Keratocyte mechanobiology

Abstract

In vivo, corneal keratocytes reside within a complex 3D extracellular matrix (ECM) consisting of highly aligned collagen lamellae, growth factors, and other extracellular matrix components, and are subjected to various mechanical stimuli during developmental morphogenesis, fluctuations in intraocular pressure, and wound healing. The process by which keratocytes convert changes in mechanical stimuli (e.g. local topography, applied force, ECM stiffness) into biochemical signaling is known as mechanotransduction. Activation of the various mechanotransductive pathways can produce changes in cell migration, proliferation, and differentiation. Here we review how corneal keratocytes respond to and integrate different biochemical and biophysical factors. We first highlight how growth factors and other cytokines regulate the activity of Rho GTPases, cytoskeletal remodeling, and ultimately the mechanical phenotype of keratocytes. We then discuss how changes in the mechanical properties of the ECM have been shown to regulate keratocyte behavior in sophisticated 2D and 3D experimental models of the corneal microenvironment. Finally, we discuss how ECM topography and protein composition can modulate cell phenotypes, and review the different methods of fabricating in vitro mimics of corneal ECM topography, novel approaches for examining topographical effects in vivo, and the impact of different ECM glycoproteins and proteoglycans on keratocyte behavior.

Keywords: Cell mechanics; Corneal keratocytes; Corneal stroma; Extracellular matrix; Mechanobiology.

Figure 1.

FEM strain maps generated using ANSYS, showing regions of matrix tension and compression surrounding a human corneal fibroblast expressing GFP-α-actinin within a 3-D collagen matrix. A. Cell-induced ECM deformation is observed following culture in media containing 10% fetal bovine serum (S+). Note that the ECM is under tension at the end of the cell, and under compression at the base of the pseudopodial process. B. The magnitudes of these deformations are reduced when the cell is switched to Y-27632 (10 μM). C. Strain on the matrix is reestablished after switching back to S+. Strain is shown relative to the “relaxed” matrix configuration determined by treating cells with Cytochalasin D and TritonX-100. Bar on right shows scale for color contour strain maps in dimensionless units ΔL/L. From (Vishwanath et al., 2003)

Figure 2.

Corneal fibroblast spreading in response PDGF, which activates Rac. A–C. Cell-induced displacement and realignment of collagen fibrils was observed during PDGF-induced spreading. Tracking of the ECM displacements showed minimal collagen displacement prior to the addition of PDGF (B, red tracks, crosses mark position at time 0:00). However, following addition of PDGF, the matrix in front of the cell was pulled inward by the extending pseudopodial processes (C), resulting in compression of the ECM (yellow arrows). From (Petroll et al., 2008)

Figure 3.

Maximum intensity projections of fluorescently labeled f-actin (green) and confocal reflection images of collagen fibrils (red) near the interface between the inner and outer matrices of nested constructs. A) Following culture in 10% fetal bovine serum, migrating cells developed a bipolar morphology with occasional stress fibers along the cell body. Collagen fibrils were compacted and aligned parallel to the long axis of pseudopodia. B) Following culture in TGFβ1 (10 ng/ml), cells developed a broad morphology and intracellular stress fibers were observed. Collagen fibrils were compacted both around and between the cells. C) Migrating cells in PDGF BB (50 ng/ml) were more elongated and had branching processes. Collagen fibrils remained more randomly aligned around the cells. From (Kim et al., 2010)

Figure 4.

Substratum stiffness modulates the mechanical phenotype of corneal keratocytes in the presence of TGF-β1. Representative traction stress maps of keratocytes cultured on either (A) 1 kPa or (B) 10 kPa polyacrylamide gels. Adapted from (Maruri et al., In Press).

Figure 5.

Fibroblast response to ECM compression using small microneedle. A-D. Human corneal fibroblast following 2 days of culture in media containing 10% fetal bovine, inside a 3-D collagen matrix. Needle was inserted axially into the matrix above the cell (A) without inducing changes in cell behavior (B). Pushing on the ECM towards the leading edge of a cell induced rapid cellular contraction and ECM compression along the cell body (C, arrows). This initial contraction was followed by re-spreading and tractional force generation (D, red tracks; crosses mark position immediately after needle push). E-H. Fibroblast response to ECM compression adjacent to cell body. Human corneal fibroblast 1 day after plating inside collagen matrix. Pushing small microneedles toward the side of the cell had no significant effect on cell morphology or tractional force generation. From (Petroll and Ma, 2008)

Figure 6.

Effect of anisotropically ordered grooves and ridges on the orientation of primary rabbit corneal keratocytes, fibroblasts and myofibroblasts cultured on collagen-coated surfaces with (A-C) planar surfaces; (D-F) a 400-nm pitch size pattern; (G-I) a 1200-nm pitch size pattern; and (J-L) a 4000-nm pitch size pattern. Double-headed arrows indicate the direction of grooves and ridges on the patterned surfaces. Keratocytes were cultured in serum-free DMEM, and 10% FBS and 10 ng/mL TGF-β1 were added to the culture medium for induction of the fibroblast and myofibroblast phenotype, respectively. Intracellular actin fibers were labeled with rhodamine-conjugated phalloidin. Scale bars, 100 μm. From (Pot et al., 2010), with permission.

Figure 7.

The effect of electrospun fiber alignment on the behavior of human corneal stromal stem cells (hCSSCs). Scanning electron micrographs of (a) aligned electrospun poly(ester urethane) urea (PEUU) fibers; (d) random PEUU fibers; and (g) PEUU cast film. Confocal laser-scanning micrographs of hCSSCs on (b, c) aligned fibers, (e, f) random fibers, and (g, h) planar films at day 1 (b, e, h) and day 3 (c, f, i). Fluorescent labeling was with the cell viability marker Calcein AM. Fibril diameter = 165 ± 55 nm. From (Wu et al., 2012), with permission.

Figure 8.

Cell patterning during wound repopulation following freeze injury on aligned collagen substrates under different culture conditions. Each panel is a montage of F-actin images collected four days after injury. “W” marks the center of the original wound area, and red lines mark the approximate location of the original wound edge. The aligned collagen fibrils are patterned vertically. The concentration of PDGF BB was 50 ng/ml and the concentration of TGFβ1 was 5 ng/ml. From (Kivanany et al., 2018b)

Figure 9.

Anterior stroma in the regenerative region of the stroma 90 days after PRK, showing overlay of native collagen labeled with DTAF (green), forward scatter SHG of lamellae (blue), and cellular F-actin labeling (red). Note the high correlation in alignment of cells and both native and newly formed collagen lamellae. From (Kivanany et al., 2018a)

Figure 10.

Fibroblasts migrating through fibrin create fibronectin tracks. Nested matrix constructs were cultured for 48h in PDGF-containing media to stimulate cell migration. Corneal fibroblasts form an interconnected network as they migrate into the outer fibrin matrix (top part of image, white line delineates edge of inner matrix). This network contained significant fibronectin. As shown by arrows, fibronectin “tracks” were sometimes present in between cells. Scale bar is 100 μm. From (Miron-Mendoza et al., 2017)