Fibromodulin-deficiency alters temporospatial expression patterns of transforming growth factor-β ligands and receptors during adult mouse skin wound healing

Abstract

Fibromodulin (FMOD) is a small leucine-rich proteoglycan required for scarless fetal cutaneous wound repair. Interestingly, increased FMOD levels have been correlated with decreased transforming growth factor (TGF)-β1 expression in multiple fetal and adult rodent models. Our previous studies demonstrated that FMOD-deficiency in adult animals results in delayed wound closure and increased scar size accompanied by loose package collagen fiber networks with increased fibril diameter. In addition, we found that FMOD modulates in vitro expression and activities of TGF-β ligands in an isoform-specific manner. In this study, temporospatial expression profiles of TGF-β ligands and receptors in FMOD-null and wild-type (WT) mice were compared by immunohistochemical staining and quantitative reverse transcriptase-polymerase chain reaction using a full-thickness, primary intention wound closure model. During the inflammatory stage, elevated inflammatory infiltration accompanied by increased type I TGF-β receptor levels in individual inflammatory cells was observed in FMOD-null wounds. This increased inflammation was correlated with accelerated epithelial migration during the proliferative stage. On the other hand, significantly more robust expression of TGF-β3 and TGF-β receptors in FMOD-null wounds during the proliferative stage was associated with delayed dermal cell migration and proliferation, which led to postponed granulation tissue formation and wound closure and increased scar size. Compared with WT controls, expression of TGF-β ligands and receptors by FMOD-null dermal cells was markedly reduced during the remodeling stage, which may have contributed to the declined collagen synthesis capability and unordinary collagen architecture. Taken together, this study demonstrates that a single missing gene, FMOD, leads to conspicuous alternations in TGF-β ligand and receptor expression at all stages of wound repair in various cell types. Therefore, FMOD critically coordinates temporospatial distribution of TGF-β ligands and receptors in vivo, suggesting that FMOD modulates TGF-β bioactivity in a complex way beyond simple physical binding to promote proper wound healing.

Figure 1. Hematoxylin and eosin (H&E) staining of wounded WT and FMOD-null adult mice skin.

(A) At day 0.5 post-injury, minimal (score: 1) inflammatory infiltrate was present at the wound edge of WT mice (upper right), while significant (score: 3) inflammatory infiltrate was detected at the wound base. On the other hand, moderate (score: 2) and significant inflammatory infiltrates were observed at the wound edge (lower right) and base of FMOD-null mice, respectively. (B) At day 1 post-injury, moderate and significant inflammatory infiltrates were seen at the wound edge (upper right) and base of WT mice, respectively. Meanwhile, high (score: 4) inflammatory infiltrate was observed at both the wound edge (lower-right) and base of FMOD-null mice. (C) Relative inflammatory infiltration (median, 25–75% quartile, min, max) in 8 animals per genotype (2 randomly chosen wound edge fields and 2 randomly chosen wound bed fields per animal; N = 32) was semi-quantitatively evaluated by three blinded reviewers. Yellow arrowheads: representative inflammatory cells (not all inflammatory cells are indicated); yellow circles: randomly chosen fields for inflammatory infiltration evaluation. Bar =  100 µm. *, significant difference determined by the Mann-Whitney test.

Figure 2. Immunohistochemical (IHC) staining of wounded WT and FMOD-null adult mice skin.

(A) TGF-β1, (B) TGF-β2, (C) TGF-β3, (D) TβRI, (E) TβRII, and (F) TβRIII. Inserts show low magnification view. Red arrowheads: inflammatory cells; open black triangles: epidermis at wound edge; solid black triangles: migrating epidermal tongues; blue arrows: dermal fibroblasts. Bar  = 100 µm.

Figure 3. Quantification of dermal protein expression (A, C, E; N = 9) and total wound RNA (B, D, F; N = 4) expression of TGF-β ligands.

(A, B) TGF-β1, (C, D) TGF-β2, and (E, F) TGF-β3. RNA expression is normalized to unwounded WT skin (blue dotted line). *, P<0.05.

Figure 4. Quantification of dermal protein expression (A, C, E; N = 9) and total wound RNA (B, D, F; N = 4) expression of TGF-β receptors.

(A, B) TβRI, (C, D) TβRII, and (E, F) TβRIII. RNA expression is normalized to unwounded WT skin (blue dotted line). *, P<0.05.

Figure 5. In vitro migration assay of primary dermal fibroblasts derived from adult WT and FMOD-null mice skin.

Cell migration was documented by photographs taken immediately after scraping, as well as 24(A). Migration was quantified by measuring the average wound gap between the wound edges before and after the treatment, and calculated as: Cell migration (%)  =  (Gap_0h-Gap_24h)/Gap_0h ×100% (B). 100 pM TGF-β3 was used to inhibit dermal fibroblast migration in vitro, while 10 µM TβRI-specific inhibitor SB-431542 was used to block TβRI-mediated signal transduction. Bar  = 200 µm. N = 6; *, P<0.05. Red stars indicate the significance that resulted from FMOD-deficiency; green stars indicate the significance that resulted from TGF-β3 application; and blue stars indicate the significance that resulted from SA-431542 blockage of TβRI.

Figure 6. A brief summary of the major effects of FMOD-deficiency on adult mouse wound healing.