| Hypertension has been the focus of cardiovascular research field, knowing that it caninduce various cardiovascular diseases including stroke, coronary heart disease and heartfailure,seriously affecting human health. Cardiovascular dysfunction in hypertension ischaracterized by sympathetic nerve overactivity and high blood pressure, which in turnpromote the development and prognosis of hypertension. The rostral ventrolateral medulla(RVLM) plays a pivotal role in the control of sympathetic outflow and basic regulation ofblood pressure, whose structural and functional abnormalities are the important mechanismsof pathological progression of hypertension. It has been documented that the cholinergicsynaptic transmission in the RVLM is significantly enhanced in spontaneously hypertensiverats (SHR).Thus, the enhancement in cholinergic synaptic transmission is a critical factorcontributing to high blood pressure and sympathetic overactivity of SHR. Effectivedepression of the cholinergic synaptic transmission is important to improve cardiovasculardysfunctions in the RVLM of hypertension.It is well known that increased renin-angiotensin system (RAS) is involved in thepathogenesis of hypertension.Angiotensin-converting enzyme2(ACE2)–Ang-(1–7)–Masreceptor axis has recently been a new axis, as an important component of RAS. ACE2is anew member of the ACE family, which predominantly metabolizes Ang II to generateAng-(1–7). Angiotensin-(1–7) exhibits an opposite effect of AngII.by acting on the specific Gprotein-coupled receptor Mas, suggesting that ACE2-Ang (1-7)-MAS axis provides a noveltarget for the treatment of hypertension. Previous studies indicated that overexpression ofACE2in the RVLM could cause long-term decrease in blood pressure in SHR, but it isunknown whether beneficial effect of ACE2in the RVLM to hypertension is associated withthe depression of cholinergic pathways.The main objective of this study was to determinewhether overexpression of ACE2in the RVLM had any impact on cholinergic synaptictransmission. Inflammation-induced enhancement of oxidative stress is an important mechanism toincrease neuronal excitability within RVLM. Ang II, a pro-inflammatory factor, has an effecton inducing inflammation, Ang II increases the activity of transcriptional factor NF-κB, AP-1,and elicits an increase in oxidative stress mediated by production of reactive oxygen species.Therefore, we will also determine whether overexpression of ACE2in the RVLM inhibits theproduction of inflammatory cytokines in SHR.[Methods]In SHRs that received lenti-ACE2injection into the bilateral RVLM, systemic arterialpressure and heart rate were measured under the conscious condition by the non-invasivetail-cuff method. The level of the24h urinary norepinephrine (NE) was detected by HPLC todefine sympathetic activity; Protein expression was evaluated by Western blot to verify thetransgene efficiency of ACE2overexpression in the RVLM. In the rats with ACE2overexpression, acetylcholine and its related receptor were tested.6weeks after gene transferof ACE2, The content of ACh in RVLM was analyzed with an Essay Kit to observe thepresynaptic release of ACh in response to overexpression of ACE2. Protein level of MasRexpression was evaluated by Western blot to verify the difference of ACE2-Ang (1-7)-MASaxis in response to ACE2overexpression in the RVLM. Expression levels of vesicular AChtransporter (VAChT), M2R, oxidative stress-related enzyme NOX4and SOD1were evaluated.Levels of the cytokine TNFα，IL-1β in RVLM were analyzed by ELISA. Effects ofacetylcholine receptor antagonist atropine microinjected bilaterally into the RVLM on bloodpressure, heart rate, renal sympathetic nerve activity were observed to define the role ofcholinergic synaptic transmission in tonically maintaining the basal cardiovascular activity..[Results]1. Effects of ACE2overexpression on mean arterial pressure and heart rate in RVLMExperiment rats were divided into three groups: WKY, SHR-GFP, SHR-ACE2. Beforetransfection of ACE2into RVLM, SHRs showed a significant increase (P<0.05) in meanarterial pressure (MAP)(182±7mmHg) and HR (321±29beats per min,bpm) compared with WKYs (138±5mmHg) and HR (351±16bpm). No difference in MAP and HR was observed atweek3of SHR-ACE2compared with SHR-GFP. Six weeks after the lenti-ACE2injection,Lenti-ACE2SHRs showed a significant decrease in MAP compared with GFP controls(149±3mmHg vs.181±8mmHg). By week5after lenti-ACE2gene delivery, SHR-ACE2showed a decrease in HR compared with SHR-GFP (289±11bpm vs.349±15bpm).2. Effects of ACE2overexpression on the content of NE in24-hour urineSix weeks after the lenti-ACE2injection, the level of24h urine norepinephrine wasmeasured in WKY and SHR groups by high performance liquid chromatography to observesympathetic activity. The data revealed that urine norepinephrine level was increasedsignificantly in SHR-GFP groups as compared with that in WKYgroup(0.166±0.021μg/24hvs.0.042±0.008μg/24h;(P<0.05)),and was decreased significantly inSHR-ACE2group (0.046±0.004μg/24h) as compared with that in SHR-GFP group, but it wasnot significantly different from that in WKY group.3. Effects of ACE2overexpression on the expression of ACE2-Ang (1-7)-Mas axis inRVLM of SHRThe results showed that SHR-GFP showed a significant decrease (P<0.05) inACE2/α-tubulin protein level compared with WKY((0.088±0.009vs.0.147±0.018).SHR-ACE2showed a significant increase in ACE2/α-tubulin protein level (0.122±0.013)compared with SHR-GFP, but it was not significantly different from that in WKY group.SHR-GFP showed a significant decrease in MasR/α-tubulin protein level comparedwith WKY ((0.482±0.008vs.0.861±0.015), while SHR-ACE2showed a significant increasein MasR/α-tubulin protein level (0.877±0.012) compared with SHR-GFP, but it was notsignificantly different from that in WKY group.4.Effects of ACE2overexpression on the release of ACh in the RVLM of SHRThe concentration of ACh in SHR-GFP was significantly increased compared with thatin WKY rat control(12.97±1.672μg/mL vs.5.69±0.517μg/mL,P<0.05). SHR-ACE2showeda significant decrease in ACh concentration (7.017±0.932μg/mL) compared with SHR-GFP,but it was not significantly different from that in WKY group.5. Effects of ACE2overexpression on protein level of VAChT, M2R, NOX4and SOD1in the RVLMThe results showed that VAChT/α-tubulin expression was increased significantly inSHR-GFP group as compared with that in WKY group (0.114±0.016vs.0.061±0.009;P<0.05),and was decreased significantly in SHR-ACE2group(0.066±0.008) ascompared with SHR-GFP, but it was not significantly different from that in WKY group.NOX4/α-tubulin expression was increased significantly in SHR-GFP as compared withthat in WKY group(P<0.05). and decreased significantly in SHR-ACE2group as comparedwith that in SHR-GFP group.SOD1/α-tubulin expression was decreased significantly in SHR-GFP group as comparedwith that in WKY group(P<0.05),and decreased significantly in SHR-ACE2group ascompared with that in SHR-GFP, but here was no statistical significance of M2R protein levelamong three groups.6.Effects of ACE2overexpression on ACh receptor subtype muscarinic in the RVLMMicroinjections of the muscarinic acetylcholine receptor inhibitor atropine (2nmol) intothe RVLM of SHR-GFP produced an decrease in MAP of （55±4）mmHg, which wassignificantly (P<0.05) higher than WKY group (15±1mmHg). while SHR-ACE2showed asignificant decrease in MAP（23±7mmHg）compared with SHR-GFP, showed a little higherthan WKYs. There was no difference in the decrease of HR of WKY (10±3bpm), SHR-GFP(12±5bpm) and SHR-ACE2(17±5bpm).7. Effects of ACE2overexpression on inflammatory cytokines in SHR RVLMThe level of cytokines TNFα，IL-1β in the RVLM was analyzed by ELISA. The datashowed that TNFα, IL-1β levels were increased significantly in SHR-GFP group as comparedwith those in WKY group (P<0.05),and decreased significantly in SHR-ACE2groupascompared with those in SHR-GFP group.[Conclusion]The results of this study showed that overexpression of ACE2within the RVLM of SHRcan reduce blood pressure, heart rate and sympathetic nerve activity. ACE2overexpressioncan significantly reduce cholinergic synaptic transmission of SHR RVLM, inflammatorycytokines and oxidative stress levels. These results suggest that the central ACE2 overexpression contributes to improve cardiovascular dysfunctions in hypertension isassociated with depression of cholinergic synaptic transmission, and may reduceinflammatory cytokines-mediated oxidative stress. |
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