Single-Cell RNA Sequencing Reveals Pericytes Acquire a Fibrotic Phenotype and Promote Mesenteric Adipose Tissue Fibrosis in Crohn’s Disease

Da Zhang,1,* Anqi Yu,1,* Mengjia He,1,* Qian Zhou,1 Shibo Sun,2 Lan Bai,1 Fang Xie1 1Guangdong Provincial Key Laboratory of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 2Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fang Xie, Guangdong Provincial Key Laboratory of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, People’s Republic of China, Tel +86-020-61642511, Email [email protected]: Creeping fat (CF) in Crohn’s disease (CD) is characterized by hyperplastic mesenteric adipose tissue (MAT) encasing fibrotic intestinal segments, marked by significant extracellular matrix (ECM) remodeling and fibrosis. Pericytes have multipotent differentiation potential, can adopt a fibrotic phenotype, and contribute to pathological ECM deposition. However, the cellular mechanisms underlying CF fibrosis remain unclear. This study aimed to elucidate the cellular origins of CF fibrosis and the involvement of pericytes.Patients and Methods: Histopathological analyses evaluated fibrosis in MAT samples and its correlation with adjacent muscularis propria thickening. Transcriptomic datasets and immunofluorescence confirmed fibrosis-related gene expression differences in MAT. Single-cell RNA sequencing (scRNA-seq) was analyzed to evaluate ECM production across cell types and identify pericyte-specific markers. Cell proportion analyses and in vitro experiments quantified vascular endothelial and mural cell populations. CytoTRACE and pseudotime analyses mapped pericyte differentiation trajectories. Primary human MAT pericytes were isolated and stimulated with transforming growth factor-β 1 (TGF-β 1) to assess fibrotic phenotype transition in vitro.Results: Fibrosis was evident in uninvolved MAT from CD patients (CD-MAT) and CF, with fibrosis severity in CF correlating positively with muscularis propria thickening. Core ECM gene COL3A1 was significantly upregulated in both CD-MAT and CF. CF exhibited increased endothelial and mural cell numbers. STEAP4 was identified as a pericyte-specific marker, with CF tissues showing higher pericyte abundance and enhanced perivascular ECM deposition. Pericytes in CD-MAT and CF adopted a fibrotic phenotype, marked by upregulation of COL3A1 and 18 other ECM genes. Furthermore, primary CD-MAT-derived pericytes treated with TGF-β 1 displayed amplified fibrotic gene expression, confirming their profibrotic potential.Conclusion: Pericytes in CF significantly expand and transition to a fibrotic phenotype, representing a key stromal cell population driving MAT fibrosis. These findings reveal an underrecognized cellular mechanism, highlighting novel therapeutic targets for MAT fibrosis. Keywords: pericytes, creeping fat, fibrosis, Crohn’s disease