circCDK13-loaded small extracellular vesicles accelerate healing in preclinical diabetic wound models.

Chronic wounds are a major complication in patients with diabetes. Here, we identify a therapeutic circRNA and load it into small extracellular vesicles (sEVs) to treat diabetic wounds in preclinical models. We show that circCDK13 can stimulate the proliferation and migration of human dermal fibroblasts and human epidermal keratinocytes by interacting with insulin-like growth factor 2 mRNA binding protein 3 in an N6-Methyladenosine-dependent manner to enhance CD44 and c-MYC expression. We engineered sEVs that overexpress circCDK13 and show that local subcutaneous injection into male db/db diabetic mouse wounds and wounds of streptozotocin-induced type I male diabetic rats could accelerate wound healing and skin appendage regeneration. Our study demonstrates that the delivery of circCDK13 in sEVs may present an option for diabetic wound treatment.

MiR-17-5p-engineered sEVs Encapsulated in GelMA Hydrogel Facilitated Diabetic Wound Healing by Targeting PTEN and p21.

Delayed wound healing is a major complication of diabetes, and is associated with impaired cellular functions. Current treatments are unsatisfactory. Based on the previous reports on microRNA expression in small extracellular vesicles (sEVs), miR-17-5p-engineered sEVs (sEVs17-OE) and encapsulated them in gelatin methacryloyl (GelMA) hydrogel for diabetic wounds treatment are fabricated. SEVs17-OE are successfully fabricated with a 16-fold increase in miR-17-5p expression. SEVs17-OE inhibited senescence and promoted the proliferation, migration, and tube formation of high glucose-induced human umbilical vein endothelial cells (HG-HUVECs). Additionally, sEVs17-OE also performs a promotive effect on high glucose-induced human dermal fibroblasts (HG-HDFs). Mechanism analysis showed the expressions of p21 and phosphatase and tensin homolog (PTEN), as the target genes of miR-17-5p, are downregulated significantly by sEVs17-OE. Accordingly, the downstream genes and pathways of p21 and PTEN, are activated. Next, sEVs17-OE are loaded in GelMA hydrogel to fabricate a novel bioactive wound dressing and to evaluate their effects on diabetic wound healing. Gel-sEVs17-OE effectively accelerated wound healing by promoting angiogenesis and collagen deposition. The cellular mechanism may be associated with local cell proliferation. Therefore, a novel bioactive wound dressing by loading sEVs17-OE in GelMA hydrogel, offering an option for chronic wound management is successfully fabricated.

MiR-141-3p-Functionalized Exosomes Loaded in Dissolvable Microneedle Arrays for Hypertrophic Scar Treatment.

Hypertrophic scar (HS) is a common fibroproliferative disease caused by abnormal wound healing after deep skin injury. However, the existing approaches have unsatisfactory therapeutic effects, which promote the exploration of newer and more effective strategies. MiRNA-modified functional exosomes delivered by dissolvable microneedle arrays (DMNAs) are expected to provide new hope for HS treatment. In this study, a miRNA, miR-141-3p, which is downregulated in skin scar tissues and in hypertrophic scar fibroblasts (HSFs), is identified. MiR-141-3p mimics inhibit the proliferation, migration, and myofibroblast transdifferentiation of HSFs in vitro by targeting TGF-ß2 to suppress the TGF-ß2/Smad pathway. Subsequently, the engineered exosomes encapsulating miR-141-3p (miR-141-3pOE -Exos) are isolated from adipose-derived mesenchymal stem cells transfected with Lv-miR-141-3p. MiR-141-3pOE -Exos show the same inhibitive effects as miR-141-3p mimics on the pathological behaviors of HSFs in vitro. The DMNAs for sustained release of miR-141-3pOE -Exos are further fabricated in vivo. MiR-141OE -Exos@DMNAs effectively decrease the thickness of HS and improve fibroblast distribution and collagen fiber arrangement, and downregulate the expression of α-SMA, COL-1, FN, TGF-ß2, and p-Smad2/3 in the HS tissue. Overall, a promising, effective, and convenient exosome@DMNA-based miRNA delivery strategy for HS treatment is provided.

MiR146a-loaded engineered exosomes released from silk fibroin patch promote diabetic wound healing by targeting IRAK1.

Unhealable diabetic wounds need to be addressed with the help of newer, more efficacious strategies. Exosomes combined with biomaterials for sustained delivery of therapeutic agents are expected to bring new hope for chronic wound treatment. Here, the engineered exosomes modified for efficiently loading miR146a and attaching to silk fibroin patch (SFP) were demonstrated to promote diabetic wound healing. Silk fibroin binding peptide (SFBP) was screened through phage display, and SFBP-Gluc-MS2 (SGM) and pac-miR146a-pac fusion protein were constructed. The designed exosomes (SGM-Exos, miR146a-Exos, and SGM-miR146a-Exos) were isolated from the engineered placental mesenchymal stem cells (PMSCs) transduced with SGM or/and pac-miR146a-pac protein. Gluc signals indicated SGM-Exo@SFP markedly increased the binding rate and the stability of SGM-Exo. Moreover, the loading efficiency of miR146a in SGM-miR146a-Exos was ten-fold higher than that in miR146a-Exos. Superior to untreated, SGM-miR146a-Exo-only treated, and SFP-only treated groups, SGM-miR146a-Exo@SFP drived wound healing associated with less inflammation, collagen deposition, and neovascularization. The transcriptomics analysis suggested anti-inflammatory and regenerative effects with SGM-miR146a-Exo@SFP treatment. Here, we show efficient exosome@biomaterial-based miRNA delivery systems for regenerative medicine and tissue engineering.

Extracellular vesicles derived from fibroblasts induced with or without high glucose exert opposite effects on wound healing and angiogenesis.

Background: Communication between fibroblasts and endothelial cells is essential for skin wound repair and regeneration. Extracellular vesicles (EVs) are crucial for intracellular communication by transporting active molecules. However, whether EVs derived from diabetic fibroblasts can perform the nomal communication function is unclear. Here, we compared the effects of EVs from human skin fibroblasts (HSFs) induced with or without HG on the angiogenic function of endothelial cells and wound healing. Methods: We first collected EVs from HSFs cultured with normal glucose concentration (NG-EVs) or with HG concentration (HG-EVs) and applied them to treat human umbilical vein endothelial cells (HUVECs). The cells were divided into three groups: control group, NG-EVs group, and HG-EVs group. We then examined the proliferation, migration, apoptosis, and tube formation of HUVECs. To illustrate the mechanism, the expression of ß-catenin, GSK-3ß, and p-GSK-3ß was detected by western-blot. Finally, NG-EVs or HG-EVs were used to treat the wounds of mice to determine their role in wound closure. Results: By DNA content detection, Annexin V/PI staining, and EdU staining, we found that NG-EVs promoted HUVEC proliferation, while HG-EVs exhibited an opposite effect (p < 0.05). Scratch assay and tube formation assay demonstrated that NG-EV promoted angiogenesis in vitro, while HG-EVs showed negative impact (p < 0.05). The expressions of ß-catenin and p-GSK-3ß in HUVECs were enhanced by NG-EVs and decreased by HG-EVs (p < 0.05). Additionally, the in vivo experiment demonstrated that NG-EVs effectively promoted wound healing by locally enhancing blood supply and angiogenesis. In contrast, HG-EVs leaded to delayed wound closure and reduced blood supply and angiogenesis (p < 0.05). Conclusion: NG-EVs and HG-EVs exert opposite effects on wound healing and angiogenesis possibly by regulating GSK-3ß/ß-catenin signaling pathway. This research may provide a new treatment strategy for wound healing and illustrate the mechanism for impaired angiogenesis in diabetics.

VH298-loaded extracellular vesicles released from gelatin methacryloyl hydrogel facilitate diabetic wound healing by HIF-1α-mediated enhancement of angiogenesis.

Endothelial malfunction is responsible for impaired angiogenesis in diabetic patients, thereby causing the delayed healing progress of diabetic wounds. Exosomes or extracellular vesicles (EVs) have emerged as potential therapeutic vectors carrying drug cargoes to diseased cells. In the present study, EVs were reported as a new treatment for diabetic wounds by delivering VH298 into endothelial cells. Firstly, EVs derived from epidermal stem cells (ESCs) were loaded with VH298 (VH-EVs), and the characteristics of VH-EVs were identified. VH-EVs showed promotive action on the function of human umbilical vein endothelial cells (HUVECs) in vitro by activating HIF-1α signaling pathway. VH-EVs were also found to have a therapeutic effect on wound healing and angiogenesis in vivo. We further fabricated gelatin methacryloyl (GelMA) hydrogel for sustained release of VH-EVs, which possessed high biocompatibility and proper mechanical properties. In diabetic mice, GelMA hydrogel containing VH-EVs (Gel-VH-EVs) effectively promoted wound healing by locally enhancing blood supply and angiogenesis. The underlying mechanism for enhanced angiogenesis was possibly associated with the activation of HIF-1α/VEGFA signaling pathway. Collectively, our findings suggest a promising EV-based strategy for the VH298 delivery to endothelial cells and provide a new bioactive dressing for diabetic wound treatment. STATEMENT OF SIGNIFICANCE: The angiogenic dysfunction is the main cause of diabetic wound unhealing. Extracellular vesicles (EVs) have been reported to be helpful but their efficacy is limited for angiogenesis in cutaneous regeneration. VH298 holds great promise to improve angiogenesis by stabilizing HIF-1α which is reported at low level in diabetic wounds. Here, we loaded EVs with VH298 (VH-EVs) to exert an on-target enhancement of proangiogenic capacity in diabetic wound. Then, we applied a photo-crosslinkable hydrogel, gelatin methacryloyl (GelMA) containing VH-EVs (Gel-VH-EVs) as a convenient biomaterial and an adaptable scaffold for sustained releasing VH-EVs. The results showed significant therapeutic effect of Gel-VH-EVs on skin defect repair. Our findings suggest a promising EVs-based drug delivery strategy and a new functional wound dressing for patients.

Extracellular Vesicles from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Diabetic Wound Healing Through MiR-17-5p-mediated Enhancement of Angiogenesis.

Endothelial dysfunction caused by persistent hyperglycemia in diabetes is responsible for impaired angiogenesis in diabetic wounds. Extracellular vehicles (EVs) are considered potential therapeutic tools to promote diabetic wound healing. The aim of this study was to investigate the effects of EVs secreted by human umbilical cord mesenchymal stem cells (hucMSC-EVs) on angiogenesis under high glucose (HG) conditions in vivo and in vitro and to explore the underlying mechanisms. In vivo, local application of hucMSC-EVs enhanced wound healing and angiogenesis. In vitro, hucMSC-EVs promoted proliferation, migration, and tube formation by inhibiting phosphatase and tensin homolog (PTEN) expression and activating the AKT/HIF-1α/VEGF pathways. MiR-17-5p was found to be highly enriched in hucMSC-EVs. In vitro, MiR-17-5p agomirs downregulated the expression of PTEN and activated the AKT/HIF-1α/VEGF pathway to promote proliferation, migration, and tube formation in HG-treated HUVECs. In vivo, miR-17-5p agomirs mimicked the effects of hucMSC-EVs on wound healing and angiogenesis, whereas miR-17-5p inhibitors reversed their effects. Our findings suggest that hucMSC-EVs have regenerative and protective effects on HG-induced endothelial cells via transfer of miR-17-5p targeting PTEN/ AKT/HIF-1α/VEGF pathway, thereby accelerating diabetic wound healing. Thus, hucMSC-EVs may be promising therapeutic candidates for improving diabetic wound angiogenesis.

Burns Impair Blood-Brain Barrier and Mesenchymal Stem Cells Can Reverse the Process in Mice.

Neurological syndromes are observed in numerous patients who suffer burns, which add to the economic burden of societies and families. Recent studies have implied that blood-brain barrier (BBB) dysfunction is the key factor that induces these central nervous system (CNS) syndromes in peripheral traumatic disease, e.g., surgery and burns. However, the effect of burns on BBB and the underlying mechanism remains, largely, to be determined. The present study aimed to investigate the effect of burns on BBB and the potential of umbilical cord-derived mesenchymal stem cells (UC-MSCs), which have strong anti-inflammatory and repairing ability, to protect the integrity of BBB. BBB permeability was evaluated using dextran tracer (immunohistochemistry imaging and spectrophotometric quantification) and western blot, interleukin (IL)-6, and IL-1ß levels in blood and brain were measured by enzyme-linked immunosorbent assay. Furthermore, transmission electron microscopy (TEM) was used to detect transcellular vesicular transport (transcytosis) in BBB. We found that burns increased mouse BBB permeability to both 10-kDa and 70-kDa dextran. IL-6 and IL-1ß levels increased in peripheral blood and CNS after burns. In addition, burns decreased the level of tight junction proteins (TJs), including claudin-5, occludin, and ZO-1, which indicated increased BBB permeability due to paracellular pathway. Moreover, increased vesicular density after burns suggested increased transcytosis in brain microvascular endothelial cells. Finally, administering UC-MSCs at 1 h after burns effectively reversed these adverse effects and protected the integrity of BBB. These results suggest that burns increase BBB permeability through both paracellular pathway and transcytosis, the potential mechanism of which might be through increasing IL-6 and IL-1ß levels and decreasing Mfsd2a level, and appropriate treatment with UC-MSCs can reverse these effects and protect the integrity of BBB after burns.

42 °C heat stress pretreatment protects human melanocytes against 308-nm laser-induced DNA damage in vitro.

Vitiligo is a common depigment of skin disorder due to loss of functional melanocytes. Recently, the phototherapy with a 308-nm xenon-chloride excimer laser (UVB laser) is wildly used in vitiligo treatment. However, excessive UVB will induce photo-damage and photo-carcinogenesis in melanocytes. Previous studies revealed a protective effect of heat on UVB-induced melanocyte damage. In this study, we combined heat stress pretreatment with UVB to evaluate whether heat stress pretreatment has an ameliorative effect on UVB-induced damage. Human primary melanocytes (HMCs) were cultured and irradiated with a 308-nm laser with/without heat treatment. MTT assay, apoptosis analysis, and comet assay were conducted to monitor the damage of HMCs. Western blot and immunofluorescence staining were performed to assess the expression and subcellular localization of HSP70. HMCs heated at 42 °C for 1 h exhibit no cytotoxicity. Furthermore, preheat treatment attenuated the UVB laser-induced injury, reduced the DNA damage, and attenuated the cell apoptosis. The level and the localization of HSP70 determined the protective effects against UVB-induced DNA damage. Combining preheat treatment with a 308-nm xenon-chloride excimer laser would be a potential therapeutic method not only promotes the repigment of vitiligo but also reduces the UVB-induced photo-damage.

Reprogramming of Keratinocytes as Donor or Target Cells Holds Great Promise for Cell Therapy and Regenerative Medicine.

One of the most crucial branches of regenerative medicine is cell therapy, in which cellular material is injected into the patient to initiate the regenerative process. Cells obtained by reprogramming of the patient's own cells offer ethical and clinical advantages could provide a new source of material for therapeutic applications. Studies to date have shown that only a subset of differentiated cell types can be reprogrammed. Among these, keratinocytes, which are the most abundant proliferating cell type in the epidermis, have gained increasing attention as both donor and target cells for reprogramming and have become a new focus of regenerative medicine. As target cells for the treatment of skin defects, keratinocytes can be differentiated or reprogrammed from embryonic stem cells, induced pluripotent stem cells, fibroblasts, adipose tissue stem cells, and mesenchymal cells. As donor cells, keratinocytes can be reprogrammed or direct reprogrammed into a number of cell types, including induced pluripotent stem cells, neural cells, and Schwann cells. In this review, we discuss recent advances in keratinocyte reprogramming, focusing on the induction methods, potential molecular mechanisms, conversion efficiency, and safety for clinical applications. Graphical Abstract KCs as target cells can be reprogrammed or differentiated from fibroblasts, iPSCs, ATSCs, and mesenchymal cells. And as donor cells, KCs can be reprogrammed or directly reprogrammded into iPSCs, neural cells, Schwann cells, and epidermal stem cells.

Are hair follicle stem cells promising candidates for wound healing?

INTRODUCTION: With the continued focus on in-depth investigations of hair follicle stem cells (HFSCs), the role of HFSCs in wound healing has attracted increasing attention from researchers. This review may afford meaningful implications for HFSC treatment of wounds. AREAS COVERED: We present the properties of HFSCs, analyze the possibility of HFSCs in wound healing, and sum up the recent studies into wound repair with HFSCs. The details of HFSCs in wound healing have been discussed. The possible mechanisms of wound healing with HFSCs have been elaborated. Additionally, the factors that influence HFSCs in wound healing are also summarized. EXPERT OPINION: Hair follicle stem cells are promising sources for wound healing. However, a further understanding of human HFSCs and the safety use of HFSCs in clinical practice still remain in relative infancy.

Puerarin inhibits iNOS, COX-2 and CRP expression via suppression of NF-κB activation in LPS-induced RAW264.7 macrophage cells.

Puerarin (7,4'-dihydroxy-8-C-glucosylisoflavone) is the most abundant isoflavone-C-glucoside extracted from Radix puerariae, and it has been used for various medicinal purposes in traditional oriental medicine for thousands of years. In the present study, the ability of the puerarin to modulate inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and C reactive protein (CRP) expression and induce changes in the nuclear factor κB (NF-κB) pathway in RAW264.7 macrophage cells was examined. The protein and mRNA levels of lipopolysaccharide (LPS)-induced iNOS, COX-2 and CRP were determined in RAW246.7 macrophage cells. Inhibitor κB (I-κB) phosphorylation and p65NF-κB expression in RAW246.7 macrophage cells were also detected under our experimental conditions. The results indicated that puerarin inhibited the expression of LPS-induced iNOS, COX-2 and CRP proteins and also suppressed their mRNAs from RT-PCR experiments in RAW264.7 cells. Subsequently, we determined that the inhibition of iNOS, COX-2 and CRP expression was due to a dose-dependent inhibition of phosphorylation and degradation of I-κB, which resulted in the reduction of p65NF-κB nuclear translocation. These data suggested that the effect of puerarin-mediated inhibition of LPS-induced iNOS, COX-2 and CRP expression is attributed to suppressed NF-κB activation at the transcriptional level.

Puerarin inhibits C-reactive protein expression via suppression of nuclear factor kappaB activation in lipopolysaccharide-induced peripheral blood mononuclear cells of patients with stable angina pectoris.

Puerarin (4'-7-dihydroxy-8-beta-D-glucosylisoflavone), the most abundant isoflavone-C-glucoside extracted from the root of the plant Pueraria lobata, has demonstrated anti-inflammatory activity in cellular models of inflammation. In this report, we examined the ability of puerarin to modulate C-reactive protein (CRP) expression and key molecules in the nuclear factor kappa B (NF-kappaB) pathway to determine its molecular target. The protein and mRNA levels of CRP were determined in lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells of patients with unstable angina pectoris. Also, we detected the I-kappaBalpha phosphorylation and the p65NF-kappaB expression in peripheral blood mononuclear cells under our experimental condition. The results indicated that puerarin inhibited the expression of the protein and mRNA levels of CRP in LPS-induced peripheral blood mononuclear cells. Subsequently, we determined that the inhibition of CRP expression was because of a dose-dependent inhibition of phosphorylation and degradation of inhibitor kappaB(I-kappaB), which resulted in a reduction of p65NF-kappaB nuclear translocation. We conclude that puerarin acts as an anti-inflammatory agent by blocking NF-kappaB signalling, and may possibly be developed as a useful agent for the chemoprevention of atherosclerosis.

PPARγ agonist rosiglitazone is neuroprotective after traumatic spinal cord injury via anti-inflammatory in adult rats.

Spinal cord injury (SCI) results in the loss of function below the lesion. Secondary injury following the primary impact includes a number of biochemical and cellular alterations leading to tissue necrosis and cell death. Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor of nuclear hormone receptor superfamily. Thiazolidinedione rosiglitazone is a potent agonist of PPARγ which is shown to induce neuroprotection in animal models of focal ischemia and traumatic brain injury. SCI is induced by the application of vascular clips (force of 24 g) to the dura via a four-level T(5)-T(8) laminectomy. To gain a better insight into the mechanism of action of the anti-inflammatory effects of rosiglitazone, the following end points of the inflammatory process were evaluated: (1) spinal cord inflammation and tissue injury (histological score); (2) neutrophil infiltration (myeloperoxidase activity); (3) apoptosis (terminal deoxynucleotidyl transferase-mediated UTP end labeling staining and electron microscopy); (4) proinflammatory cytokines TNF-α and IL-ß; (5) PPARγ, HSP70 and HSP27 expressions. To elucidate whether the protective effects of rosiglitazone were mediated via the estrogen receptors, we investigated the effect of a PPARγ antagonist, GW9662, on the protective effects of rosiglitazone. GW9662 significantly antagonized the effect of the rosiglitazone and abolished the protective effect against SCI. Taken together, our results clearly demonstrate that administration of rosiglitazone after SCI reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Puerarin inhibits adhesion molecule expression in tnf-alpha-stimulated human endothelial cells via modulation of the nuclear factor kappaB pathway.

BACKGROUND: The isoflavone puerarin is the most abundant isoflavone-C-glucoside extracted from the root (radix puerariae) of the plant Pueraria lobata and possesses many biological activities. In this report, the ability of puerarin to modulate intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and endothelial leukocyte adhesion molecule 1 (E-selectin), and to induce changes in the nuclear factor kappaB (NF-kappaB) pathway in human umbilical vein endothelial cells (HUVECs) was examined. METHODS: The protein and mRNA levels of tumor-necrosis-factor-alpha (TNF-alpha)-induced ICAM-1, VCAM-1 and E-selectin were determined in HUVECs. Inhibitor kappaB (I-kappaB) phosphorylation and p65 NF-kappaB expression in HUVECs were also examined. RESULTS: Puerarin inhibited the expression of TNF-alpha-induced ICAM-1, VCAM-1 and E-selectin proteins and mRNAs in HUVECs. Subsequently, we determined that the inhibition of ICAM-1, VCAM-1 and E-selectin expression was due to a dose-dependent suppression of phosphorylation and degradation of I-kappaB, which resulted in a reduction of p65 NF-kappaB nuclear translocation. CONCLUSION: These data suggested that the effect of puerarin-mediated inhibition of TNF-alpha-induced ICAM-1, VCAM-1 and E-selectin expression is attributed to suppressed NF-kappaB activation on the transcriptional level.

Use of phosphobetaine-type zwitterionic surfactant for the determination of alkali and alkaline earth metal ions and ammonium ion in human saliva by capillary electrophoresis.

With conventional capillary electrophoresis (CE), it was difficult to directly analyze samples containing proteins as a result of the irreversible adsorption of proteins onto the inner surface of the capillary column. This difficulty, however, was completely overcome by adding N-dodecylphosphocholine (DPC, a phosphobetaine-type zwitterionic surfactant) to the background electrolyte (BGE). DPC made two essential contributions to the determination of common inorganic cations in the protein-containing samples: protein adsorption onto the capillary walls was completely avoided, and the resolution of the analyte cations was essentially improved. The optimal BGE for analysis of biological samples was found to be 5 mM DPC, 5 mM copper(II) acetate/10 mM ethylenediamine (pH 8). The detection limits (signal-to-noise ratio =3 and UV at 215 nm) of sodium, potassium, calcium, magnesium, and ammonium ions were 25, 31, 24, 45, and 60 micro M, respectively. These five species of the common inorganic cations in human saliva samples were detected successfully within 2 min by the proposed system with direct sample injection.