| Objective: Essential hypertension is a major public health problem.The etiology of hypertension is complex,which is the result of the interaction of genetic factors and environmental factors.The pathogenesis of essential hypertension is still unclear.Some experiments have proved that inflammation is closely related to the occurrence and development of hypertension.However,the mechanism of inflammation induced hypertension is still unclear.Low grade chronic inflammation is more common in non infectious diseases.Its pathological characteristics are completely different from acute inflammation,which is usually caused by exogenous factors(such as pathogenic microorganisms).On the contrary,chronic inflammation is usually induced by endogenous substances produced by tissue damage.Chronic inflammation can last for a long time,causing tissue damage,fibrosis and irreversible organ dysfunction.Neutrophils are the first line of defense against inflammation.Neutrophils are recruited to inflammatory sites to eliminate harmful substances.Neutrophils can also form neutrophil extracellular traps(NETs)through a special form of death,continue to capture and kill microorganisms,and prevent the spread of infection.Nets are formed by chromatin reticulum,in which a variety of proteins are embedded,such as citrullinated histone H3(cit Histone H3),myeloperoxidase(MPO).The formation and existence of NETs is a chronic and low-grade inflammatory reaction.It has been confirmed that NETs have been detected in human vascular tissue,and its existence is related to vascular wall lesions,such as atherosclerosis.It is found that NETs can participate in the formation of pulmonary hypertension by promoting angiogenesis.Proteins that play a role in induction of the NETs formation,such as peptidyl arginine deiminase 4(PAD4),can cause vascular fibrosis.Vascular fibrosis is one of the pathological changes of vascular remodeling,which is involved in the occurrence and development of hypertension.These studies suggest that the formation of NETs in arteries is closely related to the occurrence of hypertension.There is no report about the role of NETs formation in hypertension.We speculate that the formation of NETs in arteries can promote the occurrence of hypertension.NETs may become a drug target for the prevention and treatment of hypertension.Therefore,the purpose of this study explored the role of NETs formation in arterial hypertension.Methods: First,the formation of NETs in superior mesenteric artery tissue of Spontaneously Hypertension Rats(SHR)and Wistar Kyoto Rats(WKY)were detected.Then,PMA was injected into the tail vein to induce the formation of NETs in the arteries of mice,and the formation of NETs and its effect on blood pressure of mice were detected.Then,the effects of NETs on the proliferation,cell cycle and apoptosis of vascular smooth muscle cells(VSMCs)were detected in vitro.The proteomic differences between VSMCs and VSMCs treated by NETs(VSMCs-NETs)were detected and compared by label-free mass spectrometry.Through differential expressed genes(DEGs)analysis,GO and GO enrichment analysis,pathway enrichment analysis and hub protein analysis,the signaling pathways and functional proteins of VSMCs affected by NETs were explored.And then the expression of functional proteins was verified by Western blot in VSMCs and VSMCs NETs.After the expression of key functional protein in VSMCs was knock down by sh RNA.Then the effect of NETs on proliferation ability of the VSMCs was observed after key functional protein knock down.Then,the exosomes derived from VSMCs and the exosomes derived from VSMCs-NETs were extracted by differential centrifugation.The effects of the two kinds of exosomes on the proliferation,cell cycle and apoptosis of VSMCs were compared.The expression of key protein in the two kinds of exosomes was detected by Western blot.The exosomes derived from VSMCs transfected with negative control(NC)plasmid(VSMCs-sh NC)and the exosomes derived from VSMCs transfected with key protein knock down plasmid(VSMCs-sh RNA)were extracted to detect the expression of key protein.Finally,the effect of the two kinds of exosomes on VSMCs proliferation was detected.Finally,the expression of key protein in the arteries and serum of mice with NETs formation in arteries induced by PMA was detected by westren blot.All values are presented as the arithmetic mean ± SEM.A two sided t-test was used for the comparison of 2 groups and ANOVA was used for that of multiple groups.P <0.05 was considered statistically significant.Results: At 6-8 weeks and 20 weeks,the blood pressure of SHR was higher than that of WKY.cit Histone H3 expression was detected in the superior mesenteric artery of SHR rats,which was significantly higher than that of WKY rats.PMA was injected into mice through tail vein to induce NETs formation in atrery.The blood pressure increased accompanied with the expression of cit Histone H3 and MPO in the superior mesenteric artery was up-regulated,which indicated that the formation of NETs participated in the increase of the blood pressure of mice.The proliferation of VSMCs-NETs was accelerated.The percentage of cells in G0-G1 phase of VSMCs-NETs decreased,while the percentage of cells in S phase increased.There was no significant difference in apoptosis between VSMCs-NETs and VSMCs.A total of 141 DEGs between VSMCs-NETs and VSMCs were screened by label-free mass spectrometry,of which 43 DEGs were expressed in both two groups.25 proteins were up-regulated in VSMCs-NETs,and 18 proteins were down regulated in VSMCs-NETs.There were 98 specific proteins,44 of which were specifically expressed in VSMCs-NETs and 54 in VSMCs.CDKN1 b is a cell cycle dependent kinase inhibitor(CKI)of G1 / S checkpoint.The expression of CDKN1 b was not detected in VSMCs-NETs s.GO annotation and GO enrichment analysis showed that growth item was one of the functions of DEGs.Pathway enrichment analysis showed that PI3K-Akt pathway was one of the related pathways of DEGs.Hub protein analysis showed that TOP2 a was protein with the most interacting protein and TK1 interacted with TOP2 a.Western blot results showed that compared with VSMCs,the expression of CDKN1 b was down regulated and the expression of TOP2 a,TK1 and p-Akt was up-regulated in VSMCs-NETs.Knockdown of TK1 expression in VSMCs slowed down the proliferation of VSMCs and blocked the proliferation promoting effect of NETs.Transmission electron microscope showed the vesicular vesicle-like structure of exosomes VSMCs;Malvern particle size analysis showed that the diameter of exosomes ranged from 30 nm to 200 nm;Western blot detected the expression of exosomes markers CD9 and Alix.Exosomes derived from VSMCs promoted the proliferation of VSMCs,while the effect of exosomes derived from VSMCs-NETs was stronger.At the same time,compared with VSMCs untreated,the proportion of G1 phase of VSMCs treated with exosomes derived from VSMCs decreased,while the proportion of S phase cells increased.Compared with VSMCs treated with exosomes derived from VMSCs,the proportion of cells in G1 phase of VSMCs treated with exosomes derived from VSMCs-NETs decreased and the proportion of cells in S phase increased.Both two kinds of exosomes did not affect the apoptosis of VSMCs.Compared with exosomes derived from VSMCs,the expression of TK1 was up-regulated in exosomes derived from VSMCs-NETs.Compared with the exosomes derived from VSMCs transfected with NC plasmid,TK1 expression was down regulated in the exosomes derived from VSMCs transfected with TK1 knockdown plasmid.The effect of TK1 knockdown exosomes on VSMCs proliferation was weakened.Finally,the expression of TK1 was up-regulated in superior mesenteric artery tissue and serum of mice with NETs formation.Conclusion: These results suggest that NETs up regulate the expression of TK1 by activating PI3K-Akt and down regulating CDKN1 b,thus promoting the G1/S phase transition and then promoting the proliferation of VSMCs.NETs can promote the proliferation of VSMCs through exosomes derived from VSMCs to transfer TK1.The formation of NETs in arterial wall participate the occurrence of hypertension by inducing VSMCs proliferation. |
