Background/Purpose: Metabolic perturbations drive fibroblast-to-myofibroblast reprogramming and tissue fibrosis. Restoring perturbed metabolism might represent a new antifibrotic strategy. Here we explored the capacity of dimethyl-alpha-ketoglutarate (dm-aKG), a core cell metabolite, to regulate myofibroblast function and skin fibrosis in SSc.

Methods: To discover dysregulated genes, pathways, and cell types in SSc skin, we integrated skin transcriptomes of 76 SSc patients and 26 healthy controls (HC) from three different cohorts (GSE:45485, 59785, 9285/32413) using limma and GSEA. We deconvoluted cell types in bulk skin transcriptomes using human skin single-cell RNA-seq data¹. We compared the dysregulated genes in SSc skin with RNA-seq data from cultured TGFβ-treated healthy human dermal fibroblasts (DF) and validated gene dysregulations with RNA-seq data from skin biopsies of the PRESS cohort with 48 early diffuse SSc patients and 33 HC. We studied the effects of dm-aKG on myofibroblast differentiation and pro-fibrotic activation in 2D and 3D cultures of SSc and HC DF treated or not with TGFβ. We analyzed gene (RNA-seq, qPCR) and protein (Western blot, ELISA) expression and contractile (collagen gel contraction), migratory (wound scratch assay, DF spheroids) and invasive (collagen gel invasion in DF spheroids) cell functions. Statistical significance was set at p< 0.05.

Results: Our deconvolution of skin transcriptomes demonstrated an enrichment of specific fibroblast populations in SSc vs. HC skin: COL6A5+COL18A1+ inflammatory fibroblasts and COL11+LAMC3+ myofibroblasts were highly abundant in SSc skin (Fig. 1A). TGFβ increased the expression of several marker genes of the COL11+LAMC3+ myofibroblasts (Fig. 1B). Meanwhile, dm-aKG decreased the TGFβ-induced LAMC3 and COL11 mRNA expression in cultured DF, suggesting that TGFβ might facilitate, while dm-aKG could repress the COL11+LAMC3+ myofibroblast expansion in SSc skin. We uncovered dysregulation of several aKG-associated metabolic pathways in SSc skin transcriptomes, such as mitochondrial respiration and oxidoreductase activity. Dm-aKG repressed the TGFβ-regulated pro-fibrotic responses and myofibroblast differentiation of DF as measured by procollagen-1, fibronectin and aSMA protein reduction. Furthermore, dm-aKG reversed the TGFβ-induced increase of genes regulating cytoskeleton organization (NEXN, CAP2, ANTXR1, LIMCH1, TGM2), cell contraction (ANKRD1, OXTR, FZD2, DYSF, CSRP2) and extracellular matrix dynamics (COL10A1, COL11A1, MMP14, MMP3, ADAMTS4, MATN3, SULF1, HAPLN1). These genes were all upregulated in skin from early diffuse SSc patients (PRESS cohort). Most importantly, functional experiments confirmed the core role of dm-aKG in suppressing contractile, migratory and invasive properties of TGFβ-activated DF.

Conclusion: Dm-aKG reverses the dysregulated expression of myofibroblast-linked gene signatures of the SSc skin and potently represses core myofibroblast functions. Thus, dm-aKG could represent a new therapy to combat skin fibrosis in SSc.

¹He H et al. J Allergy Clin Immunol 2020

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/metabolic-intermediate-dimethyl-alpha-ketoglutarate-is-a-novel-repressor-of-pathogenic-myofibroblast-reprogramming-and-skin-fibrosis-in-systemic-sclerosis/