While much is known regarding the biochemical and morphologic characteristics of corneal keratocytes and fibroblasts, less is known about how the mechanical behavior of these cells is regulated. In this study, we assessed the effects of several growth factors on the keratocyte mechanical phenotype using a standard 3-D fibrillar collagen matrix model.43–46
In this model, matrix contraction occurs by compaction of collagen fibrils through the application of cellular forces. Assessment of overall matrix contraction is a standard assay for assessing changes in cell contractility in response to different culture conditions.32
However, such global measurements can be limited, because they do not provide information on the changes in 3-D cell morphology, cytoskeletal organization, or local cell-induced matrix reorganization which reflect the mechanical state of cells. In this study, we performed a comprehensive assessment of the keratocyte mechanical response to IGF, PDGF BB, FGF2, TGFβ1, and TGFβ2, using high magnification 3-D confocal imaging. We then compared these responses to those observed on rigid 2-D substrates or within compressed collagen matrices. These growth factors were selected because they are present in the cornea and/or tear film, and have already been shown to regulate keratocyte proliferation and ECM synthesis in vitro.
Consistent with previous studies, corneal keratocytes maintained in basal, serum-free media had a dendritic morphology in both standard and compressed 3-D matrices, and do not express stress fibers or produce large amounts of matrix reorganization. At higher cell densities, these dendritic processes interconnect, producing a lattice-like network similar to that observed in vivo.29
Dermal fibroblasts form similar dendritic cell processes when contractility is blocked by inhibiting myosin II,47
and serum-cultured corneal fibroblasts develop dendritic processes in response to Rho kinase inhibition.31
Thus the dendritic cell morphology appears to be a hallmark of cells in a low tension environment.48
IGF induced elongation of keratocyte dendritic processes without producing significant collagen matrix reorganization in 3-D culture. A similar morphology and cytoskeletal organization was observed on a collagen-coated 2-D substrate, consistent with previous observations.14
Thus overall, keratocytes cultured in IGF maintained a quiescent mechanical phenotype. IGF has been shown by others to increase keratocyte proliferation and to stimulate synthesis of ECM components resembling normal corneal stroma, and also to stimulate network formation.14,15,49
Thus it has been suggested that IGF may be involved in maintenance of normal corneal structure and could contribute to a regenerative wound healing phenotype.14,15
Our data demonstrating keratocytes cultured in IGF maintain a quiescent mechanical phenotype in 3-D culture are consistent with this hypothesis.
PDGF BB also induced keratocyte elongation and formation of dendritic processes in both 2-D and 3-D culture, without producing stress fibers or significant matrix reorganization. PDGF BB has been shown previously to stimulate Rac-induced spreading of dermal and corneal fibroblasts in 3-D collagen matrices, along with significant tractional force generation by extending pseudopodial processes.50,51
However, fibroblasts in those studies were maintained in serum, and thus had a significant basal level of Rho kinase activity. When Rho kinase is inhibited, corneal fibroblasts revert to a dendritic morphology, and only small collagen displacements are observed during PDGF BB-induced cell spreading, consistent with our results in the present study using quiescent corneal keratocytes.50
Like IGF, PDGF stimulates keratocyte proliferation and has been shown to upregulate synthesis of normal stromal ECM.14,15
In addition, PDGF BB has been shown to be a potent stimulator of both corneal keratocyte and dermal fibroblast migration.8,30,52–54
Thus it is interesting to speculate that PDGF BB may contribute to stromal repopulation after injury or surgery through upregulation of both proliferation and migration, without producing fibrotic tissue or generating large forces which can alter corneal shape and transparency.
Interestingly, PDGF AB has been shown to transform corneal keratocytes to a fibroblastic phenotype in 2-D culture, as indicated by the development of stress fibers and focal adhesions14,23
; however this transformation was not observed in response to PDGF BB under any of the conditions used in the present study (including 2-D substrates). PDGF AB has also been shown to stimulate contraction of floating collagen matrices in 3-D culture. However, it should be noted that contraction of floating matrices occurs through a Rho kinase-independent mechanism, and does not require generation of significant cellular forces. In contrast, contraction of attached collagen matrices requires larger forces and has been shown to be Rho kinase-dependent.32
Previous studies indicate that FGF2 induces fibroblastic transformation of keratocytes on 2-D substrates, as indicated by changes in cell morphology and development of stress fibers and focal adhesions.14,23
We observed a similar response in this study when keratocytes were cultured on collagen-coated 2-D substrates or within compressed collagen matrices. However, in standard 3-D matrices, FGF2 appeared to stimulate ruffling of keratocyte processes without inducing major changes in cell morphology or collagen matrix organization. Furthermore, FGF2 did not induce formation of stress fibers in 3-D matrices, even at high cell density. Thus unlike PDGF BB, the response of keratocytes to FGF2 appears to be sensitive to ECM mechanical stiffness and/or ligand density. It is well known that increasing substrate stiffness can facilitate formation of actin stress fibers and focal adhesions in contractile cells.55–57
Both glass and compressed collagen matrices are several orders of magnitude stiffer than a hydrated collagen matrix, and this may underlie the difference in mechanical phenotype induced by FGF2. In the cornea, large shifts in the global distribution of ECM tension can be induced by lacerating injury, penetrating keratoplasty, or refractive surgery.7
In addition, the provisional wound healing matrix is less dense, more disorganized, and more compliant than normal corneal tissue. The response of corneal keratocytes to FGF2 during wound healing could be regulated, in part, by such changes in ECM composition, stress, and stiffness.
Consistent with previous studies, treatment with TGFβ1 and TGFβ2 increased cell contractility within standard 3-D matrices, as indicated by the formation of stress fibers and stimulation of cell-induced ECM reorganization.3,14
Interestingly, these changes were significantly enhanced at higher cell densities. Approximately 20% of cells showed positive labeling for α-SMA localized to the stress fibers at high cell density; in contrast, α-SMA labeling was negative for TGFβ1- and TGFβ2-treated cells plated at low cell density. Both connective tissue growth factor (CTGF) and PDGF have been shown to participate in TGFβ-induced myofibroblast differentiation through an autocrine feedback loop, which would be amplified at higher cell density.23,58
In addition, mechanical cross-talk between cells increases the tension within the matrix at higher cell density, and ECM stiffness is known to upregulate myofibroblast transformation.59
A recent study demonstrated that increased matrix stiffness enhances TGFβ-induced myofibroblast transformation of human tenon fibroblasts in 2-D culture.60
We observed a similar increase in stress fiber formation and myofibroblast transformation of corneal keratocytes on both rigid 2-D substrates and within compressed 3-D collagen matrix. While stress fibers were observed in all cells within compressed ECM irrespective of cell density, the percentage of cells with α-SMA incorporated into stress fibers was greater at higher cell density (approximately 60% versus approximately 20%), suggesting that both mechanical stiffness and autocrine signaling promote myofibroblast transformation. Interestingly, myofibroblasts tend to develop toward the end of the corneal wound repair process when cell density is high and the wound environment is stiffer, suggesting similar processes may be involved during in vivo healing.
We have previously shown that Rho kinase plays a central role in mediating cellular force generation and matrix reorganization by serum cultured corneal fibroblasts in 3-D culture.31
In 2-D culture, Rho kinase has also been shown to mediate fibroblastic transformation of keratocytes in response to FGF2 treatment, and myofibroblast transformation in response to TGFβ.20
In the present study, we demonstrated that Rho kinase is also required for transformation of keratocytes within compressed 3-D matrices. Interestingly, blocking Rho/Rho kinase has been shown to inhibit the decrease in KSPG synthesis normally associated with myofibroblast transformation, suggesting a linkage between increased cell contractility and fibrotic ECM synthesis.20
3-D culture models typically use either bovine dermal collagen (which is pepsinized), or rat tail tendon collagen (which is not pepsinized). Pepsin treatment reductively cleaves cross-link mediating telopeptides from collagen monomers, which alters the structural and mechanical properties of reassembled collagen matrices.36
Nonpepsinized rat tail collagen forms shorter fibrils, and has smaller pores and a higher fiber density compared with pepsin-extracted bovine collagen.36
Recent studies have shown that both the mechanics and protease dependency of migration by certain tumor cell lines is impacted by the type of collagen used.36,61,62
In the present study however, the morphologic and mechanical responses of corneal keratocytes to growth factor treatments were remarkably similar for these two matrix types.
Taken together, the data demonstrate that the keratocyte mechanical phenotypes induced by growth factors can be differentially regulated by ECM structure and/or mechanical properties. Most notably, whereas FGF2 induces a contractile fibroblastic phenotype on rigid 2-D substrate or compressed collagen ECM, a quiescent mechanical phenotype is observed in standard 3-D matrices. Furthermore, while TGFβ stimulates keratocyte contractility and myofibroblast transformation over a range of ECM environments, this transformation appears to be enhanced by both increased substrate stiffness and autocrine signaling. Keratocytes cultured in IGF or PDGF BB consistently maintain a quiescent mechanical phenotype over a range of matrix environments, and may thus have the potential to modulate migration, proliferation, and/or ECM synthesis during wound healing, without generating large contractile forces which can disrupt normal corneal structure and transparency.