BACKGROUND:
Dermal fibrosis with accompanying extracellular matrix (ECM) deposition is a common sequelae of radiation therapy. TGF-β is implicated in the radiation fibrosis cascade, it plays an essential role in regulating ECM gene expression and is dependent on Smad3, an intracellular transcription factor. The TGF- β pathway may provide novel targets for therapeutic interventions. We hypothesize transcutaneous Smad3 silencing with small interfering RNA (siRNA) will abrogate the downstream effectors of radiation in fibroblasts and mitigate the effects of irradiation on murine skin before (prevention) or after (salvage) high-dose radiation.
METHODS:
C57/BL6 wild-type murine dermal fibroblasts were irradiated with 20Gy, and mRNA was isolated at 0, 6, 12, 24, 48, and 72 hrs post-irradiation. Expression of known regulators (Smad3, IL-13, TNFα) and mediators (COL1A1, TGFβ, MMP-1, MMP-2, TIMP-1) of fibrosis were assessed. Dorsal skin from C57/BL6 mice was isolated and irradiated with 45Gy. Just prior to irradiation (prevention), Smad3 and nonsense siRNA was applied to two separate dorsal skin areas and then reapplied weekly. Skin was harvested weekly for 4 weeks. In a separate group (salvage), 4 weeks after irradiation, mice were treated in two separate areas with Smad3 and nonsense siRNA and skin was harvested 1 week later. Smad3 expression was assessed via immunohistochemistry and Western blot. Lung, liver, and spleen homogenates evaluated off-target Smad3 suppression. Radiation-induced fibrosis was measured quantitatively via tensiometry using Young's modulus. ANOVA/t-test was used to determine significance (p<0.05).
RESULTS:
In vitro: following irradiation, dermal fibroblast mRNA expression of Smad3, IL-13, TGFβ, Col1A1, MMP-2, and TIMP-1 increased steadily with peak expression at 12-24 hours and subsequent decline by 72 hrs. TNFα expression remained elevated throughout. Inhibition of Smad3 both pre- and post-irradiation significantly decreased expression of Col1A1, TGFβ, MMP-2, TIMP-1, with more pronounced effects pre-irradiation. IL-13 and TNFα expression was not affected by Smad3 silencing.
In vivo: skin treated with topical Smad3 siRNA prior to irradiation (prevention) demonstrated effective Smad3 inhibition at 1 week as determined by immunohistochemistry and Western blot with persistent suppression to week 4. Similarly, the salvage group had effective Smad3 silencing following application. No off-target Smad3 suppression was identified. The Smad3 silenced group demonstrated improved elasticity by tensiometry in both the prevention (8.8 MPa) and salvage (9.3 MPa) groups compared to that of irradiated Nonsense siRNA and untreated control (16.5 MPa). Furthermore, irradiated Smad3 silenced skin elasticity was similar to non-irradiated skin (7.8 MPa).
CONCLUSIONS:
We have characterized the early phase expression profiles of the major mediators of radiation-induced fibrosis. Furthermore, Smad3 siRNA is effective at abrogating the elevation of downstream mediators of fibrosis, Col1A1, TGFβ, TIMP-1, and MMP-2 with pre-irradiation treatment displaying the greatest efficacy in vitro. These finding suggest a therapeutic rationale for Smad3 silencing in vivo. We have also demonstrated that Smad3 expression can be effectively silenced in vivo using a novel topical delivery system. Moreover, cutaneous Smad3 inhibition mitigated radiation-induced changes in tissue elasticity restoring a near normal phenotype in both prevention and salvage groups.