Primary Investigation On The Internalization Pathway Of Microvesicles Into Human Umbilical Cord Endothelial Cells

Background:Mesenchymal stem cells(MSCs), which are characterized by their higher proliferative activity, immune regulation and multi-directional differentiation, have been widely used in regenerative medicine. Meanwhile, it may also provide a possible strategy for the treatment of ischemic cardiomyopathy. However, the specific mechanism is unclear.Recently, lines of animal experiments showed that although the majority of transplanted MSCs died of immunological rejection and maladaption to ischemic/hypoxic microenvironment within 72 hours in vivo, cardiac function was significantly restored after transplantation. However, the mechanism(s) involved in the effect of dead MSCs on cardiac function needs to be elucidated. Our previous experiment had shown that MSCs can release a large number of microvesicles(MVs) under a hypoxia and serum-deprivation condition. MVs can enhance cell proliferation, cellular migration and vessel structure formation after internalized into HUVEC in vitro and promote angiogenesis in vivo as well.Therefore, MVs derived from MSCs(MSC-MVs) may be a major mediator of MSCs function, at least in angiogenesis. Of note, the internalization of MVs is the key process in MSC-MVs mediated angiogenesis. However, there is a paucity of study about the mechanism(s) of the internalization of MSC-MVs by endothelial cells.Objects:MVs, membrance fragments released by activated or apoptotic cells, can transfer the genetic information of the parent cell to the target cells and are considered to be important mediators in the cell-to-cell communication. Our study was designed to provide theoretical foundation for the treatmental mechanisms of MSCs and the clinical replacement of stem cells by MVs.Methods:1. Firstly, density gradient centrifugation and differential adhesion method were used to isolate human bone marrow MSCs(BMSCs). The cells were identified through morphological observation, osteogenic and adipogenic differentiation assays, and flow cytometry analyzing their surface markers.2. Identified BMSCs were cultured in alpha-MEM without serum supplement under a hypoxic condition(1% O2, 5% CO2, 94% N2). Subsequently, the culture supernatants were collected to ultracentrifuge. The precipitated MVs were observed by electron microscopy.In order to identify the sources and the characters of MVs, flow cytometry was used to analyze their surface markers. The fluorescent dye CFSE or Di I labeled MVs were used to observe the cellular internalization of MSC-MVs.3. To explore the mechanisms underlying the MVs internalization, extracted MVs were pretreated with anti-human CD29 and CD44 antibodies, and/or recombinant human annexin V(Anx-V). Interfering RNAs(si RNA) targeting Anx-V and phosphatidylserine receptor(PSR) were transfected into HUVECs. Flow cytometry and confocal microscopy were used to evaluate the uptake process.Results:1. BMSCs were isolated successfully. Under the specific conditions, the adherent cells could be induced to differentiate into osteoblasts and adipocytes in vitro. Flow cytometry analysis showed that CD73 and CD44 were highly expressed on the surface of the obtained cells, while CD31 and CD45 were not.2. MVs derived from BMSCs were successfully collected by ultracentrifugation. Flow cytometry revealed that, similar to the BMSCs, the BMSC-MVs highly expressed CD73 and CD44 while not for CD31 and CD45. Additionally, high level of CD29 and PS were expressed on the surface of the BMSC-MVs as well.3. The internalization of BMSC-MVs by HUVEC was time-and dose-dependent.Anti-CD29, anti-CD44 and exogenous recombinant human Anx-V could block the internalization of BMSC-MVs through inhibition of CD29, CD44 and PS which were confirmed by Flow cytometry. Si-RNA-transfected HUVECs were co-cultured with CFSE-labeled BMSC-MVs. The positive percentage of internalization was greatly reduced in PSR si RNA-treated HUVECs, whereas down-regulation of Anx-V did not affect the incorporation of BMSC-MVs.Conclusions:1. BMSCs can release large amounts of MVs under a hypoxic and serum-deprivation condition.2. BMSCs-derived MVs can be internalized into HUVECs, which is an obviously time-and dose-dependent process.3. CD29,CD44,PS on the surface of the BMSC-MVs participate in the internalization.4. PS-PSR interaction is a critical step in the up-taking of BMSC-MVs by HUVECs.