Effects Of Renin-angiotensin System Inhibitors On ACE2-Ang-1（1-7）and Inflammatory Factors In Cultured Vascular Smooth Muscle Cells Of Spontaneous Hypertension Rats

Objective: To observe the effects of different renin-angiotensin system (RAS) inhibitors onACE2-Ang-(1-7) and inflammatory factors in cultured vascular smooth muscle cells (VSMC) ofspontaneous hypertension rats and to explore the possible mechanism that different RAS inhibitorsaffect ACE2-Ang-(1-7) and the relationship with anti-inflammation.Methods:1. VSMCs cultureVSMCs were cultured from thoracic aorta of6weeks old spontaneous hypertension rats(SHR)by modified adherence method. Cultured VSMCs before third generation were used in the study.2. Effects of AngⅡand Ang-(1-7) on proliferation of cultured VSMCsCultured VSMCs were randomly grouped as follow:control group, AngⅡgroup,ang-(1-7)group and AngⅡ+ang-(1-7) group. The cells were cultured for48hours, and then theproliferation rate of cultured vSMCs was measured by the method of3-（4,5-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay.3. Effects of different RAS inhibitors on proliferation of cultured VSMCsCultured VSMCs were randomly grouped as follow: control group,aliskiren group captoprilgroup and losartan group, and the cells were cultured for48hours, and then the proliferation rate ofculturedVSMCs was measured by the method of MTT assay.4. Effects of different RAS inhibitors on Ang(1-7),CRP, IL-6in cultured VSMCsCultured VSMCs were randomly grouped as follow: control group, aliskiren group,captoprilgroup and losartan group, and then were cultured for72hours. Ang-(1-7),interleukin-6(IL-6) andC-reactive protein (CRP) level in the supernatant of cultured VSMCs were respectively measuredby Enzyme linked immunosorbent assay (ELISA).5. Effects of different RAS inhibitors on ACE2expression in cultured VSMCs ACE2mRNA was extracted from VSMCs after culturing with different RAS inhibitors andwas measured by the method of qualitative reverse transcription-polymerase chain reaction (RT-PCR) respectively.Results:1. Cells were spindle-shape before convergence, then growed a typical state of "peak andvalley" after convergence under the microscope.2. Ang II stimulated the proliferation of VSMCs and ang-(1-7) inhibited it in a concentration-dependent manner (P<0.05). In addition, the proliferation induced by angII was completelyreversed by ang-(1-7).3. Aliskiren, captopril and losartan inhibited the proliferation of VSMCs in a concentrationdependent manner(P<0.05).4.(1) Aliskiren decereasd ang-(1-7) level in the supernatant of cultured vSMCs in aconcentration-dependent manner (0.1-100μM)(P<0.05) and on the other hand, captopril andlosartan increased the ang-(1-7) level in the supernatant of cultured vSMCs(P<0.05), in aconcentration-dependent manner(0.1-100μM), as compared with control group respectively.(2) Aliskiren,captopril and losartan decreased the concentration of CRP and IL-6in thesupernatant in a concentration-dependent manner(0.1-100μM), as compared with control grouprespectively (P<0.05).5. No significant difference of ACE2mRNA expression was found between any twoRASinhibitors separately (P>0.05).Conclusions:Ang II stimulated the proliferation of cultured VSMCs and ang-(1-7) inhibited it. Aliskiren,captopril and losartan inhibited proliferation of cultured VSMCs and reduced the expression ofinflammatory factors. Both captopril and losartan increased ang-(1-7) production, but aliskirendecreased the production. There was no significant difference of ACE2mRNA expression betweenany two RAS inhibitors. These results suggest that captopril and losartan increase and aliskirendecrease the production of ang-(1-7) is related to the higher or lower level of ang-(1-7) substrate,but not to the ACE2activity. The anti-inflammatoy effects of RAS inhibitors is also independent tothe ACE2activity in this study.