Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells
Frequency and Force Response Characteristics
Abstract—Blood vessels are continuously exposed to mechanical forces that lead to adaptive remodeling and atherosclerosis. Although there have been many studies characterizing the responses of vascular cells to mechanical stimuli, the precise mechanical characteristics of the forces applied to cells to elicit these responses are not clear. We designed a magnetic exposure system capable of producing a defined normal force on ferromagnetic beads that are specifically bound to cultured cells coated with extracellular matrix proteins or integrin-specific antibodies. Rat aortic smooth muscle cells were incubated with engineered fibronectin–coated ferromagnetic beads and then exposed to a magnetic field. With activation of extracellular signal–regulated mitogen-activated protein kinase 1/2 (ERK 1/2MAPK) used as a prototypical marker for cell responsiveness to mechanical forces, Western blot analysis demonstrated an increase in phosphorylated ERK 1/2MAPK expression reaching a maximal response of a 3.5-fold increase at a total force of ≈2.5 pN per cell. The peak response occurred after 5 minutes of exposure and slowly decreased to baseline after 30 minutes. A cyclic, rather than static, force was required for this activation, and the frequency-response curve increased ≈2-fold between 0.5 and 2.0 Hz. Vitronectin- and β3 antibody–coated beads showed a response nearly identical to those coated with engineered fibronectin, whereas forces applied to beads coated with α2 and β1 antibodies did not significantly activate ERK 1/2MAPK. Mechanical activation of the ERK 1/2MAPK system in rat aortic smooth muscle cells occurs through specific integrin receptors and requires a cyclic force with a magnitude estimated to be in the piconewton range.
Blood vessels are continuously exposed to mechanical forces that, if excessive, lead to adaptive remodeling in the form of smooth muscle hypertrophy and hyperplasia.1 2 3 Several in vitro studies have demonstrated that abnormal mechanical forces on vascular smooth muscle cells increase DNA synthesis, smooth muscle myosin protein content, and expression of immediate-early genes.4 5 6 7 In addition to adaptive remodeling, excessive wall strain causes vascular inflammation, which has been implicated in the pathogenesis of atherosclerosis.8 9 Cyclic stretch increases superoxide production via the NADH/NADPH oxidase,10 11 causes platelet-derived growth factor release,12 13 and increases the transcription of monocyte chemoattractant protein-1 and macrophage colony–stimulating factor. A recent epidemiological study from the Framingham database shows that pulse pressure (hence pulsatile wall stress) is superior to systolic or diastolic pressures in predicting the risk of coronary heart disease, which further suggests a link between wall strain and hypertension-induced atherosclerosis.14
Despite data linking specific biological responses to mechanical forces, the precise range of forces and frequencies to which cells respond remains unclear. Several devices are currently in use to study how different levels of cyclic force are translated into biochemical signals; however, all suffer from strain heterogeneity and the generation of fluid shear stresses.15 Even more problematic is the fact that these devices quantify forces in terms of elongation of cells, a measurement difficult to correlate in vivo, rather than the actual forces applied to each cell.
To address these limitations, we designed a novel system, based on a device by Ingber and Wang,16 that enables one to vary the force and frequency exerted on a cell. We designed an iron core coil magnetic field exposure system that generates a well-defined nonuniform magnetic field capable of providing either static or cyclic downward forces on ferromagnetic beads. By coating these beads with an extracellular matrix protein or an antibody directed against an integrin receptor, our device allowed us to explore how integrin receptors convert mechanical forces into biological signals. Our goal was to determine the forces and frequencies required to activate cell signaling processes and the specific receptors activated by these mechanical stimuli in vascular smooth muscle cells. We hypothesized that integrin binding is necessary for the recognition and transduction of such forces.
Materials and Methods
Materials and Reagents
“Complete Mini” protease inhibitor was from Boehringer Mannheim. Antibody against ERK 1/2MAPK was purchased from Santa Cruz Biotechnology, and antibody against phosphorylated ERK 1/2MAPK (Thr202/Tyr204) was from New England Biolabs. Specific antibodies against the amino acid terminus of the α2, β1, and β3 integrin receptors were also purchased from Santa Cruz Biotechnology. Purified human vitronectin was purchased from Biosource International. Monoclonal mouse anti-goat IgG Fc–specific antibody was purchased from OEM Concepts. Tween and the Detergent Compatible protein assay were purchased from Bio Rad Laboratories. Enhanced chemiluminescence Western blotting detection system and mouse anti-rabbit antibody were from Amersham Life Science Corp. Microcon 10-KD centrifugal filter devices were purchased from Millipore Corporation. A cell viability/cytotoxicity assay using esterase substrates was purchased from Molecular Probes. Calf serum, DMEM, fibronectin-like engineered protein, and all other chemicals were purchased from Sigma Chemical Co.
Magnetic Field Exposure System
A complete description of the magnetic field exposure system is included in an online data supplement (please see http://www.circresaha.org). Briefly, two No. 20 AWG magnetic wires were wound around an iron bar stock, 1 inch in diameter and 20 cm long, for a total of 1250 feet per wire. This length corresponds to ≈100 wrappings per layer for 25 layers. Each layer was set with epoxy to prevent vibrations from the electrical current. Sinusoidal loading waveforms from a Stanford Research Systems Model DS335, 3.1-MHz synthesized function generator were amplified using a Kepco model BOP 20-20 M power supply, which in turn delivered the prescribed current and voltage to the coil windings. The magnetic field strength was mapped using a Walker Scientific Model MG2A gaussmeter to permit calculation of the applied forces. The effective force on the cells due to the magnetic field interaction with the beads was determined by a volume force measurement in a static nonuniform field (see online data supplement).
To generate the magnetic field exposure, the output of the power supply was connected to the coil with the 2 windings connected in parallel. To provide a sham exposure, the windings were connected in anti-parallel fashion so that no net magnetic field was generated, even though current was flowing in the 2 windings. The sham exposure controls for any potential heating or vibration in the exposure system. A fixed spacer was positioned above the coil to maintain a constant distance between the coil and the cell culture dish.
Preparation of the Ferromagnetic Beads
Carboxyl ferromagnetic particles with a mean diameter of 4.5 μm were purchased from Spherotech Inc. The ferromagnetic beads were coated with different ligands according to the manufacturer’s instructions. Briefly, 2 mL of 0.05 mol/L MES buffer containing 0.5 mg of an extracellular matrix protein and 5 mg of EDAC was mixed with 5 mL of 4.5-μm ferromagnetic beads (1.0% wt/vol solution). For integrin antibodies, the beads were first coated with monoclonal mouse anti-goat IgG Fc–specific antibody using the above protocol and then incubated with 0.5 mg of integrin antibody.
Cell Culture and Strain Experiments
Rat aortic smooth muscle (RASM) cells were isolated from the thoracic aortas of rats as previously described.17 Cells were grown in DMEM supplemented with 4500 mg/L d-glucose, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 25 mmol/L HEPES, and 10% heat-inactivated calf serum. Cells between passages 3 and 15 were plated at a density of 250 000 cells per 35-mm dish and were grown for 24 hours in DMEM with 10% calf serum. Plates were then washed 3 times with serum-free DMEM and quiesced in serum-free DMEM for 72 hours. Approximately 18 million beads were added to each dish 12 hours before the experiment. The dishes were gently washed 3 times to remove unattached beads. Each dish was then placed onto the coil and subjected to the magnetic field within an incubator at 37°C with 5% CO2. Two dishes per sample were used for experiments. To calculate the bead density per cell, the number of beads per 10 cells was visualized under a 40×-microscope lens and counted. This was repeated over 4 random areas of the culture dish and averaged.
Protein Extraction and Western Blotting
For the MAPK assays, dishes were washed 3 times with cold phosphate-buffered saline, and cells were lysed with a solution containing 50 mmol/L HEPES, 5 mmol/L EDTA, 50 mmol/L NaCl and 1% Triton (Sigma). The lysis buffer also contained “Complete Mini” protease inhibitor and 50 mmol/L NaF, 1 mmol/L Na3VO4 and 10 mmol/L Na4P2O7 for phosphatase inhibition. Cells were suspended and transferred to microcentrifuge tubes, incubated at 4°C for 60 minutes, and centrifuged at 12 000g for ten minutes. The supernatant was then taken and concentrated in a Microcon 10-KD centrifugal filter device at 10 000g for 12 minutes. The concentrated protein was quantified by a modified Lowry assay.18 For MAPK Western blotting, 15 μg of protein per sample was loaded onto a 10% polyacrylamide mini-gel and size-fractionated. Total protein was transferred to a nitrocellulose membrane and blocked with 5% powdered milk, 0.5% Tween for 60 minutes. Incubation with the primary antibody was overnight, followed by three 5-minute washes in 0.2% milk, 0.2% Tween. Incubation with secondary antibodies was for 30 minutes, followed by three 5-minute washes in 0.2% milk, 0.2% Tween. Membranes were then exposed to enhanced chemiluminescence solution for 1 minute and exposed to radiograph film for 1 to 10 minutes.
The viability of the RASM cells after exposure to the magnetic field was assessed by a viability assay (Molecular Probes). Briefly, 2 μL of ethidium bromide and 0.66 μL of calcein were added per mL of culture media. Cells were incubated in this solution for 30 minutes and then examined with a fluorescent microscope. Viable cells stain green because of calcein esterfication, whereas dead cells stain red because of incorporation of ethidium bromide into the DNA.
All experiments were performed at least 3 times. All data are presented as the mean±SEM. ANOVA with the Duncan new multiple-range post hoc analysis was performed for comparison of 2 or more groups. P<0.05 was considered significant.
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
Magnetic Forces Induced on the Ferromagnetic Beads Do Not Affect Cell Viability
RASM cells exposed to 10 V (20 V, peak to peak) at 1 Hz for 5 minutes had a normal appearance without evidence of endocytosis of the ferromagnetic beads by phase-contrast microscopy. A viability assay using ethidium bromide and calcien, which stains dead and viable cells respectively under fluorescent microscopy, showed no increase in cell death for RASM cells incubated with beads coated with engineered fibronectin and exposed to a maximum applied peak-to-peak voltage of 20 V. RASM cells incubated with engineered fibronectin–coated beads but not exposed to the magnetic field were used as control.
Increasing Force Activates ERK 1/2MAPK
To determine the range of forces RASM cells sense, 4.5-μm ferromagnetic beads coated with engineered fibronectin were added to quiescent cells to give an average density of 12.9±0.7 beads per cell. When placed on top of the coil and exposed to a magnetic field, these ferromagnetic beads exert a maximum downward force proportional to the square of the applied magnetic field intensity (Figure 1⇓). By adjusting the applied coil voltage between 0 and 20 V, the estimated applied force on each cell was varied over the range from 0 to 3 pN (see online data supplement for details). Western blot analysis revealed a sharp increase in phosphorylated ERK 1/2MAPK for RASM cells over the range of voltages studied with a maximal response of a 3.5±0.68–fold (P<0.05) increase over control at a peak-to-peak voltage of 20 V (Figure 2⇓). There was no significant difference between the responses observed at 20 V and at 10 V, suggesting that a plateau in the response occurred within this range of forces. Higher forces were not used because of the potential for coil heating at higher drive voltages. No significant changes in total ERK 1/2MAPK levels were seen for these experiments.
Cyclic Force Is Necessary for Activation of ERK 1/2MAPK
To investigate whether cyclic force is necessary for activation of ERK 1/2MAPK, a static voltage of 20 V was applied to RASM cells for 5 minutes and compared with the same force at 1 Hz. Although a static force did not produce a statistically significant increase in phosphorylated ERK 1/2MAPK, a frequency of 1.0 Hz produced a 2.03±0.48–fold (P<0.02) increase compared with control (Figure 3⇓). These results compare favorably with previous work suggesting that cyclic force is necessary for maintenance of vascular smooth muscle cell phenotype.19 5 20
Frequency Response of RASM Cells at a Maximum Force
Given that cyclic force activates ERK 1/2MAPK, we next determined the frequency response characteristics of the cells to the mechanical loading. As assayed by Western blot analysis, RASM cells exposed to an applied voltage of 20 V for 5 minutes demonstrated a frequency-dependent activation of ERK 1/2MAPK with increasing activation over the 2 decades of frequency between 0.01 and 2.0 Hz. (P<0.05) (Figure 4⇓). An important detail of our system is that a ferromagnetic bead is pulled down twice during 1 sine-wave cycle. For example, a frequency of 1 Hz corresponds to a ferromagnetic bead exerting a force 120 times per minute. Thus, the maximal activation of the ERK 1/2MAPK occurs within the expected physiological heart rate range of 60 to 240 beats per minute. No significant differences in activation of ERK 1/2MAPK were seen between 0.5 and 2.0 Hz. Frequencies >2.0 Hz were not studied because of a fall-off in flux density at frequencies >2 Hz, resulting in a decrease in the applied force to the beads for which we could not effectively compensate by increasing the applied voltage.
Time Course of ERK 1/2MAPK Activation
A statistically significant increase in phosphorylated ERK 1/2MAPK was seen after applying 20 V at 1 Hz for 2.5 minutes (Figure 5⇓). This activation peaked at 5 minutes with a maximum 3.6±0.8–fold (P<0.05) increase in phosphorylated ERK 1/2MAPK compared with control and returned to baseline by 30 minutes.
Integrin Binding Is Necessary for ERK 1/2MAPK Activation
Because previous work has implicated integrins in mechanical transduction, we examined whether activation of ERK 1/2MAPK in our system occurs through mechanical stimulation of integrin receptors.21 16 22 4 We first determined that incubating RASM cells with either uncoated beads or beads coated with engineered fibronectin alone did not increase phosphorylated ERK 1/2MAPK (Figure 6⇓). For experiments with uncoated ferromagnetic beads, the washing step before exposing RASM cells to a magnetic field was eliminated. To ensure that activation of ERK 1/2MAPK was not caused by the effects of the magnetic field alone, we exposed RASM cells to the maximum magnetic field used in this study (50 mT) and found no increase in phosphorylated ERK 1/2MAPK (Figure 6⇓). The windings on the iron core coil were then connected so that current in one wire flowed in the opposite direction from current in the other. In this configuration, the net current is zero and no net magnetic field is generated. This configuration, which we refer to as “anti-parallel,” allowed us to dissect out the possible confounding effects of heat and vibration on ERK 1/2MAPK activation. As demonstrated in Figure 6⇓, no increase in phosphorylated ERK 1/2MAPK was seen with the wires in anti-parallel. Lastly, when uncoated beads were exposed to the maximum amount of force (Figure 6⇓), no significant increase in phosphorylated ERK 1/2MAPK was observed. Taken together, these results demonstrate the specificity of the ERK 1/2MAPK response in this experimental setting by showing that only mechanical forces on integrins—and not heat, current, magnetic field, or integrin clustering—are responsible for activation of ERK 1/2MAPK.
Differential Activation of ERK 1/2MAPK by Vitronectin and Integrin-Receptor Antibodies
We next examined whether different extracellular membrane proteins and integrin-specific receptor antibodies cause differential activation of ERK 1/2MAPK. Figure 7⇓ demonstrates that ferromagnetic beads coated with vitronectin or β3 integrin–receptor antibody caused a significant increase in phosphorylated ERK 1/2MAPK compared with β1 or α2 antibody–coated beads.
Although hypertension is a major risk factor for atherosclerosis, how it leads to vascular remodeling and atherosclerosis is poorly understood. Chronically abnormal mechanical forces in vessel walls are thought to trigger adaptive processes leading to hyperplasia, hypertrophy, and inflammation.23 Current cyclic stretch devices, which deliver mechanical stimuli through deformation of an elastic membrane, have been used to pursue this hypothesis, but these devices have been criticized for not accurately modeling circumferential stress. The percent stretch of the cell is presumed to correspond closely to the distension of the membrane; however, cells may experience varying degrees of stretch depending on their location and orientation on the elastic membrane. Moreover, the precise force exerted on each cell is unknown. A further drawback is the development of fluid velocity gradients leading to varying degrees of shear stress across the cells. This poses a particular problem when attempting to discern the biomechanical effects of cyclic stretch from shear stress because low shear stress may alter the expression of vasodilators, growth factors, and inflammatory mediators.15 23 Perhaps most problematic is the difficulty of correlating cyclic stretch to in vivo models. Although several studies have estimated the distention of a vessel wall in normal and disease states, these measurements are difficult and may vary widely because of differences in vascular architecture, stiffness of a vessel wall, and the dynamic release of vasoactive factors from short-term hemodynamic changes.21 24
The principle objective of this study was to design an in vitro system that applies mechanical stimuli to cells and either eliminates or mitigates the problems listed above. We therefore designed a magnetic force exposure device that delivers a quantifiable force and loading frequency to ferromagnetic beads.16 By coating these ferromagnetic beads with an extracellular matrix protein or integrin-specific antibody, the differential effects of mechanical stimuli on specific cell surface receptors can be studied. Whereas magnetic field interactions with ferromagnetic beads have been used previously to apply a transient torque (analogous to shear stress) to cells,16 our device creates a downward force on the beads, and therefore the cells, via surface integrins.
Using the magnetic field exposure device, we found that a mean total force in the piconewton range can activate ERK 1/2MAPK via integrin receptors that recognize the RGD peptide sequence or vitronectin. The maximal activation of ERK 1/2MAPK required a cyclic force for 5 minutes and demonstrated a monotonically increasing sensitivity over 2 orders of magnitude in frequency (0.02 to 4 Hz), a range that includes most of the energy content of the physiological heart rate. In our system, RASM cells seemed to sense and transduce mechanical stimuli through integrin receptors because uncoated beads subjected to a maximal cyclic force had no effect on phosphorylated ERK 1/2MAPK levels. An initial survey of integrin receptors found that β3, not α2 or β1, integrins function as the principal mechanosensors on RASM cells. These results do not support the conclusion that vitronectin is the principal extracellular matrix protein to convey mechanical stimuli to RASM cells because other extracellular matrix proteins, such as osteopontin and fibronectin, bind to β3 integrin receptors. Collagen, however, may play less of a role in mechanotransduction because it does not bind to β3 integrin receptors.25
To the best of our knowledge, this is the first study to quantify the range of cyclic forces RASM cells recognize. Schmidt et al26 found that a 30-minute application of 0.1 nN of cyclic force per cell produced an increase in phosphorylated ERK 1/2MAPK in osteoblasts. However, the precise motion of the paramagnetic beads when placed in their magnetic apparatus was not defined in their study. Furthermore, their description of the force applied to the integrin receptor in their model as a “drag force” suggests a twisting motion on integrin receptors similar to Ingber’s device.26 27 With regards to studies examining the effect of cyclic strain on vascular smooth muscle cells, our findings are consistent with previous studies examining the frequency and time course of ERK 1/2MAPK activation in vascular smooth muscle cells using cyclic stretch devices.20
Previous work suggests that cells perceive extracellular forces via integrins, heterodimeric transmembrane receptors that bind extracellular matrix proteins.28 16 4 22 Moreover, integrins are implicated in cell growth, differentiation, inflammation, and the optimal activation of tyrosine and G protein–coupled receptors, all of which play a critical role in vascular remodeling.29 30
Our results are consistent with the studies suggesting that ERK 1/2MAPK is an important link in the signal-transduction pathway of vascular tissues subjected to force. Wilson et al4 indirectly examined the role of mechanical strain in inducing DNA synthesis in RASM cells and found mechanical stimuli to be primarily transduced though αVβ3 and β3 integrin receptors. Of interest is the fact that blockade of the β1 receptor had no effect on strain-induced DNA synthesis in their study. More recently, Numaguchi et al31 found that a specific inhibitor of ERK 1/2MAPK blocked thymidine incorporation in RASM cells subjected to 20% stretch for 24 hours. Taken together, these studies suggest that ERK 1/2MAPK, activated by mechanical stimuli on β3 integrin receptors, may play an important role in strain cellular responses.
The assessment of the actual force imposed on a cell in this system is based on a number of assumptions. Implicit in the calculations are the fairly reasonable assumptions that bead density, integrin-receptor density, and receptor-ligand interactions are uniform on the cells. Perhaps more importantly, the calculation of applied forces assumes also that the bead-cell interaction does not change (ie, there is not a significant effect of receptor clustering, nor does the applied field alter the number of receptor-ligand interactions). We have some experimental data to support these assumptions in that we have demonstrated that the simple addition of coated beads does not significantly increase ERK 1/2MAPK phosphorylation, thereby indicating that receptor clustering is not relevant in this setting. Similarly, application of a static force does not appear to activate ERK 1/2MAPK, suggesting that the increase in ERK 1/2MAPK phosphorylation is not simply caused by an increase in the number of receptor-ligand interactions as the bead is depressed into the cell. Definitive evaluation of these assumptions awaits additional studies in which the physical responses of the cells to applied forces can be determined.
A concern has been raised regarding the biochemical effects of magnetic fields on cells.32 Though these studies used magnetic fields within the range generated by our device, the frequencies (60 Hz) and exposures were greater. Other than heat shock protein, the expression of no other genes or proteins has been linked to exposure to a magnetic field. It is significant that we found no increase in ERK 1/2MAPK expression in RASM cells exposed to a maximum magnetic field alone for 5 minutes.
In summary, we have demonstrated that RASM cells are capable of sensing a cyclic force in the piconewton range at frequencies that encompass the normal physiological heart rate. This loading force caused increased phosphorylation of ERK 1/2MAPK over a physiological heart rate range and is transduced primarily though β3 integrin receptors. Moreover, engagement of integrin receptors by the beads was critical for the ability of RASM cells to sense mechanical stimuli. We believe that our novel apparatus has broad applicability for studying the biomechanical effects of force in vascular biology as well as other fields. Such work should elucidate the contribution of mechanical forces to vascular remodeling and atherosclerosis.
This work was supported in part by grants from the National Institutes of Health HL10104, HL 58000, and NIEHS 07803.
Original received September 19, 2000; resubmission received February 7, 2001; revised resubmission received March 7, 2001; accepted March 7, 2001.
- © 2001 American Heart Association, Inc.
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