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

Petroll WM, Lakshman N - J Funct Biomater (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, UT Southwestern Medical Center, Dallas, TX 75390-9057, USA. matthew.petroll@utsouthwestern.edu.

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 TGFβ1, 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 TGFβ1. 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 TGFβ1. 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.

No MeSH data available.


Related in: MedlinePlus

Maximum intensity projections of F-actin organization in keratocytes within compressed rat tail collagen matrices, following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM) and/or the Rho kinase inhibitor Y-27632 (10 μM). Keratocytes within compressed collagen (which has high stiffness and low porosity) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B) and IGF (C), but developed stress fibers (arrows) in both FGF (D) and TGFβ1 (E). Rac inhibition induced fibroblastic transformation in basal media (F), PDGF (G) and IGF (H). Fibroblast and myofibroblast transformation was blocked by Rho kinase inhibition (K–O).
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jfb-06-00222-f005: Maximum intensity projections of F-actin organization in keratocytes within compressed rat tail collagen matrices, following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM) and/or the Rho kinase inhibitor Y-27632 (10 μM). Keratocytes within compressed collagen (which has high stiffness and low porosity) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B) and IGF (C), but developed stress fibers (arrows) in both FGF (D) and TGFβ1 (E). Rac inhibition induced fibroblastic transformation in basal media (F), PDGF (G) and IGF (H). Fibroblast and myofibroblast transformation was blocked by Rho kinase inhibition (K–O).

Mentions: In order to further investigate the role of ECM structure and stiffness on keratocyte responses, we plated cells within compressed collagen matrices, which provide a much stiffer 3-D culture environment than standard collagen matrices [30]. Keratocytes in compressed collagen matrices cultured in basal media (Figure 5A) or IGF (Figure 5C) generally had a stellate morphology with dendritic processes and did not develop stress fibers, as previously reported by us [27,32]. Culture in PDGF BB induced cell elongation (note different scale bars in Figure 5B, G and L), and stress fibers were only rarely observed (Figure 5B). In contrast, FGF2 induced a switch to a spread morphology, and prominent stress fiber bundles were consistently observed (Figure 5D, arrows) [27]. TGFβ also induced loss of dendritic processes and stress fiber formation (Figure 5E, arrows) [27]. Thus the growth factors induced a similar response in compressed collagen matrices as they did in cells on the bottom of uncompressed collagen ECM.


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

Petroll WM, Lakshman N - J Funct Biomater (2015)

Maximum intensity projections of F-actin organization in keratocytes within compressed rat tail collagen matrices, following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM) and/or the Rho kinase inhibitor Y-27632 (10 μM). Keratocytes within compressed collagen (which has high stiffness and low porosity) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B) and IGF (C), but developed stress fibers (arrows) in both FGF (D) and TGFβ1 (E). Rac inhibition induced fibroblastic transformation in basal media (F), PDGF (G) and IGF (H). Fibroblast and myofibroblast transformation was blocked by Rho kinase inhibition (K–O).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493509&req=5

jfb-06-00222-f005: Maximum intensity projections of F-actin organization in keratocytes within compressed rat tail collagen matrices, following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM) and/or the Rho kinase inhibitor Y-27632 (10 μM). Keratocytes within compressed collagen (which has high stiffness and low porosity) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B) and IGF (C), but developed stress fibers (arrows) in both FGF (D) and TGFβ1 (E). Rac inhibition induced fibroblastic transformation in basal media (F), PDGF (G) and IGF (H). Fibroblast and myofibroblast transformation was blocked by Rho kinase inhibition (K–O).
Mentions: In order to further investigate the role of ECM structure and stiffness on keratocyte responses, we plated cells within compressed collagen matrices, which provide a much stiffer 3-D culture environment than standard collagen matrices [30]. Keratocytes in compressed collagen matrices cultured in basal media (Figure 5A) or IGF (Figure 5C) generally had a stellate morphology with dendritic processes and did not develop stress fibers, as previously reported by us [27,32]. Culture in PDGF BB induced cell elongation (note different scale bars in Figure 5B, G and L), and stress fibers were only rarely observed (Figure 5B). In contrast, FGF2 induced a switch to a spread morphology, and prominent stress fiber bundles were consistently observed (Figure 5D, arrows) [27]. TGFβ also induced loss of dendritic processes and stress fiber formation (Figure 5E, arrows) [27]. Thus the growth factors induced a similar response in compressed collagen matrices as they did in cells on the bottom of uncompressed collagen ECM.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, UT Southwestern Medical Center, Dallas, TX 75390-9057, USA. matthew.petroll@utsouthwestern.edu.

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 TGFβ1, 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 TGFβ1. 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 TGFβ1. 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.

No MeSH data available.


Related in: MedlinePlus