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.

Revisiting roles of mast cells and neural cells in keloid: exploring their connection to disease activity.

Background: Mast cells (MCs) and neural cells (NCs) are important in a keloid microenvironment. They might contribute to fibrosis and pain sensation within the keloid. However, their involvement in pathological excessive scarring has not been adequately explored. Objectives: To elucidate roles of MCs and NCs in keloid pathogenesis and their correlation with disease activity. Methods: Keloid samples from chest and back regions were analyzed. Single-cell RNA sequencing (scRNA-seq) was conducted for six active keloids (AK) samples, four inactive keloids (IK) samples, and three mature scar (MS) samples from patients with keloids. Results: The scRNA-seq analysis demonstrated notable enrichment of MCs, lymphocytes, and macrophages in AKs, which exhibited continuous growth at the excision site when compared to IK and MS samples (P = 0.042). Expression levels of marker genes associated with activated and degranulated MCs, including FCER1G, BTK, and GATA2, were specifically elevated in keloid lesions. Notably, MCs within AK lesions exhibited elevated expression of genes such as NTRK1, S1PR1, and S1PR2 associated with neuropeptide receptors. Neural progenitor cell and non-myelinating Schwann cell (nmSC) genes were highly expressed in keloids, whereas myelinating Schwann cell (mSC) genes were specific to MS samples. Conclusions: scRNA-seq analyses of AK, IK, and MS samples unveiled substantial microenvironmental heterogeneity. Such heterogeneity might be linked to disease activity. These findings suggest the potential contribution of MCs and NCs to keloid pathogenesis. Histopathological and molecular features observed in AK and IK samples provide valuable insights into the mechanisms underlying pain and pruritus in keloid lesions.

Integrated Analysis of Single-Cell and Spatial Transcriptomics in Keloids: Highlights on Fibrovascular Interactions in Keloid Pathogenesis.

Studies on the etiopathogenesis of keloids mostly have focused on fibroblasts and their dysfunction. In this study, two cutting-edge technologies, single-cell RNA sequencing and spatial transcriptomics, were applied to uncover the underlying pathophysiology of keloids. Keloid tissue samples and normal skin control data were analyzed as well as those of patient-matched keloid and normal mature scar. Single-cell RNA sequencing revealed cellular heterogenicity such as fibroblasts, endothelial cells (ECs), and myofibroblasts within the keloids. Spatial transcriptomics results showed that disease-associated fibroblasts were enriched in the deeper keloid areas, mostly located around the spots with endothelial transcripts. Mesenchymal activation was observed in keloid ECs, characterized by dysregulation of TGF-ß/SMAD signaling. Colocalization of mesenchymal and vascular markers through multiplex immunofluorescence suggested mesenchymal activation of keloid ECs. Cell‒cell interaction analysis identified a significant network between keloid fibroblasts and ECs, and this cellular crosstalk was supported by colocalization analysis of spatial transcriptomics. This study depicted the cellular landscape of keloids at a single-cell resolution as well as the integration of single-cell and valuable spatial data of keloids using spatial transcriptomics and multiplex immunofluorescence technologies. Our findings suggested a potential role of fibrovascular communication and mesenchymal activation of ECs that might be involved in the keloid pathogenesis.

Comparative Spatial Transcriptomic and Single-Cell Analyses of Human Nail Units and Hair Follicles Show Transcriptional Similarities between the Onychodermis and Follicular Dermal Papilla.

The nail unit and hair follicle are both hard keratin-producing organs that share various biological features. In this study, we used digital spatial profiling and single-cell RNA sequencing to define a spatially resolved expression profile of the human nail unit and hair follicle. Our approach showed the presence of a nail-specific mesenchymal population called onychofibroblasts within the onychodermis. Onychodermis and follicular dermal papilla both expressed Wnt and bone morphogenetic protein signaling molecules. In addition, nail matrix epithelium and hair matrix showed very similar expressions profile, including the expression of hard keratins and HOXC13, a transcriptional regulator of the hair shaft. Integration of single-cell RNA sequencing and digital spatial profiling data through computational deconvolution methods estimated epithelial and mesenchymal cell abundance in the nail- and hair-specific regions of interest and revealed close transcriptional similarity between these major skin appendages. To analyze the function of bone morphogenetic proteins in nail differentiation, we treated cultured human nail matrix keratinocytes with BMP5, which are highly expressed by onychofibroblasts. We observed increased expressions of hard keratin and its regulator genes such as HOXC13. Collectively, our data suggest that onychodermis is the counterpart of dermal papilla and that BMP5 in onychofibroblasts plays a key role in the differentiation of nail matrix keratinocytes.