Effects And Mechanisms Of Abnormal MTORC1 Complex Activity On Offspring's Hypertension Induced By Maternal LPS Exposure

Hypertension and its complications,such as cerebral stroke,coronary heart disease and myocardial infarction,are the leading cause of human death and disability.Although massive research,lot of work and money has been spend on their prevention and treatment,the morbidity of hypertension is still on the rising year by year.This indicates there still exits uncovered mechanisms.Vascular remodeling is characterized by narrowed intima-media thickness,coarctation of the lumen diameter,and extracellular matrix deposition which involves cell hypertrophy,migration,proliferation and apoptosis and dysfunction in extracellular matrix synthesis and degradation.Previous work s of our lab have found that vascular remodeling occurs ahead of hypertension and ca n be an important cause of hypertension while conventional ideas think that vascular remodeling is a consequence of hypertension.Therefore,it's of great importance to explore the specific mechanism of vascular remodeling resulted from prenatal LPS exposure.The mTOR pathway integrates inputs from several major intracellular and extracellular signals indcluding growth factors,stress,energy status,oxygen,and amino acid to control many major processes,including protein and lipid synthesis,energy metabolism,cell survival/metabolism and cytoskeletal organization.Elevated circulating leptin,TNF-?,IL-6 and protein expression of Ang II and TNF-? on aorta accomplished by enhanced inflammatory sensitivity were observed on offspring that underwent prenatal LPS exposure.Hence,we supposed that mTOR activation might participate in hypertension of offspring that underwent prenatal LPS exposure.Therefore,this project was designed to evaluate the effects of mTOR overexpression on aortic vascular remodeling,to explore novel mechanism of hypertension and further searching possible targets for hypertension treatment.Methods1.Effects of prenatal LPS pressure on mTOR-related protein expression pattern in offspring rats.Pregnant SD rats were randomly divided into 2 groups:(1)Con group: pregnant rats received 0.5ml saline i.p.on gestation days 8,10,12;(2)LPS group: pregnant rats received 0.79 mg/kg i.p.LPS(Sigma Chemical,St.Louis,MO,USA)on gestation days 8,10 and 12.Blood pressure and body weight were measured weekly.Protein expression of p-S6ser240/244 and p-Aktser473 on aorta were detected by western blot at 1week of age(w)?3w?5w?10w?15w?26w.2.Effects of rapamycin treatment on blood pressure,vascular remolding and endothelia function in offspring.2.1 In vivo rapamycin treatment: offspring rats of 26 weeks old of age from LPS or saline treatment were divided into 4 group via rapamycin treatment or not: Con+Ve,LPS+Ve,Con+Rapa,LPS+Rapa.Rapamycin was treated i.p.at a dose of 1.5mg/kg/day for 15 days Blood pressure were and body weight were measured daily.Aorta from all group were embedded in paraffin for HE and Masson staining.2.2 Protein expression of PCNA and p-eNOS were detected by western blot after rapamycin treatment.3.Effects of prenatal LPS exposure on offspring mice aortic mTOR-related protein expression and effects of mTOR knock-down on hypertension resulted from prenatal LPS exposure.3.1 Pregnant C57 mice received 0.2ml saline or 75?g/kg LPS at gestation day 10.5.Blood pressure and body weight were measured weekly.Protein expression of p-S6K1 and p-Aktser473 on aorta were detected by western blot at 16 w.3.2 Mice conditional knock down Raptor on aorta were generated for prenatal LPS exposure.Offspring blood pressure were measure and aorta from all group were embedded in paraffin for morphological observation.4.Effects of renin-angiotensin system over-activity on mTOR over-expression prenatal resulted from prenatal LPS exposure.4.1 offspring rats of 26 weeks of age were first treated by benazepril(dissolved in drinking water,250mg/L,40mg/kg/day)for 15 days to eliminate endogenous Ang II,then detecting protein expression of p-S6ser240/244 and p-Aktser473 from offspring aorta were detected.4.2 Offspring rats of 26 weeks of age were first treated by benazepril(dissolved in drinking water,250mg/L,40mg/kg/day)for 15 days to eliminate endogenous AngII,and then exogenous Ang II were administrated by subcutaneous injection.Rats were sacrifice 0min,2min,5min,10 min,30min after exogenous AngII were administration and protein expression of p-S6ser240/244 and p-Aktser473 on aorta were detected by western.Results1.Effects of prenatal LPS pressure on mTOR-related protein expression pattern of offspring rats.Offspring rats from prenatal LPS exposure showed an tendency of elevation on aortic protein expression of p-S6ser240/244 at 1w,3w,5w,10 w,15w without statistic difference.Offspring mice showed the same elevation tendency on aortic protein expression of p-S6ser240/244 a without statistic difference.However,when compared with offspring from saline treated mother,aortic protein expression of p-S6ser240/244 of offspring that underwent prenatal LPS exposure was significantly upregulated at 26 weeks of age while aortic protein expression of p-Aktser47 stayed unchanged through all the indicated time-points.2.Effects of rapamycin treatment on blood pressure,vascular remolding and endothelia function in offspring.2.1 Compared with Con+Ve group,significantly elevated blood pressure of LPS+Ve group can be detected(p<0.01),while rapamycin treatment could partly restore the blood pressure elevation caused by prenatal LPS exposure(p<0.0001,LPS+Rapa vs.LPS+Ve).Rapamycin treatment does not affect the normal blood pressure.2.2 Compared with Con+Ve group,HE staining of aorta from LPS+Ve showed increased endothelium injury and vascular smooth muscle cells disorder,while rapamycin treatment could reverse the abnormality caused by prenatal LPS exposure.2.3 Compared with Con+Ve group,Masson staining of aorta from LPS+Ve showed increased collagen deposition of intima,while rapamycin treatment could reverse the abnormality caused by prenatal LPS exposure.2.4 Compared with Con+Ve group,prenatal LPS exposure can significantly downregulated the protein expression of p-eNOS while rapamycin treatment could restore the protein expression abnormality of p-e NOS.2.5.Compared with Con+Ve group,prenatal LPS exposure significantly upregulated the protein expression of PCNA while rapamycin treatment could restore the protein expression abnormality of PCNA.3.Effects of prenatal LPS exposure on offspring mice aortic mTOR-related protein expression and effects of mTOR knock-down on hypertension resulted from prenatal LPS exposure.3.1 prenatal LPS exposure also resulted in significant elevation of protein expression of p-S6ser240/244 on offspring mice at 16 weeks of age,while protein expression of p-Aktser473 remained unchanged.3.2 Raptor conditional knockdown on vascular smooth muscle cells significantly reversed prenatal LPS exposure induced hypertension,endothelia dysfunction and vascular smooth muscle cells disorder at 57 weeks of age.4.Effects of renin-angiotensin system over-activity on abnormal mTOR complex activity resulted from prenatal LPS exposure.4.1 Benazepril treatment for 15 days could lower prenatal LPS exposure induced blood pressure elevation to normal level without significant change in protein expression difference in p-S6ser240/244.Combined with our previous work that prenatal LPS exposure promoted aortic local rather systemical AngII expression,these results indicates that Ang II expression elevation on aorta might be a critical cause of offspring aortic abnormal mTORC1 complex activity.4.2 Exogenous Ang II administration could promote p-S6ser240/244 expression in a time-dependent manner while there existed no difference of protein expression of p-S6ser240/244 between Con+benazepril and LPS+benazepril group at the same timepoint.Exogenous Ang II administration had little effect on protein expression of p-Aktser473.This result suggeste that downstream molecules of AngII such as AT1 R and AT2 R had no effect on mTORC1 overexpression.Conclusion:1.Prenatal LPS exposure can promote the over-expression of mTORC1 rather mTORC2.2.The abnormal mTORC1 complex activity may be a key partly accounting for offspring vascular remolding and hypertension.3.AngII expression elevation on aorta may be a critical cause of offspring aorti c abnormal mTOR complex activity.