(WHS-K2.04) MICROFIBRIL ASSOCIATED PROTEIN 5 AND THE REGULATION OF SKIN SCAR FORMATION
Thursday, May 16, 2024
9:15 AM – 10:15 AM East Coast USA Time
Background: Studies in our lab have shown that Microfibril Associated Protein 5 (MFAP5, or microfibril-associated glycoprotein2/MAGP2) is upregulated in skin wound healing and modulates fibroblast phenotype. MFAP5, a 25 kD extracellular matrix (ECM) glycoprotein, is linked to fibrosis and angiogenesis in cancers and fibrotic diseases. The aim of this study was to use MFAP5 deficient (Mfap5-/-) mice to investigate MFAP5’s role in wound healing and on fibroblast transcriptome and function. Materials &
Methods: Full-thickness excisional wounds were made on dorsal skin of Mfap5-/- and C57BL/6J control (Mfap5+/+) mice. External wound closure was assessed, and wound tissue was collected during healing to assess key features of wound repair (N=9-10). Wound angiogenesis and inflammatory cell content were assessed by immunofluorescent staining of CD31 or Ly6G and CD68, respectively. Collagen deposition and maturity were assessed by Masson’s Trichrome and Herovici staining, respectively. To examine how loss of MFAP5 affects fibroblast transcriptome and phenotype, skin fibroblasts were isolated from neonatal Mfap5+/+ and Mfap5-/- mice. RNA-sequencing was performed on Mfap5+/+ and Mfap5-/- fibroblasts (N=6). All significantly downregulated genes (padj <0.05) in Mfap5-/- versus Mfap5+/+ fibroblasts underwent gene ontology enrichment analysis and annotated to biological processes (BP). Cellular migration, contractility, and proliferation were compared between Mfap5+/+ and Mfap5-/- fibroblasts (N=14-20). To examine changes to ECM composition, Mfap5+/+ and Mfap5-/- fibroblasts (N=8) were grown to confluency, treated with ascorbic acid, and then immunocytochemistry stained for ECM proteins.
Results: Mfap5-/- mice had significantly reduced rates of skin wound closure and wound angiogenesis (p < 0.05). Mfap5-/- mice also had significantly enhanced inflammatory cell content as compared to Mfap5+/+ mice (p < 0.05). Collagen deposition in Mfap5-/- mice was significantly reduced in normal skin (NS) and at 21 days post-wounding, while only NS of Mfap5-/- mice had significantly altered collagen maturity versus Mfap5+/+ mice (p < 0.05). RNA-sequencing revealed down-regulated BP related to ECM organization, cellular migration, and proliferation. Mfap5-/- fibroblasts had significantly reduced cellular migration, contractility, proliferation, and COL1A2 deposition (p < 0.05).
Conclusions: Our results suggest that MFAP5 is important for wound closure and angiogenesis. MFAP5 is also likely involved in the maturation and organization of collagen and may influence skin wound inflammation. Lastly, our in vitro assays demonstrate that MFAP5 is a regulator of fibroblast characteristics important for scar formation.