Optogenetic engineered umbilical cord MSC-derived exosomes for remodeling of the immune microenvironment in diabetic wounds and the promotion of tissue repair

Animals

Male C57BLKS-Leprdb (db/db) mice (8 weeks old) were purchased from GemPharmatech Co., Ltd. All mice were housed in a specific pathogen-free area and were maintained in a 12-h light: 12-h dark cycle with controlled temperature (24 °C ± 1 °C) and relative humidity (50–60%). All the mice had free access to food and water. All surgical procedures and methods used in the study were approved by the Ethics Committee of Zhejiang Provincial People's Hospital (No. A20220015).

To establish the model of chronic diabetic skin wounds, the mice were anesthetized with 4% (vol/vol) isoflurane (RWD Life Science, USA) before the operation. After the animals were shaved, a circular full-thickness wound of 1.5 cm in diameter was created on the back of each mouse. The wound site was observed daily and was photographed using a digital camera on days 0, 7, 14, and 21. ImageJ software was used to measure and calculate the wound dimensions.

After surgery, UCMSCs-exo/eNOS or UCMSCs-exo (20 μg dissolved in 100 μL phosphate-buffered saline) or an equal volume of phosphate-buffered saline (PBS) were injected subcutaneously into the wound every other day. On the indicated day, skin tissue surrounding the wound was collected for analysis.

Culture of fibroblasts and endothelial cells

Human vascular endothelial cells (HUVECs) and L929 mouse skin fibroblasts were purchased from the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Biological Industries, USA) and 1% penicillin–streptomycin (Thermo Fisher Scientific, USA). Cells were incubated at 37 °C with 5% CO2.

Tubule formation assay

To study capillary-like construction activity in HUVEC cells, the formation of vascular-like structures was assessed using human fibrin matrices (Corning, USA). In brief, cold Matrigel (250 µL per well) was pipetted into 24-well plates using a pre-cooled pipette tip. This was followed by the addition of 400 μL of a suspension of 3,3-Dioctadecyloxacarbocyanine perchlorate (DiO)-stained HUVECs (7.5 × 105 cells/well, untreated or treated with UCMSCs-exo/eNOS or UCMSCs-exo) on the Matrigel, and incubated at 37 °C for 12 h. Tube-forming capacity was assessed by observing the tubular structures using a fluorescence microscope (Olympus, Japan).

Reactive oxygen species (ROS) detection

Intracellular ROS production was determined by 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining (Beyotime, China). Reactive oxygen species (ROS) in cells can oxidize non-fluorescent DCFH to produce fluorescent DCF, and the fluorescence intensity of DCF can thus reflect the ROS level. Fibroblasts or endothelial cells were seeded in 6-well plates at a density of 2 × 105 cells/well. After reaching 80% confluence, the cells were incubated with UCMSCs-exo/eNOS or UCMSCs-exo for 24 h and then subjected to high-glucose (HG, 50 mM) or H2O2 (200 μM) stimulation. After the indicated times, cells were incubated with 10 μm DCFH-DA for 20 min at 37 °C in serum-free medium and then washed three times with PBS. The probe-loaded cells were observed by confocal laser microscopy and the fluorescence intensity was analyzed by ImageJ software.

Mitochondrial membrane potential measurement (JC-1 staining)

The mitochondrial membrane potential was detected by JC-1 staining (Beyotime, China). JC-1 is an ideal fluorescence probe that is widely used for the determination of the mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC -1 accumulates in the mitochondrial matrix and emits red fluorescence. At low mitochondrial membrane potentials, JC-1 produces green fluorescence in the presence of the monomer. Fibroblasts or endothelial cells were cultured in 6-well plates at densities of 2 × 105 cells per well and incubated with UCMSCs-exo/eNOS or UCMSCs-exo for 24 h. When the cells reached 80% confluence, they were treated with hydrogen peroxide (200 μM) for 12 h. The JC-1 working solution was prepared according to the manufacturer's instructions and was added to the wells and incubated at 37° C for 20 min. After three washes with buffer, the changes in fluorescence were monitored by fluorescence microscopy and the ratio of red to green fluorescence intensity was analyzed using ImageJ.

TUNEL

Apoptosis of endothelial cells treated with H2O2 (200 μM) for 12 h was assessed using a fluorescent TUNEL detection kit (Beyotime, China), according to the manufacturer's instructions. The cells were fixed with 4% paraformaldehyde at room temperature for 20 min, followed by permeabilization with 0.3% Triton X-100 for 5 min, and the addition of the TUNEL detection solution. The cells were incubated at 37 °C for 60 min in the dark. The nuclei were counterstained with DAPI. TUNEL-positive cells were evaluated and counted under confocal microscopy.

Histological analysis

The skin of the wound tissue was collected on postoperative days 7, 14, and 21, fixed with 4% paraformaldehyde, and embedded in paraffin after dehydration. The paraffin-embedded tissues were sliced into 5-μm-thick sections. Re-epithelialization and the degree of collagen maturation were observed by hematoxylin and eosin (H&E) or Masson’s trichrome staining. Images were examined under an optical microscope (Olympus, Japan).

For immunohistochemical staining, the paraffin sections were rehydrated and incubated with the primary antibody, followed by incubation with the secondary antibody and the streptavidin biotin–peroxidase complex. Finally, the samples were visualized by the chromogenic substrate diaminobenzidine (DAB). The stained sections were observed with an optical microscope.

For the assessment of immunofluorescence, the paraffin sections were rehydrated, blocked with 1.5% goat serum, and incubated with the primary antibody overnight at 4 °C. The sections were then treated with Alexa Fluor 488 and Cy3-conjugated secondary antibodies, while the nuclei were stained with DAPI. The sections were examined and imaged using a confocal microscope and the fluorescence area and intensity were evaluated by ImageJ software.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted with TRIzol reagent and complementary DNA (cDNA) was obtained by the reverse transcription of 1 μg of total RNA from each extracted sample using the PrimeScript RT reagent kit (Takara Biotechnology, Japan). Next, the SYBR Green detection reagent (Takara Biotechnology) was used for qRT-PCR analysis in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, USA). Beta-actin or GAPDH was used for normalization of the results. The sequences of the primers used in this study are provided in Additional file 4: Table S1.

Western blotting

Briefly, tissues were homogenized and lysed on ice for 30 min in pre-chilled RIPA buffer containing a phosphatase inhibitor cocktail and PMSF. The lysates of exosomes or tissues were diluted in a 1:5 ratio with protein loading buffer (5 ×) (ABclonal, China) and heated at 95 °C for 5 min. Protein extracts were separated on 4‒20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels at 120 V and blotted at 220 mA onto polyvinylidene di-fluoride (PVDF) membranes (Merck Millipore, Germany) for 90 min. The membranes were blocked with Blocking Buffer (Yoche, China) for 10 min at room temperature followed by overnight incubation at 4 °C with the primary antibodies. The following day, the membranes were incubated with horseradish peroxidase (HRP)-linked secondary antibodies (HuaBio, China) for 2 h at room temperature. Finally, protein bands were visualized using an ECL substrate kit (Bio-Rad, USA) and the expression levels of the proteins were quantified by ImageJ. All primary antibodies were from Cell Signaling Technology (USA). All protein expression was normalized to β-actin or GAPDH.

Culture and identification of human UCMSCs

Human umbilical cord-derived mesenchymal stem cells were provided by Weiwei Biomedical Technology (China) and their identification was completed with the full support of Weiwei Biomedical Technology. The UCMSC cell line was cultured in MesenCult™ MSC Basal Medium containing MesenCult™ MSC Stimulatory Supplemen (STEMCELL Technologies, Canadian). UCMSC surface marker proteins were evaluated by flow cytometry (Agilent, USA); these included three positive markers (CD90, CD105, and CD73), six negative cocktails (CD45, CD34, CD14, CD11b, CD19, and HLA-DR), and the respective isotype controls. All antibodies were obtained from BD Biosciences (San Jose, CA, USA). Osteogenesis, adipogenesis, and chondrogenesis of UCMSCs were evaluated using the MesenCult™ osteogenic, adipogenic, and chondrogenic differentiation kit (STEMCELL Technologies). Differentiation properties were identified according to the manufacturer's protocol.

UCMSCs-exo/eNOS isolation and identification

The recombinant type 5 adenovirus expressing two fusion proteins, CIBN-EGFP-CD9 and eNOS-mCherry-CRY2 was contracted for production to Shanghai Genechem Co., Ltd. To upregulate CIBN-EGFP-CD9 and eNOS-mCherry-CRY2 for protein–protein interactions using optogenetics, the adenovirus vectors carrying the fusion protein CIBN-EGFP-CD9 or eNOS-mCherry-CRY2 were transfected into UCMSCs according to the manufacturer’s protocol.

Ultracentrifugation was used for the isolation and extraction of exosomes. UCMSCs overexpressing CIBN-EGFP-CD9 and eNOS-mCherry-CRY2 were seeded into T175 flasks. After one day, the medium was carefully removed, and exosome-depleted medium was added.

Then, the cells were exposed to continuous blue light illumination from a 460: 465 nm light board in a CO2 incubator. After 72 h, the cell culture supernatant was harvested and centrifuged at 300g for 10 min and 2000g for 30 min to remove dead cells and cellular debris. After further centrifugation at 10 000g for 30 min, the supernatant was filtered through a 0.22-µm filter (Merck-Millipore). The supernatant was then centrifuged twice at 100 000g for approximately 1.5 h each. The pellets were resuspended in PBS and stored at −80 °C for further experiments. The separation and purification of UCMSCs-exo was performed using the same procedures.

To verify the ultrastructure and shape of the exosomes, the exosomes were evaluated using transmission electron microscopy (TEM). Dynamic light scattering (DLS, Malvern Instruments, UK) was used for determining the size distribution and particle concentration of the exosomes. The presence of specific exosomal surface markers (TSG101, CD9, CD63, CD81) was evaluated using Western blotting.

Exosome labeling and uptake

Exosomes were labeled with the Dil fluorescent labeling kit (Yeasen, China). Dil (10 μm) was added to an exosome suspension and incubated at room temperature for 20 min in the dark. Exosomes were collected by centrifugation at 100 000g for 90 min and then washed twice with PBS to remove any unbound dye. The Dil-labeled exosomes were then incubated with the HUVECs for 24 h. The cells were then fixed and the nuclei were stained with Hoechst. Images were obtained by confocal microscopy.

Lipid peroxidation determination

Lipid peroxidation was assessed by measuring the red byproduct of the reaction of malondialdehyde (MDA) with thiobarbituric acid (T BA). Cells were treated with UCMSCs-exo/eNOS or UCMSCs-exo for 24 h and exposed to high glucose (50 mM) for 24 h. Absorbances were measured and standard curves were established in accordance with the manufacturer's instructions (Beyotime, China).

SOD determination

The activity of superoxide dismutase (SOD) was determined by the WST-8 method. WST-8 can react with xanthine oxidase-catalyzed superoxide anion radicals to produce water-soluble formazan dye and as SOD can inhibit formazan dye production through dissimilation, the activity of SOD can be measured by colorimetric analysis. SOD activities were measured in the cells after different treatments according to the manufacturer's instructions (Beyotime, China).

Total glutathione assay

This was measured by the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) by the mitochondrial enzyme glutathione reductase. GSH reacts with the chromogenic substrate DTNB to produce yellow TNB and GSSG. The total glutathione content was calculated by adjusting the reaction system and measuring the amount of yellow TNB formed. Each group of cells was measured after 24 h of high-glucose stimulation, according to the manufacturer's instructions (Beyotime, China).

NO measurement

NO is easily oxidized to NO2- and NO3- in vivo and NO3- is reduced to NO2- by nitrate reductase. Under acidic conditions, NO2- forms diazo compounds with diazosalt sulfonamide, which can be further coupled to naphthyl vinyl diamine. The product had a characteristic absorption peak at 550 nm, allowing calculation of the NO content by measuring the absorption value. Skin tissue was collected and homogenized. According to the manufacturer's instructions, the Micro NO Content Assay Kit (Solarbio, China) was used for NO measurements.

Transwell migration assay

Cell migration was measured by the Transwell method using 24-well Transwell culture plates (Corning, USA) with 8 μm pore-sized filters was used. Approximately 1 × 104 cells were inoculated into the upper chamber and incubated with serum-free medium. Then, different treatments containing HG (50 mM), UCMSCS-exo (20 μg/ml), and UCMSCs-exo/eNOS (20 μg/ml) were added to the lower chamber. Cells were cultured for 24 or 48 h in an incubator at 37 °C, after which the medium was removed, and cells were washed three times with PBS. Subsequently, cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystal violet for several minutes. A cotton swab was used to remove cells from the top surface of the filter. Migratory activity was assessed by observing the stained cells under an optical microscope and their enumeration using ImageJ software.

Cell migration assays

Cells (2 × 105 cells per well with three replicates per group) were seeded in 6-well plates and incubated at 37 °C. After the cells reached confluency, the monolayer was manually scratched with the tip of pipette, and the detached cells were removed by washing with serum-free medium. The cells were then cultured in medium supplemented with or without HG (50 mM), UCMSCS-exo (20 μg/ml), and UCMSCs-exo/eNOS (20 μg/ml). All cells were treated with Mitomycin-C for 1 h before scratching to exclude the influence of cell proliferation on wound closure. The matched wound areas were photographed at 0, 24, 48, and 72 h after wounding. The migration area (%) was calculated as (A0-An)/A0 × 100%, where A0 represents the initial wound area and An represents the remaining area of the wound at the measured time point.

Proliferation assay

Briefly, cells (5 × 103 cells per well, six replicates per group) were seeded into 96-well culture plates and treated with UCMSCs-exo (20 μg/ml) or UCMSCs-exo/eNOS (20 μg/ml). The cell-free group served as the blank group. At 12, 24, 36, and 48 h, the Cell Counting Kit-8 reagent (CCK-8, Yeasen, China; 20 μL per well) was added to the medium (100 μL per well). After incubation at 37 °C for 2 h, the absorbance of each well was measured at 450 nm by a microplate reader. The OD values were used to assess cell proliferation.

Calcein/PI cell viability/cytotoxicity assay

The survival and death of cells were assessed using Calcein-AM (Calcein-AM) and PI (propidium iodide) double-fluorescence staining (Beyotime, China). In brief, approximately 2 × 105 HUVECs were seeded in 6-well plates and incubated in complete medium with or without UCMSCs-exo/eNOS (20 μg/ml) and UCMSCs-exo (20 μg/ml) for 24 h before stimulation with H2O2 for 12 h. The calcein AM/PI detection solution was added and incubated for 30 min at 37 °C in the dark according to the manufacturer's instructions. The nuclei were stained with Hoechst. The cells were visualized by confocal microscopy.

Flow cytometry

The attached adipose tissue was removed before harvesting the skin wound tissue and the skin samples were sliced and digested with the Opti-MEM (Invitrogen) solutions containing collagenase type I (0.5 mg/ml, BioFroxx, China), collagenase type II (0.5 mg/ml, BioFroxx, China), collagenase type IV (1 mg/ml, BioFroxx, China), hyaluronidase (1 mg/ml, BioFroxx, China), and deoxyribonuclease I (0.02 mg/ml, Biosharp, China) for 30 min on a shaker at 37 °C. The tissue was mechanically ground using a Tissue Grinder with Pestle (YiXi Bio, China), washed with a complete medium containing 10% FBS, and the red blood cells were lysed.

Cell surfaces were stained in the dark on ice for flow cytometry analysis. The cells were stained with antibodies against the surface antigens CD4 (MultiSciences, China), CD25 (MultiSciences, China), CD8 (Proteintech, China), CD69 (Proteintech), and CD103 (Proteintech). Cells were fixed and permeabilized and then incubated with anti-CD16/32 (MultiSciences, China) to block nonspecific binding. Staining of intracellular markers, such as the Treg marker FoxP3 (MultiSciences, China), were performed on ice. The cells were then analyzed by flow cytometry.

Statistical analysis

All data are presented as means ± standard deviation (SD). Independent-sample t-tests were used to compare the means between two different groups. One-way analysis of variance (ANOVA) was utilized for the evaluation of the significant difference. Multiple-group comparisons were performed using one-way ANOVA. GraphPad Prism 8 software was used for all statistical analyses. P-values < 0.05 were considered statistically significant.

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