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Exosomes originated from Nr-CWS pretreated MSC facilitate diabetic wound healing by promoting angiogenesis via circIARS1/miR-4782-5p/VEGFA axis

  • Corresponding author: Peisheng Jin Address: Department of Plastic Surgery, Affiliated Hospital of Xuzhou Medical University, Huai-hai West Road, Xuzhou, Jiangsu, P.R.China. * E-mail: 100000401006@xzhmu.edu.cn
  • Available Date: 03-Aug.-2022
  • Mesenchymal stem cell (MSC)-derived exosomes had been reported to a prospective candidate in accelerating diabetic wound healing because of the pro-angiogenic effect. MSC pretreated with chemistry or biology factors are to advance the biological activities of MSC-derived exosomes. Hence, this study intended to explore whether exosomes originated from the human umbilical cord MSC (hucMSC) preconditioned with nocardia rubra cell wall skeleton (Nr-CWS) could display superior proangiogenic effect on diabetic wound repair and its underlying molecular mechanism. We found that Nr-CWS -Exos facilitated the proliferative and migratory abilities as well as the tube formation of endothelial cells in vitro. In vivo, Nr-CWS -Exos displayed great effect on advancing the wound healing by facilitating the angiogenesis of wound tissues in contrast to Exos. Furthermore, circIARS1 expression was increased after HUVECs were treated with Nr-CWS-Exos. CircIARS1 promoted the pro-angiogenic effects of Nr-CWS -Exos on endothelial cells via miR-4782-5p/VEGFA axis. Taken together, those data reveal that exosomes derived from Nr-CWS -pretreated MSCs might serve as an underlying strategy for diabetic wound via advancing the biological function of endothelial cells through circIARS1/miR-4782-5p/VEGFA axis.
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Exosomes originated from Nr-CWS pretreated MSC facilitate diabetic wound healing by promoting angiogenesis via circIARS1/miR-4782-5p/VEGFA axis

    Corresponding author: Peisheng Jin Address: Department of Plastic Surgery, Affiliated Hospital of Xuzhou Medical University, Huai-hai West Road, Xuzhou, Jiangsu, P.R.China. * E-mail: 100000401006@xzhmu.edu.cn
  • 1. Department of Plastic Surgery, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China

Abstract: Mesenchymal stem cell (MSC)-derived exosomes had been reported to a prospective candidate in accelerating diabetic wound healing because of the pro-angiogenic effect. MSC pretreated with chemistry or biology factors are to advance the biological activities of MSC-derived exosomes. Hence, this study intended to explore whether exosomes originated from the human umbilical cord MSC (hucMSC) preconditioned with nocardia rubra cell wall skeleton (Nr-CWS) could display superior proangiogenic effect on diabetic wound repair and its underlying molecular mechanism. We found that Nr-CWS -Exos facilitated the proliferative and migratory abilities as well as the tube formation of endothelial cells in vitro. In vivo, Nr-CWS -Exos displayed great effect on advancing the wound healing by facilitating the angiogenesis of wound tissues in contrast to Exos. Furthermore, circIARS1 expression was increased after HUVECs were treated with Nr-CWS-Exos. CircIARS1 promoted the pro-angiogenic effects of Nr-CWS -Exos on endothelial cells via miR-4782-5p/VEGFA axis. Taken together, those data reveal that exosomes derived from Nr-CWS -pretreated MSCs might serve as an underlying strategy for diabetic wound via advancing the biological function of endothelial cells through circIARS1/miR-4782-5p/VEGFA axis.

    • Diabetic wounds are one of the most intractable complicating diseases of diabetes. Although a series of conventional therapies have been applied for chronic wounds, the recovery is still unsatisfactory and about 28% of those sufferers have to have their lower limbs amputated, with the death rate in this regard reaching 50-59% five-year postamputation [1]. One potential factor causing diabetes ulcerations is unsatisfactory blood vessel flow, a situation impeding wound repair [2]. Poor angiogenesis leading to delivering nutrients to wounded sites and eventually diminishes fibroblast growth, collagen syntheses, and re-epithelializsation [3]. Hence, improving revascularization is remarkably pivotal for the treatment of diabetes wounds.

      MSCs are multi-potent, non-hematopoietic adult stem cells, serving as prospective candidates for various treatments in a wide range of tissue regeneration [4]. Angiogenesis, the growing of vessels from the existent vessels and following expansion of vascular net, is pivotal for regenerating tissues [5]. It is believed that MSCs achieve a therapeutic effect in vivo primarily via paracrine signaling; MSCs are capable of releasing bioactive molecules affecting the angiogenesis of endothelial cells [6, 7]. Specifically, exosomes have been demonstrated to be a type of the biologically active molecules to achieve the mediation of the functions between MSC and targeted cells in wound repair and tissular regeneration [8, 9]. Cell-derived exosomes are becoming a novel causal link of cell-to-cell communication.

      Exosomes are exocellular vesicles produced by fusing vesicular bodies with plasmatic membranes [10]. Exosomes derived from MSCs (MSC-Exos) have become a remarkably prospective therapeutic tool due to the decreased immunogenicity and increased tissular regeneration ability capability via promoting revascularization and inducing cellular growth [11, 12]. Ding et al showed evidence that MSC-Exos accelerates diabetic wound-healing process by facilitating revascularization [13]. Shabbir et al demonstrates that MSC-Exos trigger the growth and motility of normal and persistent wound fibroblasts, and reinforce revascularization in vitro [14]. Additionally, in contrast to MSCs, their exosomes exhibit remarkable benefits such as brilliant steadiness, restricted immunorejection, convenient application and internalisation into receptor cells [15]. Therefore, MSC-Exos might be a prospective candidate as an angiogenesis-promoting treatment in diabetic wounds.

      Numerous of evidences have verified precondition MSC with physics, chemistry, and biology factors are useful to reinforce the bioactivities of MSC-Exos, elevating relevant repair potency in tissue-regenerating medicines. Liu et al. have verified that lipopolysaccharide-primed MSCs display a better treatment effect on preserving skin flap survival in a diabetes rat model in contrast to non-primed MSCs [16]. Another study indicated that exosomes originated from atorvastatin-preconditioned MSC obviously enhanced diabetic wound repair healing via reinforcing revascularization through the AKT/eNOS pathway [17]. Nr-CWS is stemmed from N. rubra, and partially comprises nocardomycolic acid, arabogalactan, and mucopeptide [18]. Nr-CWS has been reported to accelerate skin wound repair via reinforcing macrophage activation and angiogenesis[19]. However, the properties of Nr-CWS -Exos in wound repair are still elusive.

      Therefore, the aim of this study was to investigate if MSC-Exos pre-treated with Nr-CWS could facilitate the angiogenesis ability of endothelial cells in diabetic wound healing. In addition, we further explored the effects of circRNAs on facilitating wound repair of Nr-CWS -Exos.

    Materials and methods
    • The human umbilical cord MSCs and Nr-CWS were acquired from Qilu qlgxbsjjc (Jinan, China). The human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The MSCs and HUVECs were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplied with 10% fetal bovine serum (FBS). For high glucose treatment, HUVECs were cultivated with serum-free DMEM containing high glucose (25 mM) for 48 h before experiments. For Nr-CWS treatment, MSCs were treated Nr-CWS (10 μg/ml) for 48 h prior to experiments.

    • When the degree of MSC fusion reached 70-80%, the supernatant was discarded and cleaned with PBS. The culture medium was changed to 10% Exosomes-free serum medium and 10% Exosomes-free serum medium containing Nr-CWS and incubated for 48 h. Subsequently, two groups of supernatants were collected and centrifuged at 300 g for 10 min and 2000 g for 10 min to remove dead cells and large cell debris. Then, the supernatants were centrifuged at 10 000 g centrifugation for 30 min to remove cell debris. The supernatants were filtered through a 0.22 μm filter (jetbiofil, Guangzhou, China) and centrifuged at 100,000 g for 70 min. The concentration of extracted Exosomes was measured using BCA kit (KeyGEN, Nanjing, China).

      To verify Exosomes, the Tecnai transmission electron microscopy (FEI Company, Hillsboro, Oregon, USA) was used to observe morphology of Exosomes. The size distribution of exosomes was detected by Laser particle scanning analyzer (Particle Metrix, German). Western blotting was applied to detect surface markers of exosomes.

    • Exosomes were incubated with PKH26 (Sigma, MO, USA) for 10 min, and were centrifuged at 100,000 g for 70 min again to obtain PKH26-labeled exosomes without dye. HUVEC and PKH26-labeled exosomes were co-cultured for 24 h, and then fixed with 4% paraformaldehyde. The nucleic acid was stained with DAPI. The internalization of exosomes was observed Zeiss LSM880 microscope.

    • For circRNA pull-down, 107 cells were collected and subjected to lysis. Biotinylated circIARS1 probe (Tsingke, Wuhan, China) was cultivated with streptavidin magnet beads (Invitrogen, Shanghai, China) to produce beads that were coated with probes. The beads were cleaned and the bound miRNA in the pull-down substances were abstracted via Trizol reagent and studied via qRT-PCR analysis.

    • The RIP experiment was opertated with the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Boston, MA). In short, cells were subjected to lysis, and cultivated with magnetic beads conjugated with human anti-AGO2 antibody (Millipore, 03-110) or anti-mouse IgG. The RNA immunoprecipitation was identified by qRT-PCR assay.

    • A probe labeled with cy3 for identifying circIARS1 and probes labeled with FAM for identifying miR-4782-5p were prepared by GenePharma (Shanghai, China). The probe signals were identified via a Fluorescent In Situ Hybridization Kit (GenePharma) as per the supplier's specification. Results were digitally recorded using a Zeiss LSM880 microscope.

    • The diabetes mouse model was constructed as depicted in the past [20]. A serum glucose level above 16.7 mmol/L for 28 days was considered a successful diabetic mouse model. Posterior to anesthetization, a complete 1.2-cm thick cutaneous wound was created on the mice's backs and treated with PBS (Control), Exo and Nr-CWS-Exos by multisite subcutaneous injection (at least six sites per wound). The parfocal images were captured at day 0, 7, and 14 after the treatment. All procedures were performed in accordance with the Guide for Animal Care and Use Committee of Xuzhou Medical University (202108w013).

    • The data were described as the means ± standard deviation (SD). They were studied via GraphPad software r by virtue of a t-test for two-group contrasts with the controls or one-way ANOVA for multi-comparison analysis amongst groups. P<0.05 had significance on statistics.

    Results
    • The conditioned medium of MSC (no treatment) and Nr-CWS -treated MSC were collected to isolate Exos and Nr-CWS -Exos through ultra-centrifugation. The morphologies of exosomes were examined by transmission electron microscopy. As shown in Fig. 1A, the homogenized, globular, and membrane-bound vesicles were identified in both groups, which showed the typical exosomal structures (Fig. 1A). The exosome markers Alix, TSG101, and CD9 were expressed in Exos and Nr-CWS -Exos (Fig. 1B). Additionally, the particle size detected by NTA revealed that both Exos and NR-CWS-Exos exhibited one peak at about 80-120 nm (Fig. 1C, D). Importantly, the particle size, morphology, and protein expression were similar in Exos and Nr-CWS -Exos, which suggest that the exosome secretion amount by MSC was not influenced by Nr-CWS. Those data verified the successful isolation of the exosomes. Furthermore, we investigated whether HUVECs could endocytose the MSC-exos. It was revealed that the PKH26 labeled exosomes (red) were found in the perinuclear area of HUVECs (Fig. 1E), suggesting the internalization of exosomes by HUVECs.

      Figure 1.  The characterization of MSC-derived exosomes. A. The morphology of Exos and Nr-CWS -Exos was observed by TEM. B. The specific surface markers for Exos and Nr-CWS -Exos (Alix, TSG101, CD81) were assessed by Western blotting. C, D. The diameter and particle concentration of Exos and Nr-CWS -Exos were examined by NTA. E. The uptake of Exos and Nr-CWS -Exos by HUVECs was verified via laser scanning confocal microscopy. Exosomes and cell nucleus were stained red and blue, respectively.

    • To explore the impact of exons on cellular angiogenesis, we exposed MSCs to LG, HG, HG + Exos, and HG + Nr-CWS -Exos. Transwell and wound healing detection were used to examine cell migration. Data showed that HG inhibited the migration of HUVECs, while Exos and Nr-CWS -Exos facilitated the cellular migration ability of HUVECs impaired by HG. Nr-CWS -Exos displayed a better facilitating potency in cellular motility (Fig. 2A-C). Consistently, the result of tube formation showed the same pattern with the migration assay. Exos and Nr-CWS -Exos facilitated the tube forming capability of HUVECs impaired by HG. Compared with the Exos and HG group, more tube architectures in Nr-CWS -Exos-exposed HUVECs were identified (Fig. 2D, E). Moreover, cell variability assay showed that Nr-CWS -Exos-treated HUVEC exhibited a strong proliferative ability when compared with the Exos and HG group (Fig. 2F). Angiogenesis related genes have been demonstrated to promote angiogenesis. We identified the mRNA contents of these genes and discovered that Nr-CWS -Exos facilitated the expressing of VEGF, bGFG and HGF (Fig. 2G). In addition, the secretion of these factors were also increased by Nr-CWS -Exos (Fig. 2H). Taken together, these results indicate that Nr-CWS -Exos advanced HUVECs angiogenesis.

      Figure 2.  Nr-CWS-Exos facilitated endothelial cell angiogenesis in vitro. A-C. The images of migration ability as well as wound healing of HUVECs after cells were treated with Exos or Nr-CWS -Exos. In migration assay, gentian violet staining was used to stain the migration cells and the cells were observed under Olympus microscope. The numbers of cell migration per field were counted in five random fields. In wound healing assay, the wound was made in the cell monolayer using a 200 mL pipette tip, and the migration of cells and closing of the scratch wound were photographed. D, E. Representative images were captured in situ for tube formation in the supernatant of HUVECs treated with Exos or Nr-CWS -Exos. HUVECs were inoculated into Matrigel pretreated 24-well dishes at 5 × 104 cells and cultivated for 6 h. Pictures were acquired by Olympus microscope. F. The proliferation of HUVECs was detected by CCK8 kit after cells were treated with LG, HG, HG + Exos, and HG + Nr-CWS -Exos for 1, 3, and 7 days. G. The concentration of supernatant in different media was detected through ELISA kit. H. The relative expression level of VEGFA, bFGF, and HGF of HUVEC examined by qRT-PCR. LG: low glucose; HG: high glucose; HG + Exo: high glucose + MSCs arrived exosomes. **P < 0.01, ***P < 0.001.

    • To investigate the pro-angiogenic effects of Nr-CWS -Exos, a diabetes mouse model was constructed via injecting STZ and two full-thickness skin defects were formed on all mice's backs (Fig. 3A). The wounds were injected subcutaneously with PBS (control), Exos, and Nr-CWS -Exos. We first evaluated the wound healing process of diabetic mice. Digitalized images of the wounded sites presented much more rapid wound healing in the animals treated with Nr-CWS -Exos, even though Exos improved the wound healing in contrast to the controls (PBS) (Fig. 3B, C). HE and masson staining were applied to evaluate the re-epithelialization and collagen formation. The results showed that Exos and Nr-CWS -Exos promoted skin regeneration and thick collagen fiber deposition in diabetic wounds, and Nr-CWS -Exos group showed the better effect (Fig. 3D-G). Subsequently, we investigated the effect of Nr-CWS -Exos on angiogenesis in diabetic mice by staining CD31 and VEGFA. IHC and IF staining showed that more vessels were identified in Exos and Nr-CWS-Exos-exposed wounded sites in contrast to the wounded sites exposed to PBS. Nr-CWS-Exos-treated wounds showed more CD31-positive cells than Exos group, which was consistent with the expression of VEGFA (Fig. 3H-M). Taken together, the results suggest that Nr-CWS -Exos accelerates the vascularization of diabetic wounds.

      Figure 3.  Nr-CWS-Exos accelerated the wound healing of diabetic mice. A. Experimental design of the animal study. B, C. Representative images of full-thickness defects and wound healing rates of the diabetic mice receiving a multipoint injection of PBS (control), Exo, and Nr-CWS -Exos at days 0, 7, and 14 day postoperatively. D-F. H&E staining and quantification of wound length and tube formation (black arrows indicate micro vessels) at day 14. G. Masson’s trichrome staining at day 14. H-J. IHC staining of CD31 and VEGFA. K-M. IF staining of CD31 and VEGFA. **P < 0.01, ***P < 0.001.

    • CircRNAs have been proved to play crucial role in the biological functions of endothelial cells in the process of tissue injury [21]. To reveal the potential mechanism of Nr-CWS-Exos-mediated proangiogenic capacity, we treated HUVECs with HG, HG + Exos, and HG + Nr-CWS-Exos, and submitted to the whole transcriptome sequencing. A series of changed circRNAs was observed. Among the changed circRNAs, circIARS1 was the most up-regulated circRNA (Fig. 4A, B). Then, we performed qRT-PCR to detect circIARS1 expression. Our data showed the same change of circIARS1 expression (Fig. 4C). Herein, we focus on circIARS1 in the present study. Subsequently, we verified the head-to-tail splicing of circIARS1 by Sanger sequencing in HUVECs (Fig. 4D). CircIARS1 was only amplified by divergent primers in cDNA, but no amplification product in gDNA (Fig. 4E). Moreover, circIARS1 displayed resistance to RNase R, whereas IASR1 mRNA was partly degraded by RNase R (Fig. 4F).

      Figure 4.  CircIARS1 is upregulated in Nr-CWS-Exos treated HUVECs and enhances tube formation of HUVECs. A, B. CircRNA expression profile in HUVECs treated with Exos or Nr-CWS -Exos. C. CircIARS1 expression in HUVECs treated with Exos or Nr-CWS -Exos. D. Back splicing junction was verified by Sanger sequencing. E. Existence of circIARS1 was validated in HUVECs by RT-PCR. F. Expression of circIARS1 and IARS1 mRNA in HUVECs treated with or without RNase R was detected by qRT-PCR. G-I. The images of migration ability as well as wound healing of HUVECs were evaluated after cells were treated with Exos, Nr-CWS -Exos, Nr-CWS -Exos-circIARS1, or Nr-CWS -Exos-sicircIARS1 under high glucose. J, K. Tube formation assay of HUVECs was assessed after cells were treated with Exos, Nr-CWS -Exos, Nr-CWS -Exos-circIARS1, or Nr-CWS -Exos-sicircIARS1 under high glucose. L. qRT-PCR assays of VEGFA, bFGF, and HGF expression in treated HUVECs. M. ELISA analysis of VEGFA, bFGF, and HGF secretion in treated HUVECs. Data are the mean ± SD, n = 3. **P < 0.01, ***P < 0.001.

    • We investigated the effect of circIARS1 on Nr-CWS-Exos-triggered pro-angiogenic in endothelial cells. Data from transwell and wound healing assay showed that the pro-migratory effects of Nr-CWS-Exos were inhibited once circIARS1 was decreased in HUVECs. Conversely, increased circIARS1 in HUVECs suppressed the migration of endothelial cells (Fig. 4G-I). Tube formation data displayed less capillary-like structures in si-circIARS1-Exos group, while more capillary-like structures could be observed in circIARS1-Exos group (Fig. 4J, K). We also identify the expression and secretion of VEGFA, bGFG and HGF in HUVECs. As presented by Figure 4L and M, the capability of MSC-Exos to trigger VEGFA, bFGF and HGF was significantly inhibited when circIARS1 expression in HUVECs was inhibited, while increased when circIARS1 expression in HUVECs was upregulated. Collectively, the discoveries herein reveal that circIARS1 is imperative for Nr-CWS-Exos-triggered facilitation of EC revascularization.

    • To investigate the mechanism of circIARS1 in regulating angiogenesis, we first detect its location. CircIARS1 primarily localized to the cytoplasm (Fig. 5A, B), which suggest a possible function as a molecular sponge for miRNAs. We searched potential binding miRNAs and selected five candidate miRNAs to explore whether circIARS1 was capable of binding these miRNAs. The biotin-labeled probe was confirmed to pull down circIARS1 in HUVECs (Fig. 5C). Then, the circIARS1 probe was applied to pulldown the candidate miRNAs, the results revealed that miR-4782-5p was the only miRNA pulled down by circIARS1 (Fig. 5D). Next, we utilized biotin-labeled miR-4782-5p to pull down circIARS1, and the data confirmed that biotin-miR-4782-5p sponged more circIARS1 (Fig. 5E). To confirm this prediction, we established dual-luciferase reporter system in which sequence of circIARS1 was inserted into the 3′UTR of the psiCHECK2 plasmid (wild type, WT). Only the mimic miR-4782-5p significantly inhibited luciferase activity (Fig. 5F). MiRNAs suppress translation and degrade mRNA via binding their targets in an AGO2-dependent manner. We investigated whether circIARS1 served as a platform for AGO2 and miR-4782-5p. AGO2 immunoprecipitation showed that circIARS1 was specifically enriched in miR-4782-5p transfected cells (Fig. 5G). In addition, according to RNA FISH assay, circIARS1 and miR-4782-5p were colocalized in the cytoplasm (Fig. 5H). Those data above suggest that circIARS1 can directly bind to miR-4782-5p.

      Figure 5.  CircIARS1 decoys miR-4782-5p as a sponge RNA. A. Localization of circIARS1 (red). B. Relative circIARS1 expression in the cytoplasmic (cyto) and nuclear (nuc) fractions of HUVECs. C. circIARS1 in the HUVECs lysates was pulled down and enriched with circIARS1 specific probe and then detected by qRT-PCR. D. The relative levels of 5 miRNA candidates in the HUVECs lysates were detected by qRT-PCR. E. Biotinylated miR-4782-5p was transfected into cells. After streptavidin capture, circIARS1 levels were quantified by qRT-PCR. F. Relative luciferase activities of WT and MUT circIARS1. G. Anti-AGO2 RIP was executed to circIARS1 associated with AGO2. H. RNA FISH detection for circIARS1 and miR-4782-5p localization in HUVECs. I-K. The migration ability as well as wound healing of HUVECs was examined after cells were co-transfected with miR-4782-5p and VEGFA vectors. L, M. Tube formation of HUVECs after cells were co-transfected with miR-4782-5p and VEGFA vectors. N. Western blot analysis of VEGFA protein expression in cells co-transfected with miR-4782-5p and VEGFA vectors. **P < 0.01, ***P < 0.001.

    • MiRNAs post-transcriptionally modulate their target mRNA through sequence-guided recognition. According to miRDB predictions, we found that miR-4782-5p could bind to the 3′-UTR region of VEGFA. We constructed a mutated reporter (MUT) at miR-4782-5p-binding sites. Our data displayed that miR-4782-5p mimics strongly reduced the luciferase reporter activity that carrying the wild-type VEGFA 3′-UTR, but the luciferase reporter activity was unchanged in the MUT 3’-UTR reporters group (Fig. S1A). The VEGFA expression was suppressed by miR-4782-5p mimics, while inhibition of miR-4782-5p facilitated VEGFA expression (Fig. S1B). We next investigated the function of miR-4782-5p/VEGFA in the regulation of angiogenesis. MiR-4782-5p inhibited angiogenesis and migration of endothelial cells, but upregulated VEGFA expression rescued cell angiogenesis (Fig. 5I-M), which was consistent with the expression of VEGFA (Fig. 5N). Conversely, VEGFA downregulation suppressed anti-miR-4782-5p induced angiogenesis and migration of endothelial cells (Fig. S2). Collectively, these results revealed that miR-4782-5p could significantly suppress the angiogenesis of endothelial cells by targeting VEGFA.

    • To assess whether circIARS1 facilitated the angiogenesis of endothelial cells through miR-4782-5p, rescue assays were performed by co-transfecting circIARS1 and miR-4782-5p mimics into HUVECs. Introduction of circIARS1 led to increase of the migration and angiogenesis capabilities, whereas such effect could be partially weakened via ectopic expressing of miR-4782-5p (Fig. 6A-E). In addition, we found that the expressing of VEGFA was remarkably reduced in HUVECs co-transfected with circIARS1 plasmids and miR-4782-5p mimics (Fig. 6F), which was consistent with the results of cell function. Comparatively, ablation of miR-4782-5p rescued the migration and angiogenesis capabilities of HUVECs induced by knockdown of circIARS1 (Fig. 6H-L), and the expression of VEGFA showed the same change (Fig. 6G). Above all, the results demonstrated that circIARS1 promoted angiogenesis of endothelial cells via sponging miR-4782-5p and regulated VEGFA expression.

      Figure 6.  CircIARS1 regulates angiogenesis by targeting miR-4782-5p. A-C. The migration ability as well as wound healing of HUVECs were examined after cells were co-transfected with circIARS1 vector and miR-4782-5p mimics. D, E. Angiogenesis ability was evaluated by tube formation assay. F. Western blot analysis of the protein levels of VEGFA in cells co-transfected with miR-4782-5p and VEGFA vectors. G. Western blot analysis of the protein levels of VEGFA in cells co-transfected with si-circIARS1 and miR-4782-5p inhibitors. H-J. The migration ability as well as wound healing of HUVECs was examined after cells were co-transfected with si-circIARS1 and miR-4782-5p inhibitors. K, L Angiogenesis ability was evaluated by tube formation assay. Data are means of triplicate ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

    Discussion
    • Diabetic wound is a health complication and the wound-healing process was delayed because of the diabetes-induced blood vessel damage. Great efforts have been made to solve this challenge, but the methods at present are function-limited. In the present study, we investigated the angiogenesis-promoting effects of exosomes isolated from human umbilical cord MSCs (hucMSCs) pretreated with Nr-CWS to advance the diabetes wound repair in vivo and in vitro. Furthermore, we explored the possible underlying mechanism. Our results herein revealed that Nr-CWS-Exos augmented the revascularization of endothelial cells through circIARS1/miR-4782-5p/VEGFA axis, thereby leading to faster wound repair of the diabetes mice. These findings revealed that Nr-CWS pre-conditioning is a prospective way to promote the MSC-Exos therapy for diabetic cutaneous wounds.

      During wound healing depends on a balance between pro- and antiangiogenic factors, which maintains the balance between vessel growth promotion, vessel maturation, and quiescence. Nevertheless, a decrease in the pro-angiogenic stimulus in diabettic diseases induces decreased revascularization and delayed wound repair [22]. Numerous of growth factors, proteins or peptides are considered as promising growth factor for wound healing therapies and a known mediator of angiogenesis. However, diabetic patients developing resistance to angiogenic growth factors or proteins during the development of vascular disease limit its application [23]. A broad range of scaffold and hydrogel delivery systems have been employed for delivering gene therapies targeting angiogenesis in wound healing. It was demonstrated that the PC hydrogel promotes wound healing by reducing inflammation and enhancing angiogenesis in a rat type II diabetic foot model[24] .The poly-L-lactide (PLA) and polycaprolactone (PCL) composite scaffolds promoted the highest levels of cell adhesion to the scaffold, indicating that the scaffold could support cell growth to enable angiogenesis and wound healing[25]. But the adverse effects of prolonged delivery systems treatment may lead to potential unwanted cell activation and prolonged inflammation.

      Stem cell therapies are an emerging and promising approach for treating diabetic wound not well addressed by conventional therapies. Among them, MSCs, especially hucMSCs, are a promising therapeutic cell type for wound healing as cost-effectiveness, minimum invasion, easy separation, huge cellular content, fast genetic transfection. But there are still weaknesses such as immunogenicity and tumorigenic potential, limiting their clinical application [26, 27]. Comparatively, MSC-Exos exhibit smaller risks of carcinogenesis, immunorejection, and are more stable [28]. Over the last few years, increasing evidence suggests that pretreated MSC-Exos display better potency in wound regeneration. Hu et al [29] reported that exosomes from pioglitazone-pretreated MSC improved wound repair through facilitating the HUVEC angiogenesis via activating the PI3K/AKT/eNOS pathway. Another literature by Yu et al[17] reported that exosome stemmed from MSCs pre-treated with atorvastatin stimulated the cutaneous wound regeneration by reinforcing revascularization through the AKT/eNOS pathway. Therefore, we investigated the role of NR-CWS pretreated MSC-Exos in diabetic wound healing, and our data showed that NR-CWS stimulated MSC-Exos angiogenic ability of HUVECs.

      Nr-CWS is a biology reaction modifier comprising nocardomycolic acid, arabogalactan, and peptidoglycan, and it exhibits underlying anti-cancer potency in animal models [18]. Clinical researches have reveal that Nr-CWS displays remarkable anti-cancer potency against certain mankind cancers, hence realizing the upregulation of immune modulators such as interferon-γ [30, 31]. There is only one study reported that in diabetic wound animal model, Nr-CWS elevates revascularization via stimulating the expression of TGF-β1, which eventually accelerates wound healing [32]. However, the role of Nr-CWS preconditioned MSC-Exos remains unknown. In our study, the data in vitro indicated that Nr-CWS preconditioned obviously ameliorated the biofunction of HUVECs, such as growth, motility, tube forming, and VEGF excretion that were disrupted by high glucose. Angiogenesis related genes have been demonstrated to promote angiogenesis and the forming of granulation tissues, inducing faster re-epithelialisation and wound healing [33]. We demonstrated that NR-CWS-Exos promoted angiogenesis related genes VEGF, bFGF and HGF expression. In vivo, we found that Nr-CWS preconditioned MSC-Exos accelerated the vascularization of diabetic wounds, which in turn improved the wound healing process of diabetic mice. Moreover, immunohistochemistry and immunofluorescence data indicated that Nr-CWS -Exos treatment elevated the quantity of new vessels. Herein, we further explored the potential molecular mechanisms of Nr-CWS -Exos triggered revascularization in vitro.

      Circular RNAs (circRNAs) are composed of a class of natural, steady, and common RNAs stemmed from the head to tail splicing of coding RNAs or ncRNAs [34]. Increasing proofs reveal that exosomal circRNAs are vital regulators of angiogenesis and signaling pathways affecting the development of variety of illnesses. Exosomal circRNA-100338 promotes hepatocellular cancer metastasis through invasiveness and angiogenesis [35]. Exosomal circEhmt1 isolated from pericytes pretreated with hypoxia modulates high glucose-triggered microvascular function disorder through the NFIA/NLRP3 pathway [36]. Because of the sequence conservation, biostability and tissular specificity of circRNAs, they are considered to be prospective treatment targets, and may exert potential functions in the regulation of gene expression [37]. To further reveal the potential molecular mechanisms of Nr-CWS -Exos induced angiogenesis in this study, we focused on circIARS1, which was increased significantly in Nr-CWS -Exos treated HUVECs. Angiogenesis analysis revealed that Nr-CWS-Exos no longer exhibited a distinct advantage over Exos in angiogenesis-enhancement after the blockade of the circIARS1 expression. Herin, we concluded that the improved proangiogenic capability of Nr-CWS -Exos in contrast to Exos was mediated by circIARS1.

      CircIARS1, comprising eight exon (932 bp) from the IARS1 gene, was predominantly in the cytoplasm which indicated that it may sponge miRNA to produce the circRNA-miRNA-mRNA axis. The miRNA-targeting prediction analyses herein revealed that circIARS1 carried several miRNA binding sites, unveiling that circIARS1 might sponge miRNAs. Our results unraveled that circIARS1 combined with miR-4782-5p in HUVECs. Biological information prediction and luciferase reporter assay indicated that miR-4782-5p were critical negative regulators of the VEGFA pathway. VEGFA is one of the strongest proangiogenic factors in repairing wounded tissues. Main sources of VEGFA in wound repair are considered to derive from cells in the connecting tissues like macrophagus, fibroblasts, and mastocytes [38]. We found that circIARS1 could regulate the expression of VEGFA via capturing miR-4782-5p. Upregulated circIARS1 expression induced the activation of VEGFA in HUVECs, which could be mitigated by miR-4782-5p. Lastly, we demonstrated that the circIARS1-mediated angiogenesis could be weakened by overexpression of miR-4782-5p.

      Collectively, the results herein reveal that Nr-CWS precondition facilitate the function of MSC-Exos in enhancing angiogenesis via upregulating circIARS1/miR-4782-5p/VEGFA axis, resulting in accelerated diabetes wound healing. These data propose a promising therapeutic strategy for diabetic wound treatment as a cell-free therapy.

    Ethics approval and consent to participate
    • The study was approved by the Ethics Committee (full name: animal Care and Use Committee of Xuzhou Medical University) (reference number 202108w013).

    Consent for publication
    • All authors approve the final manuscript and the submission to this journal.

    Competing interests
    • The authors declare that they have no competing interests.

    Authors' contributions
    • Q.L., L.G. and J.W. conducted the experiments. Q.L. wrote the manuscript. S.T. analyzed the results. P.J. designed the experiments. All authors reviewed the manuscript.

    Availability of data and material
    • Datasets related to this article can be found at Gene Expression Omnibus accession number GSE197900, hosted at Gene Expression Omnibus.

Reference (38)

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