Natriuretic Peptide Improves Vascular Remodeling In Spontaneously Hypertensive Rats And Underlying Mechanism

Hypertension is a clinical syndrome characterized by increased systemic arterial blood pressure and is a main risk factor for cardio-cerebrovascular diseases.Long-term poor control of hypertension can lead to many complications such as stroke,myocardial infarction,atherosclerosis and renal failure.According to the latest epidemiological survey in China,there are currently 270 million hypertensive patients in China,and the prevalence of hypertension in adults aged 18 years or older is 27.9%（《the 2018 Report on Cardiovascular Diseases in China》）,the prevalence of hypertension in China continues to rise,ranking first in the prevalence of chronic diseases in large and medium-sized cities in China.Therefore,it is of great practical significance to elucidate the pathophysiological mechanism of the occurrence and development of hypertension,and then take effective prevention and treatment measures as the break point.A large number studies have shown that vascular remodeling（VR）,the change of vascular structure and function,is the pathological basis of target organ damage in hypertension.Once hypertension is formed,the abnormal mechanical force acting on the vessel wall becomes the main factor of vascular remodeling due to the increased of blood pressure.The peripheral resistance of the systemic circulation will continue to increase,and promote the continuous increase of blood pressure,which will lead to a vicious circle between hypertension and vascular remodeling.Resistance vessels refer to the vessels that is affected by all resistance in the vascular system,mainly in the small arteries and microvessels.In general,the maintenance of normal blood pressure depends mainly on peripheral resistance,which is the resistance of peripheral arterioles and microvessels to blood flow.The continued increase in resistance vascular tension also leads to an increase in blood pressure.It has been reported that in the pathophysiological process of microgravity and endothelial injury,the vasoconstriction of the mesenteric resistance artery changes,and these changes are related to blood pressure levels.In the early stage of essential hypertension,vascular smooth muscle activity and resistance arterial hemodynamic abnormalities play an important role in the occurrence and development of hypertension.If it can effectively prevent or reverse the vascular remodeling and resistance arterial hemodynamic abnormalities caused by hypertension,it will be beneficial to the control of blood pressure and reduce the complications of heart,brain and kidney caused by hypertension.Natriuretic peptides（NPs）are cardiovascular hormones that are mainly secreted by the atrium,respond to atrial tensile forces,induce diuretic,natriuretic and vasodilatation effects,it plays an important role in maintaining the water-salt balance in the body,and stabilizing blood pressure and the function of cardiovascular and renal.There are three main types of NPs currently studied:atrial natriuretic peptide（ANP）,brain natriuretic peptide（BNP）,and c-type natriuretic peptide（CNP）.Vasonatrin peptide（VNP）is a novel synthetic natriuretic peptide molecule with strong dual activity in vasodilation and natriuretic diuresis.VNP is composed of the functional domains of CNP and ANP.The secondary structure has a pair of disulfide bonds which make it unique biological activities,namely the dual effect of CNP vasodilation and ANP natriuretic diuretic.Previous studies have shown that VNP can alleviate pulmonary vascular remodeling induced by hypoxia in rats,suggesting that VNP may be superior to natural NPs in the treatment of chronic heart failure and hypertension.Adiponectin（APN）is an endogenous cytokine with genetic characteristics secreted by adipocyte which is abundant in plasma.As a polypeptide hormone,APN plays a variety of physiological functions such as anti-inflammatory,anti-atherosclerosis,anti-diabetes and cardiovascular protection through various signaling pathways.Studies have shown that APN is lower in circulating blood of hypertensive patients,and supplementation with APN can improve hypertension in APN-deficient mice or obese mice with low APN.Therefore,the increased endogenous APN levels are more beneficial to the prevention and treatment of hypertensive cardiovascular disease.Spontaneous hypertension rats（SHR）are very similar to human essential hypertension in terms of genetic characteristics,pathogenesis,changes in peripheral vascular resistance and hypertensive complications,which is recognized as the most similar animal model of the human essential hypertension,and is also the best animal model for the study of the pathogenesis of hypertension and the screening of antihypertensive drugs.Therefore,this study selected specific ages SHR as the research object,and explained the effects and its mechanism of NPs on anti-hypertensive and vascular remodeling from molecular,cellular and overall levels by pharmacological methods,it provides experimental basis for revealing the molecular etiological mechanism of hypertension vascular remodeling and a new target for the effective prevention and treatment of hypertension vascular remodeling in clinical study.Purposes1.To investigate whether VNP can improves SHR vascular remodeling.2.To study the molecular mechanism of VNP to improve vascular remodeling in hypertension,namely the effect of VNP on plasma ANP levels and the upstream mechanism.3.To investigate whether long-term aerobic exercise improves vascular remodeling by affecting the NPs sensitivity of resistance vessels in spontaneously hypertensive rats.Method1.Grouping of experimental animals and determination of blood pressure:rats were divided into four groups:model control group（SHR）,VNP low-dose treatment group（LD）,VNP high-dose treatment group（HD）,and control group（WKY）.After 8 weeks of continuous intraperitoneal injection of VNP,the rat tail artery pressure and blood glucose level were measured.2.The levels of APN,ET-1,AngII,ANP,NO,PKG and cGMP in SHR were determined by ELISA.The ratio of plasma cGMP to ANP（cGMP/ANP）was calculated and used as a marker for ANP sensitivity.3.After 8 weeks of administration,the vascular contraction method was used to detect the difference of constrictive and diastolic function in rat thoracic aorta.The effects of VNP(10^-8,10^-7,10^-6mol/L)was observed on SHR thoracic aorta.The relaxation of the aorta and the effect of the cGMP-dependent protein kinase inhibitor KT-5823 on this process.4.Histopathological changes of rat thoracic aorta were detected by HE staining,and pathological changes of vessel wall and vascular cavity were observed microscopically.5.The changes of collagen fibers in rat thoracic aorta were observed and analyzed by Masson trichrome stain.6.3T3-L1 Adipocytes were divided into control group（CON）,VNP treatment group（VNP）,VNP+KT-5823 group,VNP+8-Br-cGMP group（administerable the cell membrane cGMP analog 8-Br-cGMP treatment）,Western Blot（WB）was used to analyze the expression of APN,NPRA and PKG in adipocyte culture medium.The expression and activity of PDE5 in rat mesenteric artery were determined.7.SHR thoracic aortic vascular smooth muscle cells（VSMCs）were separated and grouped as follows:control group（CON）,VNP-treated adipocyte culture fluid group（VNP）,VNP-treated adipocyte culture fluid+Compound C（VNP+Compound C）group.The proliferation of VSMCs were determined by CCK-8 method,the apoptosis of VSMCs detected by flow cytometry,and the expression levels of AMPK and pAMPK in culture medium were detected by Western Blot.8.Experimental animal exercise training program:（1）4-week-old WKY rats;（2）4-week-old SHR;（3）16-week-old WKY rats;（4）16-week-old SHR;（5）16-week-old WKY rats trained with exercise;（6）16-week-old SHR trained with exercise.WKY rats and SHR in the exercised groups were trained,free of loading,5 days/week for 12 weeks,Rats swam for 15 min on the first day,and the swimming duration was then progressively increased to 60 min/day in 1 week.9.Using an isolated vascular perfusion method to detect the relaxation response of ANP to rat mesenteric artery:After obtaining a sustained and comparable contraction induced by NE(norepinephrine,10^-6mol/L),the functional integrity of endothelium was assessed by recording the vascular relaxation to 10^-5mol/L acetylcholine（Ach）.Vascular responses to ANP were determined by exposing mesenteric arterial segments to four concentrations of ANP(10^-9,10^-8,10^-7,10^-6mol/l),for PED5 inhibition,sildenafil（30μmol/L）was added into the bath solution 15 min before treatment of ANP,the maximal contraction amplitude induced by norepinephrine(10^-6mol/L)was set to 100%.10.Measurement of arterial blood pressure:Rats were anaesthetized by intraperitoneal injection with sodium pentobarbital（50mg/kg）.The arterial blood pressure was measured via a polyethylene catheter cannulated into the left carotid arteries.Arterial systolic and diastolic blood pressure（SBP and DBP）were continuously monitored via a data acquisition system.Result1.The blood glucose level was significantly increased in the serum of SHR.VNP treatment significantly reduced SHR blood glucose level.The non-invasive blood pressure measurement showed that the systolic and diastolic blood pressure at 12 weeks of age was significantly higher than that of normal rats.After continuous treatment with VNP for 8weeks,the systolic and diastolic blood pressure of HD group and LD group were significantly lower than that of SHR group.There was no significant difference in the WKY group.2.Experimental results of isolated vascular perfusion:VNP can significantly enhance SHR vasodilation,which was blocked by cGMP-dependent protein kinase inhibitor KT-5823.VNP(1×10^-610^-8mol/L)caused a dose-dependent relaxation of SHR thoracic aorta upon PE pre-contracted vascular annulus.The effect of VNP(10^-8mol/L)diastolic precontraction vessels was weak,and VNP(10^-6mol/L)diastolic pre-contraction was the strongest.The administration of cGMP-dependent protein kinase inhibitor KT-5823almost completely eliminated the relaxation effect of ANP.3.Results of enzyme-linked immunosorbent assay:The levels of serum APN,ANP,PKG and NO in SHR group were remarkably lower than those in WKY group.VNP treatment significantly increased serum APN,ANP,PKG and NO levels in SHR group.Serum ET-1and AngII levels in the SHR group were significantly higher than those in the WKY group.VNP treatment significantly reduced serum ET-1 and AngII levels in the SHR group.4.The results of rat thoracic aorta hematoxylin-eosin（HE）staining:The vascular morphology of rats in WKY group was normal,the boundaries of each layer were clear,the alignment was neat,the blood vessel wall was not thickened,and the blood vessel elasticity was observed.The collagen components in the wall were not proliferated,the SHR group was thicker than the WKY group,the lumen was narrow,the elastic fibers were blurred,the arrangement was disordered,and the collagen content of the tube wall was significantly proliferated;The aortic endothelial cell proliferation was mild,the morphology of VSMCs was normal,and the pathological changes of the thoracic aorta were improved to some extent,suggesting that the VNP treatment group has the improving effect of thoracic aorta.5.Masson staining results of rat thoracic aorta:Compared with WKY group,the collagen fibers in SHR aorta were significantly proliferated,coarse,arranged disorderly,interlaced into a network,unevenly arranged,and smooth muscle cell had very obvious hyperplasia and hypertrophy.Compared with the SHR group,the vascular remodeling was significantly improved in the VNP-treated group.The collagen fibers in the thoracic aorta were arranged neatly and evenly distributed.The hyperplasia of smooth muscle cell was significantly reduced,and the proliferation of collagen fibers was significantly reduced,indicating that VNP has reduced collagen deposition in the thoracic aorta.6.Western Blot results:Western Blot analysis of 3T3-L1 adipocytes showed that compared with the control group,the expression of APN,NPRA and PKG protein in VNP+8-Br-cGMP group and VNP treatment group increased significantly,The expression of APN,NPRA and PKG protein in VNP+KT-5823 group was significantly decreased,suggesting that cGMP-dependent protein kinase inhibitor KT-5823 blocked the role of VNP.Western Blot was used to detect the expression and activity of PDE5 in the mesenteric artery.It was found that the expression of PDE5 was significantly increased in young（4 weeks old）SHR compared with control WKY,and further increased in adult（16weeks old）SHR.Increased PDE5 activity is the main cause of ANP resistance in SHR,the sildenafil（non-inhibitor of PDE5）is effective in inhibiting the activity of PDE5 and attenuating the ANP resistance of SHR.7.Results of VSMCs proliferation experiment:The proliferation of VSMCs treated with VNP-treated adipocyte culture solution was significantly lower than that of the control group.The above effect of VNP was significantly attenuated by pretreatment with Compound C（an AMPK inhibitor）,suggesting that VNP significantly inhibited VSMCs proliferation through AMPK-dependent signaling pathways.8.Results of VSMCs apoptosis experiment:Compared with the control group,the apoptotic cells increased significantly after VNP-treated adipocyte culture solution was treated with VSMCs.The addition of Compound C（an AMPK inhibitor）decreased the apoptotic cells,suggesting that VNP induces VSMCs apoptosis through AMPK-dependent signaling pathway.9.Western Blot detection of AMPK and pAMPK expression levels in VSMCs:Compared with the control group,after VSMCs were subjected to VNP-treated adipocyte culture medium,AMPK showed no significant change,pAMPK level increased significantly,and after administration of Compound C（an AMPK inhibitor）,pAMPK levels were significantly reduced,suggesting that VNP increases AMPK phosphorylation and activates AMPK-dependent signaling pathways.10.The results of blood pressure measurement of exercise rats:Exercise training significantly reduced the arterial pressure of SHR,the body weights of the young（4-week-old）rats were not significantly different.In the adult（16-week-old）groups,the mean weights were again not significantly different between the two strains.The mean carotid arterial pressures of 4-week-old animals were not significantly different between the WKY rats and SHR.However,compared with WKY rats of the same age,the mean carotid arterial pressure of SHR in adults（16 weeks）was significantly higher.The mean arterial pressure in adults SHR exercise training group was significantly lower than that in untrained SHR group.The results indicated that exercise training significantly reduced the mean carotid pressure of SHR.11.The effect of ANP on the relaxation of mesenteric artery in exercise rats:the blood pressure was not yet elevated in 4-week-old SHR,in juvenile（4-week-old）WKY or spontaneously hypertensive rats,ANP induced a dose dependent relaxation of mesenteric arteries precontraction by 10^-6mol/L NE.Mesenteric arteries from 4-week-old SHR demonstrated attenuated response to ANP at the dose of 10^-8mol/L or 10^-9mol/L compared with age-matched WKY rats.Differently,relaxation of mesenteric arteries from 16-week-old SHR was even attenuated to ANP at the dose from 10^-99 mol/L to10^-6mol/L.Accordingly,plasma cGMP/plasma ANP ratio was markedly reduced in juvenile（4-week-old）SHR and even decreased in the adult（16-week-old）group,suggesting impaired cGMP generation despite high ANP stimulation.The fact that ANP resistance occurred before the formation of hypertension indicated predisposition to hypertension..Conclusion:1.Pathological vascular remodeling occurrs in the SHR thoracic aorta.2.VNP ameliorates SHR thoracic aortic vascular remodeling by promoting APN production and inhibiting VSMCs.3.Aerobic exercise improves the sensitivity of SHR resistance vessels to ANP.In this study,pharmacological methods were used to elucidate the effects of natriuretic peptides on vascular remodeling in spontaneous hypertension at the molecular and cellular levels,and to further explore its signaling pathways,mechanisms of action and significance,in order to reveal that natriuretic peptides improve vascular remodeling.The new mechanism provides experimental evidence and provides potential prevention and treatment targets for delaying the occurrence of hypertension and its complications from the perspective of improving vascular remodeling,and provides a basis for clinical effective prevention and treatment of hypertension vascular remodeling.To clarify these problems not only has important theoretical significance for understanding the mechanism of occurrence and development of hypertensive vascular remodeling,but also has important practical significance for the future use of natriuretic peptides to prevent hypertension and its complications.