Adipose tissue-derived stem cells (ADSCs) have been shown to enhance wound healing via their paracrine function. Exosomes, as one of the most important paracrine factors, play an essential role in ...this process. However, the concrete mechanisms that underlie this effect are poorly understood. In this study, we aim to explore the potential roles and molecular mechanisms of exosomes derived from ADSCs in cutaneous wound healing.
Normal human skin fibroblasts and ADSCs were isolated from patient skin and adipose tissues. ADSCs were characterized by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Exosomes were purified from human ADSCs by differential ultracentrifugation and identified by electron microscopy, nanoparticle tracking, fluorescence confocal microscopy and western blotting. Fibroblasts were treated with different concentrations of exosomes, and the synthesis of collagen was analyzed by western blotting; the levels of growth factors were analyzed by real-time quantitative PCR (RT-PCR) and ELISA; and the proliferation and migration abilities of fibroblasts were analyzed by real-time cell analysis, CCK-8 assays and scratch assays. A mouse model with a full-thickness incision wound was used to evaluate the effect of ADSC-derived exosomes on wound healing. The level of p-Akt/Akt was analyzed by western blotting. Ly294002, a phosphatidylinositol 3-kinases (PI3K) inhibitor, was used to identify the underlying mechanisms by which ADSC-derived exosomes promote wound healing.
ADSC-derived exosomes were taken up by the fibroblasts, which showed significant, dose-dependent increases in cell proliferation and migration compared to the behavior of cells without exosome treatment. More importantly, both the mRNA and protein levels of type I collagen (Col 1), type III collagen (Col 3), MMP1, bFGF, and TGF-β1 were increased in fibroblasts after stimulation with exosomes. Furthermore, exosomes significantly accelerated wound healing in vivo and increased the level of p-Akt/Akt in vitro. However, Ly294002 alleviated these exosome-induced changes, suggesting that exosomes from ADSCs could promote and optimize collagen deposition in vitro and in vivo and further promote wound healing via the PI3K/Akt signaling pathway.
This study demonstrates that ADSC-derived exosomes can promote fibroblast proliferation and migration and optimize collagen deposition via the PI3K/Akt signaling pathway to further accelerate wound healing. Our results suggest that ADSCs likely facilitate wound healing via the release of exosomes, and the PI3K/Akt pathway may play a role in this process. Our data also suggest that the clinical application of ADSC-derived exosomes may shed new light on the use of cell-free therapy to accelerate full-thickness skin wound healing and attenuate scar formation.
Schematic diagram shows how the wound healing effect of ADSC-Exos is mediated by the activation of the PI3K/Akt signaling pathways. Display omitted
•ADSC-Exos are internalized into HDFs and regulate their biological behaviors and functions.•ADSC-Exos play an important role in accelerating wound healing via activating PI3K/Akt signaling pathway.•ADSC-Exos may serve as a cell-free therapy for the potential clinical treatment of wound healing.
Fatty acid metabolism (FAM) affects the immune phenotype in a metabolically dynamic tumor microenvironment (TME), but the use of FAM-related genes (FAMGs) to predict the prognosis and immunotherapy ...response of cutaneous melanoma (CM) patients has not been investigated. In this study, we aimed to construct FAM molecular subtypes and identify key prognostic biomarkers in CM.
We used a CM dataset in The Cancer Genome Atlas (TCGA) to construct FAM molecular subtypes. We performed Kaplan-Meier (K-M) analysis, gene set enrichment analysis (GSEA), and TME analysis to assess differences in the prognosis and immune phenotype between subtypes. We used weighted gene co-expression network analysis (WGCNA) to identify key biomarkers that regulate tumor metabolism and immunity between the subtypes. We compared overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) between CM patients with high or low biomarker expression. We applied univariable and multivariable Cox analyses to verify the independent prognostic value of the FAM biomarkers. We used GSEA and TME analysis to investigate the immune-related regulation mechanism of the FAM subtype biomarker. We evaluated the immune checkpoint inhibition (ICI) response and chemotherapy sensitivity between CM patients with high or low biomarker expression. We performed real-time fluorescent quantitative PCR (qRT-PCR) and semi-quantitative analysis of the immunohistochemical (IHC) data from the Human Protein Atlas to evaluate the mRNA and protein expression levels of the FAM biomarkers in CM.
We identified 2 FAM molecular subtypes (cluster 1 and cluster 2). K-M analysis showed that cluster 2 had better OS and PFS than cluster 1 did. GSEA showed that, compared with cluster 1, cluster 2 had significantly upregulated immune response pathways. The TME analysis indicated that immune cell subpopulations and immune functions were highly enriched in cluster 2 as compared with cluster 1. WGCNA identified 6 hub genes (
) as FAM biomarkers. CM patients with high expression levels of the six biomarkers had better OS, PFS, and DSS than those with low expression levels of the biomarkers. The Cox regression analyses verified that the 6 FAM biomarkers can be independent prognostic factors for CM patients. The single-gene GSEA showed that the high expression levels of the 6 genes were mainly enriched in T-cell antigen presentation, the PD-1 signaling pathway, and tumor escape. The TME analysis confirmed that the FAM subtype biomarkers were not only related to immune infiltration but also highly correlated with immune checkpoints such as PD-1, PD-L1, and CTLA-4. TIDE scores confirmed that patients with high expression levels of the 6 biomarkers had worse immunotherapy responses. The 6 genes conveyed significant sensitivity to some chemotherapy drugs. qRT-PCR and IHC analyses verified the expression levels of the 6 biomarkers in CM cells.
Our FAM subtypes verify that different FAM reprogramming affects the function and phenotype of infiltrating immune cells in the CM TME. The FAM molecular subtype biomarkers can be independent predictors of prognosis and immunotherapy response in CM patients.
SIRT1 is reported to participate in macrophage differentiation and affect sepsis, and Notch signaling is widely reported to influence inflammation and macrophage activation. However, the specific ...mechanisms through which SIRT1 regulates sepsis and the relationship between SIRT1 and Notch signaling remain poorly elucidated. In this study, we found that SIRT1 levels were decreased in sepsis both
and
and that SIRT1 regulation of Notch signaling affected inflammation. In lipopolysaccharide (LPS)-induced sepsis, the levels of Notch signaling molecules, including Notch1, Notch2, Hes1, and intracellular domain of Notch (NICD), were increased. However, NICD could be deacetylated by SIRT1, and this led to the suppression of Notch signaling. Notably, in macrophages from myeloid-specific
mice, in which Notch signaling is inhibited, pro-inflammatory cytokines were expressed at lower levels than in macrophages from wild-type littermates and in
macrophages, and the NF-κB pathway was also inhibited. Accordingly, in the case of
mice, LPS-induced inflammation and mortality were lower than in wild-type mice. Our results indicate that SIRT1 inhibits Notch signaling through NICD deacetylation and thus ultimately alleviates sepsis.
Excessive scar formation such as hypertrophic scars and keloids, resulting from trauma or surgical procedures, present a widespread concern for causing disfigurement, discomfort, and functional ...limitations. Macrophages play pivotal roles in maintaining tissue homeostasis, orchestrating tissue development, repair, and immune responses, and its transition of function and phenotype plays a critical role in regulating the balance between inflammation and tissue regeneration, which is central to cutaneous scar formation. Recent evidence suggests the involvement of Sonic Hedgehog (SHH) in the induction of anti-inflammatory M2-like macrophage phenotypes within tumor microenvironments. In our study, we observed increased SHH expression in human hypertrophic scars, prompting an investigation into its influence on macrophage polarization, efferocytosis, and cutaneous scar formation. Our findings reveal that SHH can enhance oxidative phosphorylation (OXPHOS) in macrophages, augment macrophage efferocytosis, and promote M2 polarization, finally contributing to the progression of cutaneous scar formation. Notably, targeting SHH signaling with vismodegib exhibited promising potential in mitigating scar formation by reversing the effects of enhanced OXPHOS and M2 polarization in macrophages. In conclusion, this study underscores the critical roles of macrophage metabolism, particularly OXPHOS, efferocytosis and SHH signaling in cutaneous scar formation. Understanding these mechanisms provides new avenues for potential interventions and scar prevention strategies.
Hypertrophic scarring (HS) is a dermal fibroproliferative disorder characterized by excessive deposition of collagen and other extracellular matrix components. The aim of this study is to explore ...crucial long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) associated with HS and provide a better understanding of the molecular mechanism of HS. To investigate the lncRNA, circRNA and mRNA expression profiles, we performed RNA sequencing of human HS and normal skin tissues. After the identification of differentially expressed mRNAs (DEmRNAs), lncRNAs (DElncRNAs) and circRNAs (DEcircRNAs), we performed functional enrichment of DEmRNAs. Further on, we constructed DElncRNA/DEcircRNA–DEmRNA coexpression networks and competing endogenous RNA regulatory networks, and performed functional analyses of the DEmRNAs in the constructed networks. In total, 487 DEmRNAs, 92 DElncRNAs and 17 DEcircRNAs were identified. DEmRNAs were significantly enriched in processes such as collagen fibril organization, extracellular matrix–receptor interaction and the phosphatidylinositol 3‐kinase (PI3K)–Akt signaling pathway. In addition, we detected 580 DElncRNA–DEmRNA and 505 DEcircRNA–DEmRNA coexpression pairs. The competing endogenous RNA network contained 18 circRNA–microRNA (miRNA) pairs, 18 lncRNA–miRNA pairs and 409 miRNA–mRNA pairs, including 10 circRNAs, 5 lncRNAs, 15 miRNAs and 160 mRNAs. We concluded that MIR503HG/hsa‐miR‐204‐3p/ACAN, MIR503HG/hsa‐miR‐431‐5p/TNFRSF9, MEG3/hsa‐miR‐6884‐5p/ADAMTS14, AC000035.1‐ADAMTS14 and hsa_circ_0069865‐COMP/ADAM12 interaction pairs may play a central role in HS.
We performed RNA sequencing to acquire the expression profiles of long noncoding RNA, circular RNA and mRNA in human hypertrophic scar (HS) and normal skin tissues from three male patients with HS. We concluded that MIR503HG/hsa‐miR‐204‐3p/ACAN, MIR503HG/hsa‐miR‐431‐5p/TNFRSF9, MEG3/hsa‐miR‐6884‐5p/ADAMTS14, AC000035.1‐ADAMTS14 and hsa_circ_0069865‐COMP/ADAM12 interaction pairs may play a central role in HS.
Background/Aims: Inflammation is an essential component of innate immunity against pathogens, but is tightly regulated, such as by Krüppel-like factor 4 (Klf4), to prevent injury. Klf4 also regulates ...macrophage polarization, although the mechanisms underlying both functions are poorly understood. The aim of this study was to investigate whether and how Klf4 prevents unregulated inflammation. Methods: The bone marrow-derived macrophages and RAW264.7 cell line were used. Quantitative real-time PCR was used to determine inflammatory cytokines (IL-1β, TNF-α, IL-6 and MCP-1), Klf4 and MCPIP1 transcript levels. Extraction of nuclear and cytoplasmic proteins and Immunoblotting were used to determine Klf4, MCPIP1, relative kinases from NF-κB pathway and K63-linked polyubiquitins expression in nucleus and cytoplasm separately. Dual luciferase reporter assay was used to analyze whether Klf4 mediate MCPIP1 transcription. Immunoprecipitation was used to determine the protein interaction among Klf4, MCPIP1, TRAF6 and K63-linked polyubiquitins. Secretion of IL-1β and TNF-α into sera in mice was measured by Enzyme-linked immunosorbent assay. Results: We found that exposure to lipopolysaccharides suppresses Klf4 expression, even as it induces release of pro-inflammatory cytokines. Strikingly, Klf4 overexpression significantly lowered cytokine secretion and NF-κB signaling in the cytoplasm following exposure to lipopolysaccharide, even though Klf4 was exclusively nuclear. The cytoplasmic effects are likely mediated by MCP-1 induced protein 1 (MCPIP1), a deubiquitinase and a key modulator of inflammation that accumulates both in the nucleus and cytoplasm in response to Klf4. Indeed, binding between MCPIP1 and K63 polyubiquitins is attenuated in macrophages overexpressing Klf4, suggesting that MCPIP1 is an intermediator induced by Klf4 in the nucleus to remove K63 polyubiquitins from TRAF6 in the cytoplasm, and thereby impede NF-κB and inflammatory signaling. Importantly, Klf4 overexpression in mice alleviated sepsis symptoms following exposure to lipopolysaccharides. Conclusion: The data highlight Klf4 as an essential MCPIP1-dependent modulator of innate immunity that protects against excessive and self-destructive inflammation.
Severe tissue injuries pose a significant risk to human health. Conventional wound dressings fall short in achieving effective tissue regeneration, resulting in suboptimal postoperative healing ...outcomes. In this study, an asymmetric adhesive wound dressing (marked as SIS/PAA/LAP) was developed, originating from acrylate acid (AA) solution with laponite (LAP) nanoparticles polymerization and photo-crosslinked on the decellularized extracellular matrix small intestinal submucosa (SIS) patch. Extensive studies demonstrated that the SIS/PAA/LAP exhibited higher tissue adhesion strength (~ 33 kPa) and burst strength (~ 22 kPa) compared to conventional wound dressings like Tegaderm and tissue adhesive products. Importantly, it maintained favorable cell viability and demonstrated robust angiogenic capacity. In animal models of full-thickness skin injuries in rats and skin injuries in Bama miniature pigs, the SIS/PAA/LAP could be precisely applied to wound sites. By accelerating the formation of tissue vascularization, it displayed superior tissue repair outcomes. This asymmetrically adhesive SIS-based patch would hold promising applications in the field of wound dressings.
With the development of tissue engineering, the application of decellularized adipose matrix as scaffold material in tissue engineering has been intensively explored due to its wide source and ...excellent potential in tissue regeneration. Decellularized adipose matrix is a promising candidate for adipose tissue regeneration, while modification of decellularized adipose matrix scaffold can also allow it to transcend the limitations of adipose tissue source properties and applied to other tissue engineering fields, including cartilage and bone tissue engineering, neural tissue engineering, and skin tissue engineering. In this review, we summarized the development of the applications of decellularized adipose matrix in different tissue engineering and present future perspectives.
Level of Evidence III
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Trauma or burn injuries that affect the deep dermis often produce a hypertrophic scar, which limits patients' joint movement and generates an aesthetic problem. Inflammation is believed to be one of ...the main pathogenic mechanisms. We found that IL-17 was increased in scar tissues from patients with hypertrophic scar compared with normal skin. Recombinant mouse IL-17 was subcutaneously injected into mice that underwent full-thickness excision surgery to investigate the role of IL-17 in scar formation. Mice stimulated with IL-17 showed aggravated fibrogenesis, delayed wound healing, and increased inflammation. In addition, macrophage infiltration was also increased. According to the results of the Transwell assay, IL-17 promoted macrophage infiltration through an indirect mechanism. After depleting macrophages with clodronate liposomes, the effect of IL-17 disappeared. Levels of monocyte chemotactic protein (MCP) 1, MCP2, and MCP3 (together referred to as MCPs) were increased by IL-17 stimulation. Bindarit (an inhibitor of MCPs) was used to verify the role of MCPs. In addition, the Ly6C-low macrophages were responsible for wound fibrogenesis in mice. In this study, we detected the increased levels of IL-17 for the first time and revealed that IL-17 induced the infiltration of a specific subtype of macrophages to aggravate fibrosis through an MCP-dependent mechanism. Thus, our results provide a better understanding of scar formation and new strategies for scar prevention.
Severe inflammation may lead to multiple organs dysfunction syndrome, which has a high mortality. MicroRNA is found participated in this process. In this study we developed a ...lipopolysaccharide-induced inflammation cell model on macrophages and a lipopolysaccharide-induced inflammation mouse model. It was found that during inflammation, microRNA-9 was increased, accompanied with the up-regulation of pro-inflammatory cytokines and anti-inflammatory cytokines. Down-regulation of microRNA-9 inhibited the up-regulation of inflammatory cytokines, promoted the up-regulation of anti-inflammatory cytokines and induced the remission of organ damage, showing a protective effect in inflammation. Bioinformatics analysis combined with luciferase reporter assay showed that SIRT1 was the target gene of microRNA-9. Transfection of microRNA-9 inhibitor could increase the level of SIRT1 and decrease the activation of NF-κB pathway in macrophages. Myeloid specific sirt1 knockout mice were included and we found that lack of SIRT1 in mice macrophages led to aggravated inflammation, cell apoptosis and organ injury, and eliminated the protective property of microRNA-9 inhibitor. In conclusion, we demonstrated that inhibition of microRNA-9 could alleviate inflammation through the up-regulation of SIRT1 and then suppressed the activation of NF-κB pathway. This is a meaningful explore about the specific mechanism of microRNA-9 in inflammation.
•LPS increased miR-9 in macrophage.•MiR-9 increased inflammatory response of macrophage and promoted organ damage induced by LPS.•MiR-9 directly targeted SIRT1 to promote inflammation.
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