Saliva Exosomes-Derived UBE2O Promotes Angiogenesis in Cutaneous Wounds By Targeting SMAD6

Enhancing angiogenesis is critical for accelerating wound healing. Application of different types of exosomes (Exos) to promote angiogenesis represents a novel strategy for enhanced wound repair. Saliva is known to accelerate wound healing, but the underlying mechanisms remain unclear. In the present study, Exos were isolated from saliva, and their effect on angiogenesis was explored in vitro and in vivo. Our results have demonstrated that saliva-derived exosomes (saliva-Exos) induce HUVEC proliferation, migration, and angiogenesis in vitro, and promote cutaneous wound healing in vivo. Further experiments documented that Ubiquitin-conjugating enzyme E2O (UBE2O) is one of the main components of saliva-Exos, and activation of UBE2O has effects similar to those of saliva-Exos, both in vitro and in vivo. Mechanistically, UBE2O decreases the level of SMAD6, thereby activating BMP2, which, in turn, induces angiogenesis. The present work suggests that administration of saliva-Exos and UBE2O represents a promising strategy for enhancing wound healing through promotion of angiogenesis.


Genomes (KEGG) pathway analysis
To understand the biological function of upregulated DEGs, the Annotation, Visualization, and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/) resource was used to analyze their GO and KEGG pathway. The top ten pathways derived from functional enrichment of each GO subset and KEGG pathway were illustrated in a bubble diagram with the ggplot2 tool of R package.

EdU assay
HUVECs were seeded into 24-well culture plates at a density of 2 x 10 5 cells/well and treated with PBS, saliva, or saliva-Exos (100 μg/mL) for 24 hours. Subsequently, EdU staining was performed according to the manufacturer's protocol (Sigma-Aldrich, St. Louis, MO, USA).

Scratch wound healing assay
After treatment with the indicated reagents, a monolayer of HUVECs grown in six-well plates was scratched with a sterile 10 μl pipette tip to form wounds in the sheet of cells. The plates were then incubated for 12 and 24 hours, and the cell-free wound area was photographed under an inverted microscope.

Tube formation assay
Each well of a 96-well plate was filled with 50 μl of Growth Factor Reduced Matrigel (BD Biosciences, NJ, USA). After incubation for 30 min, 2 x 10 4 HUVECs were seeded into the wells, and tube formation was observed 12 hours later under an inverted microscope.

Western blotting
Total protein was extracted from cells, separated by 10% SDS-PAGE, and transferred onto PVDF membranes. The membranes were incubated with primary antibodies at 4℃ for overnight, and with horseradish-peroxidase-conjugated secondary antibodies at 37℃ for 1 hour. The following antibodies were used:

Quantitative real-time PCR (qRT-PCR)
Total RNA was collected using TRIzol Reagent (Invitrogen), and 1 μg of total RNA was transcribed into cDNA. qRT-PCR was performed using the StepOne™  where n is day 0, 3, 7, 10, and 14. All

Assessment of blood flow in the wound site
Ten days after the operation, laser speckle contrast imaging (LSCI) (PERIMED Ltd, Stockholm, Sweden) was used to evaluate blood flow in the wound. The mean perfusion units (MPU) ratio was calculated by comparing the MPU per mm 2 in the wound area (ROI-1) with the MPU per mm 2 in an area adjacent to the wound (ROI-2).

Hematoxylin and eosin (H&E) staining and immunohistochemistry
Tissue samples from Day 14 containing the wound region were collected, fixed in 4% buffered paraformaldehyde and embedded in paraffin. 4 μm thick sections were prepared and stained with H&E. CD31 was detected using immunofluorescence staining; briefly, sections were blocked in 1% BSA for 30 minutes and incubated overnight with anti-CD31 (1:50, Abcam, #ab28364). Subsequently, the sections were incubated with secondary antibody for 1 hour. CD31-positive cells were quantified from at least three randomly selected high power fields per section. All slides were independently evaluated by three observers blinded to the treatment.

Identification of saliva-Exos
The saliva-Exos were identified and characterized by TEM, DLS, and Western blotting. In agreement with previously reported results for Exos, the TEM and DLS demonstrated that the size of the isolated particles ranged from 30 to 150 nm ( Figure   1A, B). Western blotting documented that the particles contained exosomal marker proteins CD81 and TSG101, but did not contain non-exosomal marker calnexin ( Figure 1C), confirming that the Exos were successfully isolated from the salivary samples.

Saliva-Exos accelerate cutaneous wound healing in vivo
To determine the role of saliva-Exos in wound repair, equal amounts of PBS, saliva, and saliva-Exos were injected around the wound site. The saliva and saliva-Exos groups had a higher rate of wound healing than the control group, with the process being faster in animals treated with saliva-Exos than saliva only ( Figure   2A, 2B). The scar width was smaller in the saliva-Exos group than in the control group ( Figure 2C). The neovascularization of the wound site was significantly higher in the saliva-Exos group, as documented by increased blood flow ( Figure 2D) and higher number of CD31-positive cells ( Figure 2E, 2F). Together, these findings indicate that saliva accelerated wound healing, and this effect can be attributed to the promotion of angiogenesis by saliva-Exos.

Saliva-Exos enhanced the function of HUVECs
The
To establish the hub genes, we used DMNC algorithm and identified that UBE2O, UBA6, AREL1, MEX3C, COL4A2, LAMA4, PGF, BTRC, ASB4, COL5A are the ten hub genes among the upregulated genes ( Figure 4G). The KEGG pathway of the ten hub genes was analyzed and constructed using the online tool NetworkAnalyst (https://www.networkanalyst.ca/). The KEGG pathways of the ten hub genes were Ubiquitin mediated proteolysis, Focal adhesion, ECM-receptor interaction, PI3K-AKT signaling pathway, Small cell lung cancer, Amoebiasis, Pathway in cancer, Circadian rhythm, African trypanosomiasis, Hedgehog signaling pathway, and Shigellosis ( Figure 4H). In addition, qRT-PCR demonstrated that the levels of UBE2O mRNA in saliva-Exos samples were significantly higher than in the saliva samples ( Figure 4I). HUVECs treated with saliva-Exos exhibited a higher level of UBE2O than cells treated with PBS or saliva ( Figure 4J, 4K). Collectively, these data suggest that UBE2O is one of the hub genes in saliva-Exos upregulated genes.
Together, these findings indicate that UBE2O is the primary mediator of the beneficial effects of saliva-Exos on HUVEC function.

SMAD6/BMP2 axis is key to UBE2O-mediated HUVEC function
To explore the gene regulatory networks of UBE2O in skin tissue, we constructed skin-type specific networks and identified that sixteen genes are regulated by UBE2O, resulting in 16 putative genes ( Figure 6A). Previous studies reported that UBE2O can target SMAD6 for ubiquitination and degradation.
[13] Therefore, to determine whether UBE2O suppresses the expression of SMAD6 in HUVECs, the level of SMAD6 was assessed by Western blotting. As shown in Figure 6B, the expression level of SMAD6 in HUVECs was significantly decreased after UBE2O treatment, while its level was increased after silencing UBE2O. Moreover, SMAD6 knockdown decreased the level of SMAD6 in HUVECs ( Figure 6C). SMAD6 silencing significantly enhanced HUVEC function, as evidenced by the increased proliferation and migration of HUVECs in the siSMAD6 group ( Figure 6D-I). SMAD6 signaling also induced tube formation by HUVECs ( Figure 6J-L).
SMAD/BMP pathway has been documented to be one of the main regulators of angiogenesis.

UBE2O promotes wound healing in vivo
To investigate the effects of UBE2O on wound healing, UBE2O or siUBE2O was injected around the wound site. The rate of wound healing was faster in the UBE2O group than in the siUBE2O group ( Figure 7A, 7B). H&E staining documented that the scar width was the smallest in the UBE2O group ( Figure 7C, 7D). Laser speckle contrast imaging showed that blood flow was significantly higher in the UBE2O group than in the siUBE2O group, as supported by a higher MPU ratio in the UBE2O group ( Figure 7E). Additionally, the number of CD31-positive cells in the wound of UBE2O-treated mice was higher than in the siUBE2O-treated mice ( Figure 7F, 7G).
Collectively, these findings indicate that UBE2O promotes wound healing in vivo.
Wound healing is a complex process that involves various cells and cytokines.
The present study focused mainly on the role of saliva-Exos on HUVECs, but future studies should address the impact of saliva-Exos on other cell types, such as keratinocytes and fibroblasts. In addition, saliva-Exos contain multiple types of molecules that may play catalytic roles in wound healing. Specifically, exploring the effect of saliva-Exos and UBE2O on the healing of diabetic and chronic wounds may be promising.
In summary, the current work highlighted that saliva-Exos enhance HUVEC function through UBE2O delivery. Overexpression of UBE2O decreases the SMAD6 level, leading to upregulation of BMP2 expression and, consequently, promotion of angiogenesis in vitro and acceleration of wound healing in vivo. These findings indicate that saliva-Exos are a potential promising agent for wound therapy.
Furthermore, since upregulation of UBE2O accelerates angiogenesis, use of nanomaterials combined with UBE2O may enhance wound healing.