Upregulation of circ‐IGF1R increased therapeutic effect of hypoxia‐pretreated ADSC‐derived extracellular vesicle by regulating miR‐503‐5p/HK2/VEGFA axis

Abstract Diabetes mellitus is a major cause of blindness and chronic ulcers in the working‐age population worldwide. Wound healing is deeply dependent on neovascularization to restore blood flow. Former research has found that differentially expressed circular RNAs (circRNAs) are associated with hyperglycaemia‐induced endothelial cell damage, and hypoxia‐pretreated adipose‐derived stem cells (ADSCs)‐extracellular vesicle (HEV) transplants have a more therapeutic effect to enhance wound healing in diabetic mice by delivery circRNA. The current investigation employed high‐throughput sequencing to identify circRNAs that are abnormally expressed between EV and HEV. The regulatory mechanism and predicted targets of one differentially expressed circRNA, circ‐IGF1R, were investigated utilizing bioinformatics analyses, luciferase reporter assays, angiogenic differentiation assays, flow cytometric apoptosis analysis and RT‐qPCR. Circ‐IGF1R expression increased in HEV, and downregulation of circ‐IGF1R suppressed and reversed the promotion effect of HEV on angiogenesis in ulcerated tissue. Bioinformatics analyses and luciferase reporter assays confirmed that miR‐503‐5p was the downstream target of circ‐IGF1R, and inhibiting miR‐503‐5p restored the promotion effect of HEV on angiogenesis after circ‐IGF1R silence. The study also found that miR‐503‐5p can interact with 3'‐UTR of both HK2 and VEGFA. Overexpression of HK2 or VEGFA restored the promotion effect of HExo on angiogenesis after circ‐IGF1R silence. Overexpression miR‐503‐5p or silence HK2/VEGFA reversed the protective effect of circ‐IGF1R to MLMECs angiogenic differentiation. Overexpression of circ‐IGF1R increased the protective effect of HEV on the promotion of wound healing in mice with diabetes. Circ‐IGF1R promotes HIF‐1α expression through miR‐503‐5p sponging. Our data demonstrate that circ‐IGF1R overexpression EVs from ADSCs suppress high glucose‐induced endothelial cell damage by regulating miR‐503‐5p/HK2/VEGFA axis.


| INTRODUC TI ON
Acute and chronic skin injuries caused by burns, stress, diabetes and venous stasis have brought a huge burden to society. 1,2Normal wound healing is one of the most complex biological processes.It requires the precise coordination of many kinds of cells and accurate coordination of various biological and molecular events. 3Despite significant investment in this area, progress has been limited, especially in treating chronic wounds.
Stem cell-based therapies open a new door for tissue repair and have been widely studied in regeneration medicine. 4Adipose stem cells (ASCs) have been well documented to have therapeutic effects on skin wound healing, cardiac injury, immune disorders and other indications of ischaemia and tissue loss. 5Recent studies have shown that paracrine factors significantly promote the therapeutic effect of stem cells on tissue repair, and extracellular vesicles (EVs) may play an important role. 6Harnessing their regenerative potential could overcome many of the translation barriers to cell therapy.
Extracellular vesicles composed of exosomes and microvesicles are considered to be the mediators of intercellular communication, allowing the exchange of DNA, RNA, proteins and lipids between cells. 7Exosomes are small vesicles with a diameter of 50-200 nm, whose secretion requires multi-vesiculated endosomes to fuse with the plasma membrane. 8,9In recent years, the role of EVs from different cell types in tissue repair has been extensively studied.The release of EVs is now considered to be one of the mediators of the therapeutic activities of MSCs. 10,11Our previous research confirms that exosomes derived from mmu_circ_0000250-modified adipose-derived mesenchymal stem cells promote wound healing in diabetic mice by inducing miR-128-3p/SIRT1-mediated autophagy. 12wever, molecular mechanisms remain mysterious.
In the current investigation, we found that EVs from hypoxia adipose-derived stem cells (ADSC-HEV) are more effective in promoting wound healing compared with ADSC-Exo in mice with diabetes.Circ-IGF1R functioned in ADSC-HEV-mediated wound repair in mice with diabetes.However, the regulatory mechanism of circ-IGF1R is unclear.So, the aim of this study was to reveal the regulatory mechanism of circ-IGF1R for wound repair and clarify whether circ-IGF1R can increase the therapeutic effect of hypoxia-pretreated ADSC-derived EVs.

| ADSC-EV identifications
Technicians cultivated ADSCs that had normoxic conditions in 95% air with 20% O 2 and 5% CO 2 .For hypoxic pretreatments, technicians cultivated ADSCs in hypoxic conditions with 93% N 2 , 5% CO 2 and 2% O 2 .When getting 80%-90% confluence, technicians rinsed ADSCs using PBS, which we cultivated in EGM-2MV media without FBS and supplemented with 1× serum replacement solution (PeproTech, NJ, USA) for another 2 days.We got conditioned medium from ADSCs, which we centrifuged at 300 × g for 10 min and 2 kg for another 10 min to erase apoptosis cells and cellular debris.Lastly, post-centrifugation at 12 kg for 30 min, technicians made the procedures at 4°C.We defined EV protein content employing Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA).We stored ADSC-EV at −80°C, which we used in the following experiments: transmission electron microscopy, NTA assay and Western blotting were applied to identify the selected EVs.

| Strand-specific RNA-Seq library
Technicians obtained total RNA from ADSCs-EV as well as ADSCs-HEV employing TRIzol reagent (Invitrogen, CA, USA).We used 3 μg RNA from each specimen through VAHTS.Total RNA-Seq (H/M/R) Library Prep kits from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) were used to erase ribosomal RNA.ncRNAs and mRNAs were retained.We processed RNA using 40 U RNase R (Epicentre) at 37°C for 3 h and made TRIzol purification.We prepared the RNA-Seq library via KAPA-stranded RNA-Seq Library Prep kits (Roche, Basel, Switzerland), which were applied for highthroughput sequencing (Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China).

| Bioinformatics analysis
Predicted interactions between circRNA, miRNA and mRNA were identified using the StarBase database.
Technicians made PCR using a PCR instrument (Bio-Rad, CA, USA) with 2× Taq PCR Master Mix (Solarbio, Beijing, China) following the protocol.Our team computed fold alternations using 2 −Ct method.PCR

| Tubule formation assay
We tested neovascularization in fibrin matrices.We seeded serumstarved MLMECs in an endothelial basal medium onto plates coated with Matrigel (BD Biosciences, NJ, USA) to incubate them.Our team captured and photographed tubular structures formed in Matrigel using phase-contrast microscopy.Lengths regarding tubes newly constituted in 10 fields that were randomly selected were calculated.

| Diabetes wound induction
Our team used C57BL male mice to induce diabetes via single intraperitoneal injection of streptozotocin (STZ).We validated diabetes by taking blood from the tail vein 3 days post-STZ administration.
We regarded mice with fasting blood glucose levels >250 mg/dL as diabetic and maintained them for 1 month for experiments that followed.Technicians anaesthetized mice via intramuscular injections and eliminated hair from the dorsal leg region to sterilize the area.
The technician applied a sterile biopsy punch to make a 4-mm fullthickness excisional wound.Technicians allocated mice randomly to subcutaneous injection with 200 μg ADSC-Exos in 100 μL of PBS.Technicians euthanized mice post-15 days and harvested skin specimens.

| Immunohistochemical data
We fixed tissue specimens to embed them in paraffin.Technicians cultured sections over the night using primary antibodies against CD31 at 4°C and with secondary antibodies (Abcam) for 1 h at 37°C.
We stained sections employing 3,3-diaminobenzidine to counterstain them via haematoxylin.We conducted TUNEL staining to examine sections.

| Statistical analyses
Statisticians denoted continuous variables by means ± SD to conduct one-way variance comparisons via GraphPad Prism (GraphPad, CA, USA).The p value of ≤0.05 inferred statistical significance.

| Circ-IGF1R plays an important role in hypoxia-pretreated ADSC-derived EV (HEV) mediated promotes wound healing in diabetic mice
Our previous studies have confirmed that hypoxic ADSC-derived EV (HEV) enhance wound healing in diabetic mice. 13In this study, we also found that circRNA were different expressions between HEV and EV, including mmu_circ_0000066, mmu_ circ_0000073, mmu_circ_0000074, mmu_circ_0001582, mmu_ circ_0000079, mmu_circ_0000081 and mmu_circ_0000082 (Figure 1A).RT-qPCR detection shows that mmu_circ_0001582 expression was increased significantly in HEV compared with EV (Figure 1B).Using bioinformatics analysis (http:// www.circb ase.org/ ), we found that mmu_circ_0001582 was 546 bp and located within the IGF1R gene at chr7:75148695-75,149,241.Thus, mmu_circ_0001582 is referred to as circ-IGF1R (Figure 1C).Generation sequencing also proved that circ-IGF1R was the circRNA with back splicing.RT-qPCR detection shows that circ-IGF1R expression was decreased significantly after the silence of circ-IGF1R in HEV (Figure 1D).We

| Both HK2 and VEGFA were the downstream targets of miR-503-5p
Luciferase report analysis confirmed that only miR-503-5p can interact with 3'-UTR of both HK2 and VEGFA (Figure 3A made that both HK2 and VEGFA were the downstream targets of miR-503-5p.

| DISCUSS ION
The refractory diabetic wound has remained a worldwide challenge as one of the major health problems.The impaired angiogenesis phase during diabetic wound healing partly contributes to the pathological process. 15,16Our study found that exosomes derived from mmu_ SIRT1-mediated autophagy. 12Hypoxia adipose stem cell-derived exosomes promote high-quality healing of diabetic wounds involving activation of PI3K/Akt pathways. 17In this study, we also found that circ-IGF1R plays an important role in hypoxia-pretreated ADSCderived EV (HEV) mediated wound healing in diabetic mice.circ-IGF1R was 546 bp and located within the IGF1R gene at chr7:75148695-75149241.Under high glucose stimulation, miR-503 from M1 macrophage-derived small EVs was taken by HUVEC-targeted IGF1R in HUVECs and inhibited IGF1R expression.In HUVECs, miR-503 inhibition improved hyperglycaemia-caused HUVEC dysfunction, whereas IGF1R knockdown aggravated HUVEC dysfunction. 18,19In this study, we also found that circ-IGF1R plays an important role in hypoxia-pretreated ADSC-derived exosome (HEV) mediated wound healing in diabetic mice.Downregulation circ-IGF1R reversed the promotion effect of HEV on angiogenesis in ulcerated tissue.
Bioinformatics and luciferase report analysis confirmed that miR-503-5p was the downstream target of circ-IGF1R.
Previous studies have confirmed that miR-503-5p were 6.21-fold (p = 0.001) more highly expressed in diabetic foot patients than in healthy controls. 20,21The study also found that miR-503-5p expression was increased under high glucose condition. 22Previous studies found that hsa_circ_0022742 expression in HUVECs was decreased by high glucose treatment, and overexpression of hsa_circ_0022742 suppressed high glucose-induced endothelial dysfunction by target miR-503-5p. 23Our study also found that inhibiting miR-503-5p restored the promotion effect of HEV on angiogenesis after circ-IGF1R silence.Suggestion that miR-503-5p was the target of circ-IGF1R.
Further study also found that miR-503-5p can interact with

Animal
Care and Use Committee in the Affiliated Hospital of Nantong University approved the investigation.We conducted postoperative animal care and surgical treatment interventions according to the National Institutes of Health Guide for Laboratory Animals Care and Use.
studied ADSC-EV effects upon wound healing in full-thickness cutaneous wounds of mice having STZ-induced diabetes responding to subcutaneous injection regarding ADSC-EV and ADSC-HEV with equivalent volumes.We accelerated wound closure significantly using ADSC-HEV compared with PBS controls.However, downregulation of circ-IGF1R decreased the therapeutic effect of HEV to accelerated wound closure (Figure1E,H).Immunohistochemicals for Tunel staining show that HEV treatment decreased apoptosis in ulcerated tissue.However, circ-IGF1R silence decreased the protective effect of HExo (Figure1F,I).CD31 staining shows that HEV treatment increased angiogenesis in ulcerated tissue.But circ-IGF1R silence reversed the promotion effect of HEV on angiogenesis in ulcerated tissue (Figure1G,J).A suggestion that circ-IGF1R plays an important role in hypoxia-pretreated ADSC-derived EV (HEV) mediated promotes wound healing in diabetic mice.
-D).The detection of in vivo experiments shows that overexpression of HK2 or VEGFA restored the protective effect of HExo after circ-IGF1R silence (Figure 3E,F).Immunohistochemicals for Tunel staining show that circ-IGF1R silence decreased the protective effect of HEV.However, overexpression of HK2 or VEGFA restored the protective effect of HEV after circ-IGF1R silence (Figure 3G,I).CD31 staining shows that circ-IGF1R silence reversed the promotion effect of HExo on angiogenesis in ulcerated tissue.However, overexpression of HK2 or VEGFA restored the promotion effect of HEV on angiogenesis after circ-IGF1R silence (Figure 3H,J).A suggestion was F I G U R E 2 miR-503-5p was the downstream target of circ-IGF1R.(A) Luciferase report analysis shows the downstream target of circ-IGF1R.(B) AGO2 RNA-binding protein immunoprecipitation.All data are shown as the mean ± SD. ***p < 0.001 compared to IgG. (C) circRNA pull-down assay.**p < 0.01 compared to negative control (NC) probe.(D and E) Bioinformatics analysis and luciferase report analysis show the interactive relationship between miR-503-5p and circ-IGF1R.***p < 0.001.(F and G) Representative figures of wound-healing procedures at various time points in various groups.The wound healing rate quantification.*p < 0.05, ***p < 0.001 versus HEV.### p < 0.001 versus si-circ-IGF1R-HEV.(H and I) Immunohistochemicals for Tunel staining show the apoptosis in skin tissue from the ulcer.***p < 0.001 versus HEV.### p < 0.001 versus si-circ-IGF1R-HEV.(J and K) Immunohistochemicals for CD31 staining show the angiogenesis in skin tissue from the ulcer.***p < 0.001 versus HEV.### p < 0.001 versus si-circ-IGF1R-HEV.

3. 5 |
Circ-IGF1R upregulation increased therapeutic effect regarding ADSC-HEV on wound healing in mice with diabetesThe in vivo experiment detection shows that overexpression of circ-IGF1R increased the protective effect of HEV on promoting wound healing in mice with diabetes (Figure5A,B).Immunohistochemicals for Tunel staining show that overexpression of circ-IGF1R increased the protective effect of HEV in decreased apoptosis in ulcerated tissue in mice with diabetes (Figure5C,D).CD31 staining shows that circ-IGF1R overexpression promotes the angiogenesis of HEV in ulcerated tissue (Figure5E,F).RT-qPCR detection shows that overexpression of circ-IGF1R decreased miR-503-5p expression more than HEV treatment ulcerated tissue in mice with diabetes (Figure5G).However, overexpression of circ-IGF1R increased HK2 and VEGFA expression, and HEV treatment ulcerated tissue in mice with diabetes (Figure5H,I).