Mechanism Of SAPPα-Induced Cerebral Angiogenesis

Objective:Amyloid precursor protein（APP）is a membrane integration protein that is commonly expressed in various tissues of the human body and is mainly concentrated in the synapses of neurons.For the function of APP,the researchers focused on the fact that APP is the precursor protein that producesβ-amyloid（Aβ）,and the progressive deposition of Aβin the brain leads to the main pathological causes of Alzheimer’s disease.In the processing of APP as a precursor protein,three secretory enzymes alpha,beta,and gamma act synergistically and are responsible for the shear processing of APP.After APP is first processed byβ-secretase and then processed byγ-secretase to produce Aβ,this process is called Aβproduction pathway;in addition,APP can produce soluble sAPPαafter shear processing byα-secretase.This process is called the non-Aβproduction pathway.Recent studies have shown that the biological effects of Aβand sAPPα,two products also derived from APP,are very different.The deposition of Aβin brain causes Alzheimer’s disease,while sAPPαhas a certain degree of neuroprotective effect.Pericyte is one of the important components of neurovascular units.Pericytes are contractile and closely contact with the basal surface of brain microvascular endothelial cells and embedded in the basement membrane of brain microvascular endothelial cells.Peripheral cells regulate cerebral blood vessel permeability and blood flow through interaction with brain microvascular endothelial cells.Recent studies have shown that pericytes play an important biological role in the generation of brain microvessels,maintenance of blood-brain barrier permeability,and so on.Exosome is a membranous vesicle that can be secreted into the cell and has specific morphological features and functions.Exosomes are one of the most important carriers for the exchange of substances or information between cells.Its diameter is 30-.100nm.Exosomes have many important biological functions such as immune response,antigen presentation,exchange of cell information,and transmission of RNA and protein.Our previous research found that the expression of APP is not only limited to neurons,but also can be detected in the brain microvascular endothelial cellsAPP expression and APP processing.Therefore,this study will investigate the effect of APP processing product sAPPαon cerebral microvessels,and analyze the mechanism of sAPPαon brain microvascular endothelial cells.Furthermore,analyze the effect and mechanism of sAPPαon brain microvessels in exosomes secreted by pericytes.Methods:1.Using brain stereotaxic apparatus,sAPPαprotein was injected into the brain of 2-week-old C57 mice and supplemented once every two days.After 9 days,the brains of the rats were taken to prepare brain sections.Immunofluorescent staining of brain microvessel marker protein CD31 was performed.Confocal laser scanning microscopy was used to analyze changes in vascular density in the brain;2.Cell proliferation assay was used to analyze the effect of sAPPαon proliferation of brain microvascular endothelial cells;3.The effect of sAPPαon the migration of brain microvascular endothelial cells was analyzed by cell scratch assay.4.Using in vitro angiogenesis assay,analyze the effect of sAPPαon the in vitro angiogenesis ability of brain microvascular endothelial cells;5.Brain microvascular endothelial cells were treated with pure sAPPαprotein,and actin F-actin was stained by immunofluorescence.The effect of sAPPαon F-actin in brain microvascular endothelial cells was analyzed by confocal laser scanning microscopy.And further analyzed the blockade effect of actin polymerization blocker（Cytochalasin D）on F-actin polymerization induced by sAPPα;6.Peripheral culture of pericytes,collection of pericyte culture supernatants,extraction of exosomes in the supernatant of pericytes,and identification of exosomes by western blot;7.Simulate oxidative stress conditions（50μM H₂O₂）and detect Aβ40 and sAPPαin exosomes secreted by pericytes under oxidative stress by ELISA.8.The exosomes secreted by the pericytes were stained with PKH26 fluorescent dye.The exosomes were co-cultured with brain microvascular endothelial cells and analyzed by confocal laser scanning microscopy to detect the fusion of pericytes derived from pericytes and brain microvascular endothelial cells.HappeningResults:1.Intraventricular injection of sAPPαcan promote angiogenesis in the mouse brain;2.sAPPαcan promote angiogenesis of human brain microvascular endothelial cells;3.sAPPαcan promote the migration of human brain microvascular endothelial cells,but has no effect on the proliferation of the cells;4.sAPPαcan promote actin stress fiber formation in human brain microvascular endothelial cells,and this promotion can be blocked by cytochalasin D;5.The effect of sAPPαon angiogenesis in human brain microvascular endothelial cells can be blocked by cytochalasin D;6.Pericytes can secrete exosomes to the extracellular environment;7.Under oxidative stress conditions,the secretion of sAPPαin exosomes secreted by pericytes increased significantly,and the change of Aβ40 was not significant.8.Pericytes secreted by pericytes can fuse with brain microvascular endothelial cells into the brain microvascular endothelial cells;Conclusion:1.sAPPαpromotes the generation of brain microvessels by promoting the polymerization of actin in brain microvascular endothelial cells;2.Oxidative stress condition can increase the sAPPαcontent in exosomes secreted by pericytes;3.Exosomes secreted by pericytes can act on brain microvascular endothelial cells.