Abstract
Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.
Highlights
One of the significant causes of blindness worldwide is reduced corneal transparency due to improper fibrosis after corneal injury or inflammation[1]
The transforming growth factor (TGF)-β2–treated keratocytes were treated with 10 nM suberoylanilidehydroxamic acid (SAHA) alone and in combination with 10 ng/ml insulin-like growth factor (IGF)-1 (Fig. 1a, SAHA, combo SAHA), which showed the lowest levels of Smad[3] nuclearization, at 6.6% and 9.7%, respectively; i.e., significantly lower than both the TGF-β2 positive control and the negative control (p < 0.001)
The TGF-β2–treated keratocytes were treated with 5 ng/ml halofuginone alone and in combination with 10 ng/ml IGF-1 (Fig. 1a, HAL, combo HAL), which showed similar levels of Smad[3] nuclearization to the negative control (22.6%, 22.9%, respectively), and were significantly lower than the TGF-β2 positive control (p < 0.001)
Summary
One of the significant causes of blindness worldwide is reduced corneal transparency due to improper fibrosis after corneal injury or inflammation[1]. After IGF-1 receptor activation, the keratocytes transdifferentiate to corneal-wound-type fibroblasts[11], which express Thy-1 at the cell surface[12] and produce proteoglycans and other components of the ECM In this way, some of the intracellular transparency diminishes due to down-regulation of the crystallins[11], this scenario yields an ECM that is similar in composition to the normal cornea[11,13]. TGF-βfacilitates differentiation of the keratocytes into myofibroblasts[18,19], which are hallmarked by the formation of alpha-smooth muscle actin (α-SMA)[20] fibers These cells secrete ECM materials that form non-transparent scaffolds of scar collagen[21,22]. Smad[7] is expressed, which acts as an antagonist of this TGF-βsignal transduction, as part of a negative-feedback loop[31,33]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
