Dataset Information

Fibroblastic Transformation of Corneal Keratocytes by Rac Inhibition is Modulated by Extracellular Matrix Structure and Stiffness.

ABSTRACT: The goal of this study was to investigate how alterations in extracellular matrix (ECM) biophysical properties modulate corneal keratocyte phenotypes in response to specific wound healing cytokines and Rho GTPases. Rabbit corneal keratocytes were plated within standard collagen matrices (2.5 mg/mL) or compressed collagen matrices (~100 mg/mL) and cultured in serum-free media, PDGF BB, IGF, FGF2 or TGF?1, with or without the Rac1 inhibitor NSC23766 and/or the Rho kinase inhibitor Y-27632. After 1 to 4 days, cells were labeled for F-actin and imaged using confocal microscopy. Keratocytes within standard collagen matrices (which are highly compliant) maintained a dendritic phenotype following culture in serum-free media, PDGF, IGF and FGF, but developed stress fibers in TGF?1. Keratocytes within compressed collagen (which has high stiffness and low porosity) maintained a dendritic phenotype following culture in serum-free media, PDGF and IGF, but developed stress fibers in both FGF and TGF?1. The Rac inhibitor had no significant impact on growth factor responses in compliant matrices. Within compressed collagen matrices however, the Rac inhibitor induced fibroblastic transformation in serum-free media, PDGF and IGF. Fibroblast and myofibroblast transformation was blocked by Rho kinase inhibition. Overall, keratocyte growth factor responses appear to be regulated by both the interplay between Rho and Rac signaling, and the structural and mechanical properties of the ECM.

SUBMITTER: Petroll WM

PROVIDER: S-EPMC4493509 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Fibroblastic Transformation of Corneal Keratocytes by Rac Inhibition is Modulated by Extracellular Matrix Structure and Stiffness.

Petroll W Matthew WM Lakshman Neema N

Journal of functional biomaterials 20150414 2

The goal of this study was to investigate how alterations in extracellular matrix (ECM) biophysical properties modulate corneal keratocyte phenotypes in response to specific wound healing cytokines and Rho GTPases. Rabbit corneal keratocytes were plated within standard collagen matrices (2.5 mg/mL) or compressed collagen matrices (~100 mg/mL) and cultured in serum-free media, PDGF BB, IGF, FGF2 or TGFβ1, with or without the Rac1 inhibitor NSC23766 and/or the Rho kinase inhibitor Y-27632. After 1 ...[more]

PMID: 25874856

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Project description:After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-β1 (TGF-β1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-β1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-β1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-β1.

Project description:The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71?kPa) and tissue culture plastic (TCP; > 1?GPa) in media containing 0 or 10?ng/ml TGF?1 for 72?h. Cells were treated with 0.4??M Lat-B or DMSO for 30?min every 24?h for 72?h. RNA was collected from cells and expression of alpha-smooth muscle actin (?-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess ?-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n?=?3) or 25% DMSO (vehicle control, n?=?3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less ?SMA mRNA (P???0.007) for RCF cells grown on 22 and 71?kPa substrates as well as TCP without or with TGF?1, and significantly decreased ?-SMA protein expression in RCFs cultured on the intermediate (22?kPa) stiffness in the absence (P?=?0.028) or presence (P?=?0.018) of TGF?1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P?>?0.05) in the absence or presence of TGF?1, but RCFs grown on stiff hydrogels (71?kPa) had significantly more keratocan mRNA expression versus the 22?kPa hydrogel or TCP (P?<?0.001) without TGF?1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of ?-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.

Dataset Information

Fibroblastic Transformation of Corneal Keratocytes by Rac Inhibition is Modulated by Extracellular Matrix Structure and Stiffness.

Publications

Fibroblastic Transformation of Corneal Keratocytes by Rac Inhibition is Modulated by Extracellular Matrix Structure and Stiffness.

Similar Datasets

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