LOSS OF MFAP5 AND ITS EFFECT ON SKIN HOMEOSTASIS AND WOUND HEALING

MFAP5, also known as microfibrillar-associated glycoprotein-2 (MAGP2), may influence parameters of skin wound healing related to scar formation. To further elucidate its role in skin wound healing, we assessed skin wound repair in Mfap5-/- mice. Loss of MFAP5 significantly reduced wound closure rates and angiogenesis while enhancing neutrophil and macrophage influx into wounds. Loss of MFAP5 also reduced the deposition of total and mature collagen in uninjured normal skin (NS), but not in wounds. Furthermore, NS dermis of Mfap5-/- mice was thinner without any reduction in tensile strength. Single-cell RNA-sequencing of NS and wounds from Mfap5+/+ and Mfap5-/- mice revealed two fibroblast subclusters that express MFAP5 more highly than other subclusters. Enrichment analysis of the differentially expressed genes (DEGs) in these two subclusters suggests these fibroblasts engage in extracellular matrix (ECM) deposition and angiogenesis. Mfap5+/+ and Mfap5-/- fibroblasts also exhibit transcriptomic differences throughout in vivo wound healing, though as healing progressed, fewer differences were evident. To examine the direct effect of MFAP5 on fibroblasts outside of the wound space, fibroblasts were isolated from Mfap5+/+ and Mfap5-/- mice for in vitro analysis. MRNA-sequencing of Mfap5+/+ and Mfap5-/- fibroblasts found genes involved in cellular migration and proliferation, ECM synthesis, and angiogenesis to be downregulated in Mfap5-/- fibroblasts vs Mfap5+/+ fibroblasts. Functionally, Mfap5-/- fibroblasts exhibited reduced migration, contractility, proliferation, and ECM deposition. Our findings indicate that MFAP5 is a multifunctional glycoprotein in skin wound healing as it promotes angiogenesis and collagen deposition, inhibits inflammatory cell influx, and promotes pro-scarring fibroblast behavior. Overall design: The effects that the loss of MFAP5 has on the fibroblast transcriptome may be obscured during in vivo healing, as a myriad of cell-cell, cell-ECM, and cell-cytokine interactions may compensate for the loss of MFAP5. Thus, to assess intrinsic changes to the fibroblast transcriptome and infer mechanisms for the functional deficits observed following the loss of MFAP5, we performed mRNA-sequencing on Mfap5+/+ and Mfap5-/- fibroblasts cultured in vitro. Differential expression analysis of the raw count data generated by Novogene (Novogene America) was performed with the DESeq2 package (v1.20.0) in R [51]. Only genes with an average raw count of at least 10 across all samples were analyzed. P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate (FDR). Genes with an adjusted p-value (p.adj) < 0.05 were assigned as being differentially expressed after DESeq2 analysis. In this study, we assessed differentially regulated genes in Mfap5+/+ and Mfap5-/- fibroblasts at baseline using the following parameters: p.adj < 0.01 and log (fold change) > 0.25 or log(foldchange) < -0.25. Genes with a negative log(foldchange) were considered upregulated in Mfap5+/+ fibroblasts, while genes with a positive log(fold change) were considered upregulated in Mfap5-/- fibroblasts.