Molecular subtypes of vascular endothelial cell differentiation in keloid by single-cell sequencing

To the Editor: Keloid is a fibroproliferative disorder characterized by invasive growth beyond wound margins, causing significant physical and psychosocial morbidity. Persistent inflammation and aberrant extracellular matrix deposition by activated fibroblasts and myofibroblasts are central to keloid pathogenesis. Increasing evidence highlights the pivotal role of vascular endothelial cells (ECs) in disease progression.[1] Features of pathological angiogenesis in keloids include disorganized microvasculature, luminal flattening, and functional impairments that paradoxically induce local hypoxia despite high blood flow. This dysfunctional vasculature increases vascular permeability, promoting perivascular accumulation of inflammatory cells and mediators, thereby amplifying inflammation and fibrotic signaling. Notably, clinical associations between endothelial dysfunction—such as that observed in hypertension—and increased keloid susceptibility underscore the central role of ECs in disease predisposition. These findings position vascular ECs not only as contributors to the profibrotic microenvironment but also as potential diagnostic and prognostic indicators. Thus, assessing endothelial function holds significant clinical promise for risk prediction, molecular classification, and the development of precision interventions in keloid management. This study integrated microarray, single-cell RNA sequencing, and bulk RNA sequencing data to reconstruct EC subpopulations and their putative de novo differentiation trajectories in keloids. By combining multidimensional bioinformatic analysis with experimental validation, we established a novel molecular classification system with potential implications for prognostic assessment and precision therapeutic development. To further validate the clinical relevance of this classification framework, we performed comprehensive histopathological and immunohistochemical analyses on discarded keloid tissue specimens obtained from 19 patients undergoing day surgery, along with their matched clinical data, following approval by the Ethics Committee of Changhai Hospital, Shanghai (approval number: CHEC2023-088) and after obtaining written informed consent from all participating patients. The study design is summarized in Supplementary Figure 1, https://links.lww.com/CM9/C755. A total of 13,162 ECs were classified into six subpopulations (EC 1–6) based on the similarity of the gene expression profiles across individual cells. Significant differences in subpopulation distribution were observed between normal scars and keloids. EC 5 (highly expressing von willebrand factor, [VWF]) and EC 2 were predominantly present in normal scars, whereas EC 1 and EC 6 (highly expressing collagen type I alpha 2 [COL1A2]) each constituted more than 50% of the endothelial population in keloids. Notably, EC 3 and EC 4 exhibited similar proportional distributions in both normal scars and keloids, suggesting that they may fulfill relatively stable functional roles across these two tissue states. Robust intercellular communication was observed among the six EC clusters. EC 3 occupied a central position in the communication network and primarily mediated signaling to other cell types, with chemokine ligand 12 (CXCL12) identified as a key ligand [Supplementary Figure 2, https://links.lww.com/CM9/C755]. Pseudotime trajectory analysis reconstructed the differentiation order of the six EC subpopulations. EC 1 resides at the origin of the differentiation trajectory and progressively transitions toward EC 2; EC 2 bifurcates into two distinct differentiation branches, both of which initially share EC 3 as a common progenitor. As illustrated in Supplementary Figure 3, https://links.lww.com/CM9/C755, the upper branch ultimately differentiates into EC 6, whereas the lower branch terminally differentiates into EC 4. Key differentially expressed genes (gap junction protein alpha 4 [GJA4], hes family bHLH transcription factor 4 [HES4], lysosomal associated protein transmembrane 5 [LAPTM5], podoplanin [PDPN], retinol binding protein 4 [RBP4], and stathmin 1 [STMN1]) were found to play decisive roles in determining cell fate [Supplementary Figures 3 and 4, https://links.lww.com/CM9/C755]. Integrated analysis of single-cell and microarray transcriptomic data identified differentially expressed endothelial fate-related genes (DEEFRGs) that show significantly higher expression levels in normal scar tissues compared to keloids, suggesting potential diagnostic utility [Supplementary Figure 5, https://links.lww.com/CM9/C755]. Based on non-parametric tests and Pearson correlation analysis, we constructed a co-expression regulatory network. In this network, nodes were color-coded to highlight key genes governing cellular differentiation into five distinct trajectories: More than one-third of the genes (shown in green) were identified as critical determinants driving endothelial cell transition toward cell state 1; additionally, 7 genes (purple), 1 gene (brown), 3 genes (yellow), and 1 gene (gray) were found to specifically promote differentiation toward cell states 2, 3, 4, and 5, respectively. Furthermore, the genes extracted from this regulatory network were subjected to subsequent clustering analysis. According to the consensus clustering plot, these genes were robustly partitioned into three distinct clusters. A heatmap illustrated the expression levels of these fate-related genes across the three clusters, as well as across different time points, sex groups, and age groups. This visualization revealed significant associations between the three consensus clusters and the aforementioned clinicopathological features, further underscoring a strong correlation between endothelial cell fate determination and consensus-based clustering: Fate-determining genes were highly expressed in consensus cluster 3, whereas their expression levels in consensus clusters 1 and 2 were comparable but significantly lower than in cluster 3. Most of these genes—such as phospholipid phosphatase 3 (PLPP3), bone marrow stromal cell antigen 2 (BST2), caveolae-associated protein 2 (Cavin-2), and DIX domain-containing 1 (DIXDC1)—are linked to angiogenesis and endothelial cell (EC) function: PLPP3 enhances cell–cell adhesion and endothelial barrier integrity;[2] BST2 promotes endothelial progenitor cell adhesion;[3] Cavin-2 supports EC function via eNOS regulation;[4] and DIXDC1 mediates VEGF–Wnt/β-catenin crosstalk to coordinate vascular development.[5] The high expression of these genes is associated with enhanced angiogenesis and greater vascular stability. This supports our hypothesis that higher endothelial maturity—stronger angiogenic capacity and more stable EC function—is associated with better prognosis in patients with keloid [Supplementary Figure 6, https://links.lww.com/CM9/C755]. Chi-squared tests further confirmed significant differences among the three consensus clusters with respect to both time of diagnosis (P