Dataset Information

Growth factor regulation of corneal keratocyte differentiation and migration in compressed collagen matrices.

ABSTRACT:

Purpose

To evaluate a novel 3D culture model of the corneal stroma and apply it to investigate how key wound-healing growth factors regulate the mechanics of corneal keratocyte migration.

Methods

Rabbit corneal keratocytes were seeded within collagen matrices that were compacted using external compression. Six-millimeter-diameter buttons were then incubated in media supplemented with 10% FBS, TGFbeta1, TGFbeta2, platelet-derived growth factor (PDGF), or no growth factor (control). After 1, 3, or 7 days, matrices were labeled with phalloidin and a nucleic acid dye, and were imaged using laser confocal microscopy. To study cell migration, buttons were nested within acellular uncompressed outer collagen matrices before growth factor stimulation.

Results

Corneal keratocytes in basal media within compressed matrices had a broad, convoluted cell body and thin dendritic processes. In contrast, cells in 10% FBS developed a bipolar fibroblastic morphology. Treatment with TGFbeta induced the formation of stress fibers expressing alpha-smooth muscle actin, suggesting myofibroblast transformation. PDGF induced keratocyte elongation without inducing stress fiber formation. Both 10% FBS and PDGF stimulated significant keratocyte migration through the uncompressed outer matrix, but 10% FBS produced more cell-induced collagen matrix reorganization. TGFbeta induced the smallest increase in migration and the greatest matrix reorganization.

Conclusions

Corneal keratocytes are able to differentiate normally and respond to growth factors within compressed collagen matrices, which provide a high-stiffness, 3D environment, similar to native stromal tissue. In addition, nesting these matrices provides a unique platform for investigating the mechanics of keratocyte migration after exposure to specific wound-healing cytokines.

SUBMITTER: Kim A

PROVIDER: S-EPMC2819331 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.