Mechanisms Of Myosin 1b Regulating Vascular Endothelial Cell Senescence And Function By Mediating Autophagy

Research background:With the aging of the global population,cardiovascular disease（CVD）is becoming more prevalent.Vascular endothelial cells are continuous monolayers of flat cells lining the innermost layer of blood vessels.With the increase of age,endothelial cells undergo senescence,which leads to endothelial dysfunction such as increased reactive oxygen species（ROS）,decreased nitric oxide（NO）and increased production of inflammatory factors,resulting in vascular aging and dysfunction,and eventually leading to the development of various CVDs such as coronary artery disease,hypertension and myocardial infarction.Therefore,understanding the mechanism of endothelial cell senescence is of great importance for the prevention and treatment of CVD.Myosin 1b（Myo1b）,a type I myosin,which is a widespread intracellular kinetic protein that binds fibrils and generates kinetic energy by hydrolyzing ATP,and plays a role in cellular material transport,cell migration,and nuclear transcription.Recent studies have shown that Myo1b also plays a role in tumors,but Myo1b has not been reported in the regulation of endothelial cell senescence and function.Research purposes:1.To clarify the effect of Myo1b on the senescence and function of human umbilical vein endothelial cells（HUVEC）.2.To explore the mechanism by which Myo1b regulates HUVEC aging and dysfunction.3.To verify that Myo1b regulates vascular endothelial cell function through mediating autophagy in vivo.Research methods:1.Senescent HUVEC were obtained by in vitro passaging culture,and Myo1b was knocked down/overexpressed in senescent/young HUVEC using adenovirus,and control groups were set up accordingly.2.Myo1b expression was detected in each group of HUVEC by Western blot and q PCR assay.3.Senescence-associatedβ-galactosidase activity（SA-β-gal）and Western blot assays were performed to detect the senescence phenotype of each group of HUVEC.4.The function of each group of HUVEC was detected by ROS/NO kit,Western blot,q PCR assay and monocyte adhesion assay.5.The changes of autophagy markers LC3 and p62,lysosomal marker LAMP1 and Ca²⁺content in each group of HUVEC were detected by Western blot,immunofluorescence and fluorescent staining experiments.6.Autophagy was activated by the autophagy activator rapamycin（RAPA）,and the effect of activated autophagy on HUVEC senescence and endothelial dysfunction caused by overexpression of Myo1b was examined by Western blot,ROS/NO content and SA-β-gal assay.7.Myo1b expression was detected in aortic vascular endothelial cells of young and aged mice by immunodouble fluorescence staining of frozen sections.8.En face fluorescence staining was used to detect changes in isolated aortic vascular endothelial NO/ROS content in the young,elderly and Myo1b knockdown elderly groups.9.Autophagy markers in the aortic of three groups of mice were detected by Western blot assay.Research results:1.With HUVEC senescence,the m RNA and protein levels of Myo1b were significantly upregulated,and significant endothelial dysfunction features such as elevated ROS,decreased NO,and increased inflammatory factors were observed.However,Myo1b knockdown ameliorated HUVEC senescence and associated dysfunction,while overexpression of Myo1b in young HUVEC was found to significantly promote HUVEC senescence and endothelial dysfunction in reverse validation,consistent with our expected outcome phenomenon.2.knockdown of Myo1b in senescent HUVEC revealed a significant reduction in the expression of autophagy markers LC3 and p62 and a significant increase in co-localization of LC3 with LAMP1.In addition,the expression levels of autophagy markers LC3 and p62 as well as cytoplasmic Ca²⁺concentration were significantly increased with the upregulation of Myo1b expression in young HUVEC.RAPA treatment of Myo1b overexpressed young HUVEC significantly improved cellular senescence and dysfunction caused by up-regulation of Myo1b.3.Myo1b expression was significantly enhanced in the aortic vasculature of aged mice compared with young mice.In aged mice,adenovirus-mediated knockdown of Myo1b improved vascular endothelial function in aged mice,along with a significant increase in aortic autophagy levels.Research conclusion:In summary,this study demonstrated that Myo1b promoted vascular endothelial cell senescence through mediating autophagy,thereby inducing senescence-associated endothelial dysfunction and inflammatory responses.