Deciphering the role of wound healing genes in skin cutaneous melanoma: Insights into expression, methylation, mutations, and therapeutic implications.

Skin Cutaneous Melanoma (SKCM) is a form of cancer that originates in the pigment-producing cells, known as melanocytes, of the skin. Delay wound healing is often correlated with the occurrence of and progression of SKCM. In this comprehensive study, we investigated the intricate roles of two important wound healing genes in SKCM, including Matrix Metalloproteinase-2 (MMP2) and Matrix Metalloproteinase-9 (MMP9). Through a multi-faceted approach, we collected clinical samples, conducted molecular experiments, including RT-qPCR, bisulphite sequencing, cell culture, cell Counting Kit-8, colony formation, and wound healing assays. Beside this, we also used various other databases/tools/approaches for additional analysis including, UALCAN, GEPIA, HPA, MEXPRESS, cBioPortal, KM plotter, DrugBank, and molecular docking. Our results revealed a significant up-regulation of MMP2 and MMP9 in SKCM tissues compared to normal counterparts. Moreover, promoter methylation analysis suggested an epigenetic regulatory mechanism. Validations using TCGA datasets and immunohistochemistry emphasized the clinical relevance of MMP2 and MMP9 dysregulation. Functional assays demonstrated their synergistic impact on proliferation and migration in SKCM cells. Furthermore, we identified potential therapeutic candidates, Estradiol and Calcitriol, through drug prediction and molecular docking analyses. These compounds exhibited binding affinities, suggesting their potential as MMP2/MMP9 inhibitors. Overall, our study elucidates the diagnostic, prognostic, and therapeutic implications of MMP2 and MMP9 in SKCM, shedding light on their complex interplay in SKCM occurrence and progression.

Photobiomodulation preconditioned diabetic adipose derived stem cells with additional photobiomodulation: an additive approach for enhanced wound healing in diabetic rats with a delayed healing wound.

In this preclinical investigation, we examined the effects of combining preconditioned diabetic adipose-derived mesenchymal stem cells (AD-MSCs) and photobiomodulation (PBM) on a model of infected ischemic delayed healing wound (injury), (IIDHWM) in rats with type I diabetes (TIDM). During the stages of wound healing, we examined multiple elements such as stereology, macrophage polarization, and the mRNA expression levels of stromal cell-derived factor (SDF)-1α, vascular endothelial growth factor (VEGF), hypoxia-induced factor 1α (HIF-1α), and basic fibroblast growth factor (bFGF) to evaluate proliferation and inflammation. The rats were grouped into: (1) control group; (2) diabetic-stem cells were transversed into the injury site; (3) diabetic-stem cells were transversed into the injury site then the injury site exposed to PBM; (4) diabetic stem cells were preconditioned with PBM and implanted into the wound; (5) diabetic stem cells were preconditioned with PBM and transferred into the injury site, then the injury site exposed additional PBM. While on both days 4, and 8, there were advanced histological consequences in groups 2-5 than in group 1, we found better results in groups 3-5 than in group 2 (p < 0.05). M1 macrophages in groups 2-5 were lower than in group 1, while groups 3-5 were reduced than in group 2 (p < 0.01). M2 macrophages in groups 2-5 were greater than in group 1, and groups 3-5 were greater than in group 2. (p ≤ 0.001). Groups 2-5 revealed greater expression levels of bFGF, VEGF, SDF- 1α, and HIF- 1α genes than in group 1 (p < 0.001). Overall group 5 had the best results for histology (p < 0.05), and macrophage polarization (p < 0.001). AD-MSC, PBM, and AD-MSC + PBM treatments all enhanced the proliferative stage of injury repairing in the IIDHWM in TIDM rats. While AD-MSC + PBM was well than the single use of AD-MSC or PBM, the best results were achieved with PBM preconditioned AD-MSC, plus additional PBM of the injury.

Macrophage Polarization Dynamics in Biomaterials: Implications for in Vitro Wound Healing.

The interaction between biomaterials and the immune system plays a pivotal role in determining the success or failure of implantable devices. Macrophages, as key orchestrators of immune responses, exhibit diverse reactions that influence tissue integration or lead to implant failure. This study focuses on unraveling the intricate relationship between macrophage phenotypes and biomaterials, specifically hydrogels, by employing THP-1 cells as a model. Through a comprehensive investigation using polysaccharide, polymer, and protein-based hydrogels, our research sheds light on how the properties of hydrogels influence macrophage polarization. Phenotypic observations, biochemical assays, surface marker expression, and gene expression profiles collectively demonstrate the differential macrophage polarization abilities of polysaccharide-, polymer-, and protein-based hydrogels. Moreover, our indirect coculture studies reveal that hydrogels fostering M2 polarization exhibit exceptional wound-healing capabilities. These findings highlight the crucial role of the hydrogel microenvironment in adjusting macrophage polarization, offering a fresh avenue for refining biomaterials to bolster advantageous immune responses and improve tissue integration. This research contributes valuable insights for designing biomaterials with tailored properties that can guide macrophage behavior, ultimately improving the overall success of implantable devices.

[Analysis of the types and functions of CD34+ cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing].

Objective: To analyze the types and functions of CD34+ cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing. Methods: This study was an experimental study. The CD34+ cell lineage tracing mouse was produced, and the visualization of CD34+ cells under the fluorescent condition was realized. Six male CD34+ cell lineage tracing mice aged 7-8 weeks (designated as diabetic group) were intraperitoneally injected with streptozotocin to establish a diabetic model, and full-thickness skin defect wounds were prepared on their backs when they reached 13 weeks old. Another 6 male CD34+ cell lineage tracing mice aged 13 weeks (designated as control group) were also subjected to full-thickness skin defect wounds on their backs. On post-injury day (PID) 4, wound tissue was collected from 3 mice in control group and 2 mice in diabetic group, and digested to prepare single-cell suspensions. CD34+ cells were screened using fluorescence-activated cell sorting, followed by single-cell RNA sequencing. The Seurat 4.0.2 program in the R programming language was utilized for dimensionality reduction, visualization, and cell clustering analysis of CD34+ cell types, and to screen and annotate the marker genes for each CD34+ cell subpopulation. Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis was performed to analyze the differentially expressed genes (DEGs) of CD34+ fibroblasts (Fbs), smooth muscle cells (SMCs), keratinocytes (KCs), and chondrocyte-like cells (CLCs) in the wound tissue of two groups of mice for exploring cellular functions. Results: On PID 4, CD34+ cells in the wound tissue of both groups of mice were consisted of 7 cell types, specifically endothelial cells, Fbs, KCs, macrophages, T cells, SMCs, and CLCs. Among these, Fbs were further classified into 5 subpopulations. Compared with those in control group, the proportions of CD34+ endothelial cells, Fbs subpopulation 1, Fbs subpopulation 4, KCs, and CLCs in the wound tissue of mice were increased in diabetic group, while the proportions of CD34+ Fbs subpopulation 2, Fbs subpopulation 3, and SMCs were decreased. The marker genes for annotating CD34+ CLCs, endothelial cells, Fbs subpopulation 1, Fbs subpopulation 2, Fbs subpopulation 3, Fbs subpopulation 4, Fbs subpopulation 5, KCs, macrophages, SMCs, and T cells were respectively metastasis-associated lung adenocarcinoma transcript 1, fatty acid binding protein 4, Gremlin 1, complement component 4B, H19 imprinted maternally expressed transcript, Dickkopf Wnt signaling pathway inhibitor 2, fibromodulin, keratin 5, CD74 molecule, regulator of G protein signaling 5, and inducible T-cell co-stimulator molecule. KEGG and GO enrichment analysis revealed that, compared with those in control group, DEGs with significant differential expression (SDE) in CD34+ Fbs from the wound tissue of mice in diabetic group on PID 4 were significantly enriched in terms related to inflammatory response, extracellular matrix (ECM) organization, regulation of cell proliferation, and aging (with Pvalues all <0.05), DEGs with SDE in CD34+ SMCs were significantly enriched in terms related to cell migration, apoptotic process, positive regulation of transcription, and phagosome (with P values all <0.05), DEGs with SDE in CD34+ KCs were significantly enriched in terms related to mitochondrial function, transcription, and neurodegenerative diseases (with P values all <0.05), and DEGs with SDE in CD34+ CLCs were significantly enriched in terms related to rhythm regulation, ECM, and viral infection (with P values all <0.05). Conclusions: CD34+ cells display high heterogeneity in the healing process of full-thickness skin defect wounds in both normal mice and diabetic mice. The significantly enriched functions of DEGs with SDE in CD34+ cell subpopulations in the wound tissue of the two mouse groups are closely related to the wound healing process.

Enhancing Skin Injury Repair: Combined Application of PF-127 Hydrogel and hADSC-Exos Containing miR-148a-3p.

The use of exosomes to relieve skin injuries has received considerable attention. The PluronicF-127 hydrogel (PF-127 hydrogel) is a novel biomaterial that can be used to carry biomolecules. This study sought to investigate the impact of exosomes originating from human mesenchymal stem cells (MSCs) developed from adipose tissue (hADSC-Exos) combined with a PF-127 hydrogel on tissue repair and explore the underlying mechanism using in vitro and in vivo experiments. miR-148a-3p is the most expressed microRNA (miRNA) in hADSC-Exos. We found that exosomes combined with the PF-127 hydrogel had a better efficacy than exosomes alone; moreover, miR-148a-3p knockdown lowered its efficacy. In vitro, we observed a significant increase in the tumor-like ability of HUVECs after exosome treatment, which was attenuated after miR-148a-3p knockdown. Furthermore, the effects of miR-148a-3p on hADSC-Exos were achieved through the prevention of PTEN and the triggering of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. In conclusion, our results demonstrated that hADSC-Exos can promote angiogenesis and skin wound healing by delivering miR-148a-3p and have a better effect when combined with the PF-127 hydrogel, which may be an alternative strategy to promote wound healing.

CD64 plays a key role in diabetic wound healing.

Introduction: Wound healing poses a clinical challenge in diabetes mellitus (DM) due to compromised host immunity. CD64, an IgG-binding Fcgr1 receptor, acts as a pro-inflammatory mediator. While its presence has been identified in various inflammatory diseases, its specific role in wound healing, especially in DM, remains unclear. Objectives: We aimed to investigate the involvement of CD64 in diabetic wound healing using a DM animal model with CD64 KO mice. Methods: First, we compared CD64 expression in chronic skin ulcers from human DM and non-DM skin. Then, we monitored wound healing in a DM mouse model over 10 days, with or without CD64 KO, using macroscopic and microscopic observations, as well as immunohistochemistry. Results: CD64 expression was significantly upregulated (1.25-fold) in chronic ulcerative skin from DM patients compared to non-DM individuals. Clinical observations were consistent with animal model findings, showing a significant delay in wound healing, particularly by day 7, in CD64 KO mice compared to WT mice. Additionally, infiltrating CD163+ M2 macrophages in the wounds of DM mice decreased significantly compared to non-DM mice over time. Delayed wound healing in DM CD64 KO mice correlated with the presence of inflammatory mediators. Conclusion: CD64 seems to play a crucial role in wound healing, especially in DM conditions, where it is associated with CD163+ M2 macrophage infiltration. These data suggest that CD64 relies on host immunity during the wound healing process. Such data may provide useful information for both basic scientists and clinicians to deal with diabetic chronic wound healing.

Unraveling the Intricate Network of lncRNAs in Corneal Epithelial Wound Healing: Insights Into the Regulatory Role of linc17500.

Purpose: Epigenetic mechanisms orchestrate a harmonious process of corneal epithelial wound healing (CEWH). However, the precise role of long non-coding RNAs (lncRNAs) as key epigenetic regulators in mediating CEWH remains elusive. Here, we aimed to elucidate the functional contribution of lncRNAs in regulating CEWH. Methods: We used a microarray to characterize lncRNA expression profiling during mouse CEWH. Subsequently, the aberrant lncRNAs and their cis-associated genes were subjected to comprehensive Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot analyses were performed to determine the expression profiles of key markers during CEWH. The in vivo effects of linc17500 on this process were investigated through targeted small interfering RNA (siRNA) injection. Post-siRNA treatment, corneal re-epithelialization was assessed, alongside the expression of cytokeratins 12 and 14 (Krt12 and Krt14) and Ki67. Effects of linc17500 on mouse corneal epithelial cell (TKE2) proliferation, cell cycle, and migration were assessed by multicellular tumor spheroids (MTS), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and scratch-wound assay, respectively. Results: Microarray analysis revealed dysregulation of numerous lncRNA candidates during CEWH. Bioinformatic analysis provided valuable annotations regarding the cis-associated genes of these lncRNAs. In vivo experiments demonstrated that knockdown of linc17500 resulted in delayed CEWH. Furthermore, the knockdown of linc17500 and its cis-associated gene, CDC28 protein kinase regulatory subunit 2 (Cks2), was found to impede TKE2 cell proliferation and migration. Notably, downregulation of linc17500 in TKE2 cells led to suppression of the activation status of Akt and Rb. Conclusions: This study sheds light on the significant involvement of lncRNAs in mediating CEWH and highlights the regulatory role of linc17500 on TKE2 cell behavior. Translational Relevance: These findings provide valuable insights for future therapeutic research aimed at addressing corneal wound complications.

MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice.

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

Balancing macrophage polarization via stem cell-derived apoptotic bodies for diabetic wound healing.

BACKGROUND: Adipose tissue-derived stem cell-derived apoptotic bodies (ADSC-ABs) have shown great potential for immunomodulation and regeneration, particularly in diabetic wound therapy. However, their local application has been limited by unclear regulatory mechanisms, rapid clearance, and short tissue retention times. METHODS: We analyzed the key role molecules and regulatory pathways of ADSC-ABs in regulating inflammatory macrophages by mRNA sequencing and microRNA (miRNA) sequencing and then verified them by gene knockdown. To prevent rapid clearance, we employed microfluidics technology to prepare methacrylate-anhydride gelatin (GelMA) microspheres (GMS) for controlled release of ABs. Finally, we evaluated the effectiveness of ADSC-AB-laden GMSs (ABs@GMSs) in a diabetic rat wound model. FINDINGS: Our results demonstrated that ADSC-ABs effectively balanced macrophage inflammatory polarization through the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, mediated by miR-20a-5p. Furthermore, we showed that AB@GMSs had good biocompatibility, significantly delayed local clearance of ABs, and ameliorated diabetic wound inflammation and promoted vascularization, thus facilitating its healing. CONCLUSIONS: Our study reveals the regulatory mechanism of ADSC-ABs in balancing macrophage inflammatory polarization and highlightsthe importance of delaying their local clearance by GMSs. These findings have important implications for the development of novel therapies for diabetic wound healing. FUNDING: This research was supported by the National Key Research and Development Program of China (2020YFA0908200), National Natural Science Foundation of China (82272263, 82002053, 32000937, and 82202467), Shanghai "Rising Stars of Medical Talents" Youth Development Program (22MC1940300), Shanghai Municipal Health Commission (20204Y0354), and Shanghai Science and Technology Development Funds (22YF1421400).

miR-758-3p/ILK signaling modulated angiogenesis by regulating VEGFA in wound healing.

Chronic wounds cause physical, psychological and economic damage to patients, while therapeutic choices are limited. ILK was reported to play key roles in both fibrosis and angiogenesis, which are two important factors during wound healing. However, the function of ILK during vascularization in wounds remains unclear. In our study, we found increased ILK expression in chronic wound tissues compared to adjacent tissue, as well as a positive relationship between ILK expression and microvessel density. Moreover, fibroblasts overexpressing ILK showed an enhanced ability to promote HUVEC migration and tube formation, during which PI3K/Akt, downstream of ILK, played key roles and VEGFA was the key cytokine. Considering the important function of ILK in wound healing and the lack of an ILK activator, we investigated microRNAs targeting ILK and found that miR-758-3p could target ILK to regulate its transcription. The inhibition of miR-758-3p increased ILK expression and sequentially upregulated VEGFA and activated angiogenesis in vivo and in vitro. Taken together, these results revealed that ILK played a key role in wound healing by regulating angiogenesis and that activating ILK by inhibiting miR-758-3p was an effective way to promote wound healing. Whether miR-758-3p/ILK signaling can be utilized as a therapeutic target for wound healing requires further investigation.

Clock gene Per2 modulates epidermal tissue repair in vivo.

Wound healing can be influenced by genes that control the circadian cycle, including Per2 and BMAL1, which coordinate the functions of several organs, including the skin. The aim of the study was to evaluate the role of PER2 during experimental skin wound healing. Two groups (control and Per2-KO), consisting of 14 male mice each, were anesthetized by inhalation, and two 6 mm wounds were created on their dorsal skin using a punch biopsy. A silicone ring was sutured around the wound perimeter to restrict contraction. The wound healing process was clinically measured daily (closure index) until complete wound repair. On Day 6, histomorphometric analysis was performed using the length and thickness of the epithelial migration tongue, in addition to counting vessels underlying the lesion by immunofluorescence assay and maturation of collagen fibers through picrosirius staining. Bromodeoxyuridine (BrdU) incorporation and quantification were performed using the subcutaneous injection technique 2 h before euthanasia and through immunohistochemical analysis of the proliferative index. In addition, the qualitative analysis of myofibroblasts and periostin distribution in connective tissue was performed by immunofluorescence. Statistically significant differences were observed in the healing time between the experimental groups (means: 15.5 days for control mice and 13.5 days for Per2-KO; p = 0.001). The accelerated healing observed in the Per2-KO group (p < 0.05) was accompanied by statistical differences in wound diameter and length of the migrating epithelial tongue (p = 0.01) compared to the control group. Regarding BrdU immunoreactivity, higher expression was observed in the intact epithelium of Per2-KO animals (p = 0.01), and this difference compared to control was also present, to a lesser extent, at the wound site (p = 0.03). Immunofluorescence in the connective tissue underlying the wound showed a higher angiogenic potential in the Per2-KO group in the intact tissue area and the wound region (p < 0.01), where increased expression of myofibroblasts was also observed. Qualitative analysis revealed the distribution of periostin protein and collagen fibers in the connective tissue underlying the wound, with greater organization and maturation during the analyzed period. Our research showed that the absence of the Per2 gene positively impacts the healing time of the skin in vivo. This acceleration depends on the increase of epithelial proliferative and angiogenic capacity of cells carrying the Per2 deletion.

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.

Curcumin Promotes Diabetic Foot Ulcer Wound Healing by Inhibiting miR-152-3p and Activating the FBN1/TGF-ß Pathway.

The objective of this study was to investigate the mechanism of curcumin in diabetic foot ulcer (DFU) wound healing. A DFU rat model was established, and fibroblasts were cultured in a high-glucose (HG) environment to create a cell model. Various techniques, including Western blot, RT‒qPCR, flow cytometry, Transwell, cell scratch test and H&E staining, were employed to measure the levels of relevant genes and proteins, as well as to assess cell proliferation, apoptosis, migration, and pathological changes. The results showed that miR-152-3p was overexpressed in DFU patients, while FBN1 was underexpressed. Curcumin was found to inhibit fibroblast apoptosis, promote proliferation, migration, and angiogenesis in DFU rats, and accelerate wound healing in DFU rats. In addition, overexpression of miR-152-3p weakened the therapeutic effect of curcumin, while overexpression of FBN1 reversed the effects of the miR-152-3p mimic. Further investigations into the underlying mechanisms revealed that curcumin expedited wound healing in DFU rats by restoring the FBN1/TGF-ß pathway through the inhibition of miR-152-3p. In conclusion, curcumin can suppress the activity of miR-152-3p, which, in turn, leads to the rejuvenation of the FBN1/TGF-ß pathway and accelerates DFU wound healing.

Stem cell therapy with CRISPR/Cas9-mediated MALAT1 delivery modulates miR-142 and rescues wound healing in rats with age-associated diabetic foot ulcers.

BACKGROUND: Diabetic foot ulcer (DFU) is a serious diabetes complication, significantly impacting the quality of life, particularly in the elderly. Age-associated DFUs pose additional challenges due to impaired healing mechanisms. Our study aims to explore the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a miR-142 sponge in repairing diabetic rat foot ulcer tissue under age-associated diabetes, offering a new theoretical basis and therapeutic target for preventing and treating diabetic vascular disease in the elderly. METHODS: Using qPCR, we analyzed MALAT1 and miR-142 expression in EPCs and hUC-MSCs. Targetscan predicted potential interaction targets for MALAT1 and miR-142, confirmed by dual luciferase reporter gene assay. An age-associated diabetic rat model was established using Streptozotocin (STZ) injection. Hypoxia, apoptosis, and angiogenesis-related proteins were assessed through Western Blot. In vitro, miR-142 inhibition and MALAT1 overexpression promoted foot ulcer healing in diabetic rats. RESULTS: MALAT1 acted as a miR-142 sponge, downregulated in hUC-MSCs under high glucose, relevant to age-associated diabetic foot ulcers. MiR-142 negatively regulated SIRT1 and Nrf2. In vitro experiments demonstrated potential significance for age-related DFU treatment. CONCLUSIONS: MALAT1 in human umbilical cord mesenchymal stem cells expedited foot ulcer healing in diabetic rats, particularly in age-associated diabetes, through miR-142 sponge activity. These findings offer insights for novel therapeutic strategies targeting elderly diabetic foot ulcers, emphasizing exogenous stem cell transplantation's potential in effective DFU treatment for the elderly.

Adipose-derived Mesenchymal Stem Cells are Ideal for the Cell-based Treatment of Refractory Wounds: Strong Potential for Angiogenesis.

Although Mesenchymal Stem Cells (MSCs)-based therapy has been proposed as a promising strategy for the treatment of chronic lower-extremity ulcers, their optimal sources, amounts, and delivery methods are urgently needed to be determined. In this study, we compared the heterogeneity of the human MSCs derived from bone marrow (BMSCs), umbilical cord (UCMSCs), and adipose tissue (ADSCs) in accelerating wound healing and promoting angiogenesis and explored the underlying mechanism. Briefly, a diabetic rat model with a full-thickness cutaneous wound on the dorsal foot was developed. The wound was topically administered with three types of MSCs. Additionally, we carried out in vitro and in vivo analysis of the angiogenic properties of the MSCs. Moreover, the molecular mechanism of the heterogeneity of the MSCs derived from the three tissues was explored by transcriptome sequencing. When compared with the BMSCs- and UCMSCs-treated groups, the ADSCs-treated group exhibited markedly accelerated healing efficiency, characterized by increased wound closure rates, enhanced angiogenesis, and collagen deposition at the wound site. The three types of MSCs formed three-dimensional capillary-like structures and promoted angiogenesis in vitro and in vivo, with ADSCs exhibiting the highest capacity for tube formation and pro-angiogenesis. Furthermore, transcriptome sequencing revealed that ADSCs had higher expression levels of angiogenesis-associated genes. Our findings indicate that MSCs-based therapy accelerates the healing of ischemia- and diabetes-induced lower-extremity ulcers and that adipose tissue-derived MSCs might be ideal for therapeutic angiogenesis and treatment of chronic ischemic wounds.

The bone mesenchymal stem cell-derived exosomal miR-146a-5p promotes diabetic wound healing in mice via macrophage M1/M2 polarization.

A diabetic wound is a refractory disease that afflicts patients globally. MicroRNA-146a-5p (miR-146a-5p) is reported to represent a potential therapeutic target for diabetic wounds. However, microRNA easily degrades in the wound microenvironment. This study extracted bone marrow mesenchymal stem cell (BMSC)-derived exosomes (EXO). Electroporation technology was used to load miR-146a-5p into EXO (labeled as EXO-miR-146a). The endothelial cells (human umbilical vein endothelial cells [HUVECs]) and macrophages were cocultured in transwell chambers in the presence of high glucose. Cell proliferation, migration, and angiogenesis were measured with cell counting kit 8, scratch, and tube forming assays, respectively. Flow cytometry was introduced to validate the biomarker of macrophages and BMSCs. The expression level of macrophage polarization-related proteins and tumor necrosis factor receptor-associated factor 6 (TRAF6) was assessed with western blotting analysis. The full-thickness skin wound model was developed to verify the in vitro results. EXO-miR-146a promoted the proliferation, migration, and angiogenesis of HUVECs in the hyperglycemic state by suppressing the TRAF6 expression in vitro. Additionally, EXO-miR-146a treatment facilitated M2 but inhibited M1 macrophage polarization. Furthermore, EXO-miR-146a enhances reepithelialization, angiogenesis, and M2 macrophage polarization, thereby accelerating diabetic wound healing in vivo. The EXO-miR-146a facilitated M2 macrophage polarization, proliferation, migration, and angiogenesis of HUVECs through TRAF6, thereby ameliorating intractable diabetic wound healing. These results established the basis for using EXO to deliver drugs and revealed mediators for diabetic wound treatment.

Exploring the impact of TGF-ß family gene mutations and expression on skin wound healing and tissue repair.

Transforming Growth Factor-Beta (TGF-ß) signalling pathway is of paramount importance in the processes of wound healing, epidermal integrity maintenance and development of skin cancer. The objective of this research endeavour was to clarify the impact of gene mutations and variations in expression within TGF-ß family on mechanisms of tissue repair, as well as to identify potential targets for therapeutic purposes in non-melanoma skin cancer (NMSC). The methods utilized in this study involved obtaining RNA-seq data from 224 NMSC patients and paired normal skin tissues from the PRJNA320473 and PRJEB27606 databases. The purpose of the differential gene expression analysis was to identify genes whose expression had changed significantly. In order to evaluate the effects and interrelationships of identified gene variants, structural analysis with AlphaFold and PDB data and network analysis with the STRING database were both utilized. Critical gene expression was externally validated through the utilization of the GEPIA database. Tumour tissues exhibited a notable upregulation of genes associated with the TGF-ß pathway, specifically MMP1, MMP3, MMP9, EGF, COL3A1 and COL1A2, in comparison with normal tissues. As indicated by the central node status of these genes in the network analysis, they play a crucial role in the progression of NMSCs. The results of the structural analysis suggested that mutations might cause functional disruptions. External validation of the upregulation confirmed the expression trends and emphasized the biomarker potential of the upregulated genes. In conclusion, this research offered thorough examination of molecular modifications that occur in TGF-ß family genes, which are linked to cutaneous wound healing and NMSC. The modified expression of the identified hub genes may represent innovative targets for therapeutic intervention.

Silencing of topical proline hydroxylase domain 2 promotes the healing of rat diabetic wounds by phosphorylating AMPK.

BACKGROUND: For diabetic ulcers, the impaired response to hypoxia is a key feature associated with delayed healing. In the early phase of hypoxia, hypoxic signaling activates the AMPK system through direct phosphorylation of the PHD2 pathway, producing a significant endogenous hypoxic protective effect. METHODS: Twenty Sprague-Dawley (SD) rats were randomly divided into two groups: treatment (sh-PHD2) and control (sh-Control). Using lentiviral encapsulation of PHD2-shRNA and transfection, the silencing efficiency of PHD2 expression was verified in rat dermal fibroblasts (RDF) and in rat aortic endothelial cells (RAECs). Changes in the ability of RDF and RAECs to proliferate, migrate, and in the rate of ATP production were observed and then tested after inhibition of AMPK phosphorylation using dorsomorphin. The lentiviral preparation was injected directly into the wounds of rats and wound healing was recorded periodically to calculate the healing rate. Wounded tissues were excised after 14 days and the efficiency of PHD2 silencing, as well as the expression of growth factors, was examined using molecular biology methods. Histological examination was performed to assess CD31 expression and therefore determine effects on angiogenesis. RESULTS: Lentiviral-encapsulated PHD2-sh-RNA effectively suppressed PHD2 expression and improved the proliferation, migration, and ATP production rate of RDF and RAEC, which were restored to their previous levels after inhibition of AMPK. The rate of wound healing, vascular growth, and expression of growth factors were significantly improved in diabetic-model rats after local silencing of PHD2 expression. CONCLUSION: Silencing of PHD2 promoted wound healing in diabetic-model SD rats by activating AMPK phosphorylation.

Exosomes Derived from E2F1-/- Adipose-Derived Stem Cells Promote Skin Wound Healing via miR-130b-5p/TGFBR3 Axis.

Background: Skin wound is a widespread health problem and brings extraordinary burdens to patients. Exosomes derived from adipose-derived stem cells (ADSC-Exos) are considered promising strategies for repairing skin wounds. E2F1 is a member of the E2F family of transcription factors involved in cell growth and apoptosis. E2F1 deficiency in mice enhances wound healing by improving collagen deposition and angiogenesis. Additionally, E2F1 can regulate the transcription and paracrine activity of multiple miRNAs, which will inevitably reshape the paracrine expression profile of ADSC-Exos. This study aimed to investigate the impact of transcription factor E2F1 deficiency on the functions of ADSC-Exos in promoting wound healing. Methods: First, we obtained ADSCs from subcutaneous adipose tissues of WT and E2F1-/- C57BL/6 mice and separated their exosomes, denoted as ADSCWT-Exos and ADSCE2F1-/--Exos. The wound healing effects of ADSCWT-Exos and ADSCE2F1-/--Exos in full-thickness skin wound models were investigated by wound images, H&E staining, and immunohistochemical staining. For the in vitro study, the abilities of ADSCWT-Exos and ADSCE2F1-/--Exos to promote cell activities, collagen formation, and angiogenesis were evaluated. The potential mechanism by which ADSCE2F1-/--Exos promote wound healing was determined by miRNA sequencing, ChIP‒qPCR, and dual-luciferase assays. Results: ADSCE2F1-/--Exos accelerated wound healing by promoting collagen formation and angiogenesis. As a result, compared with the lower wound healing rate of 30.5% within 7 days in the control group and 42.3% in the ADSCWT-Exo group, ADSCE2F1-/--Exos significantly increased the wound healing rate to 72.5%. In vitro, ADSCE2F1-/--Exos activated the function of fibroblasts and vascular endothelial cells. The loss of E2F1 promoted miR-130b-5p expression in ADSCE2F1-/--Exos through transcriptional regulation. MiRNA high-throughput sequencing identified 12 differently expressed miRNAs between ADSCE2F1-/- and ADSCWT. ADSCE2F1-/--Exos enhanced fibroblast activities via the miR-130b-5p/TGFBR3 axis and TGF-ß activation. Conclusion: Our results indicated that ADSCE2F1-/--Exos effectively promoted wound healing by regulating the miR-130b-5p/TGFBR3 axis, thus providing a novel strategy of gene-engineered stem cell exosomes for accelerating wound healing.

Biomimetic nanoparticles of platelet membranes carrying bFGF and VEGFA genes promote deep burn wound healing.

INTRODUCTION: The treatment of burn wounds, especially deep burn wounds, remains a major clinical challenge. Growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) show great potential in promoting the healing of damaged tissues. This study explored wound healing following targeted delivery of bFGF and VEGFA genes into deep burn wounds through a novel platelet membrane-coated nanoparticle (PM@gene-NP) complex delivery system. METHODS: First, bFGF and VEGFA genes were inserted into plasmid (pEGFP-N1) vectors. Subsequently, the assembled plasmids were loaded onto nanoparticles to form gene-loaded nanoparticle complexes, which were then wrapped with extracted platelet membrane, fully simulating the characteristics of platelets, in order to actively target sites of inflammatory damage. After administration of PM@gene-NP complexes through the tail vein of rats, a series of experiments were conducted to evaluate wound healing. RESULTS: The PM@gene-NP complexes effectively targeted the burn sites. After the administration of the PM@gene-NP complexes, the rats exhibited increased blood flow in the burn wounds, which also healed faster than control groups. Histological results showed fewer inflammatory cells in the burned skin tissue after treatment. After the wounds healed, the production of hair follicles, sebaceous glands and other skin accessories in the skin tissue increased. CONCLUSION: Our results showed that the PM@gene-NP complexes can effectively deliver gene therapy to the injured area, and this delivery system should be considered as a potential method for treating deep burns.