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. 2011 Aug 3;101(3):622–630. doi: 10.1016/j.bpj.2011.06.051

Cell-Cell Interactions Mediate the Response of Vascular Smooth Muscle Cells to Substrate Stiffness

Olga V Sazonova †, Kristen L Lee †, Brett C Isenberg †, Celeste B Rich ‡, Matthew A Nugent †,‡,∗∗, Joyce Y Wong †,∗
PMCID: PMC3145282  PMID: 21806930

Abstract

The vessel wall experiences progressive stiffening with age and the development of cardiovascular disease, which alters the micromechanical environment experienced by resident vascular smooth muscle cells (VSMCs). In vitro studies have shown that VSMCs are sensitive to substrate stiffness, but the exact molecular mechanisms of their response to stiffness remains unknown. Studies have also shown that cell-cell interactions can affect mechanotransduction at the cell-substrate interface. Using flexible substrates, we show that the expression of proteins associated with cell-matrix adhesion and cytoskeletal tension is regulated by substrate stiffness, and that an increase in cell density selectively attenuates some of these effects. We also show that cell-cell interactions exert a strong effect on cell morphology in a substrate-stiffness dependent manner. Collectively, the data suggest that as VSMCs form cell-cell contacts, substrate stiffness becomes a less potent regulator of focal adhesion signaling. This study provides insight into the mechanisms by which VSMCs respond to the mechanical environment of the blood vessel wall, and point to cell-cell interactions as critical mediators of VSMC response to vascular injury.

Introduction

Cardiovascular disease, which includes atherosclerosis and restenosis, is the leading cause of death in the United States (1). Efforts to elucidate the mechanisms of cardiovascular disease have revealed several consistent themes: Phenotypic changes in resident vascular smooth muscle cells (VSMCs), which include enhanced proliferation, migration, and remodeling of the extracellular matrix (ECM), are hallmarks of neointimal expansion in atherosclerotic and restenotic blood vessels. Atherosclerotic lesion growth is also accompanied by vessel wall stiffening, which is itself predictive of disease progression (2–4). A growing body of work demonstrates that VSMCs are sensitive not only to the biochemical but also the mechanical properties of the ECM, and respond to changes in stiffness with systematic modifications in phenotype (5–14). Thus, it is likely that altered ECM mechanics in a diseased artery play a role in the response to injury exhibited by synthetic VSMCs, potentially contributing to both healing and disease progression.

Mechanotransduction is the process by which cells sense and respond to physical stimuli from the environment, and numerous studies point to focal adhesions (FAs) and adherens junctions (AJs) as the principal sites of mechanical signaling in cell-ECM and cell-cell interactions, respectively (15–18). In both cases, heterogeneous protein clusters form a structural link between an external anchor and the actin cytoskeleton and participate in numerous inside-out and outside-in signaling pathways. Importantly, FA and AJ dynamics can be regulated by applied force (19–21), and there is evidence that mechanotransduction through the two types of contacts is interdependent. For example, endothelial cell morphology is dependent on substrate stiffness in sparse but not confluent cultures (22), whereas varying levels of cadherin expression can directly modulate cell-substrate adhesion (23). These and similar findings (24,25) show that cells integrate biochemical and/or mechanical signals from FAs and AJs and modify the degree of each to achieve a particular phenotype.

Within an atherosclerotic lesion, VSMCs experience changes in cell-cell interactions along with mechanical and biochemical signaling. To understand the role of matrix stiffening in VSMC pathology, it is also essential to understand how communication through cell-cell junctions may affect cellular response to ECM mechanics. In this study, we quantified changes in VSMC spreading and FA and cytoskeletal protein expression as a function of substrate stiffness and cell density. Our results indicate that cell-cell interactions selectively modulate certain effects of substrate stiffness, suggesting a mechanism by which VSMCs may balance physical stimuli from cell-cell and cell-ECM contacts. This process may be critical to regulating VSMC response to local injury, driving it toward a state of repair or chronic pathology.

Materials and Methods

Substrate synthesis and characterization

Polyacrylamide (PAAM) gel substrates were synthesized and functionalized with fibronectin (Fn) using a modification of previous methods (26,27). Substrate mechanics were determined with tensile testing and nanoindentation using an atomic force microscope (AFM). For tensile testing, Hooke's law was used to determine the bulk elastic modulus for 2 × 3 inch gel samples. Nanoindentation was used to characterize the mechanics of thin gels (100 μm) on glass coverslips, where the surface elastic modulus of each sample was calculated using a linearized variant of the Hertz model (28). Control experiments verified that Fn incorporation does not affect PAAM elastic modulus (data not shown). An enzyme-linked immunosorbent assay (ELISA) was used to quantify the relative fibronectin concentration in PAAM gels. Control experiments showed that this assay is sensitive to protein content only at the gel surface: gels of uniform volume but increasing surface area produced a higher antibody signal, whereas gels of varying volume but uniform surface area did not (data not shown). Additionally, we found that the efficiency of Fn incorporation varied inversely with acrylamide concentration. To produce substrates with increasing elastic modulus but identical Fn surface concentration, we adjusted the amount of protein added to each gel solution. Detailed methods for substrate synthesis and characterization can be found in the Supporting Material.

Cell culture

Primary rat neonatal aortic smooth muscle cells were cultured on tissue culture polystyrene in Dulbecco's modified Eagle's medium supplemented with nonessential amino acids, sodium pyruvate (Mediatech, Manassas, VA), and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA). First or second passage cells were detached with a solution of 0.05% trypsin and 0.02% EDTA, centrifuged for 5 min at 1000 × g, and plated on PAAM gels at 5,000 or 12,000 cells/cm2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum that had been depleted of soluble Fn using a gelatin-sepharose column (29). Media was changed 24 h after seeding, and again every 48 h. Cells were fixed after 48 h for immunostaining, whereas samples for protein or gene expression were collected after 96–120 h.

Cell density quantification

We used a combination of phase-contrast microscopy and computational image analysis (written in MATLAB (The MathWorks, Natick, MA)) to quantify live cell density on PAAM gels 3–6 h before collecting samples for gene and protein expression studies. We averaged the computed confluence of 10–15 images from a given sample to obtain an average confluence measurement for that sample. Samples with 40–60% cell coverage were defined as low density and 80–95% cell coverage as high density. Refer to Methods and Fig. S1 in the Supporting Material for details.

Cell area quantification

Cells on PAAM substrates were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X 100 in TBS, and stained with Hoescht and rhodamine-phalloidin at a 1:400 dilution. Samples were sandwiched between two coverslips and imaged at 10× using an Olympus IX81 microscope (Olympus America, Center Valley, PA), a Hamamatsu ORCA R2 camera (Hamamatsu, Bridgewater, NJ), and Metamorph Imaging software (Universal Imaging, Downingtown PA) under appropriate fluorescent illumination. Cell area as a function of cluster size was determined by measuring cluster area and dividing by the number of nuclei to obtain average individual cell area (see Fig. 2, A and B). Refer to Methods in the Supporting Material for details.

Figure 2.

Figure 2

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Cell density modulates cell spreading on stiff but not soft substrates. (A and B) VSMC area as a function of cluster size was quantified using immunocytochemistry and fluorescent image analysis. (C) For each sample set, cell area was normalized by the average 25 kPa single cell area to produce a normalized plot of average cell area as a function of cell cluster size. Data are the product of four biological replicates, with each cluster represented by one data point on the y axis (n = 5–200). For this and all figures, asterisks (∗) indicate statistically significant differences between 135 and 25 kPa gels for a particular cell density, with p < 0.05.

Western blotting

The following antibodies were used to assess protein expression (raised in mouse and obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted): Primary anti-α-actin (clone 1A4), anti-α-tubulin (clone DM-1A), antitalin (clone 8d4), antivinculin (clone hVin-1). We used an HP-linked goat antimouse secondary antibody (Invitrogen, Carlsbad, CA) in all experiments. Gels were rinsed 3 times with ice cold phosphate buffered saline before lysis. To ensure that samples were only collected from PAAM, and not the underlying glass surface, we removed the entire gel from the coverslip before submerging it in lysis buffer. After a 30 min incubation period, gels were separated from the lysate by centrifugation through a 35 μm filter column (USB, Cleveland, OH) at 2000 × g for 2 min. After more centrifugation (10,000 × g for 10 min at 4°C), we collected the supernatant and determined the total protein concentration (BCA Protein Assay, Pierce, Rockford, IL). Electrophoresis and immunoblotting proceeded according to standard protocol (see Supporting Material).

Gene expression

PAAM gels were removed from their coverslips and submerged in guanidinium thiocyanate (4 M) for 15 min. After being separated from the gels, lysates were processed according to standard protocol for RNA isolation (see Supporting Material). RNA (500–1000 ng) from each sample was treated with DNase to remove any remaining genomic DNA. cDNA was synthesized in a reverse transcriptase (RT) polymerase chain reaction (PCR) reaction using the TaqMan EZ RT-PCR kit (Applied Biosystems, Carlsbad, CA) according to manufacturer's instructions. Real-time PCR was performed (ABI 7300; Applied Biosystems) using the following primers (Applied Biosystems): syndecan-4 (Rn00561900), β1 integrin (Mn01253227), and 18S rRNA (4308329.) Data were analyzed using the ΔΔCt method, where signal intensity was normalized to the corresponding signal for 18S rRNA expression. The resulting quantities were used to calculate an expression average for each condition relative to the 25 kPa (soft) substrate.

Focal adhesion and adherens junction analysis

VSMCs were cultured on 25 and 135 kPa substrates for 48 h in Fn-depleted medium. Samples were rinsed in ice-cold phosphate buffered saline and incubated with prepermeabilization buffer (50 mM NaCl, 30 mM sucrose, 10 mM PIPES, 3 mM MgCl2, 1 μg/ml protease inhibitor cocktail (Sigma-Aldrich), 0.05% Triton-X 100, pH 6.8) for 1 min on ice. Samples were then fixed and stained using an antivinculin or anti-p120 catenin (clone 15D2, Millipore) antibody, and imaged using confocal microscopy. Select images were processed using custom MATLAB software that identifies objects in the image based on criteria of threshold, object size (0.3–20.0 μm2), and elongation (0.7–1.0, where 0 indicates a perfect circle). Objects meeting these criteria were analyzed to quantify their number and size. Additional methods for sample preparation, confocal microscopy, and image analysis can be found in the Supporting Material.

Statistics

Statistical significance was established with ANOVA followed by the Mann-Whitney U test, or linear regression analysis; p-values <0.05 were considered significant.

Results

To model the effect of blood vessel wall stiffening on vascular smooth muscle cell behavior, we used PAAM gel substrates with tunable mechanical properties to match the range of stiffness documented in healthy and diseased vessels (30). Modifying the acrylamide/bis ratio produced substrates whose elastic modulus increased linearly with bis-acrylamide concentration (Fig. 1). Uniaxial tensile testing of the gel bulk and nanoindentation of the gel surface with AFM (Fig. 1 A) produced elastic moduli spanning 21–136 kPa. Values obtained with Hooke's law (tensile testing) and the Linearized Hertz model (nanoindentation) were in excellent agreement with one another. For simplicity, the three substrates used in this study, produced using acrylamide/bis ratios of 10%:0.1%, 10%:0.4%, and 12%:0.6%, will be referred to as having moduli of 25, 70, and 135 kPa, respectively. To promote cell adhesion, we covalently incorporated Fn throughout the bulk of the gel and quantified Fn concentration with an ELISA that was sensitive to protein at the gel surface (verified with control experiments). Efficiency of Fn incorporation proved dependent on substrate stiffness: varying Fn concentration in gel solutions produced substrates with different moduli but similar Fn surface concentrations (Fig. 1 B). Cultured VSMCs showed robust spreading and comparable levels of attachment and proliferation on all substrates (Fig. 1 C).

Figure 1.

Figure 1

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Polyacrylamide substrates with varying mechanical properties and uniform fibronectin presentation support VSMC growth. (A) PAAM mechanics were assessed with tensile testing and nanoindentation using AFM. Varying acrylamide and bis concentration from 10% to 12% and 0.1% to 0.6%, respectively, produces substrates with elastic moduli of ∼25, 70, and 135 kPa. Values represent averages of three separate experiments, n = 15–25 indentations and 2–5 tensile measurements. (B) Covalent functionalization with fibronectin produces gels with uniform ligand density. Gel surface levels of fibronectin were measured by ELISA on three separate gels for each condition; error bars in this and all figures represent standard error. (C) VSMCs show robust spreading and cell-cell interactions independently of substrate stiffness.

To investigate how cell-cell contacts affect VSMC response to substrate stiffness, we cultured cells at two different densities before assaying gene and protein expression. Cell density was quantified with a series of phase-contrast images taken 3–6 h before lysis (Fig. S1 A); these were analyzed with custom MATLAB software to compute average cell density (Fig. S1 B). We were also able to monitor the variance in cell density on each sample (Fig. S1 C).

To determine whether stiffness and cell density might exert competing effects on cell spreading, we quantified cell area as a function of cell cluster size (Fig. 2) on 25 and 135 kPa substrates. We found that single cells, paired cells, and cells in triplicate on 135 kPa substrates are ∼1.5 times larger than those on 25 kPa substrates (p < 0.05). As cell cluster size increased from 4 to 7 cells, however, average cell area on 135 kPa substrates began to decrease until it was indistinguishable from average cell area for comparable clusters on 25 kPa substrates. A linear regression fit to average cell area on 135 kPa substrates showed a statistically significant negative correlation between cell area and increasing cluster size (r2 = 0.84, slope = −0.07, p < 0.05), whereas there was no correlation for cells on 25 kPa substrates.

To confirm the link between cell density and cadherin-based cell-cell contacts on 25 and 135 kPa substrates, we examined the organization of p120 catenin in clusters of increasing size (Fig. 3). We observed a clear increase in AJ formation with increasing cell density: single cells did not exhibit any notable catenin organization, whereas clustered cells showed increasing levels of punctate adhesions at the interface of contact (white arrows). We saw no difference in this trend with substrate stiffness.

Figure 3.

Figure 3

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Increasing cell density leads to more AJs on soft and stiff substrates. VSMCs were cultured on 25 kPa (left column) and 135 kPa (right column) substrates for 48 h, then stained for p120 catenin and the cell nucleus. Confocal imaging demonstrates that single cells show negligible AJ formation, whereas cells in clusters exhibit higher levels of AJ formation (white arrows) as cluster size increases (descending rows).

To better understand how substrate mechanics and cell-cell contacts affect cell interaction with the ECM, we examined the effect of substrate stiffness and cell density on the expression of β1 integrin and syndecan-4 mRNA. At low cell density, mRNA levels for both receptors increased 2.2-fold from 25 to 135 kPa (p < 0.05), but stiffness induced no change in expression at high density (Fig. 4). We then asked whether the increase in integrin expression would correlate with changes in FA protein expression. Looking first at talin, a scaffolding protein that links β1 integrin to polymerized actin (31), we found that an increase in substrate stiffness produced a 1.5-fold increase in talin protein expression (p < 0.05), but only at low cell density (Fig. 5, top row). Similarly, vinculin, a well-studied marker of FAs that directly interacts with talin (31), was upregulated nearly twofold from 25 to 135 kPa (p < 0.05) at low but not high cell density.

Figure 4.

Figure 4

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Fibronectin receptor mRNA expression is a function of substrate stiffness only at low cell density. VSMCs were seeded onto 25, 70, and 135 kPa polyacrylamide substrates, maintained in 10% FBS depleted of Fn, and lysed for mRNA extraction after 96–120 h in culture. Each value represents an average of 2–7 biological replicates, with each sample assayed 2–3 times with qPCR.

Figure 5.

Figure 5

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Focal adhesion but not cytoskeletal protein expression is a function of cell density. VSMCs were cultured on PAAM substrates at low (40–60% confluence) and high (80–95% confluence) density for 72–96 h in 10% Fn-depleted FBS and lysed for Western blot analysis. Data were obtained with digital quantification of protein expression in ImageJ. Values represent the average of 3–14 biological replicates, with 3–4 Western blots performed per sample.

We also examined the expression of the cytoskeletal proteins smooth muscle α-actin (SMC α-actin) and α-tubulin (Fig. 5, bottom row). Unlike the expression profiles of FA elements, we found that SMC α-actin and α-tubulin were upregulated with increasing stiffness at both low and high cell density. As substrate stiffness increased from 25 to 135 kPa, SMC α-actin protein expression increased 1.5-fold (p < 0.05) and 1.3-fold (p = 0.063) in low and high density cultures, respectively, while α-tubulin exhibited a 2.0- and 1.9-fold increase (p < 0.05).

We hypothesized that changes in cell area may explain the increase in FA protein expression on stiffer substrates. Confocal imaging of FAs stained for vinculin revealed that cell area had no effect on the individual FA area (see Fig. 6 D), whereas increasing substrate stiffness from 25 to 135 kPa produced a small increase in the individual FA area (see Fig. 6 E, 25.3 to 27.0 μm2, p < 0.05). In contrast, cell area exerted a strong effect on the FA number on both soft and stiff substrates (see Fig. 6 F). The FA number increased linearly with cell area, ranging from 40 to 210 per cell on soft substrates and 50 to 250 per cell on stiff substrates.

Figure 6.

Figure 6

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Vinculin localization to FAs is regulated by cell area and substrate stiffness. VSMCs were cultured on 25 and 135 kPa substrates for 48 h in 10% Fn-depleted FBS, prepermeabilized and fixed, then stained for vinculin, the cell nucleus, and f-actin fibers. (A–C) FAs were quantified with custom MATLAB software. (D) Individual FA size plotted against cell size on 25 and 135 kPa substrates. (E) Individual FA size as a function of substrate stiffness. (F) FA number per cell plotted against cell size on 25 and 135 kPa substrates. Sample cell (A–C) on 25 kPa substrate; data were collected from four biological replicates, with 10–15 cells imaged per condition.

Discussion

Tissue elasticity is a highly regulated determinant of normal tissue development and function. Using micro- and nanoscale precision, several recent studies have documented progressive extracellular matrix stiffening in a number of diseases including cancer, cirrhosis, pulmonary fibrosis, and vascular disease (30,32–34). In particular, local calcification and remodeling of the ECM in a developing vascular lesion can lead to loss of compliance in the vessel wall. The role of tissue stiffening in disease development and progression, as well as the mechanisms by which mechanical information is transduced to alter vascular cell response, remain to be determined.

Like cell-ECM contacts, cell-cell interactions are highly dynamic sites of chemical and mechanical stimuli that govern multiple phenomena, including cell sorting, wound healing, and tissue reorganization (35,36). Recent studies show that cells are capable of exerting tension on each other through AJs and that AJ dynamics are tension dependent (19,15,37). Given the similarities between FA and AJ structure and function, it is likely that mechanisms of force sensing through cell-cell and cell-ECM adhesions are conserved and exhibit some degree of interdependence.

Studies have shown that, individually, cell-cell interactions and substrate mechanics are potent regulators of cell morphology (8,38–41). We studied the combined effects of these stimuli on VSMC response to stiffness using flexible substrates and variable cell density.

Although we did not directly control AJ formation, we showed that increasing cell density is closely correlated with higher levels of p120 catenin organization. This finding is consistent with other studies showing that cadherin expression and AJ stability increases with density in multiple cell types (42–44), confirming that varying cell density is an appropriate model for studying the effect of cadherin-based cell-cell interactions (45,46).

We report a mutual dependence between substrate stiffness and cell-cell interactions in the regulation of VSMC spreading area: On 135 kPa substrates, the stiffness-mediated increase in VSMC spread area disappeared with increasing cell cluster size, whereas cluster size had no effect on VSMC spreading on 25 kPa substrates. The first result indicates that cell-cell interactions can override the effect of substrate rigidity on VSMC spreading. Because rigidity sensing is dependent on cytoskeletal tension, this effect could be due to mechanical cues from cell-cell contacts dominating over those coming from the substrate. It is also possible that the effect is largely biochemical; FAs and AJs share a number of structural elements including vinculin and α-actinin, as well as signaling molecules such as the Rho-family GTPases (47,48). Thus, the inverse relationship between cell area and cluster size may be a consequence of shifting signaling pathways or the fact that a finite pool of resources must now be shared between the two types of adhesion complexes. However, the second finding suggests that a certain level of tension at cell-ECM adhesions is necessary for cell-cell contacts to exert their influence. If so, then this effect is not purely biochemical; whereas chemical reactions readily occur in solution, the application of force requires a physical anchor. It is possible that a 25 kPa substrate does not provide enough resistance at FAs for the cell to form mechanically active AJs. This hypothesis is consistent with two recent studies demonstrating the dependence of AJ mechanics on forces generated through integrin-ECM bonds (19,50).

Our findings are partially corroborated by a study of cell-cell and cell-matrix adhesion in endothelial cells (ECs) (51), which revealed that increasing cell density reduces EC area and FA formation by a mechanism that involves vascular endothelial cadherin engagement. Here, we show that this relationship is not unique to ECs or to vascular endothelial cadherin-based contacts. Additionally, the authors showed that inhibition of the ROCK pathway eliminated these effects, implicating cytoskeletal tension as an important variable in cell-cell signaling. This finding suggests that a microenvironment capable of regulating the level of intracellular tension would also influence the effect of cell-cell contacts on cell morphology and FA formation. Our study is consistent with this hypothesis, demonstrating that a certain threshold of ECM rigidity is necessary for cell-cell contacts to affect VSMC area. These results provide further evidence that mechanotransduction through cadherin-based contacts is subject to the properties of the extracellular microenvironment in vivo, where VSMCs and ECs are likely to encounter changes in ECM rigidity.

β1 integrin, talin, and vinculin have previously been investigated in the context of mechanotransduction, where the function of each protein has been shown to be directly modulated by force (52–54). The fibronectin receptors β1 integrin and syndecan-4 establish initial adhesion between the cell and the ECM (55), whereas talin and vinculin are recruited to the adhesion to reinforce integrin binding and form a structural link between the ECM and the actin cytoskeleton (56,57). In this study, substrate stiffness and cell density exerted similar effects on the expression of all FA proteins (β1 integrin, syndecan-4, talin, vinculin): at low cell density, expression increased with stiffness, whereas at high density, stiffness did not have an effect. Notably, we observed a striking similarity in the mRNA expression profiles of β1 integrin and syndecan-4. Although β1 integrin is strongly implicated as a mechanosensor in the literature (32,52,58), far less is known about the role of mechanics in regulating syndecan-4 expression and function (59,60). Our data indicate that both substrate stiffness and cell density can regulate syndecan-4 expression in concert with β1 integrin. Given a potential role of syndecan-4 in the development of vascular disease (61–64), our findings call for further investigation into the effect of tissue mechanics on the expression and function of this receptor.

We found that single cell size closely correlates with the FA number, with larger cells consistently forming more FAs than smaller cells, regardless of substrate stiffness. In effect, this resulted in a greater number of FAs formed on stiff substrates, as those cells tended to be nearly 1.5 times larger than cells on soft substrates. Interestingly, this increase in cell area is similar in magnitude to the stiffness-induced upregulation in gene and protein expression seen at low cell density. It thus seems likely that the observed changes in expression reflect greater cell spreading and enhanced FA formation as a result of increasing stiffness. We were not able to directly measure the effect of cell density on FA formation due to imaging artifacts created by the particular morphology of VSMC clusters. However, it has been shown that clustered ECs form fewer and smaller FAs than single cells (51). Given that clustering reduces cell area and eliminates FA upregulation on stiff substrates, we propose that, like ECs, clustered VSMCs exhibit a reduction in FA assembly as a result of increasing cell-cell interactions. Although present methods cannot quantify individual FAs within VSMC cell clusters, we expect that clustered VSMCs on 135 kPa substrates would form fewer FAs than single cells, whereas clustered and individual cells on 25 kPa substrates would be indistinguishable in this measure.

One interpretation of the results presented is that VSMCs in dense clusters are less sensitive to substrate stiffness than single cells, perhaps because cell-cell contacts provide a louder mechanical stimulus than what is perceived through cell-ECM contacts. To the contrary, we found that SMC α-actin and α-tubulin show a stiffness–mediated increase in expression at both low and high cell density. This result clearly shows that cells retain the ability to sense and respond to ECM mechanics regardless of cell density. Furthermore, it indicates that mechanotransduction in VSMCs does not require a particular degree of spreading or FA expression, because cells in dense cultures exhibit the same cell area and FA protein expression on 25 and 135 kPa substrates, yet show a clear stiffness-mediated increase in cytoskeletal protein expression.

Similarities between stiffness- and growth factor (GF)-induced cellular processes suggest that progressive stiffening of the vascular wall may contribute to VSMC pathology in a manner analogous to GF stimulation: VSMCs migrate faster and proliferate more rapidly when cultured on stiffer substrates (7–9) or in the presence of fibroblast growth factor and platelet-derived growth factor (65,66). Similarly, durotaxis, the tendency of cells to migrate up a stiffness gradient, can be accurately characterized using chemotaxis models (10). The phenotypic plasticity of VSMCs is highly sensitive to GF exposure (67), and abnormal GF levels at the site of vascular lesions have long been implicated in the pathological maintenance of the synthetic phenotype (68–70). Interestingly, cell density has been shown to regulate the response of cells to GF stimulation (71–73). In the current study, higher substrate stiffness led to enhanced cell-substrate interactions that may support higher levels of migration and proliferation, but increasing cell density attenuated this affect. Our data suggest that, in the course of normal healing, increasing cell density in the lesion could lessen certain effects of stiffness and/or GF exposure, allowing the cell to revert to a more quiescent, contractile state. The continuation of VSMC pathology may partially be due to the inability of cells to reestablish adequate AJs; there is evidence that cadherin expression contributes to the stability of atherosclerotic plaques (74), whereas GF exposure has been shown to increase cell-substrate interactions in correlation with the disruption of cell-cell adhesions (24). Thus, simultaneous GF signaling and a stiffened ECM may perpetuate vascular pathology by reducing the ability of synthetic VSMCs to reestablish cell-cell contacts.

Conclusion

Using morphological and biochemical analyses, this study revealed that substrate stiffness induces changes in VSMC morphology and gene and protein expression, but many of these effects are eliminated by increasing cell density. The vast majority of research on mechanotransduction has focused on the behavior of single or sparsely cultured cells; our study brings to light the significant and tightly regulated influence of cell-cell contacts on VSMC response to substrate mechanics, demonstrating that communication through AJs can mute the effect of substrate mechanics on FA function without fully eliminating VSMC rigidity sensing. Given the likely role of tissue mechanics in the progression of atherosclerosis, our findings provide further insight into the mechanisms by which the microenvironment of the developing lesion can influence VSMC behavior, and point to cell-cell contacts as critical mediators of VSMC response to vascular injury.

Acknowledgments

The authors thank Phil Allen and the Boston University Biomedical Engineering CORE facility for access to and assistance with confocal microscopy and AFM, as well as numerous helpful conversations regarding immunocytochemistry. We also thank Michael L. Smith, who graciously provided equipment for Fn depletion and cell area quantification, along with a critical review of the manuscript.

This work was supported in part by National Institutes of Health grants HL088672 to M.A.N. and HL072900 to J.Y.W., and by a departmental grant from the Massachusetts Lions Eye Research Fund. O.V.S. was partially supported by the National Heart, Lung, and Blood Institute training grant in Cardiovascular Biology (HL07969). K.L.L. was partially supported by the Boston University Undergraduate Research Opportunities Program.

Contributor Information

Matthew A. Nugent, Email: [email protected].

Joyce Y. Wong, Email: [email protected].

Supporting Material

Document S1. Additional materials and methods and a figure
mmc1.pdf (142.3KB, pdf)

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