Inhibition of Smad3 expression in radiation-induced fibrosis using a novel method for topical transcutaneous gene therapy.
OBJECTIVE
To attempt to mitigate the effects of irradiation on murine skin after high-dose radiation using a novel transcutaneous topical delivery system to locally inhibit gene expression with small interfering RNA (siRNA) against Smad3.


METHODS
Laboratory investigation.


METHODS
University laboratory.


METHODS
Twenty-five wild-type C57 mice.


METHODS
In an isolated skin irradiation model, the dorsal skin of C57 wild-type mice was irradiated (45 Gy). Just before irradiation, Smad3 and nonsense siRNA were applied to 2 separate dorsal skin areas and then reapplied weekly. Skin was harvested after 1 and 4 weeks. Smad3 expression were assessed by immunohistochemistry, and collagen deposition and architecture was examined using picrosirius red collagen staining.


METHODS
Epidermal thickness was measured semiquantitatively at 4 weeks. Radiation-induced fibrosis was measured quantitatively via tensiometry. The Young modulus, a measure of cutaneous rigidity inversely related to elasticity, was determined, with normal irradiated skin serving as a control specimen.


RESULTS
Murine skin treated with topical Smad3 siRNA demonstrated effective Smad3 inhibition at 1 week and persistent suppression at 4 weeks. Collagen deposition and epidermal thickness were significantly decreased in skin treated with Smad3 siRNA compared with control irradiated skin. Tensiometry demonstrated decreased tension in Smad3 siRNA-treated skin, with a Young modulus of 9.29 MPa (nonirradiated normal skin, 7.78 MPa) compared with nonsense (control) siRNA-treated skin (14.68 MPa).


CONCLUSIONS
Smad3 expression can be effectively silenced in vivo using a novel topical delivery system. Moreover, cutaneous Smad3 inhibition mitigates radiation-induced changes in tissue elasticity, restoring a near-normal phenotype.
Lee
Judy W
JW
Department of Otolaryngology-Neck Surgery, New York University School of Medicine, 550 First Ave, NBV 5E5, New York, NY 10016, USA. judy.lee@nyumc.org
Tutela
John P
JP
Zoumalan
Richard A
RA
Thanik
Vishal D
VD
Nguyen
Phuong D
PD
Varjabedian
Leon
L
Warren
Stephen M
SM
Saadeh
Pierre B
PB
D000279:Administration, Cutaneous; D000818:Animals; D004195:Disease Models, Animal; D004817:Epidermis / Q000254:growth & development / Q000473:pathology; D005355:Fibrosis / Q000473:pathology; D005782:Gels; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D034741:RNA, Small Interfering / Q000494:pharmacology*; D011832:Radiation Injuries / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D011897:Random Allocation; D012680:Sensitivity and Specificity; D012867:Skin / Q000473:pathology / Q000528:radiation effects; D051900:Smad3 Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016896:Treatment Outcome; D014945:Wound Healing / Q000235:genetics / Q000502:physiology
D005782:Gels; D034741:RNA, Small Interfering; D051900:Smad3 Protein