Role of connective tissue growth factor in the pathogenesis of conjunctival scarring in ocular cicatricial pemphigoid.
OBJECTIVE
Conjunctival fibrosis due to excessive accumulation of collagens is an important histologic feature in ocular cicatricial pemphigoid (OCP). Studies have suggested a role of transforming growth factor (TGF)-beta1 in conjunctival fibrosis in patients with OCP. Connective tissue growth factor (CTGF) is an important downstream mediator of TGF-beta1-induced collagen synthesis. CTGF usually acts synergistically with TGF-beta1 during the process of fibrosis in various organs. Hence, studying the mechanism by which CTGF influences TGF-beta1-induced synthesis of collagen in conjunctiva of patients with OCP would provide insight into the mechanism of conjunctival fibrosis in patients with OCP.


METHODS
Biopsy specimens from conjunctiva of 10 patients with OCP and 5 normal subjects, were studied, with immunohistochemistry and real-time PCR, for the expression of CTGF and interstitial type I collagen. Using fibroblasts cultured from conjunctival biopsies we determined the effects of TGF-beta1 on the induction of CTGF and type I collagen by immunostaining, and quantitative real-time PCR. The effects of blocking the bioactivity of TGF-beta1 on the expression of CTGF and type I collagen were determined in TGF-beta1-stimulated fibroblasts, before and after treatment with type II receptor neutralizing antibody.


RESULTS
An increased stromal accumulation of interstitial type I collagen with an increased expression of CTGF was observed in biopsy sections of patients with OCP, compared with the control. By quantitative real-time PCR, a 3.2-fold increase in the expression of CTGF was detected in conjunctival tissues obtained from patients with OCP, compared with control conjunctiva. Fibroblasts isolated from conjunctiva of patients with OCP expressed 4.4-fold more CTGF, compared with control conjunctival fibroblasts, by real-time PCR. When these cultured fibroblasts were immunostained, an increased expression of CTGF was detected in fibroblasts isolated from patients with OCP, compared with control. Furthermore, when conjunctival fibroblasts were treated with TGF-beta1, an approximately ninefold increase in the expression of CTGF and an approximately threefold increase in the expression of type I collagen were detected by real-time PCR, compared with unstimulated fibroblasts. Finally, when antibody to TGF-beta type II receptor was added before TGF-beta1 treatment of these fibroblasts, the expression of type I collagen and CTGF was significantly reduced.


CONCLUSIONS
In the present study, an increased expression of CTGF was recorded in conjunctiva of patients with OCP. TGF-beta1 can induce production of CTGF and type I collagen by fibroblasts obtained from conjunctiva in OCP. This induction of CTGF by TGF-beta1 can be blocked by antibody to TGF-beta type II receptors. The findings lead to the conclusion that CTGF is one of the molecules involved in the pathogenesis of conjunctival fibrosis in patients with OCP.
Razzaque
Mohammed S
MS
Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts 02115, USA. razzaque_ahmed@hms.harvard.edu
Foster
C Stephen
CS
Ahmed
A Razzaque
AR
D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis; D003228:Conjunctiva / Q000378:metabolism / Q000473:pathology*; D003231:Conjunctivitis / Q000209:etiology* / Q000378:metabolism; D055513:Connective Tissue Growth Factor; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005355:Fibrosis; D006801:Humans; D017874:Immediate-Early Proteins / Q000502:physiology*; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D010390:Pemphigoid, Benign Mucous Membrane / Q000209:etiology* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
C526317:CCN2 protein, human; D024042:Collagen Type I; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D018125:Receptors, Transforming Growth Factor beta; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II