Fibroblast Dynamics in Keloid Pathogenesis: Unraveling Cellular Crosstalk and Novel Therapeutic Targets

Cancer associated fibroblasts (CAFs) express a variety of cytokines and factors that activate and contribute to pathways that favor tumorigenesis. CAFs disrupt normal cellular functions, such as cell cycle regulation and cell death including apoptosis, or signal certain types of cells to abnormally activate pro-tumor activities. In addition, when it is abnormally activated, it is characterized by increased collagen deposition and more contractility. Likewise, This pattern of abnormal activation is often observed in keloid disease. Keloid constitutes an abnormal fibroproliferative wound healing response with excessive collagen deposition causing abnormal wound healing. To better understand the CAF subtypes with abnormal activation and cancer-promoting properties and the subtypes of keloid fibroblasts with similar activation properties, it is essential to find the master transcription factors (TFs) that determine their identity. To identify functionally important TFs in abnormal CAFs and keloid fibroblasts, we analyzed H3K27ac ChIP-seq of CAFs and keloid fibroblasts and observed that Runx2 significantly enriched the two types of abnormal fibroblasts.To investigate the biological function of Runx2 , we established CAFs from colon cancer patients and performed ShRNA-mediated knockdown of the RUNX2 gene. We confirmed that knockdown of RUNX2 in CAFs suppressed the migration or invasion of cancer cells. Additionally, using a gel contraction assay, we confirmed that fibroblast contractility was significantly reduced by RUNX2 knockdown. Similarly, in keloid fibroblasts, it was confirmed that the migration and gel contraception abilities of fibroblasts were significantly reduced following knockdown of RUNX2 [A1] gene. Last but not least, expression of ECM elements, such as collagens and fibronectin, were significantly as a result of the knockdown of the RUNX2 gene in both abnormally activated fibroblasts. To conclude, we believe that Runx2 acts as a key TF that maintains the contractility and ECM remodeling ability of abnormally activated fibroblasts. Our findings indicate that Runx2 is a attractive therapeutic target not only for pro-tumorigenesis in the cancer microenvironment, but also for keloid fibroblasts. Citation Format: Keun-Woo Lee, Insuk Sohn, Seok-Hyung Kim, So-Young Yeo. Runx2 as a key transcription factor regulating contractility and extracellular matrix remodeling in abnormally activated fibroblasts: Implications for tumorigenesis and keloid pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB320.

Green Tea Extract and (−)-Epigallocatechin-3-Gallate Inhibit Mast Cell-Stimulated Type I Collagen Expression in Keloid Fibroblasts via Blocking PI-3K/Akt Signaling Pathways

Multi-omics analysis to explore the molecular mechanisms related to keloid.

Keloids represent a dysregulated response to cutaneous wounding that results in disfiguring scars. Unique to humans, keloids are characterized by an accumulation of extracellular matrix components. The underlying molecular mechanisms of keloid pathogenesis, however, remain largely uncharacterized. Similarly, cellular signaling mechanisms, which may indicate inherent differences in the way keloid fibroblasts and normal human dermal fibroblasts interact with extracellular matrix or other cells, have not been investigated. As part of a fundamental assessment of cellular response to injury in keloid fibroblasts, phosphorylation studies were performed using three different keloid (n = 3) and normal human dermal (n = 3) fibroblast cell lines. These studies were undertaken to elucidate whether keloid and normal human dermal fibroblasts exhibit different tyrosine kinase activity. Initially, distinct tyrosine phosphorylation patterns of keloid and normal human dermal fibroblasts were demonstrated. Next, the phosphorylation patterns were correlated with known molecules that may be important to keloid pathogenesis. On the basis of molecular weight, it was hypothesized that the highly phosphorylated bands seen in keloid fibroblasts represented epidermal growth factor receptor (EGFR); discoidin domain receptor 1 (DDR1); and Shc, an adaptor protein known to bind many tyrosine kinases, including EGFR and DDR1. Individual immunoblotting using EGFR, DDR1, and Shc antibodies revealed greater expression in keloid fibroblasts compared with normal human dermal fibroblasts. These data substantiate for the first time the finding of greater phosphorylation by the above-mentioned molecules, which may be important in keloid pathogenesis.

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Keloids, partially considered as benign tumors, represent the most extreme example of cutaneous scarring that uniquely afflicts humans as a pathological response to wound healing. It is characterized by excessive deposition of collagen and other extracellular matrix components by dermal fibroblasts. Upon cutaneous injury, cocktails of chemokines, cytokines and growth factors are secreted temporally and spatially to direct appropriate responses from neutrophils, macrophages, keratinocytes and fibroblasts to facilitate normal wound healing. Signal transducer and activator of transcription 3 (Stat3) is an oncogene and a latent transcription factor activated by various cytokines and growth factors. We investigated the possible role of Stat3 in keloid scar pathogenesis by examining skin tissue and cultured fibroblasts from keloid-scarred patients. We observed enhanced expression and phosphorylation of Stat3 in keloid scar tissue, and in cultured keloid fibroblasts (KFs) in vitro. Increased activation of Janus kinase (Jak)2, but not Jak1, was detected in KFs, and suppression of Jak2 by its inhibitor repressed Stat3 Y705 phosphorylation. Inhibition of Stat3 expression and phosphorylation by short interfering RNA or Cucurbitacin I resulted in the loss of collagen production, impaired proliferation and delayed cell migration in KFs. We show, for the first time, a role of Stat3 in keloid pathogenesis. Inhibitors of Stat3 may be useful therapeutic strategies for the prospective treatment of keloid scars.

Hypertrophic scars and keloids are fibroproliferative disorders caused by abnormal wound healing. Their exact cause has not been found, but abnormalities during the wound healing process including inflammatory, immune, genetic, and other factors are thought to predispose an individual to excessive scarring. In the present study, we performed transcriptome analysis of established keloid cell lines (KEL FIB), focusing on gene expression analysis and fusion gene detection for the first time. For gene expression analysis, fragments per kilobase per million map read values were calculated, which were validated by real-time PCR and immunohistochemistry. Fusion genes were predicted by transcriptome sequence, and validated by Sanger sequence and G-banding. As a result, GPM6A was shown in the expression analysis to be upregulated in KEL FIB compared with normal fibroblasts. The GPM6A upregulation in KEL FIB was confirmed by real-time PCR, and GPM6A messenger ribonucleic acid expression was consistently significantly elevated in the tissues of hypertrophic scar and keloid compared to normal skin. Immunohistochemistry also revealed that the number of fibroblast-like spindle-shaped cells positive for GPM6A was significantly increased in keloidal tissues. GPM6A inhibition by small interfering ribonucleic acid significantly reduced the number of KEL FIB. On the other hand, although we hypothesized that fusion genes are involved in the pathogenesis of keloids, the transcriptome analysis could not prove the presence of fusion genes in KEL FIB. Taken together, GPM6A upregulation may have an inducible effect on cell proliferation in keloidal fibroblasts. GPM6A can be a novel therapeutic target in hypertrophic scars and keloids. The inflammatory nature may be more prominent in the pathogenesis of keloids, rather than being skin tumors, as proposed by Ogawa et al. Future studies using several cell lines will be required.

Keloids are pathological scars characterized by excessive collagen deposition that occurs during wound healing after skin injury. Keloid fibroblasts (KF) and keloid keratinocytes (KK) are key contributors to keloid pathogenesis. Although adipose-derived mesenchymal stromal cells (ASCs) have been investigated for keloid therapy, their therapeutic potential and underlying mechanisms require further elucidation. This study aimed to characterize the therapeutic potential of ASCs for human keloid management. Molecular profiles associated with keloid pathogenesis were characterized through integrative analyses, including gene expression profiling, functional annotation, protein-protein interaction mapping, and hub gene identification. Single-cell RNA sequencing (scRNA-seq) was used to identify ASC subpopulations with inhibitory effects on keloid development. The therapeutic efficacy of these subpopulations was subsequently assessed in a miniature pig model of hypertrophic scar. Upregulation of hub genes such as NOG and IL6 was strongly associated with KF formation, whereas increased expression of APP and NOTCH1 was implicated in KK development. Functional scRNA-seq analysis identified ASC subpopulations capable of inhibiting the development of KF, KK, or both through molecular interactions with these hub genes. Administration of porcine ASCs enriched in the identified inhibitory subpopulations effectively prevented hypertrophic scar formation in the miniature pig model. This study delineated key molecular signatures underlying keloid formation and identified ASC subpopulations with targeted inhibitory activity against pathological cell types involved in keloid development. These findings support the potential application of ASC-based interventions for prophylaxis and treatment of hypertrophic scarring in humans.

Aberrant fibroblast migration in response to fibrogenic peptides plays a significant role in keloid pathogenesis. Angiotensin II (Ang II) is an octapeptide hormone recently implicated as a mediator of organ fibrosis and cutaneous repair. Ang II promotes cell migration but its role in keloid fibroblast phenotypic behavior has not been studied. We investigated Ang II signaling in keloid fibroblast behavior as a potential mechanism of disease. Primary human keloid fibroblasts were stimulated to migrate in the presence of Ang II and Ang II receptor 1 (AT₁), Ang II receptor 2 (AT₂) or nonmuscle myosin II (NMM II) antagonists. Keloid and the surrounding normal dermis were immunostained for NMM IIA, NMM IIB, AT₂ and AT₁ expression. Primary human keloid fibroblasts were stimulated to migrate with Ang II and the increased migration was inhibited by the AT₁ antagonist EMD66684, but not the AT₂ antagonist PD123319. Inhibition of the promigratory motor protein NMM II by addition of the specific NMM II antagonist blebbistatin inhibited Ang II-stimulated migration. Ang II stimulation of NMM II protein expression was prevented by AT₁ blockade but not by AT₂ antagonists. Immunostaining demonstrated increased NMM IIA, NMM IIB and AT₁ expression in keloid fibroblasts compared with scant staining in normal surrounding dermis. AT₂ immunostaining was absent in keloid and normal human dermal fibroblasts. These results indicate that Ang II mediates keloid fibroblast migration and possibly pathogenesis through AT₁ activation and upregulation of NMM II.

CTHRC1+ fibroblasts are stimulated by macrophage-secreted SPP1 to induce excessive collagen deposition in keloids.

Knockdown of fibronectin extra domain B suppresses TGF-β1-mediated cell proliferation and collagen deposition in keloid fibroblasts via AKT/ERK signaling pathway

Keloids are characterized as an "over-exuberant" healing response resulting in a disproportionate extracellular matrix (ECM) accumulation and tissue fibrosis. In view of the integral role of inflammation and cytokines in the healing response, it is logical to assume that they may play a part in orchestrating the pathology of this "abnormal" healing process. Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine involved in activation of signaling events and transcriptional programs, such as NFkappaB. This study attempts to determine the difference in NFkappaB and its related genes expression and DNA binding activity between keloid and normal skin fibroblasts. Three keloid and normal skin tissues (NSk) and their derived fibroblasts were used to determine NFkappaB signaling pathway expression using specific cDNA microarrays, Western blot analysis and immunohistochemistry. Electrophoretic mobility gel shift assay (EMSA) was used to assess NFkappaB-binding activity, all assays were performed in the presence and absence of TNF-alpha. TNF-alpha up-regulated 15% of NFkappaB signal pathway related genes in keloid fibroblast compared to normal skin. At the protein level, keloid fibroblasts and tissues showed higher basal levels of TNF- receptor-associated factors-TRAF1, TRAF2-TNF-alpha, inhibitor of apoptosis (c-IAP-1), and NFkappaB, compared with NSk. Keloid fibroblasts showed a constitutive increase in NFkappaB-binding activity in comparison to NSk both with and without TNF-alpha treatment. NFkappaB and its targeted genes, especially the antiapoptotic genes, could play a role in keloid pathogenesis; targeting NFkappaB could help in developing therapeutic interventions for the treatment of keloid scarring.

Keloid scars are fibroproliferative disorders characterized by the accumulation of extracellular matrix (ECM) components resulting in a fibrotic condition. Several ECM promoters are regulated by Sp1. Thus, our aim was to investigate the role of Sp1 in keloid pathogenesis and investigate the antiproliferative and antifibrotic effects of Wp631 and mitoxantrone, potent inhibitors of Sp1-activated transcription. An elevated level of Sp1 was observed in tissue extracts obtained from keloid tissue. Serum stimulation elevated Sp1 levels in keloid fibroblasts (KF). Under coculture conditions Sp1 seemed to be downregulated. Wp631 and mitoxanthrone in serum growth factors resulted in a reduced expression of ECM components in KF. Both Wp631 and mitoxanthrone were also able to inhibit the proliferation of normal and keloid keratinocytes and fibroblasts significantly. As Wp631 seems to be potent in downregulating the ECM components in KF and also inhibiting the proliferation of these cells it could be explored as a possible therapeutic agent in the treatment of keloids.

Fibronectin Gene Transcription Is Enhanced in Abnormal Wound Healing

Quercetin inhibits fibronectin production by keloid-derived fibroblasts. implication for the treatment of excessive scars