Effect And Mechanism Of Metrnl On Vascular Tone And Atherosclerosis

Numerous secretory proteins play critical roles in physiological and pathological processes,and have been used as biomarkers and therapies for diseases.Thus,it’s of great significance to discover new ones and clarify their functions and mechanisms.Metrnl is a novel secretory protein homologous to the famous neurotrophic factor Meteorin.Before Metrnl’s first identification in 2012,its gene had been noticed by different research groups during their discovery of new functional proteins in the past ten years.Our lab identified Metrnl as a novel adipokine in early 2008 and since then we have been studying it.We demonstrated that Metrnl regulates white adipose biology and antagonizes insulin resistance induced by obesity.In addition,researchers from other labs showed Metrnl’s roles in processes such as neural development,white adipose browning and osteoblast differentiation.Nevertheless,more biology functions and the related mechanisms of Metrnl（like Metrnl’s function in vascular system）remains to be explored.Our preliminary experimental results had showed that Metrnl expresses highly in white adipose tissue,digestive tract and skin,while in brain Metrnl’s expression is very low.In this study,we noticed abundant Metrnl in cerebral blood vessels.We then quantified Metrnl in aorta,and found Metrnl mRNA level in aorta is as high as that in white adipose tissue.By aortas with or without endothelium,we proved Metrnl mainly expresses in endothelium within the blood vessels.We successfully detected Metrnl in the culture medium of mouse primary aortic endothelial cells,which proved that endothelial cells synthesize and secrete Metrnl.We speculated that Metrnl plays a critical role in vascular system.Therefore,we generated endothelial cell-specific Metrnl knockout(EC-Metrnl^-/-)mice to explore endothelial function of Metrnl as well as the mechanism.By detecting the circulating Metrnl level,we found that EC-Metrnl^-/-mice had significantly decreased Metrnl level compared to the wild type mice,suggesting Metrnl secreted by endothelium is one of the main contributors of circulating Metrnl.The effects of endothelial Metrnl on vascular contraction and relaxation were evaluated with thoracic aortas.Phenylephrine induced contraction exhibited no difference between EC-Metrnl^-/-mice and wild type mice.However,acetylcholine（ACh）induced endothelium dependent relaxation is dramatically attenuated in EC-Metrnl^-/-group.Meanwhile,nitroprusside,a NO donor,induced endothelium independent relaxation without difference between the two groups,suggesting Metrnl deficiency impairs endothelial function and reduces vascular relaxation induced by ACh.In the study of the eNOS-NO signaling pathway regulating ACh induced aorta relaxation,we found Metrnl deficiency in endothelial cells in vitro results in decreased phosphorylation of eNOS and reduced NO production,while it exibits no effect on the phosphorylations of AKT,AMPK,and STAT 3 as well as on the amount of PP2A.Additionally,no change was observed in eNOS interactions with Caveolin 1,Calmodulin or HSP90.Nevertheless,we found decreased tubulin expression both in mRNA and protein levels within endothelial cells with Metrnl knock down,in accordance with another result that overexpression of Metrnl in endothelial cells results in increased interaction between eNOS and tubulin.The results indicated that microtubes mediate Metrnl regulation of eNOS activity.Endothelial dysfunction is a key point in the initiation and progress of atherosclerosis.To further understand the role of Metrnl in the cardiovascular system,we studied the effect of Metrnl on atherosclerosis.We crossed the EC-Metrnl^-/-mice with the atherosclerosis-prone ApoE^-/-mice to obtain the EC-Metrnl^-/-mouse model for atherosclerosis.We found Metrnl deficiency in endothelium exacerbates atherosclerosis development with more atherosclerotic plaque and inflammation in the aortas.As further evidence,we also built the Metrnl^-/-mouse model for atherosclerosis by the same method,and the result supported the promoting effect of Metrnl deficiency on atherosclerosis development.Myocardial infarction is one of the most common complications of atherosclerosis.In our study,we detected circulating Metrnl level in patients with myocardial infarction.Due to lack of detection method,we first compared two commercially available kits for human Metrnl assay and investigated certain conditions which we thought could affect the result.In this part of our work,we found decreased circulating Metrnl level in patients with myocardial infarction compared to their controls.In conclusion,for the first time in this study,we proved that Metrnl highly expresses in vascular endothelium,and by endothelium-specific Metrnl knockout mice,we found Metrnl deficiency results in impaired endothelial function.Mechanism studies showed that Metrnl regulates vasodilation by influencing endothelial NO production,with a role of Metrnl in phosphorylation of eNOS.We speculate that tubulin mediates Metrnl regulation of eNOS activity.Moreover,our work for the first time showed that Metrnl deficiency in endothelium exacerbates atherosclerosis development,and that patients with myocardial infarction have decreased ciuculating Metrnl level.