CD74+ fibroblasts proliferate upon mechanical stretching to promote angiogenesis in keloids.

The healing of human skin wounds is susceptible to perturbation caused by excessive mechanical stretching, resulting in enlarged scars, hypertrophic scars, or even keloids in predisposed individuals. Keloids are fibro-proliferative scar tissues that extend beyond the initial wound boundary, consisting of the actively progressing periphery and the quiescent center. The stretch-associated outgrowth and enhanced angiogenesis are two features of the periphery of keloids. However, which cell population is responsible for transducing the mechanical stimulation to the progression of keloids remains unclear. Herein, through integrative analysis of single-cell RNA sequencing of keloids, we identified CD74+ fibroblasts, a previously unappreciated subset of fibroblasts with pro-angiogenic and stretch-induced proliferative capacities, as a key player in stretch-induced progression of keloids. Immunostaining of keloid cryosections depicted a predominant distribution of CD74+ fibroblasts in the periphery, interacting with the vasculature. In vitro tube formation assays on purified CD74+ fibroblasts ascertained their pro-angiogenic function. BrdU assays revealed that these cells proliferate upon stretching, through PIEZO1-mediated calcium influx and the downstream ERK and AKT signaling. Collectively, our findings propose a model wherein CD74+ fibroblasts serve as pivotal drivers of stretch-induced keloid progression, fueled by their proliferative and pro-angiogenic activities. Targeting the attributes of CD74+ fibroblasts holds promise as a therapeutic strategy for the management of keloids.

Targeting the nuclear long noncoding transcript LSP1P5 abrogates extracellular matrix deposition by trans-upregulating CEBPA in keloids.

Keloids are characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix (ECM) and are a major global health care burden among cutaneous diseases. However, the function of long noncoding RNA (lncRNA)-mediated ECM remodeling during the pathogenesis of keloids is still unclear. Herein, we identified a long noncoding transcript, namely, lymphocyte-specific protein 1 pseudogene 5 (LSP1P5), that modulates ECM component deposition in keloids. First, high-throughput transcriptome analysis showed that LSP1P5 was selectively upregulated in keloids and correlated with more severe disease in a clinical keloid cohort. Therapeutically, the attenuation of LSP1P5 significantly decreased the expression of ECM markers (COL1, COL3, and FN1) both in vitro and in vivo. Intriguingly, an antifibrotic gene, CCAAT enhancer binding protein alpha (CEBPA), is a functional downstream candidate of LSP1P5. Mechanistically, LSP1P5 represses CEBPA expression by hijacking Suppressor of Zeste 12 to the promoter of CEBPA, thereby enhancing the polycomb repressive complex 2-mediated H3K27me3 and changing the chromosomal opening status of CEBPA. Taken together, these findings indicate that targeting LSP1P5 abrogates fibrosis in keloids through epigenetic regulation of CEBPA, revealing a novel antifibrotic therapeutic strategy that bridges our current understanding of lncRNA regulation, histone modification and ECM remodeling in keloids.

BMP1 Promotes Keloid by Inducing Fibroblast Inflammation and Fibrogenesis.

Keloid is a typical fibrotic and inflammatory skin disease with unclear mechanisms and few therapeutic targets. In this study, we found that BMP1 was significantly increased in a collagen high-expressing subtype of fibroblast by reanalyzing a public single-cell RNA-sequence data set of keloid. The number of BMP1-positive fibroblast cells was increased in keloid fibrotic loci. Increased levels of BMP1 were further validated in the skin tissues and fibroblasts from keloid patients. Additionally, a positive correlation between BMP1 and the Keloid Area and Severity Index was found in keloid patients. In vitro analysis revealed collagen production, the phosphorylation levels of p65, and the IL-1ß secretion decreased in BMP1 interfered keloid fibroblasts. Besides, the knockdown of BMP1 inhibited the growth and migration of keloid fibroblast cells. Mechanistically, BMP1 inhibition downregulated the noncanonical TGF-ß pathways, including p-p38 and p-ERK1/2 signaling. Furthermore, we found the delivery of BMP1 siRNAs could significantly alleviate keloid in human keloid-bearing nude mice. Collectively, our results indicated that BMP1 exhibited various pathogenic effects on keloids as promoting cell proliferation, migration, inflammation, and ECM deposition of fibroblast cells by regulating the noncanonical TGF-ß/p38 MAPK, and TGF-ß/ERK pathways. BMP1-lowing strategies may appear as a potential new therapeutic target for keloid.

Phosphodiesterase 4 is overexpressed in keloid epidermal scars and its inhibition reduces keratinocyte fibrotic alterations.

BACKGROUND: Epidermal remodeling and hypertrophy are hallmarks of skin fibrotic disorders, and keratinocyte to mesenchymal (EMT)-like transformations drive epidermis alteration in skin fibrosis such as keloids and hypertrophic scars (HTS). While phosphodiesterase 4 (PDE4) inhibitors have shown effectiveness in various fibrotic disorders, their role in skin fibrosis is not fully understood. This study aimed to explore the specific role of PDE4B in epidermal remodeling and hypertrophy seen in skin fibrosis. METHODS: In vitro experiments examined the effects of inhibiting PDE4A-D (with Roflumilast) or PDE4B (with siRNA) on TGFß1-induced EMT differentiation and dedifferentiation in human 3D epidermis. In vivo studies investigated the impact of PDE4 inhibition on HOCl-induced skin fibrosis and epidermal hypertrophy in mice, employing both preventive and therapeutic approaches. RESULTS: The study found increased levels of PDE4B (mRNA, protein) in keloids > HTS compared to healthy epidermis, as well as in TGFß-stimulated 3D epidermis. Keloids and HTS epidermis exhibited elevated levels of collagen Iα1, fibronectin, αSMA, N-cadherin, and NOX4 mRNA, along with decreased levels of E-cadherin and ZO-1, confirming an EMT process. Inhibition of both PDE4A-D and PDE4B prevented TGFß1-induced Smad3 and ERK1/2 phosphorylation and mesenchymal differentiation in vitro. PDE4A-D inhibition also promoted mesenchymal dedifferentiation and reduced TGFß1-induced ROS and keratinocyte senescence by rescuing PPM1A, a Smad3 phosphatase. In vivo, PDE4 inhibition mitigated HOCl-induced epidermal hypertrophy in mice in both preventive and therapeutic settings. CONCLUSIONS: Overall, the study supports the potential of PDE4 inhibitors, particularly PDE4B, in treating skin fibrosis, including keloids and HTS, shedding light on their functional role in this condition.

MEG3 shuttled by exosomes released from human bone marrow mesenchymal stem cells promotes TP53 stability to regulate MCM5 transcription in keloid fibroblasts.

BACKGROUND: Despite the interest in mesenchymal stem cells (MSC), their potential to treat abnormal scarring, especially keloids, is yet to be described. The present study aimed to investigate the therapeutic potential of exosomes derived from human bone marrow MSCs (hBMSC-Exos) in alleviating keloid formation. METHODS: Exosomes were isolated from hBMSC, and keloid fibroblasts (KFs) were treated with hBMSC-Exos. Cell counting kit-8, wound healing, transwell invasion, immunofluorescence, and western blot assays were conducted to study the malignant phenotype of KFs. Mice were induced with keloids and treated with hBMSC-Exos. The effect of hBMSC-Exos on keloid formation in vivo was evaluated by hematoxylin and eosin staining, Masson staining, immunohistochemistry, and western blotting. The GSE182192 dataset was screened for differentially expressed long non-coding RNA during keloid formation. Next, maternally expressed gene 3 (MEG3) was knocked down in hBMSC to obtain hBMSC-Exossh-MEG3. The molecular mechanism of MEG3 was investigated by bioinformatic screening, and the relationship between MEG3 and TP53 or MCM5 was verified. RESULTS: hBMSC-Exos inhibited the malignant proliferation, migration, and invasion of KFs at same time as promoting their apoptosis, Moreover, hBMSC-Exos reduced the expression of fibrosis- and collagen-related proteins in the cells and the formation of keloids caused by KFs. The reduction in MEG3 enrichment in hBMSC-Exos weakened the inhibitory effect of hBMSC-Exos on KF activity. hBMSC-Exos delivered MEG3 to promote MCM5 transcription by TP53 in KFs. Overexpression of MCM5 in KFs reversed the effects of hBMSC-Exossh-MEG3, leading to reduced KF activity. CONCLUSIONS: hBMSC-Exos delivered MEG3 to promote the protein stability of TP53, thereby activating MCM5 and promoting KF activity.

Kreotoxin A administration inhibits hyperproliferation and inflammation of human ear keloid tissue and keloid-derived fibroblasts by downregulating HIF-1α expression.

Keloids represent a prevalent dermal fibroproliferative disorder. They only affect humans and exhibit several tumor characteristics, such as excessive extracellular matrix (ECM) deposition, which usually occurs after skin injury. Kreotoxin type A (KTA) can inhibit the release of acetylcholine, and thereby inhibit the proliferation of keloid fibroblasts and reducing the formation of scars. Thus, KTA could be used as a therapeutic agent for keloids. However, the mechanisms of action of KTA in keloid treatment remain unclear. In this study, we aimed to explore the underlying mechanisms of action of KTA in human keloid treatment using human tissue and a cell-based model. Integrative microarray analysis revealed that hypoxia-inducible factor 1-alpha (HIF-1α) expression was frequently upregulated in hypertrophic scar and keloid tissues, whereas it was downregulated in the KTA-treated samples. Furthermore, KTA addition to keloid-derived fibroblasts (KDFs) reduced the growth rate and viability, induced apoptosis, and decreased inflammation and oxidative stress in KDFs. However, overexpression of HIF-1α restored cell number and survival, decreased apoptosis, and promoted inflammation and oxidative stress in KTA-treated KDFs. Furthermore, KTA treatment reduced the expression of ECM proteins, including vascular endothelial growth factor (VEGF), collagen I and III, whereas HIF-1α overexpression abolished the effects of KTA on KDFs. In conclusion, our findings provide novel insights into the mechanisms of action of KTA as a potential therapeutic agent for keloids via modulating HIF-1α expression.

Serum deprivation protein response intervenes in the proliferation, motility, and extracellular matrix production in keloid fibroblasts by blocking the amplification of TGF-ß1/SMAD signal cascade via ERK1/2.

Keloid formation has been linked to abnormal fibroblast function, such as excessive proliferation and extracellular matrix (ECM) production. Serum deprivation protein response (SDPR) is a crucial regulator of cellular function under diverse pathological conditions, yet its role in keloid formation remains unknown. The current work investigated the function of SDPR in regulating the proliferation, motility, and ECM production of keloid fibroblasts (KFs), as well as to decipher the mechanisms involved. Analysis of RNA sequencing data from the GEO database demonstrated significant down-regulation of SDPR in KF compared to normal fibroblasts (NFs). This down-regulation was also observed in clinical keloid specimens and isolated KFs. Overexpression of SDPR suppressed the proliferation, motility, and ECM production of KFs, while depletion of SDPR exacerbated the enhancing impact of TGF-ß1 on the proliferation, motility, and ECM production of NFs. Mechanistic studies revealed that SDPR overexpression repressed TGF-ß/Smad signal cascade activation in KFs along with decreased levels of phosphorylated Samd2/3, while SDPR depletion exacerbated TGF-ß/Smad activation in TGF-ß1-stimulated NFs. SDPR overexpression also repressed ERK1/2 activation in KFs, while SDPR depletion exacerbated ERK1/2 activation in TGF-ß1-stimulated NFs. Inhibition of ERK1/2 abolished SDPR-depletion-induced TGF-ß1/Smad activation, cell proliferation, motility, and ECM production in NFs. In conclusion, SDPR represses the proliferation, motility, and ECM production in KFs by blocking the TGF-ß1/Smad pathway in an ERK1/2-dependent manner. The findings highlight the role of SDPR in regulating abnormal behaviors of fibroblasts associated with keloid formation and suggest it as a potential target for anti-keloid therapy development.

Revealing the pathogenesis of keloids based on the status: Active vs inactive.

Recently, the pathomechanisms of keloids have been extensively researched using transcriptomic analysis, but most studies did not consider the activity of keloids. We aimed to profile the transcriptomics of keloids according to their clinical activity and location within the keloid lesion, compared with normal and mature scars. Tissue samples were collected (keloid based on its activity (active and inactive), mature scar from keloid patients and normal scar (NS) from non-keloid patients). To reduce possible bias, all keloids assessed in this study had no treatment history and their location was limited to the upper chest or back. Multiomics assessment was performed by using single-cell RNA sequencing and multiplex immunofluorescence. Increased mesenchymal fibroblasts (FBs) was the main feature in keloid patients. Noticeably, the proportion of pro-inflammatory FBs was significantly increased in active keloids compared to inactive ones. To explore the nature of proinflammatory FBs, trajectory analysis was conducted and CCN family associated with mechanical stretch exhibited higher expression in active keloids. For vascular endothelial cells (VECs), the proportion of tip and immature cells increased in keloids compared to NS, especially at the periphery of active keloids. Also, keloid VECs highly expressed genes with characteristics of mesenchymal activation compared to NS, especially those from the active keloid center. Multiomics analysis demonstrated the distinct expression profile of active keloids. Clinically, these findings may provide the future appropriate directions for development of treatment modalities of keloids. Prevention of keloids could be possible by the suppression of mesenchymal activation between FBs and VECs and modulation of proinflammatory FBs may be the key to the control of active keloids.

STAT3-induced lncRNA GNAS-AS1 accelerates keloid formation by mediating the miR-196a-5p/CXCL12/STAT3 axis in a feedback loop.

Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence in situ hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.

UCHL1 aggravates skin fibrosis through an IGF-1-induced Akt/mTOR/HIF-1α pathway in keloid.

Keloid is a heterogeneous disease featured by the excessive production of extracellular matrix. It is a great challenge for both clinicians and patients regarding the exaggerated and uncontrolled outgrowth and the therapeutic resistance of the disease. In this study, we verified that UCHL1 was drastically upregulated in keloid fibroblasts. UCHL1 had no effects on cell proliferation and migration, but instead promoted collagen I and α-SMA expression that was inhibited by silencing UCHL1 gene and by adding in LDN-57444, a pharmacological inhibitor for UCHL1 activity as well. The pathological process was mediated by IGF-1 promoted Akt/mTOR/HIF-1α signaling pathway because inhibition of any of them could reduce the expression of collagen I and α-SMA driven by UCHL1 in fibroblasts. Also, we found that UCHL1 expression in keloid fibroblasts was promoted by M2 macrophages via TGF-ß1. These findings extend our understanding of the pathogenesis of keloid and provide potential therapeutic targets for the disease.

TEM1/endosialin/CD248 promotes pathologic scarring and TGF-ß activity through its receptor stability in dermal fibroblasts.

BACKGROUND: Pathologic scars, including keloids and hypertrophic scars, represent a common form of exaggerated cutaneous scarring that is difficult to prevent or treat effectively. Additionally, the pathobiology of pathologic scars remains poorly understood. We aim at investigating the impact of TEM1 (also known as endosialin or CD248), which is a glycosylated type I transmembrane protein, on development of pathologic scars. METHODS: To investigate the expression of TEM1, we utilized immunofluorescence staining, Western blotting, and single-cell RNA-sequencing (scRNA-seq) techniques. We conducted in vitro cell culture experiments and an in vivo stretch-induced scar mouse model to study the involvement of TEM1 in TGF-ß-mediated responses in pathologic scars. RESULTS: The levels of the protein TEM1 are elevated in both hypertrophic scars and keloids in comparison to normal skin. A re-analysis of scRNA-seq datasets reveals that a major profibrotic subpopulation of keloid and hypertrophic scar fibroblasts greatly expresses TEM1, with expression increasing during fibroblast activation. TEM1 promotes activation, proliferation, and ECM production in human dermal fibroblasts by enhancing TGF-ß1 signaling through binding with and stabilizing TGF-ß receptors. Global deletion of Tem1 markedly reduces the amount of ECM synthesis and inflammation in a scar in a mouse model of stretch-induced pathologic scarring. The intralesional administration of ontuxizumab, a humanized IgG monoclonal antibody targeting TEM1, significantly decreased both the size and collagen density of keloids. CONCLUSIONS: Our data indicate that TEM1 plays a role in pathologic scarring, with its synergistic effect on the TGF-ß signaling contributing to dermal fibroblast activation. Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity of pathologic scars.

The fibroblast heterogeneity across keloid, normal and tumor samples from single-cell resolution.

Keloids are defined as a benign dermal fibroproliferative disorder, with excessive fibroblast proliferation, and excessive overproduction of collagen. Although the heterogeneity during keloid development has been extensively studied, the heterogeneity across different skin states is still unclear. So, a global comparison across skin states is needed. In this study, we collected samples from 5 states of skin, including melanoma, cutaneous squamous cell carcinoma, keloid skin, scar skin, and healthy control samples. The heterogeneity of cell types and subtypes was analyzed and compared across 5 states, and we observed significant differences among them. Our results showed a cancer-like fibroblast, which is not in normal samples, may play an important role in antigen processing and presentation. We also noticed that the mesenchymal fibroblast increased in keloid samples, which highly expressed POSTN. And POSTN may participate in epithelial-mesenchymal transition and collagen overexpression to promote keloid growth. These findings help to understand the alteration among different skin states and provide potential genetic basis for keloid therapies.

miRSNP rs188493331: A key player in genetic control of microRNA-induced pathway activation in hypertrophic scars and keloids.

BACKGROUND: Our study aims to delineate the miRSNP-microRNA-gene-pathway interactions in the context of hypertrophic scars (HS) and keloids. MATERIALS AND METHODS: We performed a computational biology study involving differential expression analysis to identify genes and their mRNAs in HS and keloid tissues compared to normal skin, identifying key hub genes and enriching their functional roles, comprehensively analyzing microRNA-target genes and related signaling pathways through bioinformatics, identifying MiRSNPs, and constructing a pathway-based network to illustrate miRSNP-miRNA-gene-signaling pathway interactions. RESULTS: Our results revealed a total of 429 hub genes, with a strong enrichment in signaling pathways related to proteoglycans in cancer, focal adhesion, TGF-ß, PI3K/Akt, and EGFR tyrosine kinase inhibitor resistance. Particularly noteworthy was the substantial crosstalk between the focal adhesion and PI3K/Akt signaling pathways, making them more susceptible to regulation by microRNAs. We also identified specific miRNAs, including miRNA-1279, miRNA-429, and miRNA-302e, which harbored multiple SNP loci, with miRSNPs rs188493331 and rs78979933 exerting control over a significant number of miRNA target genes. Furthermore, we observed that miRSNP rs188493331 shared a location with microRNA302e, microRNA202a-3p, and microRNA20b-5p, and these three microRNAs collectively targeted the gene LAMA3, which is integral to the focal adhesion signaling pathway. CONCLUSIONS: The study successfully unveils the complex interactions between miRSNPs, miRNAs, genes, and signaling pathways, shedding light on the genetic factors contributing to HS and keloid formation.

Differential Photosensitivity of Fibroblasts Obtained from Normal Skin and Hypertrophic Scar Tissues.

It is unclear whether normal human skin tissue or abnormal scarring are photoreceptive. Therefore, this study investigated photosensitivity in normal skin tissue and hypertrophic scars. The expression of opsins, which are photoreceptor proteins, in normal dermal fibroblasts (NDFs) and hypertrophic scar fibroblasts (HSFs) was examined. After exposure to blue light (BL), changes in the expression levels of αSMA and clock-related genes, specifically PER2 and BMAL1, were examined in both fibroblast types. Opsins were expressed in both fibroblast types, with OPN3 exhibiting the highest expression levels. After peripheral circadian rhythm disruption, BL induced rhythm formation in NDFs. In contrast, although HSFs showed changes in clock-related gene expression levels, no distinct rhythm formation was observed. The expression level of αSMA was significantly higher in HSFs and decreased to the same level as that in NDFs upon BL exposure. When OPN3 knocked-down HSFs were exposed to BL, the reduction in αSMA expression was inhibited. This study showed that BL exposure directly triggers peripheral circadian synchronization in NDFs but not in HSFs. OPN3-mediated BL exposure inhibited HSFs. Although the current results did not elucidate the relationship between peripheral circadian rhythms and hypertrophic scars, they show that BL can be applied for the prevention and treatment of hypertrophic scars and keloids.

Insights into How Plant-Derived Extracts and Compounds Can Help in the Prevention and Treatment of Keloid Disease: Established and Emerging Therapeutic Targets.

Keloid is a disease in which fibroblasts abnormally proliferate and synthesize excessive amounts of extracellular matrix, including collagen and fibronectin, during the healing process of skin wounds, causing larger scars that exceed the boundaries of the original wound. Currently, surgical excision, cryotherapy, radiation, laser treatment, photodynamic therapy, pressure therapy, silicone gel sheeting, and pharmacotherapy are used alone or in combinations to treat this disease, but the outcomes are usually unsatisfactory. The purpose of this review is to examine whether natural products can help treat keloid disease. I introduce well-established therapeutic targets for this disease and various other emerging therapeutic targets that have been proposed based on the phenotypic difference between keloid-derived fibroblasts (KFs) and normal epidermal fibroblasts (NFs). We then present recent studies on the biological effects of various plant-derived extracts and compounds on KFs and NFs. Associated ex vivo, in vivo, and clinical studies are also presented. Finally, we discuss the mechanisms of action of the plant-derived extracts and compounds, the pros and cons, and the future tasks for natural product-based therapy for keloid disease, as compared with existing other therapies. Extracts of Astragalus membranaceus, Salvia miltiorrhiza, Aneilema keisak, Galla Chinensis, Lycium chinense, Physalis angulate, Allium sepa, and Camellia sinensis appear to modulate cell proliferation, migration, and/or extracellular matrix (ECM) production in KFs, supporting their therapeutic potential. Various phenolic compounds, terpenoids, alkaloids, and other plant-derived compounds could modulate different cell signaling pathways associated with the pathogenesis of keloids. For now, many studies are limited to in vitro experiments; additional research and development are needed to proceed to clinical trials. Many emerging therapeutic targets could accelerate the discovery of plant-derived substances for the prevention and treatment of keloid disease. I hope that this review will bridge past, present, and future research on this subject and provide insight into new therapeutic targets and pharmaceuticals, aiming for effective keloid treatment.

Ethyl Pyruvate Decreases Collagen Synthesis and Upregulates MMP Activity in Keloid Fibroblasts and Keloid Spheroids.

Keloids, marked by abnormal cellular proliferation and excessive extracellular matrix (ECM) accumulation, pose significant therapeutic challenges. Ethyl pyruvate (EP), an inhibitor of the high-mobility group box 1 (HMGB1) and TGF-ß1 pathways, has emerged as a potential anti-fibrotic agent. Our research evaluated EP's effects on keloid fibroblast (KF) proliferation and ECM production, employing both in vitro cell cultures and ex vivo patient-derived keloid spheroids. We also analyzed the expression levels of ECM components in keloid tissue spheroids treated with EP through immunohistochemistry. Findings revealed that EP treatment impedes the nuclear translocation of HMGB1 and diminishes KF proliferation. Additionally, EP significantly lowered mRNA and protein levels of collagen I and III by attenuating TGF-ß1 and pSmad2/3 complex expression in both human dermal fibroblasts and KFs. Moreover, metalloproteinase I (MMP-1) and MMP-3 mRNA levels saw a notable increase following EP administration. In keloid spheroids, EP induced a dose-dependent reduction in ECM component expression. Immunohistochemical and western blot analyses confirmed significant declines in collagen I, collagen III, fibronectin, elastin, TGF-ß, AKT, and ERK 1/2 expression levels. These outcomes underscore EP's antifibrotic potential, suggesting its viability as a therapeutic approach for keloids.

[Effects of low dose skin tissue derived peptides on the function and collagen expression of keloid fibroblasts].

This study aims to investigate the effects of the skin tissue derived peptides on proliferation, apoptosis, migration and collagen expressions in keloid fibroblasts. From January 2015 to January 2017, patients with hypertrophic scar who underwent surgical excision in department of plastic surgery of Nanjing maternal and child health hospital were included in this retrospective study. Four peptides were selected from the differential peptides between human hypertrophic scar and normal skin tissue. They were named as peptide deregulated in hypertrophic scar 2-5 (PDHPS2-5). Bioinformatics and functional analysis were performed. A low dose of 10 µmol/L of four peptides were respectively added to the culture medium of human primary keloid fibroblasts for 24 h. Cell counting kit-8 (CCK-8) were used to detect the changes in cell viability. Cell apoptosis was detected by flow cytometry. Cell migration ability was checked by Transwell chamber. The protein expressions of collagen COL1A2 (Collagen type I alpha 2) and the myofibroblast marker gene ACTA2 (Actin alpha 2) were analyzed by Western blot. The results showed that bioinformatics prediction analysis revealed that peptide PDHPS4 has the longest half-life and the highest thermal stability. Compared with the control group, low dose of four peptides had no significant effect on the survival rate and apoptosis of keloid fibroblasts tested by CCK-8 assay and flowcytometry. Transwell analysis showed that one peptides (PDHPS5) can significantly inhibit the cell migration ability (The optical density value in Control is 0.81±0.11, in PDHPS5 is 0.27±0.03, t=8.61, P=0.001). Western blot analysis showed that four peptides (PDHPS2, PDHPS3, PDHPS4, PDHPS5) can significantly inhibit the protein expressions of COL1A2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.21±0.04, in PDHPS3 is 0.26±0.03, in PDHPS4 is 0.53±0.04, in PDHPS5 is 0.73±0.04, t=31.38, 38.54, 18.88, 11.07 respectively, all P value are less than 0.01). Three peptides (PDHPS2, PDHPS3, PDHPS5) can significantly inhibit the protein expressions of ACTA2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.64±0.05, in PDHPS3 is 0.77±0.06, in PDHPS5 is 0.47±0.07, t=12.08, 6.38, 14.06 respectively, all P value are less than 0.01). In conclusion, the differentially expressed peptides in human hypertrophic scar tissue can affect the function of keloid fibroblasts and collagen expressions to varying degrees. Among them, two peptides (PDHPS2,PDHPS3) significantly inhibit the protein expressions of COL1A2 and ACTA2. The peptide PDHPS5 has high stability, significantly suppresses cell migration, and reduces the protein expressions of COL1A2 and ACTA2, which may provide a new strategy for scar prevention and treatment.

SUMOylation mediates the disassembly of the Smad4 nuclear export complex via RanGAP1 in KELOIDS.

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-ß/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.

Immune cells and associated molecular markers in dermal fibrosis with focus on raised cutaneous scars.

Raised dermal scars including hypertrophic, and keloid scars as well as scalp-associated fibrosing Folliculitis Keloidalis Nuchae (FKN) are a group of fibrotic raised dermal lesions that mostly occur following cutaneous injury. They are characterized by increased extracellular matrix (ECM) deposition, primarily excessive collagen type 1 production by hyperproliferative fibroblasts. The extent of ECM deposition is thought to be proportional to the severity of local skin inflammation leading to excessive fibrosis of the dermis. Due to a lack of suitable study models, therapy for raised dermal scars remains ill-defined. Immune cells and their associated markers have been strongly associated with dermal fibrosis. Therefore, modulation of the immune system and use of anti-inflammatory cytokines are of potential interest in the management of dermal fibrosis. In this review, we will discuss the importance of immune factors in the pathogenesis of raised dermal scarring. The aim here is to provide an up-to-date comprehensive review of the literature, from PubMed, Scopus, and other relevant search engines in order to describe the known immunological factors associated with raised dermal scarring. The importance of immune cells including mast cells, macrophages, lymphocytes, and relevant molecules such as cytokines, chemokines, and growth factors, antibodies, transcription factors, and other immune-associated molecules as well as tissue lymphoid aggregates identified within raised dermal scars will be presented. A growing body of evidence points to a shift from proinflammatory Th1 response to regulatory/anti-inflammatory Th2 response being associated with the development of fibrogenesis in raised dermal scarring. In summary, a better understanding of immune cells and associated molecular markers in dermal fibrosis will likely enable future development of potential immune-modulated therapeutic, diagnostic, and theranostic targets in raised dermal scarring.

Evidence of inter- and intra-keloid heterogeneity through analysis of dermal fibroblasts: A new insight in deciphering keloid physiopathology.

Keloid scars are hypertrophic and proliferating pathological scars extending beyond the initial lesion and without tendency to regression. Usually, keloids are considered and treated as a single entity but clinical observations suggest heterogeneity in keloid morphologies with distinction of superficial/extensive and nodular entities. Within a keloid, heterogeneity could also be detected between superficial and deep dermis or centre and periphery. Focusing on fibroblasts as main actors of keloid formation, we aimed at evaluating intra- and inter-keloid fibroblast heterogeneity by analysing their gene expression and functional capacities (proliferation, migration, traction forces), in order to improve our understanding of keloid pathogenesis. Fibroblasts were obtained from centre, periphery, papillary and reticular dermis from extensive or nodular keloids and were compared to control fibroblasts from healthy skin. Transcriptional profiling of fibroblasts identified a total of 834 differentially expressed genes between nodular and extensive keloids. Quantification of ECM-associated gene expression by RT-qPCR brought evidence that central reticular fibroblasts of nodular keloids are the population which synthesize higher levels of mature collagens, TGFß, HIF1α and αSMA as compared to control skin, suggesting that this central deep region is the nucleus of ECM production with a centrifuge extension in keloids. Although no significant variations were found for basal proliferation, migration of peripheral fibroblasts from extensive keloids was higher than that of central ones and from nodular cells. Moreover, these peripheral fibroblasts from extensive keloids exhibited higher traction forces than central cells, control fibroblasts and nodular ones. Altogether, studying fibroblast features demonstrate keloid heterogeneity, leading to a better understanding of keloid pathophysiology and treatment adaptation.