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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 keratocytes plated within uncompressed hydrated collagen matrices, following 1 or 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Green: F-actin, Red: Collagen (from confocal reflection imaging). Keratocytes within these collagen matrices (which are highly compliant) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B), IGF (C) and FGF (D), but developed stress fibers in TGFβ1 (E,T). Rac inhibition had no significant impact on growth factor responses in compliant matrices (F–T).
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jfb-06-00222-f001: Maximum intensity projections of keratocytes plated within uncompressed hydrated collagen matrices, following 1 or 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Green: F-actin, Red: Collagen (from confocal reflection imaging). Keratocytes within these collagen matrices (which are highly compliant) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B), IGF (C) and FGF (D), but developed stress fibers in TGFβ1 (E,T). Rac inhibition had no significant impact on growth factor responses in compliant matrices (F–T).

Mentions: Consistent with previous studies, keratocytes cultured in serum free media (basal media) within uncompressed rat tail or bovine collagen matrices maintained a broad, convoluted cell body with numerous thin dendritic processes after both 1 day (Figure 1A) and 4 days (not shown) of culture. They had a cortical, membrane associated F-actin organization, with more concentrated labeling near the ends of cell processes. Stress fibers were rarely observed. Keratocytes exposed to PDGF BB (Figure 1B), IGF (Figure 1C) and FGF2 (Figure 1D) maintained this dendritic morphology, with cortical F-actin and no stress fibers. However, keratocytes treated with PDGF BB (Figure 1B) were much more elongated. In contrast, keratocytes treated with TGFβ1 (Figure 1E) lost dendritic processes and developed a more spread morphology. Within the cell body, F-actin filament bundles (stress fibers) were observed.


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 keratocytes plated within uncompressed hydrated collagen matrices, following 1 or 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Green: F-actin, Red: Collagen (from confocal reflection imaging). Keratocytes within these collagen matrices (which are highly compliant) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B), IGF (C) and FGF (D), but developed stress fibers in TGFβ1 (E,T). Rac inhibition had no significant impact on growth factor responses in compliant matrices (F–T).
© Copyright Policy
Related In: Results  -  Collection

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

jfb-06-00222-f001: Maximum intensity projections of keratocytes plated within uncompressed hydrated collagen matrices, following 1 or 4 days of culture with the indicated growth factors, with or without the Rac1 inhibitor NSC23766 (50 μM). Green: F-actin, Red: Collagen (from confocal reflection imaging). Keratocytes within these collagen matrices (which are highly compliant) maintained a dendritic phenotype following culture in basal serum-free media (A), PDGF (B), IGF (C) and FGF (D), but developed stress fibers in TGFβ1 (E,T). Rac inhibition had no significant impact on growth factor responses in compliant matrices (F–T).
Mentions: Consistent with previous studies, keratocytes cultured in serum free media (basal media) within uncompressed rat tail or bovine collagen matrices maintained a broad, convoluted cell body with numerous thin dendritic processes after both 1 day (Figure 1A) and 4 days (not shown) of culture. They had a cortical, membrane associated F-actin organization, with more concentrated labeling near the ends of cell processes. Stress fibers were rarely observed. Keratocytes exposed to PDGF BB (Figure 1B), IGF (Figure 1C) and FGF2 (Figure 1D) maintained this dendritic morphology, with cortical F-actin and no stress fibers. However, keratocytes treated with PDGF BB (Figure 1B) were much more elongated. In contrast, keratocytes treated with TGFβ1 (Figure 1E) lost dendritic processes and developed a more spread morphology. Within the cell body, F-actin filament bundles (stress fibers) were observed.

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