| Cardiovascular diseases(CVD)have now become the leading cause of death from noncommunicable diseases.Hypertension is a major risk factor for cardiovascular disease,affecting the health of more than one billion people worldwide.It is predicted that by 2025,the number of hypertensive patients is expected to be between 15.4-15.5 billion.Ninety percent of hypertension with unknown origin is called essential hypertension(EH),with the characteristics of multigenerational inheritance,and its'current mechanism remains unclear.Recent studies have shown that unfavorable prenatal conditions promote EH in offspring.However,whether and how it affects the multigenerational inheritance of hypertension remains unknown.Epidemiological investigations have shown that prenatal inflammation exposure(PIE)leads to an increased incidence of cardiovascular disease,including hypertension.PIE include,but are not limited to,pathogen infections(such as viruses,bacteria,and parasites)that remain a significant and uncontrolled event during pregnancy.However,whether and how PIE affects the multigenerational inheritance of CVD is unclear.In the past,our laboratory has established a PIE model by treating pregnant animals in the mid-gestation with lipopolysaccharide(LPS)and polyriboinosinic polyribocytidylic acid(poly I:C).A stable animal model of CVD in which F1 offspring develops high blood pressure,structural damage to the cardiovascular system,and dysfunction.However,i t is unclear whether the incidence of hypertension in the F1 offspring will be transmitted to F2or even multiple generations.Therefore,this study verified the transgenerational effects of PIE-induced hypertension by constructing transgenerational model of prenatal inflammation exposure.Considering that the occurrence of EH is the result of the interaction between genetic and environmental factors,this study intend ed to start from the epigenetic inheritance to study the molecular mechanism of intergenerational genetics of blood pressure in the offspring of prenatal inflammation exposure.Considering,it provided new experimental data and theoretical basis for the prevention and treatment of EH and new drug research for it.1.Methods:1.1 PIE transgenerational model offsprings'phenotype detection1.1.1 Preparation of PIE transgenerational rat model:SD pregnant rats(F0)were randomly divided into 5 groups,and intraperitoneal injection of LPS or Ploy I:C was given on days 8,10,and 12(Persistent Treatment)or day 8(Transient Treatmen).Male offspring(F1)were bred with normal SD female rats at 16 weeks of age to obtain F2 offspring,which were named as control group,LPS persistent treatment group(F2-LPS-P)and LPS transient treatment group(F2-LPS-T),Ploy I:C persistent treatment group(F2-Ploy I:C-P)and Ploy I:C transient treatment group(F2-Ploy I:C-T).The LPS persistent treatment model was chosen as a representative for the multigenerational research.The 16th-week F2-Contol and F2-LPS-P male rats were bred with the normal SD female rats for the F3 offspring(F3-LPS-P)rats,and the F3 offspring(F4-LPS-P)rats was continued according to this method.1.1.2 Blood pressure,body weight,blood glucose and blood lipids of F2 offspring were measured to evaluate the transgenerational effect of PIE phenotype.There were no significant transgenerational genetic metabolic phenotypes other than blood pressure in the F2 offspring.Therefore,this study used LPS persistent treatment model as a representative to investigate the multigenerational inheritance effects of blood pressure in the F3?F4generation.1.1.3 PIE offspring disease sensitivity study:We used LPS persistent treatment model as a representative to re-stimulate F2 offspring by giving high-fat diet feeding,streptozotocin(STZ)injection and angiotensin II(Ang II)sustained-release infusion.Blood pressure,body weight,blood glucose and blood lipids of F2 offspring were detected to evaluate the sensitivity of F2-LPS-P rats to disease risk.1.2 PIE transgenerational model offsprings'vascular structure and function testThis study used LPS persistent treatment model as a representative to examine the underlying mechanisms of changed vascular structure,vascular function and elevated blood pressure.1.2.1 HE staining and tissue immunofluorescence were used to detect changes in intima and wall structures of the thoracic aorta and superior mesenteric artery.1.2.2 The in vitro vessel rings experiment was used to determine the contractile response to PE,the relaxation response to Ach and SNP,and the sensitivity to e NOS inhibitor L-NAME in the thoracic aortic tissue and superior mesenteric artery of F2offspring.1.2.3 HE staining and in vitro vessel rings experiment of thoracic aorta and superior mesenteric artery in Ang II re-stimulation model were performed to determine the sensitivity of PIE offspring arteries to hypertensive risk factors.1.3 PIE transgenerational model offsprings'epigenetic mechanism research1.3.1 The whole genome distribution of methylation in thoracic aortic tissue was detected by Me DIP-seq,and the reads were compared by MEDIPS package to obtain DMRs.Six DMRs were tested using another classical methylation assay,Bisulfite Sequencing PCR(BSP),to verify the results of the Me DIP-seq analysis.1.3.2 Functional analysis of DMRs was performed by using DAVID,KEGG,and IPA.Associations of functional changes such as F2-LPS-P vascular structure and elevated blood pressure were investigated based on enriched GO and Pathway.1.3.3 We verified the TOP 4 Pathway–G??,which was closely related to the vascular remodeling signal pathway and obtained by IPA analysis.Real-time PCR was used to verify the m RNA levels of genes related hypomethylated DMRs in the G??signaling pathway including Sos1,Sos2,Prkacz,Prkci,Prkd1,Prk3c3.Gng10,Gnaq,and Gng5 in the F2offspring thoracic aorta.Methylation of promoter regions of the differentially expressed genes included Sos1,Sos2,Prkacb,Gng10,and Prk3c3 in F2 thoracic aorta was detected by bisulfite-transformed PCR(BSP).In order to search for evidence of transgenerational epigenetic inheritance,the methylation rate of the above genes in F1 sperm was examined.1.3.4 G??directly activates the PI3K-Akt signaling pathway and plays a key role in the regulation of vascular smooth muscle cell proliferation and vascular tone resp onse.Therefore,Western-blot method was used to detect the protein expression level of Akt,p-Akt,S6 and p-S6 in the thoracic aorta of F2 offspring to verify the activity of PI3K pathway of PIE F2 offspring.The effect of PI3K activity on the contractile reactivity of thoracic aorta from F2 offspring was evaluated by an in vitro vascular ring experiment with PI3K inhibitor LY20094.2.Results:2.1 PIE transgenerational model offsprings'phenotype changes2.1.1 F0 pregnant rats were given intraperitoneal injection of LPS or Ploy I:C for inflammatory stimulation during pregnancy,whether continuous stimulation or transient stimulation,the systolic blood pressure of F2 offspring was higher than the control group and there was a statistical difference.The systolic blood pressure of F3-LPS-P and F4-LPS-P also showed a significant increase compared with the control group.This suggested that the phenotype of elevated blood pressure of PIE F1 offspring in our previous study was transgenerational inherited to F2-F4.2.1.2 Although the PIE F1 generation showed metabolic phenotypic changes in elevated weight,blood glucose and blood lipids,metabolic phenotype was not transmitted to the F2 generation.The body weight,fasting blood glucose,and blood lipid level s of the PIE F2 offspring were not different from the control group.2.1.3 PIE transgenerational model offsprings'sensitivity to high-fat diet,STZ model diabetes,Ang II model hypertension(disease risk factors).In order to investigate the sensitivity of PIE offspring to different environmental risk factors in adulthood,the F2-LPS-P offspring was used as representative to construct several re-stimulation models.No significant difference in blood pressure,body weight and blood lipids between F2-LPS-P and control group after re-stimulation of high-fat diet was observed.The diabetic model was successfully constructed after injection of STZ in F2offspring.After re-stimulation,the changes in body weight and blood glucose of F2-LPS-P and control group were the same.After re-stimulation with Ang II sustained-release infusion,the blood pressure of F2-LPS-P and control group increased significantly,but the increase of F2-LPS-P blood pressure was significantly higher from 8th to 10th day after modeling.The results suggested that the response of F2-LPS-P to the high-fat diet and STZ was not significantly different from the control group,while the PIE F2 offspring ha d a stronger sensitivity to Ang II-the risk factor of hypertension.2.2 PIE transgenerational model offsprings'vascular structure and function change2.2.1 From the HE results,when compared with control group,the F2-LPS-P rat thoracic aorta showed obvious structural damage,wall thickening and increased wall thickness/lumen diameter ratio,while the lumen diameter was not significantly different,which suggested a phenomenon of vascular remodeling.The superior mesenteric artery of F2-LPS-P rats only showed an increase in lumen diameter compared with the control group,and there was no difference in vessel wall thickness and wall thickness/lumen diameter ratio.Histochemical immunofluorescence staining of the thoracic aorta and superior mesenteric artery showed structural changes of the F2-LPS-P vascular endothelium.These results suggested the more significant vascular remodeling in the conductive artery of PIE F2 offspring.2.2.2 In the vessel rings experiment,the contractile reactivity of the thoracic aorta and superior mesenteric artery of F2-LPS-P to PE was significantly higher than control group,but there was no difference of vasodilation caused by Ach between two groups.This indicated that the PIE F2 offspring was characterized by an increase in the reactivity of arteries to vasoconstrictors.Without endothelium,the contractile reactivity was enhanced and the diastolic reactivity was weakened.The removal of the endothelial layer eliminated the difference in vasoconstriction between F2-LPS-P and the control artery,indicating that this effect was endothelial-dependent.In the vessel rings experiment with the addition of e NOS inhibitor L-NAME,inhibition of e NOS by L-NAME increased the contractile strength of F2-LPS-P and control rats'aorta.However,the vasoconstriction of F2-LPS-P responded to this inhibition more weakly than the control group.These suggested that the activity and buffering capacity of e NOS in F2-LPS-P vessels decreased.2.2.3 The method of constructing hypertension with Ang II sustained-release perfusion model was used to re-stimulate F2 offspring.The vasoconstriction intensity of thoracic aorta and superior mesenteric artery in F2-LPS-P offspring still significantly increase than control group,suggesteding PIE F2 offspring still had strong sensitivity to Ang II-a risk factor for hypertension.2.3 PIE transgenerational model offsprings'epigenetic mechanism of increased blood pressure2.3.1 Angiotensin-converting enzyme(ACE)was significantly increased in the thoracic aorta,superior mesenteric artery,and heart tissue of F2-LPS-P,and there was obvious vascular remodeling and functional changes in thoracic aorta,which suggested the pathological changes were more obvious in thoracic aorta.Therefore,we used the thoracic aorta as representative to research the epigenetic state of F2 offspring.After Me DIP-seq analysis,the whole genome methylation status of F2-LPS-P was changed compared with the control group,with 1696 hypermethylated DMRs and 1111 hypomethylated DMRs(Methylation changes>1.5 fold and edge-P-value<1×10-4 loci).BSP verified the DNA methylation results of F2 control and F2-LPS-P thoracic aortic tissue,showing that methylation status of the three hypermethylated(Fgfr2,Pfdn1 and Srpk2)and three hypomethylated(Cfhr1,Ndrg2 and Cox7a2l)DMRs with the largest difference or in the promoter region were consistent with the Me DIP-seq results.Most hypermethylated and hypomethylated DMRs are located in the relevant regions of intron elements.Compared to hypermethylated DMRs,hypomethylated DMRs were more distributed in the proximal promotor region of-1 kb upstream and+500 bp downstream of the transcriptional start site(Promoter-TSS)and transcription termination site(TTS).2.3.2 David GO bioprocess enrichment and KEGG analysis showed that hypermethylated DMRs-related genes were mainly enriched in brain and nervous system development,synapse assembly,c AMP and TGF-?signaling pathway.Hypomethylated DMRs-related genes were mainly enriched in positive regulation of RNA transcription and gene expression,endothelial cell migration,NF-kappa B transcription factor activity,MAPK activation,Ras and Jak/STAT signaling pathway,which were related to the activation of cardiovascular pathology and vascular remodeling signaling pathways.These results suggested that hypomethylated DMRs-related genes were mainly involved in the development of transgenerational epigenetic inheritance hypertension in PIE offspring.2.3.3 The Ingenuity(?)pathway analysis(IPA)software package was used to further identify the classical pathways and IPA interaction networks of hypomethylated DMRs closely related to the mechanism of increased blood pressure.The results showed that among the genes related to hypomethylated DMRs,there were several significantly enriched signaling pathways associated with vascular remodeling and the pathogenesis of hypertension,such as G Beta Gamma(G??)signaling,JAK/STAT signaling,Renin-angiotensin signaling,LPS-stimulated MAPK Signaling,p53 signaling,p70S6K signaling,G-protein coupled receptor(GPCR)signaling and ERK/MAPK signaling pathways.Among them,G??signaling pathway was the fourth most important classical pathway of hypomethylated DMRs.IPA network interaction analysis showed that G??signaling pathway was closely related to GPCR,JAK/STAT,LPS-stimulated MAPK,ERK/MAPK signaling pathway.This suggested that hypomethylated DMRs in the G??signaling pathway might play a key role in the development of vascular lesions and hypertension in the of PIE F2 offspring.2.3.4 m RNA expression levels and DNA methylation status in the gene promoter region of the G??signaling pathway were verified by Real-time PCR and BSP,respectively.The data showed that the m RNA expression of hypomethylated DMRs-related genes in the G??signaling pathway of F2-LPS-P rats had the tendency of increase,and the expression of Sos1,Sos2,Gng10,Prkacb and Pik3c3 was statistically different.BSP data showed a statistically decrease of the methylation rate in the DNA promoter regions of Sos1,Sos2,Gng10 and Prkacb in the thoracic aorta from PIE F2-LPS-P offspring compared to control.Evidence for transgenerational inheritance was verified by identifying methylation status in F1 offspring sperm.The sperm was isolated from the F1 male tail epididymis.The methylation rate of DNA promoter region in the G??signaling pathway was verified by BSP.The data showed the methylation pattern of Sos1,Sos2,Gng10 and Prkacb in PIE F1-LPS-P sperm was similar to that in the thoracic aorta of the PIE F2-LPS-P offspring.These data suggested that a reduced DNA methylation ratio across generations in the promoter region of genes clustered in the G??signaling pathway might play a key role in the development of multigenerational hereditary hypertension diseases.2.3.5 The G??signaling pathway combines with PI3K Class Ip110?to activate the PI3K signaling pathway.Activation of the PI3K-Akt signaling pathway plays a key role in the regulation of vascular smooth muscle cell proliferation and vascular tone response,and its overactivation is often associated with vascular remodeling and pathological changes.The activity of key downstream effector molecules of PI3K-Akt signaling pathway was analyzed.The Western-blot data showed that the phosphorylation levels of Akt and S6,the substrates of m TORC1 and m TORC2,in the thoracic aorta of PIE F2-LPS-P were significantly increased than those of control group.Total protein levels of Akt and S6 were not significantly different from the control group,indicating that PI3K-Akt signaling activity is enhanced in PIE F2 offspring.The role of PI3K-Akt activation in the enhancement of thoracic aorta contraction in the PIE F2-LPS-P offspring was evaluated by in vitro vascular ring reaction experiment.The experiment showed that the treatment of the PI3K inhibitor LY294002 significantly reversed the enhanced P IE-induced F2-LPS-P offspring thoracic aorta contraction response,the contraction intensity of two groups decreased significantly and the original differences between groups disappeared.In addition,there was no difference in Ach-induced thoracic aortic vasodilation between PIE F2-LPS-P offspring and F2 control group after LY290042 treatment.These results confirmed the important role of the PI3K signaling pathway in the enhancement of vasoconstriction and the pathogenesis of hypertension in F2-LPS-P offspring.3.Conclusion:3.1 Based on the previous LPS or Poly(I:C)-induced PIE hypertension model in our laboratory,a transgenerational inheritance model of LPS/Poly(I:C)of PIE was established.3.2 F2-LPS-P offspring showed changes in vascular reactivity characterized by increased responsiveness of arteries to vasoconstrictors.Compared with the superior mesenteric artery,the phenomenon of vascular remodeling in the thoracic aorta was more serious.3.3 Compared with control group,the sensitivity of PIE F2 offspring to Ang II,a r isk factor for hypertension,was significantly increased,which may be an important cause of hypertension.3.4 The whole methylation of PIE F2-LPS-P offspring changed,and the GO and signaling pathways of hypomethylated DMRs were closely related to vascular remodeling and the mechanism of hypertension.3.5 The consistency of hypomethylation in the promoter region of G??signaling pathway gene of PIE F2 offspring thoracic aorta and the F1 offspring sperm revealed transgenerational epigenetic inheritance.The activity of the PI3K/Akt signaling pathway,which was closely related to the G??signaling,was enhanced in the F2 offspring.This played a key role in the enhance of vascular reactivity and the development of hypertension in PIE offspring. |
PDF Full Text Request