Adipocyte-Conditioned Medium Induces a Transcriptionally Distinct Myofibroblast Phenotype with Enhanced Remodeling and Reduced Fibrosis

Introduction: : Human dermal fibroblasts (HDFs) are key regulators of tissue repair, responsible for regulating extracellular matrix (ECM) remodeling, and supporting wound closure. Typically, fibroblast activation is driven by TGF-β1 signaling, which promotes fibroblast differentiation into contractile myofibroblasts that repair the wound site. Chronically active myofibroblasts can also lead to excessive ECM deposition, matrix stiffening, and fibrosis. Previous studies, including our own, have shown that adipose-derived factors can influence fibroblast behavior. Our studies indicate that adipocyte conditioned medium (ACM) promotes the conversion of HDFs into highly contractile, alpha-smooth muscle actin positive myofibroblasts through a TGF-β1 independent mechanism. Here, we used RNA-Seq to compare the transcriptome of ACM-induced myofibroblasts to TGF-β1-induced myofibroblasts, focusing on the expression of ECM remodeling markers and typical myofibroblast contractile genes.

Materials and

Methods: : Adipocyte conditioned medium was generated by culturing 3D adipocyte spheroids in differentiation medium (PDM-2, Lonza) for 10 days, followed by a 48-hour incubation in Rooster CollectTM-EV medium, a protein-free, low-particle formulation designed to enable clean collection of conditioned media and minimize interference from extraneous proteins as a treatment medium. Human dermal fibroblasts (HDFs) were cultured in DMEM with 10% FBS for 48 hours, then treated for an additional 48 hours under the following conditions: (1) ACM, (2) Rooster Collect with 1 ng/mL TGF-β1 and 50 μg/mL ascorbate-2-phosphate (A2P) (positive control), (3) Rooster Collect alone (control) (n = 5 per culture condition). Total RNA was extracted using the Qiagen RNeasy Mini Kit with on-column DNase digestion (Zymo RNA clean & Concentrator-5 Kit). RNA libraries were prepared using poly(A) enrichment and sequenced on the Element Aviti24 platform to generate paired-end reads (2 × 75 bp). Sequencing quality was assessed using FastQC and MultiQC; adapter trimming was performed with Flexbar, and rRNA contamination was removed using SortMeRNA. Clean reads were aligned to the human transcriptome (GRCh38 reference) using HISAT2, and gene-level counts were quantified with featureCounts. Differential expression and enrichment analyses were conducted using DESeq2 and clusterProfiler.

Results, Conclusions, and Discussions:: Principal component analysis (PCA) revealed clear differences among control, positive control, and ACM exposed HDFs, with PC1 (88% of the variation) indicating that major changes to the transcriptome were due to media treatment, while PC2 (9% of the variation) reflected smaller biological variability between the groups (Fig. 1A). Both the ACM and TGF-β1 groups differed significantly compared to the control group; however, ACM and TGF-β1 fibroblasts also clustered independently from each other, signifying that the two treatments triggered different transcriptional programs. Next, we examined fibrosis- and ECM-related gene expression based on differentially expressed genes identified by DESeq2 through pairwise comparisons between treatment conditions (Fig. 1B). Fibrosis-related genes such as MYL9 (myosin light chain), TAGLN (transgelin), and PALLD (palladin) were elevated in ACM-treated fibroblasts compared to controls, and at levels comparable to those in TGF-β1-treated cells, consistent with our prior work that showed ACM can functionally induce myofibroblast activation. However, classical contractile markers, such as ACTA2 (α-SMA) and VIM (vimentin), were surprisingly lower in the ACM group compared to both TGF-β1-treated cells and controls. In addition, ECM remodeling markers TNC (tenascin C) and PTX3 (pentraxin-3) were significantly upregulated in ACM-treated fibroblasts, with expression levels even higher than those in TGF-β1-treated cells. Gene Ontology (GO) and KEGG pathway enrichment analysis further revealed that ACM selectively enriched biological processes related to focal adhesion, cytoskeletal organization, and epithelial morphogenesis. These pathways were associated with cell migration, proliferation, and matrix interactions, key features of tissue repair. In contrast, TGF-β1 treatment enriched profibrotic pathways including ECM organization, contractility, and fibrosis progression.

These results clearly show that ACM induces a transcriptionally distinct myofibroblast phenotype compared to TGF-β1 induced myofibroblasts. ACM appears to promote ECM remodeling and enhance migratory potential while avoiding the same level of fibrotic gene expression seen in TGF-β1 exposed HDFs, which suggests adipocyte secreted factors could favor a more balanced, wound-healing fibroblast state. These findings provide new insights into adipose-fibroblast interactions and highlight the therapeutic potential of ACM-derived factors in modulating fibroblast behavior, which may offer new strategies for promoting healthy tissue repair.

Acknowledgements and/or References (Optional):: Support for this work was provided by the National Institutes of Health, NIGMS R01GM145626. Adipose was supplied by the Tissue Procurement Core at the University of Iowa which is supported by an award from NIH (NCI award number P30CA086862) and the University of Iowa Carver College of Medicine.