Decreasing Wound Edge Stress Enhances Leader Cell Formation

Article pubs.acs.org/journal/abseba

Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Decreasing Wound Edge Stress Enhances Leader Cell Formation during Collective Smooth Muscle Cell Migration Zachary S. Dean,†,‡ Nima Jamilpour,§ Marvin J. Slepian,†,⊥ and Pak Kin Wong*,†,§,∥ †

Department of Biomedical Engineering, §Department of Aerospace and Mechanical Engineering, and ⊥Sarver Heart Center, College of Medicine, The University of Arizona, Tucson, Arizona 85721, United States ∥ Departments of Biomedical Engineering, Mechanical Engineering, and Surgery, The Pennsylvania State University, University Park, Pennsylvania 16802, United States

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S Supporting Information *

ABSTRACT: Collective cell migration is vital to tissue remodeling in wound repair, development, and cancer invasion. Nevertheless, studies on collective cell migration have largely focused on epithelial growth and repair mechanisms and have only recently expanded to explore coordinated metastatic cancer and smooth muscle cell behaviors. The regulatory mechanisms of smooth muscle cell collective migration, such as leader−follower organization and mechanosensitivity, remain poorly understood. In this study, we demonstrate the involvement of leader cells during collective smooth muscle cell migration using dynamic cell tracking and single cell gene expression analysis. Engineered wound models, including ingrowth, outgrowth, and straight edge geometries, along with traction force microscopy and finite element stress mapping reveal that smooth muscle leader cells are enhanced at the wound edge when the intercellular tension near the cell wound boundary is reduced. Pharmacological perturbation further supports the notion that mechanical force negatively regulates the formation of leader cells. The mechanical regulation of collective smooth muscle cell migration via the formation of leader cells may lead to novel treatment strategies for pathogenic smooth muscle cell conditions in the future. KEYWORDS: collective cell migration, leader cell, single cell, smooth muscle, traction force, wound healing

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INTRODUCTION Cardiovascular disease is the leading cause of death in the United States, ahead of other debilitating diseases such as cancer.1 During significant cardiovascular disease processes, such as atherosclerosis and restenosis, normally quiescent vascular smooth muscle cells physiologically change from a contractile phenotype to a proliferative phenotype and migrate from the media to the intima.2−4 Following injury and stiffening in an atherosclerotic blood vessel, smooth muscle cells initiate proliferation and migration in response to the cues in the surrounding microenvironment. Substrate stiffness, cell− substrate interaction, cell−cell contact, and cytoskeletal tension modulate the migration of vascular smooth muscle cells,5−8 collectively underscoring that the mechanoregulation of smooth muscle cell migration is a coordinated, multicellular process. Collective cell migration is widely implicated in numerous pathological and physiological processes, including disease progression, tissue regeneration, and embryogenesis.9,10 Collective migration is a highly coordinated process, where cells exhibit a gradient of responses from the wound edge to the inner region of the monolayer with graded gene expression, varying proliferation rate, and leader−follower organization.11−16 In particular, mechanical force and Notch1-Dll4 signaling are shown to autonomously regulate the formation leader cells near the wound edge.17,18 These specialized leader cells exhibit enlarged cell size, broad integrin-based focal © XXXX American Chemical Society

adhesions, and ruffling actin-based lamellipodia. Nevertheless, the majority of reports describing the leader−follower coordination of collective migration and their responses to biophysical cues to date focus on epithelial sheet migration. Elucidating the mechanotransduction intricacies of collective smooth muscle cell migration will provide valuable insight into the mechanisms behind vascular communication during the pathogenesis of atherosclerosis and restenosis as well as during vascular tissue engineering. In the present study, the mechanoregulation of smooth muscle cell migration is investigated by engineering the wound model and the associated intracellular stress on cells with geometrical controls: Ingrowth, outgrowth, and straight edge wound geometries are incorporated into a wound healing assay to investigate the biomechanical coordination of smooth muscle cells (Figure 1a).19 Single-cell tracking is applied to characterize the migratory behaviors of smooth muscle cells within the migrating monolayer. The expression profiles of β3 integrin, β-actin, Notch1, and Dll4 are characterized near the wound edge using a previously characterized double-stranded locked nucleic acid (dsLNA) probe that enables real-time Special Issue: Biomaterials for Mechanobiology Received: October 6, 2018 Accepted: March 5, 2019

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DOI: 10.1021/acsbiomaterials.8b01222 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. Geometric control of leader cells during collective smooth muscle cell migration. (a) Schematic illustrating the ingrowth, outgrowth, and straight edge migration assays. Geometries were formed using a PDMS or acrylic blocker. (b) Brightfield images showing leader cells (*) at the leading edge during collective smooth muscle cell migration. Images are representative of three independent experiments. Scale bars, 100 μm.

spatiotemporal gene monitoring.20,21 The mechanoregulation of the smooth muscle cell migration is further perturbed using force-modulating drugs, including nocodazole and Y-27632, to decipher the roles of mechanical force in the coordination of smooth muscle cell migration. Finally, in addition to the single cell analysis and pharmacological mechanisms, traction force microscopy and computational biomechanical analysis of the stress distribution in the migrating wound front are performed to elucidate the influence of geometrical wound controls. Our findings suggest a mechanoregulation scheme used by smooth muscle cells to coordinate the formation of leader cells during collective migration under diverse physiological conditions.

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oligonucleotide strands with alternating DNA and LNA monomers. One strand, the donor strand, is 20 nucleotides long and designed complementary to the target RNA of interest. 6-FAM, the fluorophore used for fluorescence detection, is located at the 5′ end. The second strand, the quencher strand, is ten nucleotides in length. Iowa Black FQ, a dark fluorescence quencher, is located at the 3′ end of the quenching strand immediately adjacent to the 6-FAM donor fluorophore on the 5′ end of the donor strand. Two dsLNA probes were developed to monitor β-actin mRNA and β3 integrin mRNA expression along with a probe for Dll4 and a probe for Notch1 (Table S1). The probe sequence for each mRNA target was verified using the NCBI GenBank database. A random, scrambled probe was also developed with no known intracellular targets as a negative control. The sequences of all dsLNA probes were also additionally verified through NCBI Basic Local Alignment Search Tool for nucleotides (BLASTn). Transfection was performed by dissolving the donor and quencher strands in 10 mM Tris-EDTA buffer and 0.2 M NaCl before mixing them at a 1:2 donor-toquencher ratio. The probes were heated at 95 °C for 5 min in a dry block heater before cooling to room temperature gradually over the course of the next two and a half hours. To stain cells for the table of contents image, 50 mL of 1× PBS was heated for 30 min to 37 °C, and the DAPI staining solution (Invitrogen, Carlsbad, CA) was simultaneously warmed to room temperature. Cells prewounded were fixed for 15 min with 1.5% paraformaldehyde (Sigma, St. Louis, MO) diluted in 1X PBS, then were immediately washed twice with PBS for 5 min. Cells were then permeabilized with 0.4% Triton X-100 for 15 min before washing with PBS three times for 5 min. The cells were then blocked with 1% BSA (Sigma, St. Louis, MO) in PBS for 30 min to prevent nonspecific antibody binding. Rhodamine phalloidin (Invitrogen, Molecular Probes, Eugene, OR, 1:400 dilution) was incubated with cells for 1

MATERIALS AND METHODS

1.1. Cell Culture and Reagents. Rat smooth muscle cells were acquired from the aorta of Sprague−Dawley rats. The cells were used in between passages P2 and P9. The smooth muscle cells were grown in Dulbecco’s Modified Eagle Medium (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Corning, Manassas, VA), 1% L-glutamine (Sigma-Aldrich, St. Louis, MO), and 1% antibioticantimycotic (Life Technologies, Carlsbad, CA). Cells were incubated at 37 °C and 5% CO2 in a tissue culture incubator. Nocodazole (Sigma-Aldrich, St. Louis, MO) and Y-27632 (Calbiochem, San Diego, CA), were dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO). To perturb mechanical forces and evaluate the effects on leader cell formation cells were incubated with either 1 μM nocodazole to increase cell traction force or 5 μM Y27632 to reduce cell traction force, similar to previously reported experiments.22 1.2. dsLNA Probes and Cell Staining for Single-Cell Gene Expression Analysis. The dsLNA probe consists of two B


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Figure 2. Single cell tracking of smooth muscle cell migration at the wound edge. (a) Imaris tracks for cells 0−50 μm from the wound edge for each wound shape scenario. The arrows indicate straight line displacement. Scale bars, 100 μm. (b) Straightness of migration, (c) mean migration speed, and (d) perpendicular and (e) parallel migration distance for cells at each distance from the wound edge. All three geometries were analyzed. *, p < 0.05; **, p < 0.01; NS, not significant. Approximately 50 cells were analyzed for each distance from the wound edge, and three independent experiments were performed to acquire the results. transfection. 0.8 μg probe was transfected per well, with 2.4 μL Lipofectamine 2000 transfection reagent used per well in 24-well plates. 1.4. Particle Image Velocimetry and Live Cell Tracking. To gather cell migration characteristics over time, cells were first seeded and studied in an ingrowth, outgrowth, or straight edge pattern as described above.19 After creation of the cell-free region for collective cell migration, a 1:2000 dilution of Hoechst 33342 was added to the cells and incubated for 20 min. Then, the cells were washed three times with culture media and placed in a live cell imaging system (Oko Laboratories, Pozzuoli, Italy) at 37 °C with 5% CO2. Images were acquired once every 5 min for 12 h. For analyzing cell movement over time, particle image velocimetry (PIV), the Imaris (Bitplane, Zurich, Switzerland) spot track feature was used. The initial wound edge was also marked using the Imaris surface tool. To improve tracking, we converted the original image pixel values to binary using

h to visualize the actin cytoskeleton. Primary antibodies against vinculin (Sigma, St. Louis, MO, 1:400 dilution) were then incubated with the cells for 1 h at room temperature followed by Alexa Flour 488-conjugated secondary IgG antibodies (Invitrogen, Molecular Probes, Eugene, OR, 1:400 dilution) for 1 h at room temperature. Finally, nuclei were then stained with DAPI (Invitrogen, Carlsbad, CA). 1.3. Probe Transfection and Wound Healing Assays. In wound healing experiments, cells were seeded at 250,000 cells/mL and allowed to settle for 6 h before wounding. Plastic (PDMS or acrylic) blockers were used to create the wound area based on previous studies.19 Cells were then allowed to grow for 12 h to form a dense monolayer before washing twice with 1X PBS before proceeding with migration experiments. In the case of a transfection, cells were immediately transfected after washing. The dsLNA probe and Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) were combined using the manufacturer’s protocol prior to C


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Figure 3. Migration analysis at the wound edge. Imaris cell tracking data analyzed by the R package “mixtools,” with the “normalmixEM” function. Cell subpopulations (green), which include a combination of leader and follower cells, migrate straighter and faster than the majority of cells (red). For this analysis, data from the first 100 μm from the wound edge were used to obtain the bimodal plots. Approximately 100 cells were analyzed for each plot, and three independent experiments were performed to acquire the results. ImageJ software. For dsLNA and PIV fluorescence images, approximately 350 cells were measured per well. At least three samples were used per experiment. The table of contents image was acquired with a Leica SP8 confocal microscope equipped with HyD photomultiplier tubes. Mean ± SEM was used to evaluate data. Independent group comparisons were performed using the Student’s t-test, and the comparison of multiple groups involved a one-way ANOVA analysis with post hoc Tukey’s multiple comparison test. P < 0.05 was accepted as statistically significant. For the analyzing the bimodal distributions, the R package “mixtools” was used to break down the data acquired both from cell migration processing using PIV and Imaris as well as the dsLNA probe fluorescence data. The first 100 μm from the wound edge were analyzed in each case. The data were analyzed by the R package “mixtools,” with the “normalmixEM” function, which isolates mixtures of normal distributions. To do this, mixtools uses a technique known as “unsupervised clustering,” where subgroups from a sample of measurements is discerned using an expectation-maximization (EM) algorithm, an iterative method that finds maximum likelihood estimates of parameters. The distributions of migration characteristics were illustrated in graphical form, and the bimodal Gaussian peak area (λ), the peak mean (μ) of the two peaks, and the standard deviation (σ) of the two peaks was compared.

the NIH ImageJ default threshold setting to determine objects from background before analyzing the images.23 1.5. Computational Biomechanical Analysis of Wound Geometry Stress. Finite element analysis was performed with ANSYS 13 as previously described.24−26 Briefly, a three-dimensional computational model was constructed with two separate layers: a fixed layer at the bottom with a contractile layer on top. The fixed layer properties are E = 500 Pa, Poisson’s ratio = 0.499. The contractile layer properties are E = 100 Pa, Poisson’s ratio = 0.499. Because the stiffness of the well plates is on the order of GPa (∼9 orders of magnitudeshigher than the cell), it was assumed that the substrate is rigid, and therefore the surfaces of monolayer in contact with the well plate (the sides and bottom) were fixed. Contraction was replicated by applying a uniform thermal strain. To study the effects of geometric constraints on cell migration, the cell monolayer area was considered constant for all geometries. The geometrical constraint areas were 2.2 mm × 6.5 mm for the straight edge shape (with the long edge used to measure stress and the outer circle diameter of 15.6 mm, or equivalent to a 24-well plate diameter), 5 mm diameter for outgrowth, and 2 mm diameter for ingrowth. The simulation was static. To keep an equal mesh density, we maintained the element size at 4 μm. The first principal stress was calculated at the cell−substrate interface. 1.6. Imaging and Data Analysis. Bright-field, fluorescence, PIV, and traction force images were taken with an inverted fluorescence microscope (TE2000-U, Nikon Instruments Inc., Minato, Japan) and acquired with a CCD camera (SensiCamQE, Cooke Corp., Romulus, MI). All fluorescence images were taken with the same exposure time for comparison purposes. Data analysis was performed using NIH

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RESULTS 2.1. Geometric Control of Leader Cells during Collective Migration. Ingrowth, outgrowth, and straight edge geometries were incorporated into the migration assay to D


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Figure 4. Upregulation of β-actin and β3-integrin at the wound edge. (a, c) Single cell gene expression analysis of smooth muscle cell near the wound edge by dsLNA probes. Fluorescence images illustrating single cell gene expression profiles of (a) β-actin and (c) β3 integrin mRNA during smooth muscle cell collective migration. Scale bars, 100 μm. (b, d) Spatial expression profiles of (b) β-actin and (d) β3 integrin mRNA. Approximately 35 cells were analyzed for each distance from the wound edge, and 4 independent experiments were performed to acquire the results. **, p < 0.001.

Cells in the outgrowth geometry, overall, also displayed a higher migration speed compared to ingrowth and straight edge geometries (Figure 2c). We also examined the perpendicular and parallel components of cell displacement (Figure 2d, e). In all geometries, cells near (0−50 μm) the wound edge migrated more perpendicularly away, the x-component straight-line distance from the initial wound edge. Contrarily cells in the inner region of the monolayer (>50 μm) tended to migrate less perpendicularly to the initial wound, resulting in spatial gradients of perpendicular displacement (Figure 2d). Smooth muscle cells in the outgrowth geometry also had a larger gradient of perpendicular displacement component compared to cells in the straight edge and ingrowth scenarios. In contrast, cells in all cases and regardless of position within the monolayer did not significantly move parallel, the y-component straight-line distance, to the initial wound edge over the course of 12 h of migration (Figure 2e). 2.2. Outgrowth Geometry Enhances the Formation of Leader Cells. The data distribution for migration straightness and speed were analyzed to investigate the coordination of smooth muscle cells and the function of

investigate the mechanoregulation of leader cell formation during collective smooth muscle cell migration (Figure 1a). The formation of leader cells with a migratory phenotype protruding from the wound boundary was observed near the wound edge in all geometries (Figure 1b). Single cell tracking, particle image velocimetry (PIV), was performed to extract information about individual cell movement in each geometry over a 12 h period. In Figure 2a, the straight, red arrows illustrate the straight-line displacement of each cell, measured from the start to the finish of the experiment. The multicolored, curved traces represent the actual path each cell took during the 12 h cell tracking experiment. The graph in Figure 2b illustrates the migration straightness of each cell. In this study, the migration straightness is defined as the ratio between the straight-line displacement and the total distance traveled. Overall, gradients of migration straightness were observed for all cases. In particular, cells beginning closer to the wound edges (0−50 μm) migrated straighter than cells beginning farther from the wound edges (>50 μm). Furthermore, cells near the wound edge for the outgrowth assay migrated significantly straighter than cells near the wound edge in either the ingrowth or the straight edge assays. E


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Figure 5. Notch1 and Dll4 mRNA expressions at the wound edge. (a, b) Fluorescence images illustrating Notch1 and Dll4 expressions in migrating smooth muscle cells in the straight edge assay. (c, d) Dll4 and Notch1 mRNA expression profiles in migrating smooth muscle cells from the wound edge as measured by the dsLNA probe. (e, f) Fluorescence signal was measured in the cells at the first, second, and third rows from the wound edge. The signal was then measured deep into the monolayer, at least 500 μm from the wound edge. ***, p < 0.001; **, p < 0.01; *, p < 0.05. Approximately 15 cells were analyzed for each row, and 35 cells were analyzed for each monolayer for each independent experiment. Nine independent experiments were performed to acquire the results for both Dll4 and Notch1 dsLNA analysis.

leader cells. Generally, the cell migration probability distribution functions of cell migration straightness and speed for the three geometries did not follow Gaussian distribution and exhibited bimodal-like distribution characteristics (Figure 3). Table S2 shows the corresponding characteristics of each graph including the mean (μ), the standard deviation (σ), and the area for the Gaussian peaks (λ). The outgrowth geometry exhibited a larger group of leader cells, represented by the second, higher peak, moving with a higher straightness, 32.8%, compared to ingrowth, 16.1%, and straight edge, 24.4%. Interestingly, both peaks of migration speed were higher in the outgrowth geometry (approximately 16 and 25 μm/h) compared to the ingrowth and straight edge geometries (approximately 12 and 21 μm/h for each). The increase in migration speed in the outgrowth geometry, which correlated

with the formation of leader cells, was contributed by an overall increase in migration speed for all cells near the leading edge. 2.3. Upregulation of β-Actin and β3-Integrin during Smooth Muscle Cell Migration. To investigate the molecular mechanisms responsible for the increase in migration speed of the outgrowth case, we measured the smooth muscle cell gene expression profiles in the three wounding scenarios. The dsLNA probe transfection protocol was optimized to both maximize the transfection efficiency and the fluorescence signal-to-noise ratio according to previously published protocols.20,21 Because focal adhesion formation and cytoskeleton reorganization are characteristics of leader− follower organization,27 the expression of both β-actin mRNA and β3-integrin mRNA in ingrowth, outgrowth, and F


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Figure 6. Biomechanical analysis at the wound edge. (a) Representative bright-field image and traction force distribution in the experiment. The direction of perpendicular traction force is illustrated by the arrow in the upper-right corner of the brigh-tfield image. (b−d) Traction force analysis showing the average perpendicular force generated by cells 0−400 μm from the wound edge for each wound geometry. In the graph, the frequency (Y-axis) corresponds to the number of cells expressing traction force within each range of 25 Pa intervals (i.e., 0−25 Pa, 25−50 Pa, and so on). Three independent experiments were performed to acquire the results.

straight edge smooth muscle migration geometries was first analyzed (Figure 4). In general, gradients in gene expression were formed near the wound edge for all geometries. In agreement with the migration data, the signal of both β-actin and β3-integrin mRNA was higher at the wound edge compared to the inner region of the monolayer. Additionally, for both β-actin and β3-integrin mRNAs outgrowth expression was significantly increased at the wound edge (0−50 μm) compared to the ingrowth and straight edge cases. As a control, a random scrambled probe was also transfected into cell for all wound scenarios, and the signal was not significant at any point (Figure S1). 2.4. Notch1-Dll4 Lateral Inhibition at the Wound Edge. We have previously demonstrated Dll4 is indicative of leader cells.17 Notch1-Dll4 signaling near the leading edge was, therefore, examined to study its regulatory roles in leaderfollower organization during smooth muscle collective cell migration. Notch1 and Dll4 mRNAs were measured 0−500 μm from the wound edge for ingrowth, outgrowth, and straight edge wound geometries (Figure 5a−d). Similar to β-actin and β3-integrin mRNA, gradients of Notch1 mRNA and Dll4 mRNA expressions were formed near the wound edge for all wound geometries (Figure 5c, d). A comparison of Dll4expressing leader cells over a constant area of initial wound showed that the outgrowth geometry contains a higher density

of leader cells than ingrowth or scratch assays (Figure S2). Close examination of the smooth muscle cells reveals local cell−cell coordination of Notch1-Dll4 genes and the formation of leader cells at the wound edge (Figure 5e, f). In particular, distinctive gene expression profiles were observed for Dll4 and Notch1 mRNAs in the first few rows from the wound edge. Dll4 mRNA was typically increased in cells at the first row of the migrating front, whereas Notch1 mRNA was maximized at the second row of the monolayer, supporting lateral inhibition of Notch1-Dll4 signaling in the regulation of smooth muscle leader cells.28 2.5. Biomechanical Analysis of Smooth Muscle Cell Migration Geometric Control. In this study, ingrowth, outgrowth, and straight edge wound geometries were applied to investigate the biomechanical coordination of smooth muscle leader cells. Traction force microscopy was used to study the physical interactions that cells in the migrating monolayer have with each other and their substrate.29,30 Figure 6a shows a representative bright-field image of smooth muscle cells in the traction force assay as well as a heat map image demonstrating the range of forces generated by cells; note that in Figure 6a the force shown only represents the positive perpendicular component (y-axis-component). Cells at the wound edge in the field of view were seen creating strong focal contacts while migrating in contact with the monolayer. Figure G


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ACS Biomaterials Science & Engineering 6b−d shows the positive perpendicular component (y-axiscomponent) of traction force near the wound edge for the ingrowth, outgrowth and straight edge geometries. Despite the large fluctuation of traction force, a nonzero average cell traction force was observed for each geometry at the wound edge. The average cell traction force had a magnitude of approximately 90 Pa for both the straight edge and the ingrowth scenarios while the outgrowth scenario produced a lower mean traction force, 78 Pa, at the wound edge. In silico biomechanical analysis was also performed to illustrate the effects of wound geometries on the intercellular stress distribution. The geometries were the same as the experiments with the cells: 5 mm outgrowth diameter, 2 mm ingrowth inner diameter, 15.6 mm ingrowth outer diameter (24-well plate well diameter), and a 2.2 mm by 6.5 mm rectangle (again, with an outer diameter of 15.6 mm). Figure 7 shows computational finite element modeling of the cellular stress distribution in each wound geometry out to 1.5 mm

from the wound boundary. Figure 7a shows representative illustrations for the wound geometries in each scenario. The normalized first principal stress for each wound scenario is shown in Figure 7b. The stress at the wound edge for all scenarios rapidly decreased at the wound edge before gradually reducing over the next millimeter from the wound edge. In agreement with traction force microscopy, the outgrowth geometry showed lower stress at the wound edge compared to the ingrowth and straight edge, which were similar in stress distribution. These results, in combination with the traction force experiment outcome, confirm that cells migrating in the outgrowth scenario generate decreased force overall while migrating away from the original wound location. Therefore, these results support the notion that a reduction of the wound edge stress due to the wound geometry can enhance the formation of smooth muscle leader cells. 2.6. Biomechanical Perturbation through Y-27632 and Nocodazole Addition. Traction force and computational modeling data suggest geometry regulates leader cell formation during collective migration via a mechanoregulation scheme. To independently investigate the role of mechanical force on collective migration of smooth muscle cells, we pharmacologically modulated the intercellular tension originating from cell traction force.22 By adjusting the force generating capability of the smooth muscle cells via drugs that increase and decrease cellular traction force, the relationship between force and leader cell development can be better understood. ROCK inhibitor, Y-27632, was employed to decrease traction force and microtubule inhibitor, nocodazole, was used to increase traction force. To quantify the formation of leader cells, which form protruding structures and increased monolayer waviness, we measured the curvature of the wound edge under different drug treatments (Figure 8a−c). The curvature is defined as the ratio of the total free wound edge distance over the straight-line wound distance. A straight (uniform) wound edge using this definition will have a value of 1, whereas the formation of fingerlike structures by leader cells will increase the value. Nocodazole produced a straighter wound edge, while Y-27632 increased the wound edge curvature (Figure 8d-f). For both the outgrowth and the straight edge case, 1 μM nocodazole produced significantly less curvature than 5 μM Y-27632 (***, P < 0.001, and *, P < 0.05). As the ingrowth and outgrowth scenarios already present with curved monolayers prior to wounding their curvature was greater than that of the straight edge case. The overall effects of nocodazole and Y-27632, nevertheless, were consistent in all cases. The formation of leader cells as indicated by the curvature ratio also correlated with the migration rate of the monolayer. Figure 8g shows the migration rate decreased with nocodazole addition. In contrast, Y-27632 treatment resulted in an increase of migration rate for all three wound geometries (Figure 8h). The cell area was also examined to study the cellular mechanism responsible for the changes in migration rate under drug treatment (Figure 9 and Figure S3). Without drug addition, gradients of cell area were observed in all three geometries. In particular, smooth muscle cells with enlarged cell size spreading on the surface were observed at the wound edge and the cell size gradually decreased toward the inner region of the monolayer. Similar gradients of cell area were previously observed in epithelial cells supporting the coordination of migratory and proliferative phenotypes during collective cell migration.15 With Y-27632, the area of all cells,

Figure 7. Computation analysis of the wound edge stress. (a) Finite element analysis mapping the stress distribution from the wound edge for each wound geometry. The cell monolayer consists of two separate layers: a fixed layer at the bottom with a contractile layer on top. The fixed layer properties are E = 500 Pa, Poisson’s ratio = 0.499. The contractile layer properties are E = 100 Pa, Poisson’s ratio = 0.499. Because the stiffness of the well-plates is on the order of GPa (∼9 orders of magnitude higher than the cell), it was assumed that the substrate is rigid, and therefore the surfaces of monolayer in contact with the well-plate (the sides and bottom) were fixed. The second and third rows are zoomed-in on the edge of the simulation. (b) Graphical representation of the stress distribution illustrating that the outgrowth geometry has a lower stress at the wound edge compared to the ingrowth and straight edge. H


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Figure 8. Perturbation of the wound edge stress through Y-27632 and nocodazole addition. (a−c) Bright-field images illustrating the increasing smooth muscle cell wound edge with Y-27632 addition along with the decreasing smooth muscle cell wound edge with nocodazole addition. (d−f) Wound edge curvature with drug addition quantified in graphical form. The curvature is the ratio of the total wound edge free area divided by the straight line distance across the wound edge. (g, h) Migration also increased with Y-27632 addition and decreased with nocodazole addition in a dose dependent fashion. **, p < 0.01; p < 0.001. Three independent experiments were performed to acquire the results, with at least five wound edges evaluated for curvature for d−f. For g and h, at least four wounds were evaluated for growth over the course of three independent experiments.

cell types,31 smooth muscle leader cells processed a large size and protruded from the wound boundary to direct follower cells during collective migration. Single cell tracking and a bimodal distribution of the migratory behaviors throughout the monolayer characterized the leader cell subpopulation at the wound edge with a large migration speed. Functionally, leader cells migrated in a more directionally persistent manner at the wound edge and the migration was steadfastly perpendicular to the wound edge. Our single cell gene expression analysis also revealed the upregulation of migration associated genes, including β-actin and β3-integrin, which may contribute to the functions of leader cells.27 The outgrowth geometry, in particular, showed not only a straighter, or more persistent, migration front, but also a faster migration speed overall. As leader cells pull on the cells behind them (the follower cells also pull on the leader cells),32 the increased number of leader cells from the outgrowth geometry additionally produces a more persistent, straighter migration.33 Our results also reveal the regulatory role of mechanical force in collective smooth muscle cell migration. Previous studies have employed traction force microscopy near the front of a migrating epithelium and proposed a tug-of-war model. In

regardless of position in the monolayer, increased in all three geometries. In contrast, nocodazole notably reduced the cell size. These results collectively support the notion that traction force modulates the coordination of smooth muscle cells as well as the formation of leader cells during collective cell migration.

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DISCUSSION In this study, we investigate the formation of leader cells during collective smooth muscle cell migration. In the experiments, gradients of cell size, migration straightness, speed, and gene expression were observed near the wound edge to coordinate the smooth muscle cells during collective cell migration. We observed a correlation between the formation of smooth muscle leader cells and increased migration speed and straightness as well as increased β-actin and β3-integrin. Consistent with the lateral inhibition mechanism, Dll4 and Notch1 mRNA were maximized in the first and second rows, respectively, creating checkered patterns. These results support the existence of smooth muscle leader cells, a finding that suggests the applicability of leader-follower organization beyond epithelial cells. Consistent with studies using other I


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Figure 9. Size of cells versus location in relation to the wound edge was measured. The size of cells was evaluated in (a) control (no drug), (b) (5 μM) Y-27632 addition, and (c) nocodazole addition (1 μM). At least 25 cells per wound range (100 μm interval) were evaluated over the course of three independent experiments.

also increased the curvature of the monolayer in all cases by at least 8%. One μM nocodazole, which increases the traction force generation of the cells, decreased the monolayer curvature by at least 15%. As the collective migrating front begins to form finger-like structures with the advent of leader cells the curvature of the migration front increases as more leader cells form. Conversely, the migrating front is expected to decrease in curvature as fewer leader cells form. Therefore, the migrating front increasing in curvature with Y-27632 addition or decreasing the traction force generation capability of the cells directly matches our wound experiments using the outgrowth scenario, where the traction force is the lowest compared to the other migration geometries. In addition, the migration speed of the cells was increased with Y-27632 in all scenarios and decreased with nocodazole addition, results that also agree directly with the hypothesis that reducing wound edge stress enhances the formation of smooth muscle leader cells.

the tug-of-war model, the reduction of intercellular tension at the leading edge may provide a signal to initiate the formation of leader cells.17,18 To investigate the mechanoregulation of leader cell formation and coordination of the migrating monolayer we incorporated, ingrowth, outgrowth, and straight edge geometries in the wound healing assay. The average traction force in the outgrowth wound scenario in the first 0 to 50 μm from the wound edge was 12 Pa less than the ingrowth and the straight edge scenarios (78 Pa versus 90 Pa). This also matches the finite element analysis that we performed at the same time on the three wound scenariosthe ingrowth and straight edge had a similar stress distribution while the outgrowth had a decreased stress distribution in comparison. Our technique provides a noninvasive, nonpharmacological approach for probing the mechanoregulation of collective cell migration. Our data, including migration straightness, speed, cell size, and gene expression profiles, collectively indicate that the outgrowth case enhances the formation of leader cells, supporting a mechanoregulation scheme in the coordination of smooth muscle cell migration. To independently examine whether decreased stress in migrating smooth muscle cells causes increased leader cell formation, we employed traction-force modulating drugs, Y27632 and nocodazole, to modulate the cells’ ability to generate traction force.22 In accordance with our results, 5 μM Y-27632, which decreases the traction force generation in cells,

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CONCLUSIONS This study demonstrates that decreasing stress on cells at the wound edge in collective smooth muscle cell migration enhances the formation of leader cells at the edge of a migrating monolayer. Our data collectively suggest the involvement of leader cells in smooth muscle cell migration and support the functional role of leader cells to physically pull J


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on the cells behind them and lead collective migration. Overall, our study reveals leader cells as an important regulator of smooth muscle cell collective migration, results that may provide expanded treatment options in pathogenic smooth muscle cell conditions, such as atherosclerosis and wound healing.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b01222.

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Random dsLNA probe data for migrating smooth muscle cells; chart evaluating the number of cells versus the location of cells in the monolayer for all wound geometries and for nocodazole and Y-27632 addition; table listing the sequence as well as the Gibbs free energy for the probes in the presence and absence of target; table listing the mean, standard deviation, and overlap of the bimodal peaks analyzed (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +1-814-863-5267. ORCID

Pak Kin Wong: 0000-0001-7388-2110 Present Address

‡ Z.S.D. is currently at Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California, USA

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Kaitlyn Ammann and Katrina DeCook for preparing smooth muscle cells. The work was funded and carried out under NIH New Director’s New Innovator Award project 1DP2OD007161-01.

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REFERENCES

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Decreasing Wound Edge Stress Enhances Leader Cell Formation

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