| Angiotensin (Ang)-converting enzyme (ACE)2is a homolog of angiotensin-convertingenzyme(ACE),which cleaves the decapeptide Ang-I and octapeptide AngⅡ to Ang-(1–9) andAng-(1–7) to lead to effection of vasodilatation and hypotension. The rostral ventrolateralmedulla（RVLM） plays a critical role in the regulation of BP and cardiovascular function.The enhancement in excitatory mechanism including AngII-induced oxidative stress andglutamatergic inputs in the RVLM contributes to increased sympathetic outflow, which isimportant in processing of pathophysiological course of hypertension. It has beendemonstrated that increase in endogenous Ang1-7by overexpression of ACE2in the RVLMcauses a long-term decrease in blood pressure in the sponstanously hypertensive rats (SHR),but the exact mechanisms is not certainly clear. So the present objective was to study thepotential mechanisms by which overexpression of ACE2in the RVLM reduces sympatheticoutflow in the SHR.ObjectiveThe present study aimed to study the effects of Lenti-ACE2over-expression in theRVLM of both WKY rats and SHR on changes in cardiovacular activities. Both concentrationof NE in24-h urine and glutamate (GLU) in microdialysis liquid from the RVLM weredetected by high performance liquid chromatography (HPLC). The changes of proteinexpression such as ACE2, AT1receptor, NOX4and superoxide dismutase (SOD) weredetected by western-blot analysis. Glu recepoter antagonist microinjected into the RVLM to determine the functional changes of Glu receptor involved in cardiovascular activity.According to the above experiments, the present work was carried out for detecting therelationship between ACE2overexpression and AT1R、NOX4、SOD overexpression in theRVLM, and determining the mechanism by which overexpression of ACE2in the RVLMreduces sympathetic outflow in SHR.MethodsTo transfection of ACE2into the RVLM, overexpression of Lentivirus was taken as thevector. Blood pressure and heart rate were detected by a non-invasive blood pressure system.Both concentrations of NE in urine or GLU in microdialysis liquid from the RVLM weredetected by HPLC. Protein expressions such as ACE2, AT1receptor, NOX4, SOD, glutamatereceptor in the RVLM were detected by western-blot. GLu receptor antagonist wasmicroinjected into the RVLM to test the cardiovascular function of the Glu receptors byrecording of blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity(RSNA)6weeks after tansfection of ACE2in the RVLM.Results1. Effects of Lenti-ACE2over-expression in the RVLM on BP and HR:Before transfection in the WKY, systolic blood pressure (SBP,160±13mmHg),diastolic blood pressure (DBP,(114±11mmHg)，mean arterial blood pressure (MBP,126±12mmHg)，heart rate HR (357±24bpm) were significantly lower than those of SHR (SBP,200±11mmHg; DBP,150±8mmHg; MAP,167±9mmHg; HR,401±32bpm). Four weeksafter RVLM transfection of Lenti-ACE2in SHR, SBP (190±12mmHg), DBP (136±9mmHg),MAP (151±9mmHg) was significantly lower than those of Lenti-GFP group (SBP,202±11mmHg; DBP,149±9mmHg; MAP,166±10mmHg). Five weeks after RVLM transfection of Lenti-ACE2in SHR, HR（397±29bpm）was significantly lower than that of SHR transfectionof Lenti-GFP（435±37bpm）.2. Effects of Lenti-ACE2over-expression in the RVLM on sympathetic toneSix weeks after RVLM transfection of Lenti-ACE2, the content of NE in24hour’ urine(0.367±0.075μg/24h) was significantly lower than that of Lenti-GFP group (0.534±0.058μg/24h) in SHR. There was no significantly difference between normal WKY(0.426±0.044μg/24h) and SHR, which received RVLM transfection of ACE2.3. Effects of Lenti-ACE2over-expression in the RVLM on protein expression3.1Six weeks after RVLM transfection of Lenti-ACE2in SHR, expression of ACE2（0.103±0.0047, ratio to β-actin）was significantly higher than that of transfection Lenti-GFP（0.062±0.0051）. There was no significant difference of Lenti-ACE2expression betweenSHR and WKY, which received transfection of Lenti-ACE2. ACE2expression aftertransfection of Lenti-GFP was significantly lower in SHR than in WKY.3.2Six weeks after RVLM transfection of Lenti-ACE2in SHR, AT1R expression（0.173±0.041） was significantly lower compared wtih that of Lenti-GFP group（0.5460±0.061）,whereas it was still higher than that of WKY group (0.0835±0.0043).3.3Six weeks after RVLM transfection of Lenti-ACE2in SHR, SOD1expression（0.247±0.044）was significantly higher than that of Lenti-GFP group（0.138±0.053）, but itwas still lower than that of WKY group (0.663±0.046).3.4Six weeks after RVLM transfection of Lenti-ACE2in SHR, NOX4expression（0.137±0.023）was significantly lower than that of Lenti-GFP group（0.182±0.034）. Therewas no significantly difference of NOX4expression between SHR and WKY, which receivedransfection of Lenti-ACE2(0.143±0.029).4. The effects of Lenti-ACE2over-expression in the RVLM on concentration of Glu inmicrodialysis liquidSxi weeks after RVLM transfection of Lenti-ACE2in SHR, concentration of Glu in microdialysis liquid from the RVLM (621.±121g/L) was significantly lower than that ofLenti-GFP group’s (1586±285g/L), whereas it was still higer than that of WKY group’s(279.82±145.20g/L).5. Effect of Glu receptor blockade in the RVLM on cardiovascular activitySix weeks after RVLM transfection of Lenti-ACE2in SHR, decreases in BP（16±3mmHg）,HR（20±4bpm）, and RSNA（16.5±4.6%）evoked by unilateral microinjection of the Glureceptor antagonist kynurenic acid (Kyn,2.7nmol) into the RVLM were significantly reducedthan those of Lenti-GFP group (BP,26±4mmHg; HR,30±6bpm; and RSNA,21.3±4.6%）.However, injection of Kyn into the RVLM had no effect on resting BP（2±1mmHg）,HR（4±5bpm）, and RSNA（3.1±5.4%）in normal WKY rats.ConclusionsOverexpression of ACE2in the RVLM caused a long-term decrease in bloodpressure,heart rate and sympathetic outflow in SHR. Expression of AT1R in the RVLM wasdownregulated, suggesting that functionof ACE-AngⅡ-AT1R axis is attenuated in SHRreceived ACE2overexpression in the RVLM. Expression of NOX4was down-regulated andexpression of superoxide dismutase(SOD1) was upregulated after overexpression of ACE2inthe RVLM, suggesting that production of ROS was attenuated and clearance of reactiveoxygen species (ROS) was enhanced. Excitatory inputs to the RVLM were attenuated afteoverexpression of ACE2in the RVLM. Overexpression of ACE2in the RVLM reduced therelease of the excitatory neurotransmitter Glu in the RVLM. Collectively, the current datasuggest that overexpression of ACE2in the RVLM is capable of reducing sympatheticoutflow and blood pressure in hypertension via attenuating excitatory factors such asAT1R-ROS and glutamatergic inputs in the RVLM. |
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