Stem cell-derived exosomes from human exfoliated deciduous teeth promote angiogenesis in hyperglycemic-induced human umbilical vein endothelial cells

Abstract Objective To investigate the angiogenesis in human umbilical vein endothelial cells (HUVEC) under high glucose concentration, treated with exosomes derived from stem cells from human exfoliated deciduous teeth (SHED). Methodology SHED-derived exosomes were isolated by differential centrifugation and were characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometric assays. We conducted in vitro experiments to examine the angiogenesis in HUVEC under high glucose concentration. Cell Counting Kit-8, migration assay, tube formation assay, quantitative real-time PCR, and immunostaining were performed to study the role of SHED-derived exosomes in cell proliferation, migration, and angiogenic activities. Results The characterization confirmed SHED-derived exosomes: size ranged from 60–150 nm with a mode of 134 nm, cup-shaped morphology, and stained positively for CD9, CD63, and CD81. SHED-exosome significantly enhanced the proliferation and migration of high glucose-treated HUVEC. A significant reduction was observed in tube formation and a weak CD31 staining compared to the untreated-hyperglycemic-induced group. Interestingly, exosome treatment improved tube formation qualitatively and demonstrated a significant increase in tube formation in the covered area, total branching points, total tube length, and total loop parameters. Moreover, SHED-exosome upregulates angiogenesis-related factors, including the GATA2 gene and CD31 protein. Conclusions Our data suggest that the use of SHED-derived exosomes potentially increases angiogenesis in HUVEC under hyperglycemic conditions, which includes increased cell proliferation, migration, tubular structures formation, GATA2 gene, and CD31 protein expression. SHED-exosome usage may provide a new treatment strategy for periodontal patients with diabetes mellitus.


Introduction
Diabetes is a group of metabolic disorders characterized by high blood glucose levels and potentially debilitating diseases, ranked ninth among the leading causes of death worldwide. 1 In 2019, 463 million people were diagnosed with diabetes, disregarding undiagnosed cases. 2 The long-term complications of diabetes are associated with the destruction of blood vessels, leading to cardiovascular disease, chronic kidney disease, retinopathy, neuropathy, and periodontitis. 3 Diabetes has a bidirectional relationship with periodontitis, especially in poorly controlled diabetes patients.
The data from the US National Health and Nutrition Examination Survey (NHANES) III showed that adults with HbA1C levels higher than 9% had a significantly higher prevalence of severe periodontitis than those without diabetes. 4 Periodontitis is a chronic inflammatory disease involving the destruction of the periodontal tissue, which comprises the gingiva, cementum, periodontal ligaments, and alveolar bone.
Periodontal therapy aims to regenerate damaged tissues. 5 Angiogenesis is a critical part of regenerative therapy since an established vasculature is critical for supplying nutrients, minerals, and oxygen for proper tissue development and functionality. 3,5 Moreover, vascularization aids in growth factor production that helps modulate the function of various cells in periodontal tissue, such as osteoblasts, osteoclasts, and related mesenchymal stem cells (MSCs). 6 Notably, diabetes contributes to endothelial cell dysfunction (ECD). 7 In diabetic vasculature, the hyperglycemic condition can cause non-enzymatic glycosylation of proteins and lipids leading to the interference of normal protein function. 8 Hyperglycemia also increases oxidative stress through several pathways.
A major mechanism appears to be superoxide (O 2 •− ) overproduction by the mitochondrial electron transport chain. Moreover, hyperglycemia can promote inflammation via induction of cytokine secretion by several cell types. 8 Undoubtedly, the regenerative impact of ECD on angiogenesis leads to a poor response of diabetic patients to regenerative therapy.
Diabetic patients had poor responses to periodontal treatment, possibly due to ECD involvement. 9 In many situations, conventional periodontal therapy involving root surface debridement to induce healing, guided tissue regeneration, and bone graft placement cannot achieve tissue regeneration effectively. The traditional treatment for periodontitis is associated with a relatively high degree of variability in clinical outcome, and the curative effect remains unsatisfactory. 10

Isolation and characterization of SHEDexosome
Methods for exosome isolation and characterization were performed as described previously with minor modifications. 25,31 SHED was cultured until approximately 80% confluent, washed three times with phosphate-buffer saline (PBS), and then incubated in DMEM without FBS and antibiotics. After incubation for 48 hours, the CM was collected and centrifuged at 1,000 rpm for 5 minutes, followed by filtering through 0.2 µm filters. The CM was then concentrated in Amicon ® Ultra-15 10 kDa nominal molecular weight centrifugal filter at 5,000 g for 40 minutes. The exosomes were isolated using the differential centrifugation method. Following ultracentrifugation at 100,000 × g for 70 minutes at 4°C, the exosomes were resuspended in PBS and then stored at −80°C.
The concentration of exosomes was measured using Nanodrop spectrophotometers. The characteristics of the exosomes derived from SHED cells were further identified. First, the particle size distribution

Exosomes internalization
Exosomes derived from SHED cells were labeled with PHK67 green fluorescent cell linker kit (Sigma-Aldrich Corp., St. Louis, MO, USA). In brief, 1.5 μl PKH67 dye was added to 10 μg exosomes in a total of 250 μl diluent C provided in the kit and incubated at room temperature for 5 minutes. Exosomes without PKH67 staining were used as the negative control.
Excessive dye was removed by centrifugation at 190,000 × g for 2 hours at 4°C. The mixture was resuspended in a complete medium and incubated with HUVEC at 37°C for 4 hours. A laser confocal microscope was used to visualize the incorporation of exosomes into HUVEC.

Treating the HUVEC with high glucose concentration
After reaching the desired confluence, HUVEC Negative control: HUVEC were cultured in 5.5 mM of glucose in a DMEM.
All groups were cultured for four days and were then investigated for angiogenesis.

Effects of SHED-exosome on angiogenesis
Cell counting Kit-8 assay The Cell Counting Kit-8 assay (CCK-8 assay, Dojindo, Kumamoto, Jp) was used to evaluate cell proliferation according to the manufacturer's instructions. Briefly, 3×10 3 HUVEC per well were seeded in a 96-well plate and incubated with exosomes (10 μg/ml) or EGM (control). A total of six wells were designed for each group. On days 0, 2, 4, 6, and 7, the CCK-8 working solution was added to each well and incubated for 2 hours. The optical density at 450 nm was subsequently measured using a microplate reader.

Migration assay
Scratch assays were used to evaluate the cell migration. Briefly, 1×10 5 HUVEC were seeded in 24-well plates. Cells at 90%-100% confluency were subjected to single vertical scratches using a 200 µL sterile pipette tip and then washed with EGM to remove detached cells. SHED-exosome (10 μg/ml) or EGM medium (control) were added, and images were recorded at 0, 6, and 12 hours after scratching using an optical microscope. The closure distance was analyzed using Image Analysis J (Olympus, Alexandra, SG). The rate of wound closure was estimated as follows: Rate of wound closure =

Tube formation assay
Matrigel ® Matrix (60 μl, Corning, Glendale, AZ, USA) was added to pre-cooled 96-well plates and were incubated at 37°C for 1 hour. The HUVEC cells were seeded at 1.5×10 4 cells per well into each Matrigel-coated well. The network structures in 6 hours were captured using phase contrast microscopy.

Characterization and internalization of SHEDexosomes
The diameter of the exosome isolated from SHED CM was accessed by NTA with the camera level set to 14 and the detection threshold to 5 and was shown to range from 60 to 150 nm with a peak of 134 nm ( Figure 1A). The negative staining and morphology of the SHED-exosomes via a TEM microscope showed the typical cup-shaped particles ( Figure   1B). Flow cytometry of SHED-exosomes showed the positive expression of the exosome-specific markers CD9, CD63, and CD81 ( Figure 1C). These findings confirmed that the extracellular vesicles isolated from SHED CM are exosomes. Subsequently, the exosomes were labeled with PKH67 dye to test the interaction between the exosome and HUVEC. The Effects of SHED-exosome on the migration of high glucose-treated HUVEC A scratch assay was used to test if SHED-exosome could enhance the migration behavior of high glucose-treated HUVEC as shown in Figure 2B and p=0.01) and 12 hours (87.1±4.9; p=0.03) ( Figure   2B and Figure 2C). The HUVEC cultured in DMEM (negative control) showed a lower percentage of relative closure wounds than those other groups (23±1.5 at 6 hours and 39.5±1.3 at 12 hours). These results indicate that SHED-exosome can enhance the migration of hyperglycemic-induced endothelial cells.

Effect of SHED-exosome on the tube formation
Tube formation experiments on Matrigel-coated wells were conducted to study the proangiogenic effects of SHED-exosome on glucose-treated HUVEC, as shown in Figure 3. As expected, at 6 hours HUVEC The covered area, total tube length, total branching points, and total loops of each group were then quantified using WimTube image analysis ( Figure 3B).

Mesh-like structures in the hyperglycemia-induced
group were significantly lower in all aspects (p<0.001) compared to the positive control group, confirming increased glucose concentration's detrimental effects on angiogenesis. However, mesh-like structures in the HUVEC-supplemented SHED-exosomes in the high glucose concentration-treated group were substantially higher than those in the high glucosetreated group (p=0.03; Figure 3B). greater organized network formation, loop formation, branching points, thicker tubes, and a faster rate of tube formation were observed in the exosomestreated endothelial cells group. In contrast, the high glucose-treated HUVEC formed more disorganized networks, fewer loops and branching points, thinner tubes, and tube formation at a slower rate ( Figure 3C and video link at shorturl.at/bjwEZ). After 11 hours, while the tubes of the HUVEC treated with exosomes maintained the thickness and had complex networks, the tubes of the HUVEC that were not treated with exosomes began to gradually disconnect ( Figure   3C and video link at shorturl.at/bjwEZ). Together, these findings imply that SHED-exosomes induce  Figure 4C). Most evidently, the intensity of CD31 fluorescence in the high glucose concentrationtreated group was significantly lower than the positive control (p=0.03), having a similar intensity to the negative control. Compared to the group treated with high glucose, the intensity of the CD31 positively stained in HUVEC treated with SHED-exosome was significantly higher (p=0.03: Figure 4B and Figure   4C). The results indicate that SHED-exosomes help to recover the GATA2 gene and CD31 glycoprotein expression angiogenesis in hyperglycemic-induced HUVEC.