Background:Diabetic foot ulcer(DFU)is one of the most common and complex complications of diabetes,but the underlying pathophysiology remains unclear.Single-cell RNA sequencing(scRNA-seq)has been conducted to explore ...Background:Diabetic foot ulcer(DFU)is one of the most common and complex complications of diabetes,but the underlying pathophysiology remains unclear.Single-cell RNA sequencing(scRNA-seq)has been conducted to explore novel cell types or molecular profiles of DFU from various perspectives.This study aimed to comprehensively analyze the potential mechanisms underlying impaired re-epithelization of DFU in a single-cell perspective.Methods:We conducted scRNA-seq on tissues from human normal skin,acute wound,and DFU to investigate the potential mechanisms underlying impaired epidermal differentiation and the pathological microenvironment.Pseudo-time and lineage inference analyses revealed the distinct states and transition trajectories of epidermal cells under different conditions.Transcription factor analysis revealed the potential regulatory mechanism of key subtypes of keratinocytes.Cell-cell interaction analysis revealed the regulatory network between the proinflammatory microenvironment and epidermal cells.Laser-capture microscopy coupled with RNA sequencing(LCM-seq)and multiplex immunohistochemistry were used to validate the expression and location of key subtypes of keratinocytes.Results:Our research provided a comprehensive map of the phenotypic and dynamic changes that occur during epidermal differentiation,alongside the corresponding regulatory networks in DFU.Importantly,we identified two subtypes of keratinocytes:basal cells(BC-2)and diabetes-associated keratinocytes(DAK)that might play crucial roles in the impairment of epidermal homeostasis.BC-2 and DAK showed a marked increase in DFU,with an inactive state and insufficient motivation for epidermal differentiation.BC-2 was involved in the cellular response and apoptosis processes,with high expression of TXNIP,IFITM1,and IL1R2.Additionally,the pro-differentiation transcription factors were downregulated in BC-2 in DFU,indicating that the differentiation process might be inhibited in BC-2 in DFU.DAK was associated with cellular glucose homeostasis.Furthermore,increased CCL2+CXCL2+fibroblasts,VWA1+vascular endothelial cells,and GZMA+CD8+T cells were detected in DFU.These changes in the wound microenvironment could regulate the fate of epidermal cells through the TNFSF12-TNFRSF12A,IFNG-IFNGR1/2,and IL-1B-IL1R2 pathways,which might result in persistent inflammation and impaired epidermal differentiation in DFU.Conclusions:Our findings offer novel insights into the pathophysiology of DFU and present potential therapeutic targets that could improve wound care and treatment outcomes for DFU patients.展开更多
Background:Refractory diabetic wounds are a common occurrence in patients with diabetes and epidermis-specific macroautophagy/autophagy impairment has been implicated in their pathogenesis.Therefore,identifying and de...Background:Refractory diabetic wounds are a common occurrence in patients with diabetes and epidermis-specific macroautophagy/autophagy impairment has been implicated in their pathogenesis.Therefore,identifying and developing treatment strategies capable of normalizing epidermisspecific macroautophagy/autophagy could facilitate diabetic wound healing.The study aims to investigate the potential of bone marrow mesenchymal stem cell-derived exosomes(BMSC-exos)from hypoxic conditions as a treatment to normalize epidermis-specific autophagy for diabetic wound healing.Methods:We compared the effects of bone marrow mesenchymal stem cell(BMSC)-sourced exosomes(BMSC-Exos)from hypoxic conditions to those of BMSC in normoxic conditions(noBMSC-Exos).Our studies involved morphometric assessment of the exosomes,identification of the microRNA(miRNA)responsible for the effects,evaluation of keratinocyte functions and examination of effects of the exosomes on several molecules involved in the autophagy pathway such as microtubule-associated protein 1 light chain 3 beta,beclin 1,sequestosome 1,autophagyrelated 5 and autophagy-related 5.The experiments used human BMSCs from the American Type Culture Collection,an in vivo mouse model of diabetes(db/db)to assess wound healing,as well as the human keratinocyte HaCaT cell line.In the methodology,the authors utilized an array of approaches that included electron microscopy,small interfering RNA(siRNA)studies,RNA in situ hybridization,quantitative real-time reverse transcription PCR(qRT-PCR),the isolation,sequencing and differential expression of miRNAs,as well as the use of miR-4645-5p-specific knockdown with an inhibitor.Results:Hypoxia affected the release of exosomes from hypoxic BMSCs(hy-BMSCs)and influenced the size and morphology of the exosomes.Moreover,hyBMSC-Exo treatment markedly improved keratinocyte function,including keratinocyte autophagy,proliferation and migration.miRNA microarray and bioinformatics analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in‘autophagy’and‘process utilizing autophagic mechanism’in the‘biological process’category and miR-4645-5p as a major contributor to the proautophagy effect of hyBMSC-Exos.Moreover,mitogen-activated protein kinase-activated protein kinase 2(MAPKAPK2)was identified as a potential target of exosomal miR-4645-5p;this was confirmed using a dual luciferase assay.Exosomal miR-4645-5p mediates the inactivation of the MAPKAPK2-induced AKT kinase group(comprising AKT1,AKT2,and AKT3),which in turn suppresses AKT-mTORC1 signaling,thereby facilitating miR-4645-5p-mediated autophagy.Conclusions:Overall,the results of this study showed that hyBMSC-Exo-mediated transfer of miR-4645-5p inactivated MAPKAPK2-induced AKT-mTORC1 signaling in keratinocytes,which activated keratinocyte autophagy,proliferation and migration,resulting in diabetic wound healing in mice.Collectively,the findings could aid in the development of a novel therapeutic strategy for diabetic wounds.展开更多
基金supported by the National Natural Science Foundation of China(Nos 82256147,82072178)Support Program for Growth Factor Research(No.SZYZ-TR-06)GuangDong Basic and Applied Basic Research Foundation(No.2023A1515111087).
文摘Background:Diabetic foot ulcer(DFU)is one of the most common and complex complications of diabetes,but the underlying pathophysiology remains unclear.Single-cell RNA sequencing(scRNA-seq)has been conducted to explore novel cell types or molecular profiles of DFU from various perspectives.This study aimed to comprehensively analyze the potential mechanisms underlying impaired re-epithelization of DFU in a single-cell perspective.Methods:We conducted scRNA-seq on tissues from human normal skin,acute wound,and DFU to investigate the potential mechanisms underlying impaired epidermal differentiation and the pathological microenvironment.Pseudo-time and lineage inference analyses revealed the distinct states and transition trajectories of epidermal cells under different conditions.Transcription factor analysis revealed the potential regulatory mechanism of key subtypes of keratinocytes.Cell-cell interaction analysis revealed the regulatory network between the proinflammatory microenvironment and epidermal cells.Laser-capture microscopy coupled with RNA sequencing(LCM-seq)and multiplex immunohistochemistry were used to validate the expression and location of key subtypes of keratinocytes.Results:Our research provided a comprehensive map of the phenotypic and dynamic changes that occur during epidermal differentiation,alongside the corresponding regulatory networks in DFU.Importantly,we identified two subtypes of keratinocytes:basal cells(BC-2)and diabetes-associated keratinocytes(DAK)that might play crucial roles in the impairment of epidermal homeostasis.BC-2 and DAK showed a marked increase in DFU,with an inactive state and insufficient motivation for epidermal differentiation.BC-2 was involved in the cellular response and apoptosis processes,with high expression of TXNIP,IFITM1,and IL1R2.Additionally,the pro-differentiation transcription factors were downregulated in BC-2 in DFU,indicating that the differentiation process might be inhibited in BC-2 in DFU.DAK was associated with cellular glucose homeostasis.Furthermore,increased CCL2+CXCL2+fibroblasts,VWA1+vascular endothelial cells,and GZMA+CD8+T cells were detected in DFU.These changes in the wound microenvironment could regulate the fate of epidermal cells through the TNFSF12-TNFRSF12A,IFNG-IFNGR1/2,and IL-1B-IL1R2 pathways,which might result in persistent inflammation and impaired epidermal differentiation in DFU.Conclusions:Our findings offer novel insights into the pathophysiology of DFU and present potential therapeutic targets that could improve wound care and treatment outcomes for DFU patients.
基金supported by the National Natural Science Foundation of China(No.82060350,No.82002272,No.82272276)China Postdoctoral Science Foundation(No.2022 M711335,No.2021 M701434)+2 种基金GuangDong Basic and Applied Basic Research Foundation(No.2022A1515110490,No.2021A1515011453,No.2022A1515011380,No.2022A1515012160)Industry-university-research Innovation Fund of Higher Education of China(No.2021JH028)the Science and Technology Innovation Committee of Shenzhen(No.JCYJ20220530152015036).
文摘Background:Refractory diabetic wounds are a common occurrence in patients with diabetes and epidermis-specific macroautophagy/autophagy impairment has been implicated in their pathogenesis.Therefore,identifying and developing treatment strategies capable of normalizing epidermisspecific macroautophagy/autophagy could facilitate diabetic wound healing.The study aims to investigate the potential of bone marrow mesenchymal stem cell-derived exosomes(BMSC-exos)from hypoxic conditions as a treatment to normalize epidermis-specific autophagy for diabetic wound healing.Methods:We compared the effects of bone marrow mesenchymal stem cell(BMSC)-sourced exosomes(BMSC-Exos)from hypoxic conditions to those of BMSC in normoxic conditions(noBMSC-Exos).Our studies involved morphometric assessment of the exosomes,identification of the microRNA(miRNA)responsible for the effects,evaluation of keratinocyte functions and examination of effects of the exosomes on several molecules involved in the autophagy pathway such as microtubule-associated protein 1 light chain 3 beta,beclin 1,sequestosome 1,autophagyrelated 5 and autophagy-related 5.The experiments used human BMSCs from the American Type Culture Collection,an in vivo mouse model of diabetes(db/db)to assess wound healing,as well as the human keratinocyte HaCaT cell line.In the methodology,the authors utilized an array of approaches that included electron microscopy,small interfering RNA(siRNA)studies,RNA in situ hybridization,quantitative real-time reverse transcription PCR(qRT-PCR),the isolation,sequencing and differential expression of miRNAs,as well as the use of miR-4645-5p-specific knockdown with an inhibitor.Results:Hypoxia affected the release of exosomes from hypoxic BMSCs(hy-BMSCs)and influenced the size and morphology of the exosomes.Moreover,hyBMSC-Exo treatment markedly improved keratinocyte function,including keratinocyte autophagy,proliferation and migration.miRNA microarray and bioinformatics analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in‘autophagy’and‘process utilizing autophagic mechanism’in the‘biological process’category and miR-4645-5p as a major contributor to the proautophagy effect of hyBMSC-Exos.Moreover,mitogen-activated protein kinase-activated protein kinase 2(MAPKAPK2)was identified as a potential target of exosomal miR-4645-5p;this was confirmed using a dual luciferase assay.Exosomal miR-4645-5p mediates the inactivation of the MAPKAPK2-induced AKT kinase group(comprising AKT1,AKT2,and AKT3),which in turn suppresses AKT-mTORC1 signaling,thereby facilitating miR-4645-5p-mediated autophagy.Conclusions:Overall,the results of this study showed that hyBMSC-Exo-mediated transfer of miR-4645-5p inactivated MAPKAPK2-induced AKT-mTORC1 signaling in keratinocytes,which activated keratinocyte autophagy,proliferation and migration,resulting in diabetic wound healing in mice.Collectively,the findings could aid in the development of a novel therapeutic strategy for diabetic wounds.
