CTGF Binds To Growth Factors Of The Cysteine Knot Superfamily And Is Critical For Corneal Wound Healing Through Modulation Of PDGF-B Signaling

Abstract

Purpose: This study aimed to determine whether CTGF had differential affinity to growth factors of the cysteine knot superfamily including vascular endothelial growth factor (VEGF)-A, transforming growth factor (TGF)-β, platelet derived growth factor (PDGF)-B, and bone morphogenic protein (BMP)-4. The modulation of CTGF on PDGF-B signaling was further investigated in the setting of corneal wound closure.

Methods: The binding between CTGF and tested growth factors was quantitatively measured using yeast two-hybrid analysis and surface plasma resonance (SPR). The effects of CTGF on PDGF-B signaling during rabbit corneal fibroblast migration were determined in Western analysis and in vitro migration assay. In addition, conditional deletion of Ctgf gene was induced after intraperitoneal injection with tamoxifen on mice that were homozygous for floxed Ctgf allele and carried an ubc-Cre/Ert2 transgene. Corneas of the resulting conditional knockouts and wild type mice received laser excimer ablation followed by PDGF-BB treatment at wounded sites. Corneal re-epithelialization was assessed by fluorescein staining. The percentage of wound closure was quantified after imaging analysis.

Results: The yeast two-hybrid analysis showed that the affinity to CTGF was VEGF-A>BMP4>PDGF-B>TGF-β. SPR analysis determined that dissociation constant (Kd) for CTGF and VEGF-A interaction was 1.8 nM and Kd for CTGF and PDGF-B binding was 43 nM. In addition, recombinant CTGF protein could enhance PDGF-B binding to PDGFRβ receptor and downstream AKT activation in Western analysis. CTGF and PDGF-B worked in concert to promote rabbit corneal fibroblast migration in vitro. Tropical application of PDGF-BB protein as low as 100 ng was able to accelerate corneal re-epithelialization in control mice. In contrast, deletion of Ctgf gene in conditional knockout mice delayed corneal wound closure even after tropical application of PDGF-BB (100 ng) at 30 hours after laser injury.

Conclusions: CTGF has differential affinity to key regulators of wound healing. Its strong binding to VEGF-A is consistent with previous reports about sequestration of VEGF-A and inhibition of VEGF-A signaling by CTGF for anti-angiogenesis, whereas its modest binding to PDGF-B can potentiate PDGFRβ activation and downstream PDGF-B signaling. CTGF enhances PDGF-B signaling and is critical for corneal re-epithelialization.

Keywords: 543 growth factors/growth factor receptors • 480 cornea: basic science • 519 extracellular matrix