IOVS Papers in Press. Published on March 3, 2015 as Manuscript iovs.15-16388
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The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective
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Migration of Corneal Fibroblasts in Different ECM Environments
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Miguel Miron-Mendoza, Eric Graham, Pouriska Kivanany, Jonathan Quiring
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and W. Matthew Petroll
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Short Title: Fibroblast Clustering and Collective Migration
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Corresponding Author:
W. Matthew Petroll, Ph.D. Department of Ophthalmology Southwestern Medical Center 5323 Harry Hines Blvd. Dallas, TX 75390-9057 Phone: 214-648-7216 FAX: 214-648-4507 E-mail: [email protected]
This study was supported in part by NIH R01 EY 013322, NIH P30 EY020799, and an unrestricted grant from Research to Prevent Blindness, Inc., NY, NY.
Keywords:
Extracellular Matrix Corneal Keratocytes Cell Mechanics Thrombin 3-D Culture
1 Copyright 2015 by The Association for Research in Vision and Ophthalmology, Inc.
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ABSTRACT
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Purpose: We previously reported that extracellular matrix composition (fibrin versus collagen)
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modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study,
36
we investigate the role of thrombin and cell contractility in mediating these differences in cell
37
behavior.
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Methods: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen
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and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded
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with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs
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were cultured in serum-free media containing PDGF, with or without thrombin, the Rho kinase
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inhibitor Y-27632 and/or the myosin II inhibitor blebbistatin. 3-dimensional and 4-dimensional
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imaging were used to assess cell mechanical behavior, connectivity and cytoskeletal
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organization.
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Results: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also
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induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices,
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but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when
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Rho kinase was inhibited. In nested matrices, cells always migrated independently through
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collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an
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interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but
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cells continued to migrate collectively.
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Conclusions: The results suggest that while thrombin-induced actomyosin contraction can
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induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce
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collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is
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not required for clustering or collective migration of corneal fibroblasts interacting with fibin.
2
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INTRODUCTION
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The cornea is an optically clear tissue that forms the front surface of the eye, and accounts for
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nearly two-thirds of its refractive power. The corneal stroma, which makes up 90% of corneal
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thickness, is a highly ordered structure with lamellae consisting of uniformly thin collagen fibrils
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organized into a pseudo-hexagonal lattice that is critical to maintenance of corneal transparency.
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Corneal stromal cells (keratocytes) reside between the collagen lamellae, and are responsible for
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secreting ECM components required to maintain normal corneal structure and function (i.e.
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transparency and biomechanical stability).1-3 From a mechanical standpoint, resting keratocytes
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are considered quiescent; they do not express stress fibers or generate substantial contractile
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forces.4, 5 Since it is directly exposed to environmental conditions, the cornea is susceptible to
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physical and chemical injuries. Because of its accessibility and optical power, it is also the target
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for numerous refractive surgical procedures, such as photorefractive keratectomy (PRK) and
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LASIK.
69 70
During wound healing following injury or surgery, quiescent corneal keratocytes generally
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become activated, and transform into fibroblast and myofibroblast repair phenotypes.6-9 Corneal
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fibroblasts proliferate, develop intracellular stress fibers, migrate into the wound and reorganize
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the extracellular matrix (ECM) through the application of mechanical forces. Corneal
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myofibroblasts express α-smooth muscle actin, generate even stronger forces on the matrix and
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synthesize a disorganized fibrotic ECM.8, 9 Together these processes can alter corneal shape and
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transparency.10-12 Thus a better understanding of the underlying cellular and molecular
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mechanisms that regulate the transformation and biomechanical activities of corneal keratocytes
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could lead to more effective approaches to modulating the wound healing response in vivo.
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Most studies investigating corneal keratocyte differentiation have been performed using rigid, 2-
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dimensional (2-D) substrates. However, keratocytes reside within a complex 3-dimensional (3-
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D) extracellular matrix in vivo, and significant differences in cell morphology, adhesion
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organization, and mechanical behavior have been identified between 2-D and 3-D culture
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models.13-15 Furthermore, unlike rigid 2-D substrates, 3-D models also allow assessment of
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cellular force generation and cell-induced matrix reorganization; biomechanical activities that are
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critically involved in the migratory, contractile and remodeling phases of wound healing. In a
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recent study, we demonstrated for the first time that ECM composition can modulate the
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mechanism of corneal fibroblast spreading and migration in 3-D culture. Specifically, whereas
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corneal fibroblasts generally move independently within 3-D collagen matrices, fibrin induces a
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switch to an interconnected, collective mode of cell spreading and migration which is
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independent of differences in ECM stiffness.16 The mode of cell migration (individual versus
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collective) has been shown to have a dramatic influence on the pattern of polarization, force
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generation and tissue organization in other systems, and also plays a pivotal role in cancer
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invasion.17-19 Thus a better understanding of the factors that mediate the switch between
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individual and collective cell migration in 3-D culture could have broad significance.
96 97
Previous studies by others have shown that stimulating cell contractility using serum or the
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phospholipid lysophosphatidic acid (LPA) can induce clustering of dermal fibroblasts plated on
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top of compliant collagen matrices;20, 21 however, the role of cell contractility in fibrin-induced
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clustering and collective cell migration has not been investigated. In addition to cleaving
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fibrinogen to form fibrin fibers, thrombin has been shown to stimulate contractility in a variety of
4
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cell types.22-26 Previous studies have confirmed that the cornea contains and synthesizes
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prothrombin, as well as factors required for conversion of prothrombin to form thrombin.27
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However, the impact of thrombin on corneal keratocyte mechanical behavior has not been
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assessed previously.
106 107
The goal of this study was to assess the roles of thrombin and cell contractility in mediating
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collective spreading and migration of fibroblasts interacting with fibrin and collagen matrices.
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The results suggest that while thrombin-induced actomyosin contraction can induce clustering of
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fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell
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migration inside a 3-D collagen construct. Furthermore, increased contractility is not required
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for clustering or collective migration of corneal keratocytes or fibroblasts interacting with fibin.
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This interplay between cell contractility, and matrix composition, stiffness and dimensionality
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may influence cell behavior during wound healing, development, tumor invasion and
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repopulation of engineered tissues.
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METHODS
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Materials
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Dulbecco's modified Eagle medium (DMEM) and 0.25% trypsin/EDTA solution were purchased
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from Invitrogen (Gaithersburg, MD). Platelet-derived growth factor BB isotype (PDGF) was
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obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Fetal bovine serum (FBS), fatty
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acid-free and fraction V bovine serum albumin (BSA), RPMI 1640 vitamin solution, HEPES,
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Sodium bicarbonate, and thrombin from human plasma were obtained from Sigma-Aldrich (St.
5
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Louis, MO). Penicillin, streptomycin, and amphotericin B were obtained from. Type I rat tail
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collagen was purchased from BD Biosciences (Bedford, MA). Alexa Fluor Phalloidin 488 and
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Propidium Iodide (PI) were obtained from Molecular Probes, Inc. (Eugene, OR). RNase (DNase
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free) was purchased from Roche (Indianapolis, IN). Y-27632 and blebbistatin was purchased
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from Calbiochem (San Diego, CA).
129 130
Cell Culture
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A previously published human corneal fibroblast cell line (HTK cells) was used.28, 29 Fibroblasts
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were maintained in tissue culture flasks with DMEM containing 10% FBS and supplemented
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with 1% penicillin/streptomycin/amphotericin B. In some experiments, primary cultures of
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corneal keratocytes (NRK cells) were also used. Corneal keratocytes were isolated from rabbit
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eyes obtained from a slaughterhouse (Pel Freez, Rogers, AR, USA) as previously described 4, 30
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NRK cells were maintained in serum-free media composed of DMEM containing pyruvate,
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HEPES, 1% RPMI vitamin mix, 1% 100x MEM non-essential amino acids, 100 μg/mL ascorbic
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acid and 1% penicillin/streptomycin/amphotericin B to maintain the keratocyte phenotype.4, 31
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Global Matrix Contraction Assay
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To assess the effects of thrombin on cell contractility, contraction of restrained collagen matrices
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was measured.32 A neutralized collagen solution was prepared by mixing high concentration rat
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tail type I collagen with 0.1 N NaOH, 10X DMEM, and H2O to achieve a final concentration of
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2 mg/ml. A 14-mm-diameter circular score was made within each well of 12-well culture plates,
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and 200µl of the collagen solution containing 5x104 cells was poured into each circle. Samples
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were then allowed to polymerize for 30 min in a humidified incubator (37°C, 5% CO2), and
6
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basal media supplemented with 5 mg/ml BSA and 50 ng/ml PDGF was added to stimulate cell
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spreading and migration. After overnight incubation to allow cell spreading, the media was
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changed to basal media or basal media supplemented with 0.5 U/ml thrombin, 50 ng/ml PDGF,
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or both thrombin and PDGF. Since the bottom of the matrix remained attached to the dish, cell
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induced contraction resulted in a decrease in matrix height.33 Height was measured by focusing
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on the top and bottom of each matrix after 0, 1 and 24 hours of culture using phase contrast
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imaging. The percentage decrease in matrix height over time was then calculated. Duplicate
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samples were analyzed for each condition in each experiment. Final results for each condition
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are the mean and standard deviation of three separate experiments.
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Assessment of 2-D Cell Clustering on Fibrin or Collagen ECM
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To investigate the effects of thrombin-induced contractility on fibroblast clustering, cells were
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plated on top of compliant 3-D collagen or fibrin matrices, or rigid, collagen- or fibrin-coated
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glass substrates. For experiments with cells on top of 3-D matrices, 100 µl neutralized solutions
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of collagen (2 mg/ml) or fibrin (1 mg/ml) were poured onto glass bottom dishes (MatTek, model
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P35GC-1.5-14-C, Ashland, MA). The collagen solution was prepared by mixing high
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concentration rat tail type I collagen with 0.1 N NaOH, 10X DMEM and H2O to achieve a final
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concentration of 2 mg/ml. For fibrin matrices, fibrinogen was warmed for 20 minutes and mixed
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with DMEM to achieve a final concentration of 1 mg/ml, and the solution was mixed with 0.5
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U/ml thrombin to initiate polymerization. Samples were then placed for 30 minutes in a
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humidified incubator (37°C, 5% CO2) to polymerize. Matrices were then gently rinsed twice
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with DMEM to remove excess thrombin. For experiments with cells plated on rigid 2-D
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substrates, glass bottom dishes were coated by adding a 50 µg/ml neutralized solution of
7
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collagen, and incubating for 1 hour (37°C, 5% CO2). For cell seeding, 3 ml of basal media
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supplemented with 5 mg/ml BSA and 50 ng/ml PDGF containing 6x104 cells was added to each
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dish. After overnight culture to allow cell spreading, media was replaced with serum-free media
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supplemented with 5 mg/ml BSA and 50 ng/ml PDGF with or without thrombin and/or the Rho
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kinase inhibitor Y-27632 (10 μM).
175 176
After 24 hours of culture, f-actin and nuclei were fluorescently labeled as detailed below.
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Fluorescence images were collected using an inverted microscope (Leica DMI 4000B, Leica
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Microsystems, Wetzlar, Germany) equipped with a digital camera (Hamamatsu Flash 4.0,
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Hamamatsu City, Japan). Four fields were imaged at random on each dish using a 10x objective
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lens. In order to quantify cell clustering, nearest-neighbor and connected component analysis
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were performed using ImageJ (Supplemental Figure 1).34 Using the “Find Maxima” tool, point
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selections were automatically marked at the center of each cell nucleus. Nuclei missed by the
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automated process were then manually marked with the “Point Tool.” The marked points were
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subsequently converted to a binary mask and the coordinates written to the “Results” table in
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ImageJ. Using the “Graph” plugin, distances between points were computed to produce a map
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of connected components (chains of cells within a prescribed distance of each other) and the
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corresponding adjacency list, and a distance matrix. In Microsoft Excel, the distribution of
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different cluster sizes was extracted from the adjacency lists, while the distribution of nearest-
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neighbor distances was taken from the distance matrices. Duplicate samples were analyzed for
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each condition in each experiment. Final results for each condition are the mean and standard
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deviation of three separate experiments.
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Assessment of 3-D Cell Migration
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In order to study the pattern and amount of 3-D cell migration, cell-populated compressed ECM
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constructs were nested within acellular uncompressed matrices as previously described.16, 32, 35 In
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this model, 2ml of a neutralized collagen solution (4 mg/ml) was poured into a rectangular metal
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mold (3 cm length, 2 cm width, 1 cm height) and placed in a humidified incubator (37°C, 5%
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CO2) for 30 minutes for polymerization. Cells (6x106) were mixed with a second 2ml collagen
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solution, which was added on top of the first collagen layer. After 30 minutes to allow collagen
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polymerization, the sandwich construct was compressed as previously described.32, 35-37 This
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produced a ~200 μm thick construct with an acellular ECM on the bottom and a cell-populated
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ECM on top. The first collagen layer serves as a spacer that prevents cells from contacting the
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glass substrate as they migrate out of the matrix, thus ensuring that they interact with the outer
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fibrin or collagen ECM.
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To prepare the nested constructs, 6 mm diameter buttons from compressed sandwiched matrices
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were cut with a trephine blade and gently placed on glass bottom dishes. Buttons were then
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covered with a 100 µl solution of collagen (2 mg/ml) or fibrin (1 mg/ml). After polymerization,
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constructs were rinsed twice with DMEM to remove excess thrombin, and 2 ml of experimental
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media was then added to each sample. Experiments were carried out using serum-free media
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supplemented with 5 mg/ml BSA and 50 ng/ml PDGF, with or without 0.5 U/ml thrombin and/or
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Rho kinase inhibitor Y-27632 (10 μM) and the myosin II inhibitor blebbistatin (20 μM).
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Experiments were performed using duplicate matrices for each condition, and repeated three
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times.
9
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Quantitative analysis of cell migration was carried out as previously described.16, 38 After 24
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hours of culture, f-actin and nuclei were fluorescently labeled as detailed below. Images were
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then collected with a laser confocal microscope (Leica SP2, Heidelberg, Germany). A HeNe
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laser (633nm) was used for confocal reflection imaging of collagen and fibrin fibrils, and Argon
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(488nm) and GreNe (543nm) lasers were used for fluorescence imaging of f-actin and Propidium
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Iodide, respectively. Images were acquired sequentially to avoid cross talk between fluorescence
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channels. Stacks of optical sections were acquired by changing the position of the focal plane in
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the z-direction with a step size of 2–5µm using a 20X dry objective, or a step size of 1 μm using
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a 63X water immersion objective (1.2 NA, 220 µm free working distance). Maximum intensity
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projection images were generated from Propidium Iodide image stacks using Metamorph, and
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overlaid with reflection images. An index of cell migration was then determined by counting the
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number of cells (based on nuclear staining) that migrated from the inner matrix into the outer
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matrix of the nested constructs. Counts were collected from up to four 750μm wide strips in each
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construct, at approximately 90° intervals. Each strip included the border of the button and the
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furthest moving cell.
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Time-Lapse Imaging of Cell Spreading and Migration
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For dynamic assessment of cell spreading and migration mechanics, time-lapse imaging was
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performed on additional samples using a Nikon TE300 inverted microscope (TE300; Nikon,
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Tokyo, Japan) equipped with an environmental chamber (In Vivo Scientific, MO) as previously
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described.39, 40 Hardware was controlled using a PC running Nikon Elements software. Z-stacks
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of images were taken at the border between the inner and outer matrices of nested constructs at
10
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10 minute intervals using a 20X dry objective with Nomarski DIC, or a 10X dry phase contrast
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objective. Imaging was carried out for up to 72 hours. To create movies of cell movements,
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single z-plane images were selected at each time point, so that the same cells were in focus for
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the entire sequence. MetaMorph software version 7.7 (Molecular Devices Inc.) was used to
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generate movies of cell movements.
243 244
Fluorescent Labeling
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Cell labeling was carried out by fixing cells with 3% paraformaldehyde in phosphate buffer,
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PBS, for 10 min, and permeabilizing with 0.5% Triton X-100 in PBS for 15 min. Subsequently
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samples were washed for 30 minutes with PBS. For f-actin labeling, cells were incubated with
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Alexa Fluor 488 Phalloidin (1:150 ratio) for 60 minutes and then washed for 30 minutes to
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remove excess label. For nuclear staining, after f-actin labeling, samples were incubated for 30
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minutes with Propidium Iodide (1:100 ratio) in PBS containing 1:100 RNase (DNase free). All
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staining procedures were performed in the MatTek culture dishes to avoid cell or matrix
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distortion.
253 254
Statistical Analysis
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Statistical analysis was performed using the analysis module within Sigmaplot (version 12.5,
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Systat Software, Inc., San Jose, CA). Analysis of variance (ANOVA) was used to compare group
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means. Post-hoc multiple comparisons between groups were performed using the Holm–Sidak
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method.
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RESULTS
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Thrombin Stimulates Fibroblast Contractility and Induces Clustering on Top of Compliant
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Collagen Matrices
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Previous studies have confirmed that the cornea contains and synthesizes prothrombin, as well as
265
factors required for conversion of prothrombin to form thrombin.27 However, the impact of
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thrombin on keratocyte contractility and mechanical behavior has not been assessed previously.
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As shown in Figure 1A, the addition of thrombin to basal media or media containing PDGF
268
induced a significant increase in global matrix contraction of restrained 3-D collagen matrices.
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To assess cell morphology and cytoskeletal organization, corneal fibroblasts were also cultured
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on collagen coated dishes. As shown in Figure 1B, cells incubated with thrombin had a broader
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morphology and developed more stress fibers than cells cultured in PDGF alone, consistent with
272
an increase in cell contractility.
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275 276 277 278 279 280 281 282
Figure 1. Thrombin stimulates corneal fibroblast contractility. A. Global matrix contraction (decrease in matrix height) was significantly higher at both 1 hour and 24 hours after adding thrombin to the media. (* P < 0.05, ** P < 0.01, repeated measures ANOVA). B. When fibroblasts were plated on rigid substrates, f-actin labeling showed an increase in stress fiber formation and a decrease in the number of dendritic processes in thrombin-containing media. Scale bar is 50 μm.
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Despite stimulating fibroblast contractility, cell clusters did not form in response to thrombin on
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rigid 2-D substrates. In contrast, when corneal fibroblasts were cultured on top of 2 mg/ml
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collagen matrices, the addition of thrombin induced cell clustering (Figure 2A, compare columns
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1 and 2). For quantitative analysis, nearest neighbor distances and cluster sizes were calculated.
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Thrombin induced a shift in the histogram of nearest neighbor distances to smaller values (Fig.
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2A, second row), and the formation of larger cell groups (Fig. 2A, third row); both of these are
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indicators of cell clustering.34 13
290 291 292 293 294 295 296
Figure 2. Cell contractility induces clustering on compliant collagen matrices. A. Representative images of cells grown on top of collagen matrices and cultured with PDGF, PDGF + thrombin, and PDGF + thrombin + Y-27632 are shown in the first row. Each image corresponds to the two graphs below it. Cells incubated with PDGF+Thrombin clustered, however when Y-27632 was present, cluster formation was blocked. Scale bar is 100 µm. The distribution of nearest-neighbor distances is displayed in the first row of graphs. A nearest-
14
297 298 299 300 301 302 303 304
neighbor distance is the distance between the center of one cell nucleus and that of its closest neighbor. The frequency of group sizes is displayed in the second row of graphs. Chains of neighboring cells within a distance of 40 µm were grouped together. All data are means ± standard deviation (n=5 experiments). B. Summary of nearest neighbor analysis for cells on collagen matrices (all 5 experiments combined). The average nearest-neighbor distances was less in PDGF+thrombin (*p < 0.05, ANOVA). C. Summary of cluster analysis for cells on collagen matrices (all 5 experiments combined). The fraction of cells with no neighbors closer than 40 μm was less in PDGF+thrombin (*p < 0.05, ANOVA).
305 306 307
Thrombin-Induced Clustering is Dependent on Rho Kinase In order to evaluate if thrombin-induced clustering of corneal fibroblasts was dependent on Rho
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activation, we used the specific Rho kinase inhibitor Y-27632. As shown in Figure 2A,
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thrombin-induced cluster formation was inhibited by Y-27632 (top row, compare columns 2 and
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3). Both the shift in the histogram of nearest neighbor distances and the formation of larger cell
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clusters induced by thrombin were blocked by inhibiting Rho kinase (rows 2 and 3). These
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quantitative results are summarized in Figure 2B and 2C, which show a statistically significant
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decrease in both the average nearest neighbor distance and the number of isolated (non-
314
clustered) cells in thrombin as compared to all other conditions tested.
315 316
To gain further insights into the mechanism of thrombin-induced clustering, time-lapse DIC
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imaging was performed. Cells on collagen matrices incubated with PDGF moved randomly and
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did not form stable clusters (Figure 3A, Supplemental Movie 1). However, following addition of
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thrombin, cells gradually moved toward each other to form clusters (Figure 3B and 4C;
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Supplemental Movie 2). During cluster formation, collagen fibers were displaced, and lines of
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tension between and around cells were observed, indicating an increase in cell contractile force
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(Figure 3B, arrows). Following addition of Y-27632, cells that were grouped began to separate
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and move apart (Figure 3D, Supplemental Movie 3). Cells also become elongated and develop 15
324
dendritic process following Rho kinase inhibition. Taken together, these results demonstrate that
325
Rho kinase-dependent contractile forces are necessary to form and maintain corneal fibroblast
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clusters in response to thrombin.
327
328 329 330 331 332 333 334 335 336 337
Figure 3. Dynamic assessment of thrombin induced clustering. When observed under DIC time-lapse imaging, transient collagen fibril reorganization appears to directly impact the process of fibroblast clustering on top of collagen matrices. Image A was taken just prior to the addition of thrombin after 24 hr of incubation in PDGF. Images B and C were taken at 30 and 38 hours respectively. The thrombin-induced cellular force generation displaces the matrix substrate so as to pull cells toward each other. Arrows in B denote regions of aligned collagen that form between cells during clustering. D. Subsequent treatment with Y-27632 (at 48 hr.) induces the breakup of clusters and development of a more dendritic morphology. Scale bar is 50 μm.
16
338
Thrombin Does Not Induce Collective Cell Migration in 3-D Collagen Matrices
339
Since thrombin induces cluster formation on top of collagen matrices, we decided to investigate
340
whether thrombin could stimulate collective cell migration within nested collagen matrices.
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Nested collagen matrices were prepared and cultured with media containing PDGF or PDGF
342
plus thrombin. Interestingly, cells migrated individually through the collagen ECM, even in the
343
presence of thrombin (Figure 4). Once cells escaped from the inner matrix, they moved in a
344
random walk pattern and neither stable interactions nor grouping of cells were observed
345
(Supplemental Movie 4). These data demonstrate that although thrombin stimulates Rho kinase
346
dependent clustering of corneal fibroblasts on top of collagen matrices, it does not induce
347
collective cell migration through 3-D collagen matrices.
348
349 350 351 352 353 354 355
Figure 4. Thrombin does not induce collective cell migration in 3-D collagen matrices. Phase contrast images from time-lapse videos of fibroblasts incubated in PDGF+thrombin in nested collagen matrices. Scale bar is 100 µm. Cells migrated independently and no cell streaming or collective migration was observed. Arrows denote isolated cells within the migratory front.
17
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Cell Clustering on Fibrin Matrices is Not Dependent on Cell Contractility
357
As shown above, thrombin can stimulate fibroblast clustering on top of compliant collagen
358
matrices by increasing cell contractility. Our previously published results show that cells
359
cultured on fibrin matrices also form clusters, and this was associated with an increase in stress
360
fiber formation. Thus to determine whether fibrin-induced clustering is dependent on cell
361
contractility, cells were cultured on top of fibrin matrices and incubated with and without the
362
addition of Y-27632. Blocking contractility of cells on fibrin matrices inhibited stress fiber
363
formation; however, cells still formed clusters and remained interconnected (Figure 5). Both the
364
nearest neighbor distances and group sizes were similar for cells cultured with PDGF and those
365
cultured with PDGF plus Y-27632. Taken together, these results suggest that cell contractility is
366
not required for corneal fibroblast clustering on top of fibrin ECM.
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367 368 369 370 371 372
Figure 5. Blocking cell contractility does not inhibit clustering of cells on fibrin matrices. Factin and nuclear staining of cells cultured overnight on top of fibrin matrices with media containing PDGF +/- Y-27632. Graphs show the distribution of cluster sizes and nearest neighbor distances under the two different culture conditions. No significant change is observed when contractility is blocked with the Y-27632. Data are the average +/- SD of three separate
373
experiments with duplicate matrices for each condition. Scale bar is 50 μm.
19
374
Fibrin-Induced Collective Cell Migration is Not Dependent on Cell Contractility
375
We previously demonstrated that fibrin induces an interconnected, collective mode of cell
376
spreading and migration in 3-D culture.16 In order to evaluate if cell contractility could play a
377
role inducing collective cell migration through fibrin ECM, we used Y-27632 and blebbistatin (a
378
myosin II inhibitor) to block cellular force generation. Although the amount of cell migration
379
decreased when matrices were cultured with Y-27632 or blebbistatin (Figure 6A and 6B), the
380
pattern of migration did not change; cells still remained interconnected during migration (Figure
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6C, Supplemental Movie 5). Under all conditions tested, corneal fibroblasts migrating into 3-D
382
fibrin matrices moved into the outer fibrin ECM while maintaining connection to cells in the
383
inner matrix at the rear. Other cells followed behind these cells along the same paths, producing
384
lines of interconnected cells. As migration into fibrin proceeded, adjacent cells having lateral
385
protrusions became interconnected, resulting in the formation of a mesh-like structure
386
(Supplemental Movie 5).
387 388
To further assess the role of cell contractility on fibrin-induced collective cell migration, we
389
carried out experiments using primary rabbit corneal keratocytes maintained in serum-free
390
media, which do not generate significant forces on the ECM when cultured in PDGF,32, 38
391
Corneal keratocytes also migrated collectively and remained interconnected within the fibrin
392
matrix, even in the presence of Y-27632. These data further demonstrate that collective cell
393
migration induced by fibrin matrices is not induced by increased cell contractility.
20
394 395 396 397 398 399 400 401 402
Figure 6. Blocking cell contractility does not change the pattern of cell migration through fibrin matrices. Maximum intensity projection images of migrating cells in nested fibrin matrices. (A) F-actin staining of migrating cells in fibrin nested matrices. Cells were cultured with PDGF, PDGF+Y27632, or PDGF+blebbistatin for 48 hours. Inhibitors reduced the cell migration rate but they did not change the pattern of migration. Scale bars are 50 µm. (B) Graph from the same experiment showing cell counts in the outer matrix after 48 hours of incubation (*P
1
The Role of Thrombin and Cell Contractility in Regulating Clustering and Collective
2
Migration of Corneal Fibroblasts in Different ECM Environments
3 4
Miguel Miron-Mendoza, Eric Graham, Pouriska Kivanany, Jonathan Quiring
5
and W. Matthew Petroll
6 7
Short Title: Fibroblast Clustering and Collective Migration
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Corresponding Author:
W. Matthew Petroll, Ph.D. Department of Ophthalmology Southwestern Medical Center 5323 Harry Hines Blvd. Dallas, TX 75390-9057 Phone: 214-648-7216 FAX: 214-648-4507 E-mail: [email protected]
This study was supported in part by NIH R01 EY 013322, NIH P30 EY020799, and an unrestricted grant from Research to Prevent Blindness, Inc., NY, NY.
Keywords:
Extracellular Matrix Corneal Keratocytes Cell Mechanics Thrombin 3-D Culture
1 Copyright 2015 by The Association for Research in Vision and Ophthalmology, Inc.
33
ABSTRACT
34
Purpose: We previously reported that extracellular matrix composition (fibrin versus collagen)
35
modulates the pattern of corneal fibroblast spreading and migration in 3-D culture. In this study,
36
we investigate the role of thrombin and cell contractility in mediating these differences in cell
37
behavior.
38
Methods: To assess cell spreading, corneal fibroblasts were plated on top of fibrillar collagen
39
and fibrin matrices. To assess 3-dimensional cell migration, compacted collagen matrices seeded
40
with corneal fibroblasts were embedded inside acellular collagen or fibrin matrices. Constructs
41
were cultured in serum-free media containing PDGF, with or without thrombin, the Rho kinase
42
inhibitor Y-27632 and/or the myosin II inhibitor blebbistatin. 3-dimensional and 4-dimensional
43
imaging were used to assess cell mechanical behavior, connectivity and cytoskeletal
44
organization.
45
Results: Thrombin stimulated increased contractility of corneal fibroblasts. Thrombin also
46
induced Rho kinase-dependent clustering of cells plated on top of compliant collagen matrices,
47
but not on rigid substrates. In contrast, cells on fibrin matrices coalesced into clusters even when
48
Rho kinase was inhibited. In nested matrices, cells always migrated independently through
49
collagen, even in the presence of thrombin. In contrast, cells migrating into fibrin formed an
50
interconnected network. Both Y-27632 and blebbistatin reduced the migration rate in fibrin, but
51
cells continued to migrate collectively.
52
Conclusions: The results suggest that while thrombin-induced actomyosin contraction can
53
induce clustering of fibroblasts plated on top of compliant collagen matrices, it does not induce
54
collective cell migration inside 3-D collagen constructs. Furthermore, increased contractility is
55
not required for clustering or collective migration of corneal fibroblasts interacting with fibin.
2
56
INTRODUCTION
57
The cornea is an optically clear tissue that forms the front surface of the eye, and accounts for
58
nearly two-thirds of its refractive power. The corneal stroma, which makes up 90% of corneal
59
thickness, is a highly ordered structure with lamellae consisting of uniformly thin collagen fibrils
60
organized into a pseudo-hexagonal lattice that is critical to maintenance of corneal transparency.
61
Corneal stromal cells (keratocytes) reside between the collagen lamellae, and are responsible for
62
secreting ECM components required to maintain normal corneal structure and function (i.e.
63
transparency and biomechanical stability).1-3 From a mechanical standpoint, resting keratocytes
64
are considered quiescent; they do not express stress fibers or generate substantial contractile
65
forces.4, 5 Since it is directly exposed to environmental conditions, the cornea is susceptible to
66
physical and chemical injuries. Because of its accessibility and optical power, it is also the target
67
for numerous refractive surgical procedures, such as photorefractive keratectomy (PRK) and
68
LASIK.
69 70
During wound healing following injury or surgery, quiescent corneal keratocytes generally
71
become activated, and transform into fibroblast and myofibroblast repair phenotypes.6-9 Corneal
72
fibroblasts proliferate, develop intracellular stress fibers, migrate into the wound and reorganize
73
the extracellular matrix (ECM) through the application of mechanical forces. Corneal
74
myofibroblasts express α-smooth muscle actin, generate even stronger forces on the matrix and
75
synthesize a disorganized fibrotic ECM.8, 9 Together these processes can alter corneal shape and
76
transparency.10-12 Thus a better understanding of the underlying cellular and molecular
77
mechanisms that regulate the transformation and biomechanical activities of corneal keratocytes
78
could lead to more effective approaches to modulating the wound healing response in vivo.
3
79 80
Most studies investigating corneal keratocyte differentiation have been performed using rigid, 2-
81
dimensional (2-D) substrates. However, keratocytes reside within a complex 3-dimensional (3-
82
D) extracellular matrix in vivo, and significant differences in cell morphology, adhesion
83
organization, and mechanical behavior have been identified between 2-D and 3-D culture
84
models.13-15 Furthermore, unlike rigid 2-D substrates, 3-D models also allow assessment of
85
cellular force generation and cell-induced matrix reorganization; biomechanical activities that are
86
critically involved in the migratory, contractile and remodeling phases of wound healing. In a
87
recent study, we demonstrated for the first time that ECM composition can modulate the
88
mechanism of corneal fibroblast spreading and migration in 3-D culture. Specifically, whereas
89
corneal fibroblasts generally move independently within 3-D collagen matrices, fibrin induces a
90
switch to an interconnected, collective mode of cell spreading and migration which is
91
independent of differences in ECM stiffness.16 The mode of cell migration (individual versus
92
collective) has been shown to have a dramatic influence on the pattern of polarization, force
93
generation and tissue organization in other systems, and also plays a pivotal role in cancer
94
invasion.17-19 Thus a better understanding of the factors that mediate the switch between
95
individual and collective cell migration in 3-D culture could have broad significance.
96 97
Previous studies by others have shown that stimulating cell contractility using serum or the
98
phospholipid lysophosphatidic acid (LPA) can induce clustering of dermal fibroblasts plated on
99
top of compliant collagen matrices;20, 21 however, the role of cell contractility in fibrin-induced
100
clustering and collective cell migration has not been investigated. In addition to cleaving
101
fibrinogen to form fibrin fibers, thrombin has been shown to stimulate contractility in a variety of
4
102
cell types.22-26 Previous studies have confirmed that the cornea contains and synthesizes
103
prothrombin, as well as factors required for conversion of prothrombin to form thrombin.27
104
However, the impact of thrombin on corneal keratocyte mechanical behavior has not been
105
assessed previously.
106 107
The goal of this study was to assess the roles of thrombin and cell contractility in mediating
108
collective spreading and migration of fibroblasts interacting with fibrin and collagen matrices.
109
The results suggest that while thrombin-induced actomyosin contraction can induce clustering of
110
fibroblasts plated on top of compliant collagen matrices, it does not induce collective cell
111
migration inside a 3-D collagen construct. Furthermore, increased contractility is not required
112
for clustering or collective migration of corneal keratocytes or fibroblasts interacting with fibin.
113
This interplay between cell contractility, and matrix composition, stiffness and dimensionality
114
may influence cell behavior during wound healing, development, tumor invasion and
115
repopulation of engineered tissues.
116 117
METHODS
118
Materials
119
Dulbecco's modified Eagle medium (DMEM) and 0.25% trypsin/EDTA solution were purchased
120
from Invitrogen (Gaithersburg, MD). Platelet-derived growth factor BB isotype (PDGF) was
121
obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Fetal bovine serum (FBS), fatty
122
acid-free and fraction V bovine serum albumin (BSA), RPMI 1640 vitamin solution, HEPES,
123
Sodium bicarbonate, and thrombin from human plasma were obtained from Sigma-Aldrich (St.
5
124
Louis, MO). Penicillin, streptomycin, and amphotericin B were obtained from. Type I rat tail
125
collagen was purchased from BD Biosciences (Bedford, MA). Alexa Fluor Phalloidin 488 and
126
Propidium Iodide (PI) were obtained from Molecular Probes, Inc. (Eugene, OR). RNase (DNase
127
free) was purchased from Roche (Indianapolis, IN). Y-27632 and blebbistatin was purchased
128
from Calbiochem (San Diego, CA).
129 130
Cell Culture
131
A previously published human corneal fibroblast cell line (HTK cells) was used.28, 29 Fibroblasts
132
were maintained in tissue culture flasks with DMEM containing 10% FBS and supplemented
133
with 1% penicillin/streptomycin/amphotericin B. In some experiments, primary cultures of
134
corneal keratocytes (NRK cells) were also used. Corneal keratocytes were isolated from rabbit
135
eyes obtained from a slaughterhouse (Pel Freez, Rogers, AR, USA) as previously described 4, 30
136
NRK cells were maintained in serum-free media composed of DMEM containing pyruvate,
137
HEPES, 1% RPMI vitamin mix, 1% 100x MEM non-essential amino acids, 100 μg/mL ascorbic
138
acid and 1% penicillin/streptomycin/amphotericin B to maintain the keratocyte phenotype.4, 31
139 140
Global Matrix Contraction Assay
141
To assess the effects of thrombin on cell contractility, contraction of restrained collagen matrices
142
was measured.32 A neutralized collagen solution was prepared by mixing high concentration rat
143
tail type I collagen with 0.1 N NaOH, 10X DMEM, and H2O to achieve a final concentration of
144
2 mg/ml. A 14-mm-diameter circular score was made within each well of 12-well culture plates,
145
and 200µl of the collagen solution containing 5x104 cells was poured into each circle. Samples
146
were then allowed to polymerize for 30 min in a humidified incubator (37°C, 5% CO2), and
6
147
basal media supplemented with 5 mg/ml BSA and 50 ng/ml PDGF was added to stimulate cell
148
spreading and migration. After overnight incubation to allow cell spreading, the media was
149
changed to basal media or basal media supplemented with 0.5 U/ml thrombin, 50 ng/ml PDGF,
150
or both thrombin and PDGF. Since the bottom of the matrix remained attached to the dish, cell
151
induced contraction resulted in a decrease in matrix height.33 Height was measured by focusing
152
on the top and bottom of each matrix after 0, 1 and 24 hours of culture using phase contrast
153
imaging. The percentage decrease in matrix height over time was then calculated. Duplicate
154
samples were analyzed for each condition in each experiment. Final results for each condition
155
are the mean and standard deviation of three separate experiments.
156 157
Assessment of 2-D Cell Clustering on Fibrin or Collagen ECM
158
To investigate the effects of thrombin-induced contractility on fibroblast clustering, cells were
159
plated on top of compliant 3-D collagen or fibrin matrices, or rigid, collagen- or fibrin-coated
160
glass substrates. For experiments with cells on top of 3-D matrices, 100 µl neutralized solutions
161
of collagen (2 mg/ml) or fibrin (1 mg/ml) were poured onto glass bottom dishes (MatTek, model
162
P35GC-1.5-14-C, Ashland, MA). The collagen solution was prepared by mixing high
163
concentration rat tail type I collagen with 0.1 N NaOH, 10X DMEM and H2O to achieve a final
164
concentration of 2 mg/ml. For fibrin matrices, fibrinogen was warmed for 20 minutes and mixed
165
with DMEM to achieve a final concentration of 1 mg/ml, and the solution was mixed with 0.5
166
U/ml thrombin to initiate polymerization. Samples were then placed for 30 minutes in a
167
humidified incubator (37°C, 5% CO2) to polymerize. Matrices were then gently rinsed twice
168
with DMEM to remove excess thrombin. For experiments with cells plated on rigid 2-D
169
substrates, glass bottom dishes were coated by adding a 50 µg/ml neutralized solution of
7
170
collagen, and incubating for 1 hour (37°C, 5% CO2). For cell seeding, 3 ml of basal media
171
supplemented with 5 mg/ml BSA and 50 ng/ml PDGF containing 6x104 cells was added to each
172
dish. After overnight culture to allow cell spreading, media was replaced with serum-free media
173
supplemented with 5 mg/ml BSA and 50 ng/ml PDGF with or without thrombin and/or the Rho
174
kinase inhibitor Y-27632 (10 μM).
175 176
After 24 hours of culture, f-actin and nuclei were fluorescently labeled as detailed below.
177
Fluorescence images were collected using an inverted microscope (Leica DMI 4000B, Leica
178
Microsystems, Wetzlar, Germany) equipped with a digital camera (Hamamatsu Flash 4.0,
179
Hamamatsu City, Japan). Four fields were imaged at random on each dish using a 10x objective
180
lens. In order to quantify cell clustering, nearest-neighbor and connected component analysis
181
were performed using ImageJ (Supplemental Figure 1).34 Using the “Find Maxima” tool, point
182
selections were automatically marked at the center of each cell nucleus. Nuclei missed by the
183
automated process were then manually marked with the “Point Tool.” The marked points were
184
subsequently converted to a binary mask and the coordinates written to the “Results” table in
185
ImageJ. Using the “Graph” plugin, distances between points were computed to produce a map
186
of connected components (chains of cells within a prescribed distance of each other) and the
187
corresponding adjacency list, and a distance matrix. In Microsoft Excel, the distribution of
188
different cluster sizes was extracted from the adjacency lists, while the distribution of nearest-
189
neighbor distances was taken from the distance matrices. Duplicate samples were analyzed for
190
each condition in each experiment. Final results for each condition are the mean and standard
191
deviation of three separate experiments.
8
192 193
Assessment of 3-D Cell Migration
194
In order to study the pattern and amount of 3-D cell migration, cell-populated compressed ECM
195
constructs were nested within acellular uncompressed matrices as previously described.16, 32, 35 In
196
this model, 2ml of a neutralized collagen solution (4 mg/ml) was poured into a rectangular metal
197
mold (3 cm length, 2 cm width, 1 cm height) and placed in a humidified incubator (37°C, 5%
198
CO2) for 30 minutes for polymerization. Cells (6x106) were mixed with a second 2ml collagen
199
solution, which was added on top of the first collagen layer. After 30 minutes to allow collagen
200
polymerization, the sandwich construct was compressed as previously described.32, 35-37 This
201
produced a ~200 μm thick construct with an acellular ECM on the bottom and a cell-populated
202
ECM on top. The first collagen layer serves as a spacer that prevents cells from contacting the
203
glass substrate as they migrate out of the matrix, thus ensuring that they interact with the outer
204
fibrin or collagen ECM.
205 206
To prepare the nested constructs, 6 mm diameter buttons from compressed sandwiched matrices
207
were cut with a trephine blade and gently placed on glass bottom dishes. Buttons were then
208
covered with a 100 µl solution of collagen (2 mg/ml) or fibrin (1 mg/ml). After polymerization,
209
constructs were rinsed twice with DMEM to remove excess thrombin, and 2 ml of experimental
210
media was then added to each sample. Experiments were carried out using serum-free media
211
supplemented with 5 mg/ml BSA and 50 ng/ml PDGF, with or without 0.5 U/ml thrombin and/or
212
Rho kinase inhibitor Y-27632 (10 μM) and the myosin II inhibitor blebbistatin (20 μM).
213
Experiments were performed using duplicate matrices for each condition, and repeated three
214
times.
9
215 216
Quantitative analysis of cell migration was carried out as previously described.16, 38 After 24
217
hours of culture, f-actin and nuclei were fluorescently labeled as detailed below. Images were
218
then collected with a laser confocal microscope (Leica SP2, Heidelberg, Germany). A HeNe
219
laser (633nm) was used for confocal reflection imaging of collagen and fibrin fibrils, and Argon
220
(488nm) and GreNe (543nm) lasers were used for fluorescence imaging of f-actin and Propidium
221
Iodide, respectively. Images were acquired sequentially to avoid cross talk between fluorescence
222
channels. Stacks of optical sections were acquired by changing the position of the focal plane in
223
the z-direction with a step size of 2–5µm using a 20X dry objective, or a step size of 1 μm using
224
a 63X water immersion objective (1.2 NA, 220 µm free working distance). Maximum intensity
225
projection images were generated from Propidium Iodide image stacks using Metamorph, and
226
overlaid with reflection images. An index of cell migration was then determined by counting the
227
number of cells (based on nuclear staining) that migrated from the inner matrix into the outer
228
matrix of the nested constructs. Counts were collected from up to four 750μm wide strips in each
229
construct, at approximately 90° intervals. Each strip included the border of the button and the
230
furthest moving cell.
231 232
Time-Lapse Imaging of Cell Spreading and Migration
233
For dynamic assessment of cell spreading and migration mechanics, time-lapse imaging was
234
performed on additional samples using a Nikon TE300 inverted microscope (TE300; Nikon,
235
Tokyo, Japan) equipped with an environmental chamber (In Vivo Scientific, MO) as previously
236
described.39, 40 Hardware was controlled using a PC running Nikon Elements software. Z-stacks
237
of images were taken at the border between the inner and outer matrices of nested constructs at
10
238
10 minute intervals using a 20X dry objective with Nomarski DIC, or a 10X dry phase contrast
239
objective. Imaging was carried out for up to 72 hours. To create movies of cell movements,
240
single z-plane images were selected at each time point, so that the same cells were in focus for
241
the entire sequence. MetaMorph software version 7.7 (Molecular Devices Inc.) was used to
242
generate movies of cell movements.
243 244
Fluorescent Labeling
245
Cell labeling was carried out by fixing cells with 3% paraformaldehyde in phosphate buffer,
246
PBS, for 10 min, and permeabilizing with 0.5% Triton X-100 in PBS for 15 min. Subsequently
247
samples were washed for 30 minutes with PBS. For f-actin labeling, cells were incubated with
248
Alexa Fluor 488 Phalloidin (1:150 ratio) for 60 minutes and then washed for 30 minutes to
249
remove excess label. For nuclear staining, after f-actin labeling, samples were incubated for 30
250
minutes with Propidium Iodide (1:100 ratio) in PBS containing 1:100 RNase (DNase free). All
251
staining procedures were performed in the MatTek culture dishes to avoid cell or matrix
252
distortion.
253 254
Statistical Analysis
255
Statistical analysis was performed using the analysis module within Sigmaplot (version 12.5,
256
Systat Software, Inc., San Jose, CA). Analysis of variance (ANOVA) was used to compare group
257
means. Post-hoc multiple comparisons between groups were performed using the Holm–Sidak
258
method.
259 260
11
261
RESULTS
262
Thrombin Stimulates Fibroblast Contractility and Induces Clustering on Top of Compliant
263
Collagen Matrices
264
Previous studies have confirmed that the cornea contains and synthesizes prothrombin, as well as
265
factors required for conversion of prothrombin to form thrombin.27 However, the impact of
266
thrombin on keratocyte contractility and mechanical behavior has not been assessed previously.
267
As shown in Figure 1A, the addition of thrombin to basal media or media containing PDGF
268
induced a significant increase in global matrix contraction of restrained 3-D collagen matrices.
269
To assess cell morphology and cytoskeletal organization, corneal fibroblasts were also cultured
270
on collagen coated dishes. As shown in Figure 1B, cells incubated with thrombin had a broader
271
morphology and developed more stress fibers than cells cultured in PDGF alone, consistent with
272
an increase in cell contractility.
273
12
274
275 276 277 278 279 280 281 282
Figure 1. Thrombin stimulates corneal fibroblast contractility. A. Global matrix contraction (decrease in matrix height) was significantly higher at both 1 hour and 24 hours after adding thrombin to the media. (* P < 0.05, ** P < 0.01, repeated measures ANOVA). B. When fibroblasts were plated on rigid substrates, f-actin labeling showed an increase in stress fiber formation and a decrease in the number of dendritic processes in thrombin-containing media. Scale bar is 50 μm.
283
Despite stimulating fibroblast contractility, cell clusters did not form in response to thrombin on
284
rigid 2-D substrates. In contrast, when corneal fibroblasts were cultured on top of 2 mg/ml
285
collagen matrices, the addition of thrombin induced cell clustering (Figure 2A, compare columns
286
1 and 2). For quantitative analysis, nearest neighbor distances and cluster sizes were calculated.
287
Thrombin induced a shift in the histogram of nearest neighbor distances to smaller values (Fig.
288
2A, second row), and the formation of larger cell groups (Fig. 2A, third row); both of these are
289
indicators of cell clustering.34 13
290 291 292 293 294 295 296
Figure 2. Cell contractility induces clustering on compliant collagen matrices. A. Representative images of cells grown on top of collagen matrices and cultured with PDGF, PDGF + thrombin, and PDGF + thrombin + Y-27632 are shown in the first row. Each image corresponds to the two graphs below it. Cells incubated with PDGF+Thrombin clustered, however when Y-27632 was present, cluster formation was blocked. Scale bar is 100 µm. The distribution of nearest-neighbor distances is displayed in the first row of graphs. A nearest-
14
297 298 299 300 301 302 303 304
neighbor distance is the distance between the center of one cell nucleus and that of its closest neighbor. The frequency of group sizes is displayed in the second row of graphs. Chains of neighboring cells within a distance of 40 µm were grouped together. All data are means ± standard deviation (n=5 experiments). B. Summary of nearest neighbor analysis for cells on collagen matrices (all 5 experiments combined). The average nearest-neighbor distances was less in PDGF+thrombin (*p < 0.05, ANOVA). C. Summary of cluster analysis for cells on collagen matrices (all 5 experiments combined). The fraction of cells with no neighbors closer than 40 μm was less in PDGF+thrombin (*p < 0.05, ANOVA).
305 306 307
Thrombin-Induced Clustering is Dependent on Rho Kinase In order to evaluate if thrombin-induced clustering of corneal fibroblasts was dependent on Rho
308
activation, we used the specific Rho kinase inhibitor Y-27632. As shown in Figure 2A,
309
thrombin-induced cluster formation was inhibited by Y-27632 (top row, compare columns 2 and
310
3). Both the shift in the histogram of nearest neighbor distances and the formation of larger cell
311
clusters induced by thrombin were blocked by inhibiting Rho kinase (rows 2 and 3). These
312
quantitative results are summarized in Figure 2B and 2C, which show a statistically significant
313
decrease in both the average nearest neighbor distance and the number of isolated (non-
314
clustered) cells in thrombin as compared to all other conditions tested.
315 316
To gain further insights into the mechanism of thrombin-induced clustering, time-lapse DIC
317
imaging was performed. Cells on collagen matrices incubated with PDGF moved randomly and
318
did not form stable clusters (Figure 3A, Supplemental Movie 1). However, following addition of
319
thrombin, cells gradually moved toward each other to form clusters (Figure 3B and 4C;
320
Supplemental Movie 2). During cluster formation, collagen fibers were displaced, and lines of
321
tension between and around cells were observed, indicating an increase in cell contractile force
322
(Figure 3B, arrows). Following addition of Y-27632, cells that were grouped began to separate
323
and move apart (Figure 3D, Supplemental Movie 3). Cells also become elongated and develop 15
324
dendritic process following Rho kinase inhibition. Taken together, these results demonstrate that
325
Rho kinase-dependent contractile forces are necessary to form and maintain corneal fibroblast
326
clusters in response to thrombin.
327
328 329 330 331 332 333 334 335 336 337
Figure 3. Dynamic assessment of thrombin induced clustering. When observed under DIC time-lapse imaging, transient collagen fibril reorganization appears to directly impact the process of fibroblast clustering on top of collagen matrices. Image A was taken just prior to the addition of thrombin after 24 hr of incubation in PDGF. Images B and C were taken at 30 and 38 hours respectively. The thrombin-induced cellular force generation displaces the matrix substrate so as to pull cells toward each other. Arrows in B denote regions of aligned collagen that form between cells during clustering. D. Subsequent treatment with Y-27632 (at 48 hr.) induces the breakup of clusters and development of a more dendritic morphology. Scale bar is 50 μm.
16
338
Thrombin Does Not Induce Collective Cell Migration in 3-D Collagen Matrices
339
Since thrombin induces cluster formation on top of collagen matrices, we decided to investigate
340
whether thrombin could stimulate collective cell migration within nested collagen matrices.
341
Nested collagen matrices were prepared and cultured with media containing PDGF or PDGF
342
plus thrombin. Interestingly, cells migrated individually through the collagen ECM, even in the
343
presence of thrombin (Figure 4). Once cells escaped from the inner matrix, they moved in a
344
random walk pattern and neither stable interactions nor grouping of cells were observed
345
(Supplemental Movie 4). These data demonstrate that although thrombin stimulates Rho kinase
346
dependent clustering of corneal fibroblasts on top of collagen matrices, it does not induce
347
collective cell migration through 3-D collagen matrices.
348
349 350 351 352 353 354 355
Figure 4. Thrombin does not induce collective cell migration in 3-D collagen matrices. Phase contrast images from time-lapse videos of fibroblasts incubated in PDGF+thrombin in nested collagen matrices. Scale bar is 100 µm. Cells migrated independently and no cell streaming or collective migration was observed. Arrows denote isolated cells within the migratory front.
17
356
Cell Clustering on Fibrin Matrices is Not Dependent on Cell Contractility
357
As shown above, thrombin can stimulate fibroblast clustering on top of compliant collagen
358
matrices by increasing cell contractility. Our previously published results show that cells
359
cultured on fibrin matrices also form clusters, and this was associated with an increase in stress
360
fiber formation. Thus to determine whether fibrin-induced clustering is dependent on cell
361
contractility, cells were cultured on top of fibrin matrices and incubated with and without the
362
addition of Y-27632. Blocking contractility of cells on fibrin matrices inhibited stress fiber
363
formation; however, cells still formed clusters and remained interconnected (Figure 5). Both the
364
nearest neighbor distances and group sizes were similar for cells cultured with PDGF and those
365
cultured with PDGF plus Y-27632. Taken together, these results suggest that cell contractility is
366
not required for corneal fibroblast clustering on top of fibrin ECM.
18
367 368 369 370 371 372
Figure 5. Blocking cell contractility does not inhibit clustering of cells on fibrin matrices. Factin and nuclear staining of cells cultured overnight on top of fibrin matrices with media containing PDGF +/- Y-27632. Graphs show the distribution of cluster sizes and nearest neighbor distances under the two different culture conditions. No significant change is observed when contractility is blocked with the Y-27632. Data are the average +/- SD of three separate
373
experiments with duplicate matrices for each condition. Scale bar is 50 μm.
19
374
Fibrin-Induced Collective Cell Migration is Not Dependent on Cell Contractility
375
We previously demonstrated that fibrin induces an interconnected, collective mode of cell
376
spreading and migration in 3-D culture.16 In order to evaluate if cell contractility could play a
377
role inducing collective cell migration through fibrin ECM, we used Y-27632 and blebbistatin (a
378
myosin II inhibitor) to block cellular force generation. Although the amount of cell migration
379
decreased when matrices were cultured with Y-27632 or blebbistatin (Figure 6A and 6B), the
380
pattern of migration did not change; cells still remained interconnected during migration (Figure
381
6C, Supplemental Movie 5). Under all conditions tested, corneal fibroblasts migrating into 3-D
382
fibrin matrices moved into the outer fibrin ECM while maintaining connection to cells in the
383
inner matrix at the rear. Other cells followed behind these cells along the same paths, producing
384
lines of interconnected cells. As migration into fibrin proceeded, adjacent cells having lateral
385
protrusions became interconnected, resulting in the formation of a mesh-like structure
386
(Supplemental Movie 5).
387 388
To further assess the role of cell contractility on fibrin-induced collective cell migration, we
389
carried out experiments using primary rabbit corneal keratocytes maintained in serum-free
390
media, which do not generate significant forces on the ECM when cultured in PDGF,32, 38
391
Corneal keratocytes also migrated collectively and remained interconnected within the fibrin
392
matrix, even in the presence of Y-27632. These data further demonstrate that collective cell
393
migration induced by fibrin matrices is not induced by increased cell contractility.
20
394 395 396 397 398 399 400 401 402
Figure 6. Blocking cell contractility does not change the pattern of cell migration through fibrin matrices. Maximum intensity projection images of migrating cells in nested fibrin matrices. (A) F-actin staining of migrating cells in fibrin nested matrices. Cells were cultured with PDGF, PDGF+Y27632, or PDGF+blebbistatin for 48 hours. Inhibitors reduced the cell migration rate but they did not change the pattern of migration. Scale bars are 50 µm. (B) Graph from the same experiment showing cell counts in the outer matrix after 48 hours of incubation (*P
