Interference with transforming growth factor-beta/ Smad3 signaling results in accelerated healing of wounds in previously irradiated skin

Abstract

Transforming growth factor (TGF)-beta regulates many aspects of wound repair including inflammation, chemotaxis, and deposition of extracellular matrix. We previously showed that epithelialization of incisional wounds is accelerated in mice null for Smad3, a key cytoplasmic mediator of TGF-beta signaling. Here, we investigated the effects of loss of Smad3 on healing of wounds in skin previously exposed to ionizing radiation, in which scarring fibrosis complicates healing. Cutaneous wounds made in Smad3-null mice 6 weeks after irradiation showed decreased wound widths, enhanced epithelialization, and reduced numbers of neutrophils and myofibroblasts compared to wounds in irradiated wild-type littermates. Differences in breaking strength of wild-type and Smad3-null wounds were not significant. As shown previously for neutrophils, chemotaxis of primary dermal fibroblasts to TGF-beta required Smad3, but differentiation of fibroblasts to myofibroblasts by TGF-beta was independent of Smad3. Previous irradiation-enhanced induction of connective tissue growth factor mRNA in wild-type, but not Smad3-null fibroblasts, suggested that this may contribute to the heightened scarring in irradiated wild-type skin as demonstrated by Picrosirius red staining. Overall, the data suggest that attenuation of Smad3 signaling might improve the healing of wounds in previously irradiated skin commensurate with an inhibition of fibrosis.

Figure 1.

Smad3-null mice are resistant to the injurious effects of ionizing irradiation. A and B: Dramatic differences are apparent in the appearance of skin exposed to 45 Gy of ionizing radiation dependent on the Smad3 genotype at 30 days after irradiation. C and D: Histology of wounds 3 days after making 1-cm incisions in skin irradiated with 30 Gy 6 weeks before wounding as visualized by H&E staining. Blue arrow marks the edge of the wound; green arrow marks the edge of the migrating epithelial tongue. A and C, WT; B and D, KO. E: Phenotypic score of effects of 30-Gy irradiation on flank skin of mice of different Smad3 genotypes. −/− (KO, black bars), +/− (HT, gray bars), and +/+ (WT, striped bars) mice were irradiated with 30 Gy as described. At the indicated time after irradiation, mice were evaluated for a skin reaction according to a phenotypic scale. 1, normal; 2, hair loss; 3, erythema; 4, dry desquamation; 5, <30% moist desquamation; 6, >30% moist desquamation. Values were averaged from 10 KO, 6 HT, and 9 WT mice scoring two irradiated flanks per mouse. Original magnifications, ×50.

Figure 2.

Smad3-null mice show a smaller wound width, accelerated epithelial migration, but reduced bursting strength compared to littermate controls. WT, HT, and KO mice were irradiated with 30 Gy and wounded as described. A–C: Three days after wounding, wounds were excised and samples were prepared as described. Wound width (A), epithelial migration (B), and the percent epithelialization (C) were determined as described in Materials and Methods. n = 9 to 13 wounds for each genotype for all measurements. *, P < 0.05 versus WT. D: Bursting strength of wounds in irradiated (30 Gy, black bars) or sham-irradiated (gray bars) skin was determined 7 days after wounding as described. n = 8 to 18 wounds analyzed.

Figure 3.

The number of neutrophils and myofibroblasts is significantly reduced in wounds of irradiated KO mice compared to WT. Sections from wounds in irradiated flank skin of WT (A, C) and KO (B, D) were stained with rat anti-mouse neutrophil antibody (A, B) or anti-α-SMA to identify myofibroblasts (C, D). Peroxidase, with Carazzi hematoxylin counterstain. E: Dermal fibroblasts prepared from WT or KO neonatal mice were treated with TGF-β1 (5 ng/ml) for 4 days. Cell lysates were subjected to Western blotting using anti-SMA or antibody that recognizes all actin isoforms as described in Materials and Methods. F: Smad3 WT fibroblasts (gray bars) migrate in response to TGF-β, whereas KO fibroblasts (black bars) do not. Results are representative of four experiments in which 3.2 to 3.8 times more WT fibroblasts migrated in response to TGF-β than to vehicle, whereas KO fibroblasts did not migrate in response to TGF-β, but did migrate toward 10% serum. n = 4 to 6 wells/treatment. *, P < 0.0002 versus WT, vehicle treated. ⁺, P < 0.00007 versus KO, vehicle treated. Original magnifications, ×400 (A–D).

Figure 4.

Levels of immunohistochemical staining for TGF-β and CTGF are higher in the granulation tissue of irradiated WT compared to KO wounds 3 days after wounding. Wound cross-sections from nonirradiated (A, E) and irradiated (C, G) WT and KO (B and F, D and H, respectively) mice were stained with antibodies against extracellular TGF-β1 (A–D) or CTGF (E–H) as described. A–D are ×200 magnification photographs taken immediately beneath the epithelium. The arrow marks the edge of the migrating epithelium and S marks the position of the scab. Peroxidase with Carazzi hematoxylin counterstain. E–H are ×400 magnification photographs taken deeper in the dermis at the edge of the wound bed. Red alkaline phosphatase.

Figure 5.

Irradiation augments the effects of TGF-β on autoinduction and induction of CTGF. Dermal fibroblasts prepared from WT or KO neonatal mice were subjected to 5 Gy of γ-irradiation (Irrad) followed 24 hours later by treatment with TGF-β1 as described in Materials and Methods. A: Northern blotting of RNA isolated from these cells using the indicated probe; bottom panel shows ethidium bromide staining of the gel. B and C: Fold-change in TGF-β or CTGF mRNA levels. For each genotype the level of hybridization of the nonirradiated, untreated cells was set to 1 and hybridization levels (normalized to correct for loading differences) were compared to these levels. No irradiation, gray bars; with irradiation, black bars. D: WT (gray bars) or KO (black bars) dermal fibroblasts were irradiated at the indicated doses followed 24 hours later by treatment with TGF-β. Northern blotting was performed on RNA prepared from these cells using a CTGF probe and data normalized to the nonirradiated sample for each genotype. E: Western blotting of lysates from dermal fibroblasts treated as indicated and probed with anti-CTGF or anti-actin.

Figure 6.

Picrosirius-red staining shows similar matrix production in the wound bed of WT and KO mice 5 weeks after wounding, but a reduced scarring phenotype in the dermis at the wound edge of KO mice after irradiation. Skin sections from wounded, nonirradiated (A) and irradiated (C) WT and KO (B and D, respectively) mice were stained with Picrosirius red and photographed under polarized light. The arrow marks the edge of the wound. Inset is a higher magnification of the granulation tissue. Scar index as described in Materials and Methods; three to five wounds analyzed per treatment with two edge measurements, one on either side of the wound bed. *, P < 0.03 versus wound bed of WT Rad, edge of WT Non, and edge of KO Rad. Original magnifications: ×200 (A–D); ×400 (inset).