Full‐thickness wounding of the mouse tail as a model for delayed wound healing: accelerated wound closure in Smad3 knock‐out mice

Abstract

Experimentally induced wounds in animal models are useful in gaining a better understanding of the cellular and molecular processes of wound healing, and in the initial evaluation of the safety and effectiveness of potential therapeutic agents. However, studying delayed healing has proved difficult in animals, whose wounds heal within a few days. In this report, we describe a novel method for establishing mouse wounds that require up to 3 weeks or more for complete closure, and we show the validity of this model in Smad3 null mice, which are known to display accelerated healing. Full-thickness wounds, measuring 0.3 by 1.0 cm, were made down to fascia on the dorsal aspect of the mouse tail in Smad3 knock-out mice and control littermates, approximately 1 cm distal to the body of the animal. The wounds were left to heal by secondary intention and were assessed histologically by computerized planimetry for wound closure at various times after wounding. The wounds in wild-type mice displayed delayed healing, with full closure occurring between 14 and 25 days after wounding. Complete closure of similar wounds in Smad3 null mice healed 30 percent faster (p < 0.01). By immunostaining for ki67, a marker for proliferation, Smad3 null animals also showed increased proliferation of dermal wound cells by day 4 after wounding. Cultured dermal fibroblasts from Smad3 null mice had increased baseline DNA synthesis and, interestingly, an enhanced response to transforming growth factor-beta1. By Western blot analysis, Smad3 null mice fibroblasts showed a compensatory increase in mitogen-activated protein kinase phosphorylation in response to transforming growth factor-beta1, suggesting that mitogen-activated protein kinase overcompensation together with loss of Smad3 may be involved in the modulation of faster healing. We conclude that this novel tail-wounding model may be useful for studying delayed wound closure.

Smad3 deficiency alters key structural elements of the extracellular matrix and mechanotransduction of wound closure

“…Contrary to expectations, improved healing of incisional wounds is observed in mice lacking either TGF␤1 (2) or Smad3 (3,4), a key cytoplasmic signaling intermediate downstream of the TGF␤ receptors. This finding has been based primarily on more rapid closure of these wounds due to effects of loss of TGF␤͞Smad3 signaling on keratinocyte proliferation and migration, whereas other aspects of wound healing, including granulation tissue formation and inflammation, are reduced in these models (2)(3)(4).…”

Activation of Smad2 but not Smad3 is required to mediate TGF-β signaling during axolotl limb regeneration

“…Various Smad knockout (KO) mice have been generated: Smad2 and Smad4 KOs were lethal, whereas the Smad3 KO was viable (Nomura and Li, 1998;Sirard et al, 1998;Waldrip et al, 1998;Weinstein et al, 1998;Zhu et al, 1998;Datto et al, 1999;Yang et al, 1999b). The phenotype of Smad3 KO mice was interesting in multiple ways: (1) mice were viable and relatively normal (Datto and Wang, 2000); (2) the TGF-β response was somewhat amplified in fibroblasts, which is contrary to what one would expect for an R-Smad KO (Piek et al, 2001); (3) they displayed improved wound healing capacities for various types of injury, which were marked by an increased rate of re-epithelialization and significantly reduced scarring (Ashcroft et al, 1999;Flanders et al, 2003;Falanga et al, 2004); and (4) they had less inflammation following skin wounding (Ashcroft et al, 1999;Yang et al, 1999b;Ashcroft and Roberts, 2000). All of these changes observed in the Smad3 KO mice, as compared with their wild-type littermates, actually display a striking resemblance to the early phases of regeneration in axolotls (Roy and Lévesque, 2006).…”

The integrin αv-TGFβ signaling axis is necessary for epidermal proliferation during cutaneous wound healing

“…9,10,[16][17][18][19] However, the specific TGFb role in keratinocytes during re-epithelialization is unclear, as genetic ablation of TGFb signaling pathway elements accelerates wound re-epithelialization in some settings. [20][21][22][23][24] We previously showed that integrin av is necessary for keratinocyte proliferation in organotypic skin, and thus we hypothesized that av integrins are essential for wound re-epithelialization. 6 Here, we develop a novel, human organotypic wound re-epithelialization assay and show, both in vivo and in vitro, that av integrin function in keratinocytes is required for keratinocyte proliferation and efficient wound reepithelialization.…”