Bone marrow mesenchymal stem cell-derived exosomal miR-221-3p promotes angiogenesis and wound healing in diabetes via the downregulation of forkhead box P1.

AIM: Impaired wound healing in patients with diabetes can develop into nonhealing ulcerations. Because bone marrow mesenchymal stem cells (BMSCs) exosomes can promote wound healing, this study aims to investigate the mechanism of BMSCs-isolated exosomal miR-221-3p in angiogenesis and diabetic wound healing. METHODS: To mimic diabetes in vitro, human umbilical vein endothelial cells (HUVECs) were subjected to high glucose (HG). Exosomes were derived from BMSCs and identified by transmission electron microscopy (TEM), western blot analysis and dynamic light scattering (DLS). The ability to differentiate BMSCs was assessed via Oil red O staining, alkaline phosphatase (ALP) staining and alizarin red staining. The ability to internalise PKH26-labelled exosomes was assessed using confocal microscopy. Migration, cell viability and angiogenesis were tested by scratch, MTT and tube formation assays separately. The miRNA and protein levels were analysed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or western blotting. The relationship among miR-221-3p, FOXP1 and SPRY1 was determined using the dual-luciferase reporter, ChIP and RIP assays. RESULTS: Exosomal miR-221-3p was successfully isolated from BMSCs and delivered into HUVECs. HG was found to suppress the angiogenesis, cell viability and migration of HUVECs and exosomal miR-221-3p separated from BMSCs inhibited the above phenomenon. FOXP1 could transcriptionally upregulate SPRY1, and the silencing of FOXP1 reversed the HG-stimulated angiogenesis inhibition, cell viability and migration in HUVECs via the downregulation of SPRY1. Meanwhile, miR-221-3p directly targeted FOXP1 and the overexpression of FOXP1 reversed the positive effect of exosomal miR-221-3p on HUVEC angiogenesis. CONCLUSION: Exosomal miR-221-3p isolated from BMSCs promoted angiogenesis in diabetic wounds through the mediation of the FOXP1/SPRY1 axis. Furthermore, the findings of this study can provide new insights into probing strategies against diabetes.

UCMSCs-derived exosomal circHIPK3 promotes ulcer wound angiogenesis of diabetes mellitus via miR-20b-5p/Nrf2/VEGFA axis.

AIMS: Experiments confirmed that circular RNAs contributed to the pathogenesis of diabetic foot ulcers (DFUs). CircHIPK3 was upregulated in type 2 diabetes mellitus (T2DM), but its role in DFU remained unknown. Our study aimed to investigate the regulatory functions of exosomal circHIPK3 and its potential mechanisms in DFU. METHODS: Exosomal size and distribution, marker proteins, and circHIPK3 levels were evaluated by transmission electron microscope, ExoView R200, western blot, and qRT-PCR. Flow cytometry, MTT, Wound healing assays, and tube formation assays were used to assess the roles of exosomal circHIPK3 in high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). The relationships between Nrf2/VEGFA/circHIPK3 and miR-20b-5p, and between Nrf2 and VEGFA were determined by luciferase reporter assay and RNA immunoprecipitation. We used cell and mice models to investigate the mechanisms of exosomal circHIPK3 under diabetic conditions. RESULTS: CircHIPK3 was significantly upregulated in exo-circHIPK3 rather than exo-vector. Exo-circHIPK3 remarkably inhibited cell apoptosis but promoted cell proliferation, migration, and tube formation in HG-treated HUVECs. Luciferase reporter and RIP assays showed that miR-20b-5p targeted and inhibited Nrf2 and VEGFA, and circHIPK3 acted as a ceRNA of miR-20b-5p to inhibit the binding to its downstream genes Nrf2 and VEGFA. Mechanistically, circHIPK3 promoted cell proliferation, migration, and angiogenesis via downregulating miR-20b-5p to upregulate Nrf2 and VEGFA. However, the overexpressed miR-20b-5p could abolish the promoting effects of circHIPK3 overexpression on cell proliferation, migration, and tube formation under HG conditions. CONCLUSION: UCMSCs-derived exosomal circHIPK3 protected HG-treated HUVECs via miR-20b-5p/Nrf2/VEGFA axis. The exosomal circHIPK3 might be a therapeutic candidate to treat DFU.

LncRNA GNAS-AS1 knockdown inhibits keloid cells growth by mediating the miR-188-5p/RUNX2 axis.

Keloid is a common dermis tumor, occurring repeatedly, affecting the quality of patients' life. Long non-coding RNAs (lncRNAs) have crucial regulatory capacities in skin scarring formation and subsequent scar carcinogenesis. The intention of this study was to investigate the mechanism and function of GNAS antisense-1 (GNAS-AS1) in keloids. Clinical samples were collected to evaluate the expression of GNAS-AS1, RUNX2, and miR-188-5p by qRT-PCR. The proliferation, migration, and invasion of HKF cells were detected by CCK-8, wound healing, and Transwell assays. The expression levels of mRNA and protein were examined through qRT-PCR and Western blot assay. Luciferase reporter assay was used to identify the binding relationship among GNAS-AS1, miR-188-5p, and Runt-related transcription factor 2 (RUNX2). GNAS-AS1 and RUNX2 expressions were remarkably enhanced, and miR-188-5p expression was decreased in keloid clinical tissues and HKF cells. GNAS-AS1 overexpression promoted cells proliferation, migration, and invasion, while GNAS-AS1 knockdown had the opposite trend. Furthermore, overexpression of GNAS-AS1 reversed the inhibitory effect of 5-FU on cell proliferation, migration, and invasion. MiR-188-5p inhibition or RUNX2 overexpression could enhance the proliferation, migration, and invasion of HKF cells. GNAS-AS1 targeted miR-188-5p to regulate RUNX2 expression. In addition, the inhibition effects of GNAS-AS1 knockdown on HKF cells could be reversed by inhibition of miR-188-5p or overexpression of RUNX2, while RUNX2 overexpression eliminated the suppressive efficaciousness of miR-188-5p mimics on HKF cells growth. GNAS-AS1 knockdown could regulate the miR-188-5p/RUNX2 signaling axis to inhibit the growth and migration in keloid cells. It is suggested that GNAS-AS1 may become a new target for the prevention and treatment of keloid.

GLI family zinc finger protein 2 promotes skin fibroblast proliferation and DNA damage repair by targeting the miR-200/ataxia telangiectasia mutated axis in diabetic wound healing.

Diabetic foot ulcer (DFU) is a serious complication of diabetic patients which negatively affects their foot health. This study aimed to estimate the role and mechanism of the miR-200 family in DNA damage of diabetic wound healing. Human foreskin fibroblasts (HFF-1 cells) were stimulated with high glucose (HG). Db/db mice were utilized to conduct the DFU in vivo model. Cell viability was evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays. Superoxide dismutase activity was determined using detection kits. Reactive oxygen species determination was conducted via dichlorodihydrofluorescein-diacetate assays. Enzyme-linked immunosorbent assay was used to evaluate 8-oxo-7,8-dihydro-2'deoxyguanosine levels. Genes and protein expression were analyzed by quantitative real-time polymerase chain reaction, western blotting, or immunohistochemical analyses. Luciferase reporter gene and RNA immunoprecipitation assays determined the interaction with miR-200a/b/c-3p and GLI family zinc finger protein 2 (GLI2) or ataxia telangiectasia mutated (ATM) kinase. HG repressed cell proliferation and DNA damage repair, promoted miR-200a/b/c-3p expression, and suppressed ATM and GLI2. MiR-200a/b/c-3p inhibition ameliorated HG-induced cell proliferation and DNA damage repair repression. MiR-200a/b/c-3p targeted ATM. Then, the silenced ATM reversed the miR-200a/b/c-3p inhibition-mediated alleviative effects under HG. Next, GLI2 overexpression alleviated the HG-induced cell proliferation and DNA damage repair inhibition via miR-200a/b/c-3p. MiR-200a/b/c-3p inhibition significantly promoted DNA damage repair and wound healing in DFU mice. GLI2 promoted cell proliferation and DNA damage repair by regulating the miR-200/ATM axis to enhance diabetic wound healing in DFU.

Exosomal circHIPK3 derived from umbilical cord-derived mesenchymal stem cells enhances skin fibroblast autophagy by blocking miR-20b-5p/ULK1/Atg13 axis.

BACKGROUND: Umbilical cord-derived mesenchymal stem cells (UCMSCs) could alleviate diabetes-induced injury. Hence, this investigation aimed to explore the role and mechanism of UCMSCs-derived exosomal circHIPK3 (exo-circHIPK3) in diabetes mellitus (DM). METHODS: HFF-1 cells were cultured in high glucose (HG) medium or normal medium, and treated with UCMSCs-derived exo-circHIPK3 or miR-20b-5p mimics or Unc-51-like autophagy activating kinase 1 (ULK1) overexpression vector. The surface markers of UCMSCs were analyzed using a flow cytometer. The differentiation potential of UCMSCs was evaluated using oil red O staining, alizarin red staining and alkaline phosphatase (ALP) staining. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The miRNA expressions were analyzed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR). Protein levels were quantified by western blot. An immunofluorescence staining was used for observing LC3 expression. The interaction between miR-20b-5p and circHIPK3, and between miR-20b-5b and ULK1 were identified by a RNA immunoprecipitation (RIP) assay and a luciferase reporter assay. RESULTS: Up-regulation of circHIPK3 was found in UCMSCs-derived exosomes. Exo-circHIPK3 decreased the miR-20b-5p level while increasing the contents of ULK1 and autophagy-related gene 13 (Atg13) in HG-induced fibroblasts. In addition, exo-circHIPK3 activated HG-induced fibroblast autophagy and proliferation. Overexpressed miR-20b-5p promoted fibroblast injury by inhibiting cell autophagy via the ULK1/Atg13 axis in HG conditions of high glucose. Moreover, exo-circHIPK3 enhanced autophagy and cell viability in HG-induced fibroblasts through the miR-20b-5p/ULK1/Atg13 axis. CONCLUSION: UCMSCs-derived exosomal circHIPK3 promoted cell autophagy and proliferation and accelerated the fibroblast injury repair by the miR-20b-5p/ULK1/Atg13 axis.

Exosomes from mmu_circ_0001052-modified adipose-derived stem cells promote angiogenesis of DFU via miR-106a-5p and FGF4/p38MAPK pathway.

BACKGROUND: Diabetic foot ulcer (DFU) is a chronic infectious disease caused by diabetes mellitus (DM). Angiogenesis plays the decisive role in wound healing of DFU. Adipose-derived stem cells (ADSCs) can ameliorate angiogenesis in DFU by exosomes. This study aims to determine the mechanism of exosomes from mmu_circ_0001052-modified ADSCs in angiogenesis of DFU. METHODS: HUVECs were induced by high glucose and mice stimulated using STZ injection during high-fat feeding, which were treated with exosomes derived from mmu_circ_0001052-modified ADSCs. Real-time PCR determined the expression of gene and western blot determined protein levels. Proliferation, migration, apoptosis and angiogenesis of HUVECs were studied by MTT assay, transwell test, flow cytometry and tube formation experiment, respectively. Histological lesion of wound was determined by HE staining. RESULTS: The expression of circ_0001052 was upregulated in ADSCs and miR-106a-5p elevated in high glucose-induced HUVECs. Exosomal mmu_circ_0001052 significantly accelerated wound healing in mice with DFU. Also, exosomal mmu_circ_0001052 evoked the reduction of miR-106a-5p and the elevation of FGF4 in high glucose-induced HUVECs and wound tissue of DFU mice. Exosomal mmu_circ_0001052 was determined to sponge miR-106a-5p that targeted FGF4 in DFU. In high glucose-induced HUVECs, exosomal mmu_circ_0001052 inhibited apoptosis and miR-106a-5p expression, and meanwhile promoted proliferation, migration, angiogenesis and expressions of FGF4, VEGF and p-p38/p38, which were reversed by miR-106a-5p elevation. CONCLUSION: Mmu_circ_0001052 in ADSCs-derived exosomes promote angiogenesis of DFU via miR-106a-5p and FGF4/p38MAPK pathway.

LncRNA MALAT1 promotes wound healing via regulating miR-141-3p/ZNF217 axis.

BACKGROUND: The process of wound healing is complex. Increasing evidences have shown that lncRNA MALAT1 is abundant in fibroblasts and may be engaged in wound healing process. Therefore, we explored the mechanism of MALAT1 affecting wound healing. METHODS: The expression levels of MALAT1, miR-141-3p as well as ZNF217 in human fibroblast cells (HFF-1) were quantified by qRT-PCR. HFF-1 proliferation was measured by MTT, while migration was detected by wound healing assay. SMAD2 activation and matrix proteins expression were detected by western blotting. The interaction between miR-141-3p and MALAT1 or ZNF217 was further confirmed using the luciferase reporter gene assay. In vivo wound healing was assessed by full-thickness wound healing model on C57BL/6 mice. RESULT: Knockdown of MALAT1 as well as overexpression miR-141-3p remarkably inhibited the proliferation, migration and matrix protein expression in HFF-1 cells. MALAT1 directly targeted and inhibited the expression of miR-141-3p. MiR-141-3p suppressed the activation of TGF-ß2/SMAD2 signaling pathway by targeting ZNF217. Knockdown of MALAT1 inhibited wound healing process in mice. CONCLUSIONS: MALAT1 up-regulates ZNF217 expression by targeting miR-141-3p, thus enhances the activity of TGF-ß2/SMAD2 signaling pathway and promotes wound healing process. This investigation shed new light on the understanding of the role of MALAT1 in wound healing, and may provide potential target for the diagnosis or therapy of chronic wounds.

Preparation and properties of a novel carbon nanotubes/poly(vinyl alcohol)/epidermal growth factor composite biological dressing.

Wound dressings with drug delivery system have drawn increasing attention in skin damage recombination. Herein, a novel composite biological dressing was prepared and based on poly(vinyl alcohol) (PVA) combined with carbon nanotubes (CNTs) and epidermal growth factor (EGF) by electrospinning on gauze. The properties of the CNTs/PVA/EGF composite dressing were systemically investigated by general observation, and scanning electron microscopy (SEM). In vitro, the cytotoxicity of this dressing was investigated using a methyl thiazolyl tetrazolium (MTT) assay on L929 fibroblasts. In order to study the sustained release of EGF from this dressing, the concentration of EGF at different times was tested by ELISA. Furthermore, the biological activity of the released EGF was also evaluated using the MTT assay. Moreover, an in vivo experiment was conducted to observe whether this dressing was capable of improving healing in the model of wounded skin on rats. It was revealed that this dressing had a well-distributed microstructure by SEM. Additionally, the grade of cytotoxicity was low, and the EGF had a sustained release rate from this dressing. Furthermore, a maximum accumulative release rate of 12.47% was identified at 12 h, and was retained at 9.4% after 48 h. Simultaneously, the relative growth rate of L929 fibroblasts in the 12 h experimental group and 48 h group was 291.24 and 211.3%, respectively. Next, the efficacy of these products was evaluated in vivo using Sprague-Dawley rats with a skin injury model. The healing of wounded skin of rats was sped up by this dressing based on the gross and histological appearances. From 7 to 10 days, the wounds in the experimental group were almost healed. In conclusion, this CNTs/PVA/EGF dressing had a well-distributed structure and an ability to release EGF at a sustained rate with the activity being favorable. On the basis of those results, a positive influence of designed dressing for accelerated wound healing was confirmed.

[The clinical application on composite graft of acellular allo-dermal matrix and auto-microskin on escharectomy after deep burns].

OBJECTIVE: To present the clinical application of composite graft of acellular allo-dermis matrix (ADM) with thin auto-microskin on burn wound. METHODS: 8 inpatients with 18 full thickness skin burn wounds were transplanted with allo-ADM after eschar was excised, then the auto-microskin and allo-human skin were covered on the area of the matrix, the wound where no allo-ADM grafting were covered as control groups only with auto-microskin and allo-human skin. The area of donor to wound is 1:5 - 1:8. RESULTS: Survived rate of 18 pieces composite skin that allo-ADM with auto-microskin were grafted were 94%. After following up for 3 to 13 months, the skins of complex grifting had well elastic and smooth texture compared to auto-microskin grafted, they appeared less cicatrisation and ulceration. 3 months after operation, it was indicated by histological examination that tightknit the epithelial-dermal conjunction and epidermal papilla structure could be identified in the allo-ADM skin and there were orderly collagenous fibres, but scar skin structure was observed in that auto-microskin grifted area. CONCLUSION: The graft effectiveness of allo-ADM and auto-microskin was better than that of auto-microskin, and this method could be used on major deep burn wound healing.

[Epidemiological investigation of burn inpatients in 6 hospitals of Hainan province: a retrospective analysis in 8 years].

OBJECTIVE: To analyze epidemiological characteristics of burn inpatients in Hainan province over 8 years. METHODS: Six thousand and ninety-nine burn patients admitted to 6 hospitals of Hainan province from January 2002 to December 2009 were enrolled in the study. The clinical data of these patients were analyzed retrospectively, including age, gender, injury cause, wound position, burn area, ailment prior to admission, admission time, medical insurance, length of hospital stay, and mortality rate, relationship among inpatient distribution, admission time, and ambient temperature at the time of admission. Data were processed with SPSS 13.0 software. RESULTS: There were more burn male patients than female, with ratio of 2.1: 1.0. Most patients were younger than 13 years (57.2%, 3488/6099). The most common burn area was smaller than or equal to 10% TBSA (67.4%, 4108/6099), and the fewest patients had burn areas of over 50% TBSA (2.0%, 121/6099). The main causative agents were hot liquid and flame, accounting for 71.5% (4358/6099), 17.9% (1092/6099), respectively. Most patients had injuries of more than two body areas (60.7%, 3705/6099), and lower extremity injury (17.1%, 1042/6099) was predominant in wound of single body area. Among 703 cases who had other ailments prior to admission (11.5%), the highest rate of prior ailments was found in patients older than 60 years (18.5%, 48/260), it was lowest in children younger than 1 year (8.0%, 32/398). The length of hospital stay was 1 to 375 day, and the admission time was 10 minutes to 90 days after burn. Total mortality rate was 0.4% (26 cases). The number of inpatients aged from 19 to 59 was obviously higher in months with high ambient temperature (from June to August), and for inpatients younger than 13 years the incidence of burn injury showed no obvious seasonal change. The inpatients who had medical insurance accounted for 10.9% (66/603) to 19.5% (121/619) from 2002 to 2005, which increased to 46.0% (372/808) in 2007 and 79.1% (869/1098) in 2009. CONCLUSIONS: For burn inpatients in Hainan province, the main injury cause of burn injury is hot liquid, the number of burn adults aged from 19 to 59 seems to increase in months with high ambient temperature, while the incidence of burn in children showed no obvious seasonal change. The number of inpatients and those with medical insurance showed a tendency of increase from 2005 to 2009 in Hainan province.

[Follow-up review on the long-term effect of composite transplantation of allogeneic acellular dermal matrix and split thickness skin autograft].

OBJECTIVE: To review the long-term clinical effect of composite transplantation of allogeneic acellular dermal matrix (ADM) and split thickness skin autograft (STSG). METHODS: Nineteen patients with 34 wounds transplanted with allogeneic ADM combined with STSG who were hospitalized from March 2001 to October 2008 were enrolled as composite transplantation group (CT). Another 9 patients with 11 wounds transplanted with STSG admitted within the same time frame were enrolled as control group (C). All patients were followed up for longer than 2 years. Color, evenness, texture, contracture, sensation, and complications of transplanted skin were assessed using a modified Manchester Scar Scale (1-4 scores, the higher the score, the poorer the situation). The scar formation on skin donor sites was assessed by the Vancouver Scar Scale. Patients' degree of satisfaction and health status during the transplantation period were investigated in the form of questionnaire. The skin tissue structure of 4 patients was observed with histological method. The joint range of motion was assessed by the neutral position before and after operation and at follow-up. Data were processed with nonparametric test, chi-square test or t test. RESULTS: (1) The evenness, contracture, and texture of transplanted skin in CT group scored (1.6 ± 0.5), (1.8 ± 0.8), and (1.5 ± 0.8), respectively, which were significantly lower than those in C group [(2.0 ± 0.7), (2.2 ± 0.9), and (2.3 ± 0.7), with Z value respectively -2.058, -2.220, -2.323, P values all below 0.05]. Scores of color, sensation, and complications of transplanted skin in two groups were close to each other (with Z value respectively -0.628, -0.428, -2.520, P values all above 0.05). (2) Mild scar formation was observed in one of the skin donor sites in CT group. (3) Information as obtained from questionnaire showed no statistical difference between two groups in pinching, itching, and satisfaction degree (with χ(2) value respectively 0.187, 0.019, 2.628, P values all above 0.05). (4) Nerve fibers were seen in hand tissue 2 years after operation. ADM did not induce severe inflammatory responses in the site of grafting. (5) Eleven joints in CT group recovered or improved in function; while the other two joints required secondary surgery. Obvious contracture was observed in the two joints in C group. CONCLUSIONS: Allogeneic ADM combined with STSG transplantation prevents scar contracture and has obvious effect in improving function and appearance. There is no problem in regard to safety for its existence in either adult or children.

[Survey on the bacterial flora and their resistance to antibiotics in hospitalized burn patients on tropical islands].

OBJECTIVE: To investigate the bacterial flora and their drug resistance in hospitalized burn patients on tropical islands. METHODS: Retrospective study was carried out to analyze pathogenic microorganisms and their drug resistance characteristics in 392 burn patients hospitalized during 2000-2005. RESULTS: (1) Totally 671 strains of bacteria were isolated, among which Pseudomonas aeruginosa, Staphylococcus aureus, Aerobacter cloacae and Acinetobacter species were predominant, but the isolation rate of Pseudomonas aeruginosa was declining compared with that in 1990's. (2) The resistance rate of Pseudomonas aeruginosa to imipenem and cefepime was 32.2% and 36.7% , respectively, while that to other antibiotics was above 80%. (3) Among 141 strains of Staphylococcus aureus, 89 strains (63.1%) of MRSA were isolated, and none of them were resistant to Vancomycin. CONCLUSION: Pseudomonas aeruginosa, Staphylococcus aureus, Aerobacter cloacae and Acinetobacter species were predominant in tropical islands. The species of the pathogens are changing . The drug resistance rates of Pseudomonas aeruginosa and Staphylococcus aureus are rather high.

[Repair of electric burn wound in the upper limbs with lateral intercostal perforator-based pedicled flap].

OBJECTIVE: To describe an operative method for the repair of electric burn wound in the upper limbs with lateral intercostal perforator-based pedicled flap, and to observe its clinical effect. METHODS: Intercostal artery perforator-based pedicled abdominal flap with the blood supply originating from the lateral perforator branches of the 7th-10th intercostal arteries were used to repair the wounds of 6 patients with burn wounds in elbows, forearm, wrists and palms. The pedicles were (16. 0 cm x 12. 0 cm) - (9. 0 cm x 7.0 cm) in area, and the pedicles were severed 18 to 21 days after the operation. The survival and the appearance of the flaps were observed after operation. RESULTS: The procedure was easy and safe, and there was reliable and adequate blood supply in the lateral intercostal perforator-based pedicled flap. All the flaps survived in 5 patients, except marginal necrosis (3.5 cm x 2. 0 cm) was found in the distal portion of flap because flap cutting exceeded the paraumbilical line. The appearance was satisfactory after operation. CONCLUSION: This flap is suitable for the repair of deep wounds in hands, forearms, and elbows.