Biogenerated Oxygen‐Related Environmental Stressed Apoptotic Vesicle Targets Endothelial Cells

Abstract The dynamic balance between hypoxia and oxidative stress constitutes the oxygen‐related microenvironment in injured tissues. Due to variability, oxygen homeostasis is usually not a therapeutic target for injured tissues. It is found that when administered intravenously, mesenchymal stem cells (MSCs) and in vitro induced apoptotic vesicles (ApoVs) exhibit similar apoptotic markers in the wound microenvironment where hypoxia and oxidative stress co‐existed, but MSCs exhibited better effects in promoting angiogenesis and wound healing. The derivation pathway of ApoVs by inducing hypoxia or oxidative stress in MSCs to simulate oxygen homeostasis in injured tissues is improved. Two types of oxygen‐related environmental stressed ApoVs are identified that directly target endothelial cells (ECs) for the accurate regulation of vascularization. Compared to normoxic and hypoxic ones, oxidatively stressed ApoVs (Oxi‐ApoVs) showed the strongest tube formation capacity. Different oxygen‐stressed ApoVs deliver similar miRNAs, which leads to the broad upregulation of EC phosphokinase activity. Finally, local delivery of Oxi‐ApoVs‐loaded hydrogel microspheres promotes wound healing. Oxi‐ApoV‐loaded microspheres achieve controlled ApoV release, targeting ECs by reducing the consumption of inflammatory cells and adapting to the proliferative phase of wound healing. Thus, the biogenerated apoptotic vesicles responding to oxygen‐related environmental stress can target ECs to promote vascularization.


Cell biocompatibility test:
The ApoVs were co-cultured with endothelial cells (HUVECs, FDCC, China) or vascular smooth muscle cells (HA-VSMCs, FDCC, China) in DMEM with 10% FBS.Cell viability was assessed using the CCK-8 kit (CK04, Dojindo, Kumamoto, Japan).Hypoxic endothelial cells were incubated with 100 μmol/L cobalt chloride for 24 h.Optical density (OD) values of the various groups were measured at 450 nm using an enzyme marker (Varioskan Flash 3001, Thermo Fisher Scientific, Massachusetts, USA).On day 1, 2 and 3, cells were stained using a live/dead cell double staining kit (Sigma-Aldrich, St. Louis, Missouri, USA) for 30 min at room temperature and then examined under a invert fluorescence microscope.Live and dead cell quantifications were performed using ImageJ software and cell viability was calculated as the ratio of the number of live cells to the total number of cells.The biocompatibility of different concentrations and types of ApoVs with ECs was assessed in a similar way.

Microarray analysis of miRNAs:
The miRNA microarray assay (Agilent Human miRNA Microarray Kit, Release 21.0,8x60K, DesignID:070156; Agilent Technologies Deutschland GmbH, Waldbronn, Germany) and data analysis of the nine ApoVs samples were performed by OE Biotechnology Co. (Shanghai, China).Feature Extraction software (version 10.7.1.1;Agilent Technologies Deutschland GmbH,Waldbronn,Germany) was used to analyze the array images to obtain raw data.Raw data were normalized using quantification algorithms.Differently expressed ApoV miRNAs were subsequently identified based on their fold change as well as p value.Thresholds for up-and down-regulated genes were set at FC≥2.0 and p≤0.05.Target genes were predicted based on miRWalk 3.0 and miRDB database.
Based on the hypergeometric distribution, the screened miRNA target genes were analyzed for GO and KEGG pathway enrichment.

Synthesis of GelMA:
GelMA was synthesized according to the previous description [1] .
Briefly, 20 g of gelatin (Sigma-Aldrich, St. Louis, MO) was dissolved in 200 ml of carbonate-bicarbonate buffer (0.1 M) at 60 °C with stirring.2 ml of methacrylic anhydride (Aladdin, Shanghai, China) was added dropwise at 0.2 ml/min to the gelatin solution and reacted for 3 h at 50 °C.The gelatin solution was then mixed with 100 ml of DPBS at 40 °C to stop the reaction.The solution was dialyzed in deionized water at 40 °C for 1 week and then the final product was freeze-dried for 3 days to form a white foam.The GelMA foam was dissolved in deuterium oxide and the chemical shifts were measured using 1H NMR (Bruker Avance NEO 400MHz, Billerica, MA,) and the degree of methacrylation was determined, which is the ratio of the number of reacted methacrylamide groups to the number of amine groups in the unreacted gelatin.

Characterization of microspheres:
The morphology of microspheres was observed using an invert microscope and 100 microspheres were randomly selected for particle size analysis.The encapsulation and release of PKH26-labelled ApoVs from GA-MSP@Oxi-ApoVs were monitored at several time points (days 0, 1, 3, and 5) using an inverted fluorescence microscope.To obtain release curves of GA-MSP@Oxi-ApoVs, the protein concentration of ApoVs was initially determined using the BCA protein assay kit, and the fluorescence intensity was measured using the microplate reader (BioTek Synergy H1, Winooski, VT) to generate a standard curve.Microspheres prepared from 1 mL of GelMAsodium alginate solution were then immersed in PBS and incubated in a horizontal shaker at 37 ± 1°C, 90 rpm for 9 days.100 μL of the leachate was collected daily and replaced with fresh PBS.The concentration of ApoVs was obtained by detecting the fluorescence intensity of the leachate.
In vivo imaging: Fluorescence imaging analysis of living animals, MSCs or ApoVs were labeled with DiD dye (V22887, Vybrant, Thermo Fisher) following the manufacturer's instructions.MSCs or ApoVs were injection through the tail vein of nude mice.The in vivo distribution was observed on day 1, 3, and 7 by IVIS Lumina Kinetic series in vivo imaging system with a cooled CCD camera (PerkinElmer, Waltham, MA, USA).

Histopathology and immunofluorescence staining:
The collected samples were immediately stored in dry ice for frozen sections or fixed in 4% paraformaldehyde for 48 hours and then paraffin embedded.HE staining and Masson staining were performed for histological analysis in each group.ROS production at the wound site was detected using an intracellular ROS detection kit (MAK143, MAK145, Sigma-Aldrich, St. Louis, Missouri, USA).Immunostaining for CD31, α-SMA, Col I and Col III was then analyzed.The results of immunofluorescence staining for CD31 and α-SMA were calculated based on the number of new vessels in each group on days 7 and 14.Additionally, areas of neocollagen deposition in three random areas were calculated based on immunofluorescence staining for Col I/Col III using Image J software (National Institutes of Health, USA).

Figure S3 .
Figure S3.miRNA microarray analysis of oxygen-related environmental stressed ApoVs and ApoVs-treated EC phosphokinase microarrays.(A) Heatmap of DEmiR analysis of Hypo-ApoVs and Oxi-ApoVs compared to Con-ApoVs, respectively.(B) Wayne plots of miRNA predictions by miRDB and miRWalk databases.(C) The top 10 most significant GO entries of the three categories of Hypo-ApoVs and Oxi-ApoVs compared to Con-ApoVs, respectively.(D) Original images of phosphorylated protein arrays.