Angiotensin-Converting Enzyme And Angiotensin-Converting Enzyme 2 In Spontaneously Hypertensive Rats And The Effects Of Enalapril On Them

Objective(1)To observe the blood pressure,body weight and ventricular remodeling in spontaneously hypertensive rats(SHR) and wistar-kyoto rats(WKY),whereby to search the pathophysiological features of SHR.(2) To search the effects of enalapril on blood pressure,ventricular remodeling in SHR.( 3 ) To observe the cardiac expression of mRNA and protein of angiotensin-converting enzyme(ACE) and angiotensin-converting enzyme 2(ACE2) in SHR and WKY rats,whereby to explore the role played by ACE and ACE2 in the development of hypertension in SHR.(4) To search the effects of enalapril on the cardiac expression of mRNA and protein of ACE and ACE2 in SHR,whereby to explore the function of ACE and ACE2 in enalapril's curative mechanisms.Methods(1) Animal protocol: 15 SHR were randomly assigned to two groups: SHR control group(n=7)which were treated with vehicle and SHR enalapril group(n=8)which were treated with enalapril (15mg.kg-1.d-1).The treatment lasted 4 weeks.During this process,the body weight was measured once per week,and the dose was adjusted in accordance with the body weight. 10 WKY rats surved as the normotensive control group which were also treated with vehicle.(2) Blood pressure: the systolic pressure was measured using the non-invasive tail cuff method once per week during the procedure. (3) Sample collection:after the treatment, sodium pentobarbital was injected transperitoneally to anaesthetize the rats. The thoracic cavity was cut open and the heart was separated. Then the left ventricle was dissected carefully. A thin transverse slice was removed from the midline and fixed in 10% buffered formalin for immunohistorical staining.The remaining left ventricular pieces were frozen preserved in liquified nitrogen for in vitro reverse transcription-polymerase chain reaction (RT-PCR) and western blot protein staining.(4)Left ventricular mass(LVM) and left ventricular mass index(LVMI):LVM and LVMI can be parameters indicating left ventricular remodeling.LVMI is the ratio of left ventricular mass to body mass.The body mass was measured before animals were killed and left ventricular mass was weighed after being dissected. LVMI was obtained by calculating the ratio of left ventricular mass to body mass.(5)Quantification of ACE and ACE2 mRNA:the total RNA was isolated by using Trizol and the concentrations of total RNA were examined. 4μg of total RNA was reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase in a mixture containing deoxyribonucleotides,random hexamers,and Rnase inhibitor in reverse transcriptase buffer.3μl of the resulting cDNA was used for amplification of the gene of ACE,ACE2 and EF1α(elongation factor 1α) which served as control gene. Amplification product was separated on a agarose gel.The optical density of the bands was quantified by computerized densitometry.The target mRNA concentration was expressed as the ratio of target to control EF1α.(6)Western blot:the membrane protein was isolated using cell lysis solution containing proteinase inhibitor. The concentrations of the total protein were measured using Bradford method. 10μg total protein per sample was used for SDS-PAGE gel electrophoresis.Then the proteins were transferred onto nitrocellulose membranes. Western blotting of ACE,ACE2 and controlβ-actin were performed and the the membranes were visualized by chemoluminescence and photographed by X ray. The protein bands were examined and analyzed by densography. The target protein was expressed as the ratio of target to controlβ-actin.(7) Immunohistochemistry:the method of streptavidin-biotin complex was used for ACE and ACE2 immunohistochemical staining.Sections were dewaxed and hydrated.Then endogenous peroxidase activity was quenched in 3% H2O2. After blotting sections using rabbit serum,the primary antibody followed by second antibody and SABC succesively were added and incubated.The diaminobenzidine (DAB) was used to visualize antibody binding.Then the sections were dehydrated by ethonal and were cover-slipped according to conventional procedures.Finally,sections were examined under a Olympus microscope.(8) Statistics: Data were anlyzed using SPSS 11.0 software.Each value was expressed as mean±SEM.Significant differences were obtained when P<0.05.Differences between two groups were analyzed using t-test.One-way ANOVA was used to analyze the differences between multiple groups.Results(1) Body mass and blood pressure: the body weight of SHR was lower and blood pressure significanly higher than the counterpart of WKY not only at week 16(380±23g vs 339±21g, P<0.05; 191±19 mmHg vs 122±14mmHg,P<0.01),but at week 20 as well(415±25g vs 376±20g,P<0.01; 187±11 mmHg vs 116±4mmHg, P<0.01).(2) Ventricular remodeling and the effect of enalapril on it in SHR:at week 20, both LVM and LVMI in SHR were obviously higher than that in WKY(782±24 mg vs 1102±81 mg,P<0.01;1.88±0.12 mg/g vs 2.96±0.17 mg/g,P<0. 01).Both LVM and LVMI in enalapril group were lower than that in SHR cntrol group(906±128mg vs 1102±81 mg,P<0.01; 2.43 mg/g±0.29 vs 2.96±0.17 mg/g,P<0. 01),but still higher than that in WKY group(906±128mg vs 782±24mg,P<0.05;2.43±0.29 mg/g vs 1.88±0.12 mg/g,P<0. 01).HE staining showed that there was obvious cardiac hypertrophy in SHR when compared with WKY.But there was no obvious appearance of cardiac hypertrophy in enalapril group whose cardiac myocyte was almost as fine as WKY's.(3) The evolvement of blood pressure during the period from week 16 to week 20:The five values of blood pressure in SHR control group were succesively 188±24mmHg,187±17 mmHg,189±18 mmHg,185±15 mmHg,187±11 mmHg. There was no significant difference between these values (P>0.05).The five values of blood pressure in WKY group were succesively 122±14 mmHg,124±10 mmHg,118±13 mmHg, 120±15 mmHg,116±4 mmHg and there was also no significant difference between them(P>0.05).One week after the treatment had begun, the blood pressure of enalapril group was reduced from 195±16mmHg to 150±11 mmHg,the greatest decrease during that4 weeks(P<0.01). The third, fourth and fifth blood pressure in enalapril group were respectively 140±9 mmHg,140±12 mmHg and 138±11 mmHg and there was no significant difference between adjacent blood values (P>0.05).The last blood pressure in enalapril group was significantly decreased when compared with SHR control group(138±11 mmHg vs 187±11 mmHg, (P<0.01),but was still higher than that of WKY group(138±11 mmHg vs 116±4mmHg,P<0.01).(4) The expression of ACE and ACE2:the expression of cardiac ACE mRNA and protein in SHR was obviously higher than that in WKY(ACE/EF1α:1.68±0.34 vs 0.33±0.12,P<0.01;ACE/β-actin:1.21±0.14 vs 0.71±0.11,P<0.01),whereas the levels of ACE2 mRNA and protein were significantly lower than that in WKY(ACE2/EF1α:0.50±0.15 vs 1.16±0.24,P<0.05;ACE2/β-actin:0.71±0.24 vs 1.22±0.14,P<0.05). (5) Effect of enalapril on ACE and ACE2:the expression of both ACE mRNA and ACE protein was obviously decreased after enalapril treatment when compared with the SHR control group(ACE/EF1α:0.44±0.19 vs 1.68±0.34,P<0.01;ACE/β-actin:0.87±0.13 vs 1.21±0.14,P<0.05 ).As for ACE2,the expression of mRNA was significantly elevated after enalapril treatment when compared with SHR control group(ACE2/EF1α: 1.77±0.49 vs 1.16±0.24,P<0.05),but ACE2 protein remained unchanged ( ACE2/β-actin:0.42±0.22 vs 0.71±0.24,P>0.05 ) ,still markedly lower than that in WKY( ACE2/β-actin:0.42±0.22 vs 1.22±0.14,P<0.01 )。(6)In cardiac tissue of SHR and WKY,ACE and ACE2 immunoreactivity was predominantly localized to cardiomyocytes,vascular endothelium and was faintly seen in smooth muscle cells.The cardiac ACE immunoreactivity was stronger and ACE2 immunoreactivity lower in SHR than the counterpart in WKY.Enalapril could qualitatively reduce ACE immunoreactivity , but didn't result in obvious change of ACE2 immunoreactivity. Conclusions(1) The blood pressure of SHR was higher than that of WKY at the same age.During the period from week 16 to week 20,the blood pressure of SHR was stable and there was no obvious evolvement.(2) At the age of week 20,there was an obvious ventricular remodeling with significantly elevated LVM and LVMI in SHR compared with WKY rats.(3) Enalapril could significantly reduce the blood pressure and reverse the ventrilular remodeling of SHR.(4) The expression of cardiac ACE mRNA and protein were remarkably increased,whereas ACE2 mRNA and protein were notably decreased in SHR.This kind of disbalance between ACE and ACE2 might take part in the development of hypertension of SHR.(5) Enalapril could markedly reduce the expression of ACE mRNA and protein and could significantly elevate the level of ACE2 mRNA,but had no effect on the expression of ACE2 protein.This kind of effect of enalapril on ACE and ACE2 might play a role in the enalapril's functional mechanisms.(6) In cardiac tissue of SHR and WKY,Both ACE and ACE2 were expressed predominantly on the membrane of cardiomyocytes,vascular endothelium and less on the membrane of smooth muscle cells.