Effects And Mechanism Of Exosomes Derived From Human Amniotic Epithelial Cells In Facilitating Diabetic Wound Healing

Objective:With the aging of the population and the frequent occurrence of the obesity,diabetes and other chronic diseases,the number of with is increasing.In the United States,there are 3 to 6 million new chronic wounds patients each year,and the treatment costs are increased nearly $5 to $10 billion.In China,chronic wounds patients accounted for 1.5% to 20.3% of inpatients in surgery.Therefore,chronic wounds have become a worldwide problem and a heavy burden on the entire social health care system as well.Diabetic ulcer is a frequent and difficult chronic wound in diabetic patients characterized by local skin breakage,which occurs on the basis of the peripheral neurovascular lesions.According to statistics,the incidence of diabetic ulcers accounts for about 2% of the total number of diabetics in China.For every diabetic,there is a 25% chance of developing lower limb ulcers.Although there are clinically standardized treatments for diabetic ulcers,such as blood sugar control,local blood circulation improvement,infection control and the necrotic tissue removement,about 7-20% patients with diabetic foot ulcers eventually need amputation.Therefore,it is of great clinical and social significance to manage diabetic ulcers effectively,develop new treatment methods,improve the ability of wound repairment,and promote the healing of diabetic wounds.Human Amniotic Epithelial Cells(hAECs)are derived from the innermost layer of amniotic membranes and belong to embryonic stem cells,which have the advantages of non-tumorigenicity,low-immunogenicity,immune regulation,easy acquirement and non-ethical restrictions.Our research group confirmed that both cryopreserved living micronized amnion and fresh human amniotic membrane epithelial cells could promote diabetic wound healing by regulating the local microenvironment and vascularization process of the wound.Exosomes are a kind of extracellular vesicles that almost all cells can secrete,and their formation process selectively loading functional or non-functional lipids,proteins,genetic material,etc.,is one of the important carriers for the transmission of intercellular signals.Numerous studies have confirmed that exosome mechanism may be a key pathway for stem cells to play the role in tissues/organs regeneration.Literatures reported that,similar to the exosomes derived from endothelial progenitor cells,fat stem cells and umbilical cord protoplasmic stem cells,the exosomes from human amniotic epithelial cells(hAECs-Exos)could accelerate the speed of acute wound healing and improve the quality of wound by regulating the repairment cells function and the wound microenvironment as well.However,whether it is beneficial to the diabetic wound healing process is unknow.This paper aims to explore the role and mechanisms of the hAECs-Exos on the wound healing-associated cells in the high-sugar environment and diabetic wounds,and provide a new idea for the effective management of diabetic wounds.Methods:1.hAECs separation and identification(1)Amniotic tissues were collected and rinsed and hAECs were separated by trypsin enzyme-digesting technique;(2)Inverted microscope and flow cytometry were applied to identify the general view and cell phenotype of hAECs respectively;(3)Directional differentiation experiments and protein immunofluorescence technique were used to identify the stem cell characteristics of hAECs.2.hAECs-Exos separation and identification(1)The condition medium of hAECs was collected and ultracentrifugation was conducted to collect hAECs-Exos;(2)Transmission Electron Microscope(TEM)was applied to observe the general view of hAECs-Exos;(3)Dynamic Light Scattering(DLS)was conducted to detect the particle size distribution of hAECs-Exos;(4)Flow Cytometer(FCM)was used for examining exosome phenotypes.3.hAECs-Exos regulate the function of the wound healing-associated cells in high-glucose environmentThe wound healing-associated cells(HFBs/HUVECs/HUVECs)were randomly divided into hAEC-Exos and control groups(PBS).(1)CCK-8 was used to detecte the proliferative effect of hAECs-Exos on wound healing-associated cells;(2)Transwell and scratch experiments were used to evaluate the migration effect of hAECs-Exos on wound healing-associated cells;(3)Tube formation experiment was carried out to assess the angiogenic effect of HUVECs by hAECs-Exos.4.hAECs-Exos promotes the healing speed of diabetic woundsDiabetic mice were anesthetized with 1% pentasorbital sodium(0.1 ml/20g)by intraperitoneal injection and two full-thickness wounds with diameter of 8mm were prepared on both sides of the spine for each diabetic mouse.And then they were randomly assigned to either subcutaneously inject with hAEC-Exos,or equal volume of PBS was subcutaneously injected for control group around the wounds.We took regular photos to record the general view of wounds and to statistical analysis the healing rate at different points.5.hAECs-Exos enhanced the functions of wound healing-associated cells by activating PI3K-AKT-m TOR(1)High-throughput sequencing predicted the enrichment signaling pathway for the target genes of hAECs-Exos-mi RNAs,and WB assay was used to verify the result in wound healing-associated cells.(2)The wound healing-associated cells were randomly divided into hAEC-Exos +DMSO group,PBS + DMSO group and hAEC-Exos + LY294002 group:(1)WB was used to verify the activation or inhibition of PI3K-AKT-m TOR pathway;(2)CCK-8 was carried out to detect the proliferation of wound healing-associated cells;(3)Transwell and scratch assay were applied to detect the migration of wound healing-associated cells;(4)Tubular formation analysis was used to detect the tubule-forming ability of HUVECs.6.hAECs-Exos accelerated the healing speed and quality of diabetic wounds by activating PI3K-AKT-m TORDiabetic mice were anesthetized with 1% pentasorbital sodium(0.1 ml/20g)by intraperitoneal injection and two full-thickness wounds with diameter of 8mm were prepared on both sides of the spine for each diabetic mouse.And then they were randomly divided into hAEC-Exos+DMSO,PBS+DMSO and hAECExos+LY294002 group.(1)Photos were taken to record the general view of wounds and healing rate at different points was analyzed;(2)H&E and Masson staining were used to observe the pathological structure of the wound tissue;(3)Immunohistochemical staining and sirius red staining were conducted to examine the degree of tissue vascularization,cell proliferation and extracellular matrix collagen deposition.Results:1.hAECs obtained by trypsin enzyme-digesting technique are oval,rich in cytoplasm,uniform in size,arranged in a typical pebble-like monolayer,and have obvious epithelial-like cell morphology.They highly express in CD73,CD90 and CD29 on the surface,but not CD34 and HLA-DR.At the same time,hAECs can induce differentiation into bone tissue,cartilage tissue and adipose tissue,and highly express in embryonic antigens SSEA-3,SSEA-4 and the transcription factor Oct-4,which is very important to maintain the pluripotency and self-renewal of embryonic stem cells;2.hAECs-Exos obtained by ultracentrifugation are spherical,disc-shaped or cupshaped,the main peak of the particle size is near 100 nm,and the surface markers of CD63 and TSG101 are strongly positive;3.hAECs-Exos can promote the proliferation and migration of wound healingrelated cells and the tube formation of HUVECs in high-sugar environment;4.Local injection of hAECs-Exos can shorten the wound healing time of diabetic mice;5.hAECs-Exos-mi RNA target genes are highly enriched in PI3K-AKT-m TOR signaling pathway;6.hAECs-Exos can activate the PI3K-AKT-m TOR signaling pathway in wound healing-related cells in high-glucose environment.When this pathway is blocked,the effects of hAEC-Exos on wound healing-related cells will be partially suppressed;7.hAECs-Exos can activate the PI3K-AKT-m TOR signaling pathway in the wound tissues of diabetic mice.When this pathway is blocked,hAEC-Exosmediated functions that promotion in the healing speed and quality of diabetic wounds will be partially suppressed.Conclusions:1.hAECs with high purity and specificity can be obtained by trypsin enzymedigesting technique;2.hAECs-Exos meeting the experimental requirements can be successfully separated by ultracentrifuge method;3.hAECs-Exos accelerate the healing speed and quality of diabetic wounds by improving the function of wound healing-related cells in high-glucose environment;4.PI3K-AKT-mTOR mediates the effects of hAEC-Exos on healing-related cells function and the healing speed and quality of diabetic wounds;5.hAEC-Exos treatment is expected to become a promising new strategy for the management of diabetes wounds.