Anti-Atherosclerosis Effects And Mechanisms Of Biologically Active Short Peptide ELA-11 By Modulating Macrophage Function

1.BackgroundAtherosclerosis is a complex chronic inflammatory disease.Macrophages are the core cells of vascular inflammation and injury,which make the inflammatory cycle continue by secreting inflammatory cytokines.Therefore,inhibition of macrophage proliferation,foaming or apoptosis may be a promising treatment to inhibit the progression of atherosclerosis.Elabela/Toddler(ELA)is the second endogenous ligand of Apelin receptor(APJ)encoded by APELA gene.Some studies have proved that ELA may play an important protective role in atherosclerotic diseases.ELA-11 fragment has low molecular weight and high fat solubility,which can directly activate APJ and its downstream signals.This study confirmed that the development and optimization of ELA-11 peptide structure is expected to become a new drug for the prevention and treatment of atherosclerosis.2.ObjectiveTo verify whether ELA-11 can inhibit the development of atherosclerosis by regulating the function of macrophages in vivo and in vitro,and to explore the potential mechanisms of its anti-atherosclerosis effects.3.MethodsThe level of ELA in human serum was detected by ELISA and the expression of APELA gene in human carotid atherosclerotic plaque was detected by RT-q PCR.Mouse foaming macrophages were induced by ox-LDL,oil red O staining and flow cytometry were used to verify the effects of ELA-11 on macrophage foaming,apoptosis and M1 polarization.Through rescue experiments with WB technique we verified that the ELA-11,only with combination of APJ,can inhibit atherosclerosis through AKT and endoplasmic reticulum stress pathways.The atherosclerotic model of Apo E-/-gene knockout mice was established,and the anti-atherosclerotic effects and toxicity of ELA-11 were evaluated by HE staining,oil red O staining,Masson staining,immunohistochemistry and immunofluorescence techniques.m PEG@ELA-11,a kind of passive targeting nanoparticles designed by us,can not only prolong the half-life of ELA-11 in vivo,but also automatically degrade into ELA-11 in the low p H micro-environment of unstable plaques.The polymers were characterized by TEM,FT-IR,NMR,DLS and other techniques,and the targeting effect was verified by fluorescence imaging.Finally,it is verified whether the modified ELA-11 has a better therapeutic effect on inhibiting macrophage foaming and reducing plaque area.4.ResultsThe level of ELA in serum of healthy people was negatively correlated with age,and the expression of APELA gene in human carotid atherosclerotic plaque was down-regulated,and the expression of APELA gene in unstable plaque was lower than that in stable plaque.ELA-11 can inhibit the mouse macrophages foaming,M1 polarization and apoptosis,which induced by ox-LDL through AKTendoplasmic reticulum stress pathway,thus inhibiting the development of plaque.p H-responsive m PEG@ELA-11 polymers can significantly inhibit the progression of atherosclerotic plaque in mice,and the effect is better than that of ELA-11 alone.5.Conclusionas an endogenous APJ receptor agonist,ELA-11 can inhibit macrophage foaming and apoptosis by down-regulating the factors related to AKT-ERS pathway,thus playing an anti-atherosclerotic effect.The designed and synthesized p H-responsive polymers,m PEG@ELA-11,can passively target atherosclerotic plaques and enhance the anti-atherosclerotic effect of ELA-11.ELA-11 has the prospect of further developing into anti-atherosclerotic drugs.