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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 (A,C) and collagen (B,D) in cells on the bottom of hydrated bovine collagen matrices, following 4 days of culture in PDGF BB with or without the Rac1 inhibitor NSC23766 (50 μM). Cells develop stress fibers and compact the collagen on their apical surface following Rac1 inhibition.
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jfb-06-00222-f003: Maximum intensity projections of F-actin (A,C) and collagen (B,D) in cells on the bottom of hydrated bovine collagen matrices, following 4 days of culture in PDGF BB with or without the Rac1 inhibitor NSC23766 (50 μM). Cells develop stress fibers and compact the collagen on their apical surface following Rac1 inhibition.

Mentions: Addition of the Rac1 inhibitor NSC23766 had no visible impact on the effects of basal media (Figure 2F), IGF (Figure 2H), FGF (Figure 2I) or TGFβ1 (Figure 2J) on cells at the bottom of 3-D collagen matrices. In contrast, for cells cultured in PDGF BB, inhibition of Rac induced a switch from a dendritic morphology to a spread morphology, and the development of stress fiber bundles in most cells (Figure 2G, arrows). To further assess this fibroblastic transformation, cell-induced matrix reorganization was assessed by imaging the collagen on the apical surface of the cells. Whereas minimal compaction of collagen fibrils was observed surrounding cells cultured in PDGF alone (Figure 3A,B), significant cell-induced compaction of the collagen was observed when NSC23766 was added to inhibit Rac activation (Figure 3C,D).


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 (A,C) and collagen (B,D) in cells on the bottom of hydrated bovine collagen matrices, following 4 days of culture in PDGF BB with or without the Rac1 inhibitor NSC23766 (50 μM). Cells develop stress fibers and compact the collagen on their apical surface following Rac1 inhibition.
© Copyright Policy
Related In: Results  -  Collection

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

jfb-06-00222-f003: Maximum intensity projections of F-actin (A,C) and collagen (B,D) in cells on the bottom of hydrated bovine collagen matrices, following 4 days of culture in PDGF BB with or without the Rac1 inhibitor NSC23766 (50 μM). Cells develop stress fibers and compact the collagen on their apical surface following Rac1 inhibition.
Mentions: Addition of the Rac1 inhibitor NSC23766 had no visible impact on the effects of basal media (Figure 2F), IGF (Figure 2H), FGF (Figure 2I) or TGFβ1 (Figure 2J) on cells at the bottom of 3-D collagen matrices. In contrast, for cells cultured in PDGF BB, inhibition of Rac induced a switch from a dendritic morphology to a spread morphology, and the development of stress fiber bundles in most cells (Figure 2G, arrows). To further assess this fibroblastic transformation, cell-induced matrix reorganization was assessed by imaging the collagen on the apical surface of the cells. Whereas minimal compaction of collagen fibrils was observed surrounding cells cultured in PDGF alone (Figure 3A,B), significant cell-induced compaction of the collagen was observed when NSC23766 was added to inhibit Rac activation (Figure 3C,D).

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