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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.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.

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 on the bottom of uncompressed hydrated bovine collagen matrices (attached to the rigid glass substrate) following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Keratocytes maintained a dendritic phenotype following culture in basal 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 PDGF (G), but had no effect in serum-free basal media (F) or IGF (H).
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jfb-06-00222-f002: Maximum intensity projections of F-actin organization in keratocytes on the bottom of uncompressed hydrated bovine collagen matrices (attached to the rigid glass substrate) following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Keratocytes maintained a dendritic phenotype following culture in basal 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 PDGF (G), but had no effect in serum-free basal media (F) or IGF (H).

Mentions: To assess the effect of substrate stiffness on the keratocyte response to growth factors and Rac inhibition, we studied cells on the bottom of the 3-D collagen matrices, where the effective stiffness is similar to that of a rigid (glass) substrate. This was accomplished by plating the unpolymerized bovine collagen solution immediately after adding the cell suspension, which allows the cells to sink to the bottom of bovine collagen and attach to the glass substrate (note that normally a 5 min pre-incubation is used to allow partial polymerization of the collagen, which prevents the cells from sinking to the bottom of the collagen solution). The growth factor responses observed on the bottom of 3-D collagen matrices were similar to our previously published results using keratocytes plated on 2-D collagen-coated glass substrates [27]. Specifically, in basal media, keratocytes developed a dendritic morphology with membrane associated F-actin labeling (Figure 2A). Keratocytes exposed to PDGF BB (Figure 2B) and IGF (Figure 2C) maintained this dendritic morphology, with cortical F-actin and no stress fibers. Keratocytes treated with PDGF BB (Figure 2B) were also more elongated. In contrast, FGF2 induced a switch from a dendritic morphology to a spread morphology, and prominent stress fiber bundles were observed in most cells (Figure 2D, arrows). TGFβ induced myofibroblast transformation, as indicated by loss of dendritic processes, development of stress fibers and α-smooth muscle actin labeling (not shown) (Figure 2E).


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 on the bottom of uncompressed hydrated bovine collagen matrices (attached to the rigid glass substrate) following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Keratocytes maintained a dendritic phenotype following culture in basal 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 PDGF (G), but had no effect in serum-free basal media (F) or IGF (H).
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Related In: Results  -  Collection

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

jfb-06-00222-f002: Maximum intensity projections of F-actin organization in keratocytes on the bottom of uncompressed hydrated bovine collagen matrices (attached to the rigid glass substrate) following 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Keratocytes maintained a dendritic phenotype following culture in basal 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 PDGF (G), but had no effect in serum-free basal media (F) or IGF (H).
Mentions: To assess the effect of substrate stiffness on the keratocyte response to growth factors and Rac inhibition, we studied cells on the bottom of the 3-D collagen matrices, where the effective stiffness is similar to that of a rigid (glass) substrate. This was accomplished by plating the unpolymerized bovine collagen solution immediately after adding the cell suspension, which allows the cells to sink to the bottom of bovine collagen and attach to the glass substrate (note that normally a 5 min pre-incubation is used to allow partial polymerization of the collagen, which prevents the cells from sinking to the bottom of the collagen solution). The growth factor responses observed on the bottom of 3-D collagen matrices were similar to our previously published results using keratocytes plated on 2-D collagen-coated glass substrates [27]. Specifically, in basal media, keratocytes developed a dendritic morphology with membrane associated F-actin labeling (Figure 2A). Keratocytes exposed to PDGF BB (Figure 2B) and IGF (Figure 2C) maintained this dendritic morphology, with cortical F-actin and no stress fibers. Keratocytes treated with PDGF BB (Figure 2B) were also more elongated. In contrast, FGF2 induced a switch from a dendritic morphology to a spread morphology, and prominent stress fiber bundles were observed in most cells (Figure 2D, arrows). TGFβ induced myofibroblast transformation, as indicated by loss of dendritic processes, development of stress fibers and α-smooth muscle actin labeling (not shown) (Figure 2E).

Bottom Line: The Rac inhibitor had no significant impact on growth factor responses in compliant matrices.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.

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