) targeting AT1a receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C (2-kidney, 1-clip) renovascular hypertension. This is our first step to explore the possibility of RNAi as a gene interference strategy for hypertension and the related target organ damage.Methods1 Two RNA interference plasmids (pAT1a-shRNA1 and pAT1a-shRNA2) targeting AT1a receptor gene (Agtr1a) and a control plamids pGenesil-Control (pCon) not targeting any gene of rats were designed, constructed, identified, prepared and purified successively. In which, pAT1a-shRNA1 carried an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 928-946; pAT1a-shRNA2 carried an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 978-996; and pCon carried an U6 promoter and a nonspecific shRNA-coding sequence.2 In vitro experiments: The primary cultured rat aortic vascular smooth muscle cells ( VSMCs ) and neonatal rat ventricular myocytes (NRVMs) were transfected by plasmids pAT1a-shRNA1, pAT1a-shRNA2, the control plasmid pCon or nothing (Blank control). 48 hours later, the expression of AT1a, AT2 mRNA and protein were analyzed by semi-quantified RT-PCR and Western-blot respectively, normalized to the internal control geneβ-actin. A colorimetric assay (A490nm) with MTT was adopted to test cellular viability and proliferation of VSMCs.3 In vivo experiments:The renovascular hypertensive rats models were constructed by two-kidney one-clip (2K1C) methods. Rats with renovascular hypertension were randomly divided into 5 equal groups: pAT1a-shRNA1 group,pAT1a-shRNA2 group and pCon group (injected with plasmids pAT1a-shRNA1,pAT1a-shRNA2 and pCon 4mg/kg respectively via tail vein just only once), valsartan group (perfused into the stomach with valsartan, a AT1 receptor inhibitor, 30mg·kg-1·d-1) and blank group (without any treatment). Three weeks later, the systolic pressure of the caudal artery was measured, catheterization through carotid artery was conducted to measure the the systolic blood pressure (SBP), diastolic blood pressure (DBP), and draw the left ventricular pressure curve. Then the rats were killed; the weight of the heart was measured, the ratio of left ventricular weight to body weight (LV/BW) was calculated, and pathological examination of the heart and thoracic aorta was performed. Western blotting was used to detect the protein expression of AT1 in the ventricle and aorta.Another 18 age-matched healthy male rats were used and randomly divided into 3 groups, named as"Normal blank"group (without any intervention) ,"normal pAT1a-shRNA1"group and"normal pAT1a-shRNA2"group. The latter two groups were injected with plasmids pAT1a-shRNA1 and pAT1a-shRNA2 4mg/kg respectively via tail vein just only once.Results1. Three plasmids pAT1a-shRNA1, pAT1a-shRNA2 and pCon, each containing a shRNA-expressing template, were successfully constructed, prepared and purified.2. In vitro experiments:In cultured VSMCs, plasmid pAT1a-shRNA1 resulted in 82% reduction of AT1a mRNA and 69% reduction of AT1 protein, pAT1a-shRNA2 resulted in 77% and 56% reduction in AT1a mRNA and protein levels respectively. In cultured NRVMs, pAT1a-shRNA1 resulted in 70% reduction of AT1a mRNA and 67% reduction of AT1 protein, pAT1a-shRNA2 resulted in 66% and 52% reduction in AT1a mRNA and protein levels respectively. On the contrary, the plasmid pCon didn't induce significant reduction of AT1a mRNA and protein in the two kinds of cells and in which AT2 receptor level was not affected by any of the plasmids.The A490nm values of the VSMCs were similar within groups absence of AngII but decreased significantly in pAT1a-shRNA1 and pAT1a-shRNA2 groups within groups presence of AngII.3. In vivo experiments:There was no significant difference in the caudal artery pressure among the 5 groups (all:P>0.05) before intervention. Three weeks later: the caudal artery pressures of the blank group and pCon group continued to increase by about 25mmHg compared to the values before intervention (both P <0.001) and had no significant difference between these two groups; However, the caudal artery pressures of the pAT1a-shRNA1,pAT1a-shRNA2 and Valsartan groups were 15.1mmHg±5.4 mmHg, 16.4mmHg±8.4 mmHg, 30.6mmHg±18.2 mmHg lower than those before intervention respectively (all P <0.01) and were also significantly lower than those of the blank group (P <0.01 or P <0.05); The carotid pressure of the pAT1a-shRNA1,pAT1a-shRNA2 and Valsartan groups were194 mmHg±5mmHg, 200 mmHg±5mmHg, 164 mmHg±5mmHg, all significantly lower than those of the blank and the pCon groups (234 mmHg±10mmHg and 232 mmHg±7mmHg respectively; all P <0.01); There was no significant difference in the±dp/dt max value of left ventricle and indicators of renal function among the groups; The LV/BW of the pAT1a-shRNA1,pAT1a-shRNA2 and Valsartan groups were 2.27±0.37, 2.31±0.26, 2.26±0.39, all significantly lower than that of the blank and the pCon groups (3.24±0.38 and 2.94±0.06 respectively; all P <0.01), similar to that of the"normal blank"group (P >0.05). The myocardiocytes were significantly hypertrophic and the arterial tunica media was significantly thickened in the blank group and such changes were all improved to different degrees in the pAT1a-shRNA1,pAT1a-shRNA2 and Valsartan groups; The protein expression of AT1 receptor in the myocardium of the pAT1a-shRNA1 and pAT1a-shRNA2 groups were 53.3% and 47.8% lower than that of the blank group respectively, and that in the thoracic aorta of the pAT1a-shRNA1 and pAT1a-shRNA2 groups were 58.7% and 49.3% lower than that of the blank group respectively (all P <0.01); however, there was no significant difference in the protein expression of AT1 receptor in the myocardium and thoracic aorta between the valsartan and blank groups (both P >0.05). There was no significant difference in the caudal artery pressure before and after injection with the plasmid in both of the"normal pAT1a-shRNA1"and"normal pAT1a-shRNA2"groups; The carotid pressure of the"normal pAT1a-shRNA1"and"normal pAT1a-shRNA2"groups were not significantly different from that of the"Normal blank"group.ConclusionRNAi can specifically knockdown AT1a expression on primary cultured VSMCs and neonatal rat ventricular myocytes (NRVMs) and relieve the cell proliferation of VSMCs stimulated by AngII without injury on cellular viability. RNA interference targeting AT1a receptor inhibits the development of the renovascular hypertension and the accompanying cardiac hypertrophy, however, it doesn't affect the blood pressure of normal rats significantly. The RNAi technology may become a new strategy of gene therapy for hypertension and other cardiovascular diseases.