RNA interference targeting transforming growth factor-beta type II receptor suppresses ocular inflammation and fibrosis.
OBJECTIVE
Transforming growth factor-beta(TGF-beta) is an important mediator of wound healing responses. In the eye, TGF-beta activity has been implicated in causing corneal haze after laser surgery and subconjunctival scarring following glaucoma surgery. The purpose of the study was to determine whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-beta (TbetaRII) could be used to suppress the TGF-beta action.


METHODS
TbetaRII specific siRNAs designed from the human gene sequence were transfected into cultured human corneal fibroblasts. The protein and transcript levels of the receptor were determined by immunofluorescence, western blotting, and real time PCR. Immunofluorescence and immunoblotting were carried out to examine fibronectin assembly. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. In addition, the in vivo effects of TbetaRII siRNA were evaluated in a mouse model of ocular inflammation and fibrosis generated by subconjunctival injection of phosphate buffered saline and latex beads. Mouse TbetaRII siRNA was introduced into experimental eyes. Cellularity on tissue sections was evaluated after staining with hematoxylin and eosin. Collagen deposition was visualized by picrosirius red staining.


RESULTS
TbetaRII siRNAs abrogated the receptor transcript and protein expression in cultured corneal fibroblasts. The gene knockdown inhibited fibronectin assembly and retarded cell migration. In the mouse model, introduction of TbetaRII specific siRNA significantly reduced the inflammatory response and matrix deposition.


CONCLUSIONS
TbetaRII specific siRNAs were efficacious both in vitro and in vivo in knocking down the TGF-beta action. A direct application of siRNA into eyes to downregulate TbetaRII expression may provide a novel therapy for preventing ocular inflammation and scarring.
Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, 60612, USA.
Hiroshi
H
Nakamura
Shahid S
SS
Siddiqui
Xiang
X
Shen
Asrar B
AB
Malik
Jose S
JS
Pulido
Nalin M
NM
Kumar
Beatrice Y J T
BY
Yue
D000293:Adolescent; D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003228:Conjunctiva / Q000473:pathology*; D003231:Conjunctivitis / Q000473:pathology / Q000517:prevention & control*; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D004195:Disease Models, Animal; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005355:Fibrosis / Q000517:prevention & control; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D017346:Protein Serine-Threonine Kinases; D034622:RNA Interference / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism
D005353:Fibronectins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II