| Background and ObjectiveEssential hypertension is a critical factor in cardiovascular diseases,which extremely increases the risks of disorders of the brain,heart,kidney,and other organs,leading to high mortality.The sympathetic nerve system plays a vital role in stabilizing cardiovascular function and always is hyperactive in hypertension.As numerous studies suggest that the rostral ventrolateral medulla(RVLM)participates in the central regulation of sympathetic activity and blood pressure,where C1 neurons receive the inputs from several central key cardiovascular areas and integrate the information from peripheral cardiovascular activity.After the complex information integration,pre-sympathetic neurons from RVLM are projected onto the intermediolateral column of the spinal cord and regulate the function of the heart,kidney,and vessels by sympathetic nerves.Based on the above theories,it’s crucial to clarify the mechanism and prevention strategies of sympathetic hyperactivity in hypertension from the view of RVLM structural and functional abnormality.The central renin-angiotensin system(RAS),a major part of the cardiovascular and fluid homeostasis regulation system,contains all necessary components of peripheral RAS,such as the precursors,enzymes,and metabolic substances.Among them,angiotensin Ⅱ(Ang Ⅱ)in the hypothalamus and medulla can lead to an increase in blood pressure,but angiotensin-(1-7)(Ang-(1-7))hydrolyzed from Ang Ⅱ plays a role opposite to Ang Ⅱ by targeting Mas receptor(Mas R).Hence,the balance between Ang Ⅱ and Ang-(1-7)in the brain makes a big difference in the regulation of blood pressure and the progress of hypertension.Angiotensin-converting enzyme inhibitor(ACEI)and angiotensin Ⅱ type 1receptor blocker(ARB)are prevalent in treating hypertension by blocking the production and function of Ang Ⅱ.Therefore,the balance of the RAS system is crucial for the regulation of blood pressure,and promoting the production of Ang-(1-7)or inhibiting its degradation is another key way to regulate RAS balance.However,the current strategy of synthetic Ang-(1-7)analogues to delay the degradation is not ideal for treating hypertension.Herein,the broken of the balance between Ang Ⅱ and Ang-(1-7)may be regulated in other pathways,which provides a new insight into the central regulation mechanism of blood pressure and the treatment strategy of hypertensionDipeptidyl peptidase 3(DPP3),one kind of zinc-dependent hydrolase found in the bovine anterior pituitary gland at first,specially hydrolyzes oligopeptides with 4-12 amino acid residues,including Ang Ⅱ,Ang-(1-7),enkephalins,endorphins,etc.What’s more,DPP3 participates in the hydrolysis of not only Ang Ⅱ but also Ang-(1-7)and generates angiotensin-(3-7)(Ang-(3-7))and Angiotensin-(5-7)(Ang-(5-7)).According to many studies,the circulating level of DPP3 is inextricably bound with various pathophysiological processes(such as the regulation of blood pressure,signal transduction of pain,and oxidation stress of cancer cells).Based on the RNA transcriptome maps of 11 organs in rats published by the Rat Genome Database,it was found that DPP3 is expressed in the nervous system.However,it is unclear whether DPP3 takes part in the regulation of blood pressure and sympathetic activity by altering the balance between Ang Ⅱ and Ang-(1-7).Therefore,we can’t neglect the function of central DPP3 in regulating blood pressure and sympathetic activity,when it comes to the pathogenesis of hypertension.As the important components of RAS,both Ang Ⅱ and Ang-(1-7)can be hydrolyzed by DPP3,and Ang-(3-7)hydrolyzed from Ang-(1-7)has been identified that it promotes the release of dopamine and gamma-aminobutyric acid in the corpus striatum and increases blood pressure by activating neurons in RVLM.The above adequately shows that we cannot emphasize enough how important Ang-(3-7)produced by DPP3 is in terms of treating hypertension.Ang-(5-7)can further be hydrolyzed from Ang-(3-7),but whether it participates in the central regulation of blood pressure the same as Ang-(3-7)is unidentified.Based on the above backgrounds and previous experimental results,the whole hypothesis is as following: central DPP3 promoting the up-regulation of Ang-(5-7)participates in sympathetic hyperactivity in hypertension.And this study aims at identify the role of DPP3 in regulating blood pressure,to provide new direction and insights into the mechanism and prevention strategies of hypertension.MethodsThis study was carried out through clinical samples,animal,and molecular experiments.Firstly,the amount of DPP3 and norepinephrine(NE)in plasma samples of patients with cardiovascular diseases were determined by Enzyme-linked Immunosorbent Assay(ELISA).Then,the determination of DPP3 and NE levels in plasma was also performed in the WKY rats and spontaneously hypertensive rats(SHR)with different age gradients.The changes of DPP3 in different cardiovascular-related organs were detected using Western Blot(WB).After overexpression or inhibition of DPP3 by microinjecting the adeno-associated virus(AAV)into the RVLM,the function of DPP3 in regulating sympathetic activity and blood pressure was monitored with Power Lab recording system,WB and immunofluorescence.Furthermore,the role of DPP3 in regulating central RAS was detected through ELISA,enzyme activity test kit,and liquid chromatography-mass spectrometry.In ways of the intracisternal infusion and microinjection of DPP3 or Ang-(5-7),the mechanism of DPP3 promoting the elevation of Ang-(5-7)in the RVLM was further clarified by detecting the changes of blood pressure,heart rate,sympathetic activity,oxidative stress and neuronal activity.Results1.The roles and effects of DPP3 on cardiovascular function.(1)The relationship between DPP3 and sympathetic hyperactivity,and the expression and distribution of DPP31.1 The concentration of DPP3 and NE were positively correlated in the plasma of patients with cardiovascular diseases and rats.1.2 The expression of DPP3 was increased in both kidney and RVLM of SHR by comparison with WKY rats.The level of DPP3 in the RVLM of SHR elevated with the increase of age,and it was higher than that of WKY rats in all age groups.The existence of DPP3 was directly seen by immunofluorescence.(2)DPP3 in the RVLM promoted sympathetic activity and blood pressure2.1 After the microinjection of DPP3,the blood pressure and renal sympathetic activity were elevated with an increase in the dose of DPP3.2.2 The content of DPP3 and the mean arterial pressure(MAP)was elevated after DPP3 over-expressed in the RVLM of WKY rats(blood pressure in the awake state:123.5±1.3 vs.161.0±1.2 mm Hg;blood pressure under anesthesia:114.6±4.7 vs. 143.3 ± 6.2 mm Hg,P<0.05).And the change of MAP(ΔMAP)was significantly increased after sympathetic ganglia blocked by hexamethonium bromide(43.7±4.3 vs. 83.2±2.1 mm Hg,P<0.05).2.3 After DPP3 expression was inhibited in the RVLM of SHR,MAP and ΔMAP were both lower than the control group(P<0.05).2.DPP3 in the RVLM regulating the production of Ang-(5-7)promotes sympathetic hypertension and high blood pressure.(1)Among DPP3’s major substrates(endorphin,enkephalin,Ang Ⅱ,and Ang-(1-7)),it was found that the content of Ang Ⅱ was increased,while the content of Ang-(1-7)and the ratio of Ang-(1-7)to Ang Ⅱ was decreased,but no significant difference in the contents of β-endorphins and enkephalins after DPP3 over-expressed in bilateral RVLM of WKY rats.For other components of RAS,after DPP3 over-expressed in bilateral RVLM,the activities of ACE and ACE2,the content of Ang-(3-7),and the content of Ang-(5-7)were elevated,whereas the content of AT1 R,AT2R,and Mas R remained unchanged.(2)We further identified that the effects of DPP3 knockdown in the RVLM of SHR,the contents of Ang Ⅱ and enkephalins were decreased.However,the content of Ang-(1-7) and the ratio of Ang-(1-7)to Ang Ⅱ were increased,while the content of β-endorphins was unchanged.Besides,the content of Ang-(5-7)was significantly decreased,while the activities of ACE and ACE2 as well as the content of AT1 R,AT2R,Mas R and Ang-(3-7) were unchanged.3.Ang-(5-7)in the RVLM increased cardiovascular activity.(1)The sympathetic outflow and MAP were elevated after microinjection of Ang-(5-7) nto the RVLM.(2)MAP and heart rate variability(HRV)were increased after the intracisternal infusion of Ang-(5-7)in WKY rats.However,the baroreflex sensitivity were further inhibited by Ang-(5-7),compared to Ang Ⅱ.4.The mechanism of Ang-(5-7)in regulating cardiovascular activity.(1)Overexpression of DPP3 in the RVLM of WKY rats significantly increased the level of ROS,as well as the expression level of c-fos,a marker of neuronal activity.However, the level of ROS was decreased after DPP3 knockdown in the RVLM of SHR,along with the reduction of c-fos expression.(2)After pre-microinjection of the tempol,the mimic of superoxide dismutase,into the RVLM of WKY rats,the pressor effects of Ang-(5-7)were partially blocked.ConclusionThe expression of DPP3 within the RVLM is closely related to the progression of hypertension.DPP3 in the RVLM promotes the production of Ang-(5-7)and enhances oxidative stress,leading to sympathetic hyperactivity and high blood pressure.This study clarified the mechanism of central DPP3 affecting sympathetic hyperactivity by regulating RAS,and provided a new insight into the central regulatory mechanism of hypertension. |
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