Bovine trachea were collected from a local slaughterhouse (Dale T Smith & Sons Inc., Draper, UT) and transported to the laboratory in cold (4°C) bicarbonate buffer containing 120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3 (pH 7.4). Tissue culture reagents were obtained from Sigma (St. Louis, MO) with the exception of Dulbecco's modified Eagles's medium (DMEM)-Ham's F-12 (1:1) which was purchased from GIBCO (Grand Island, NY). The synthetic arginine-glycine-aspartic acid (RGD) containing peptide was purchased from American Peptide Company (Sunnyvale, CA). Primary antibodies against HSP20, cofilin, phosphorylated cofilin and 14-3-3 γ proteins, as well as the appropriate secondary antibodies, were obtained from Millipore (Billerica, MA). Unless otherwise noted, all other reagents were obtained from Sigma. Acetylcholine, histamine, serotonin, isoproterenol, and N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cAMP) were reconstituted in sterile distilled water, frozen in aliquots, and diluted appropriately in serum-free media on the day of use.
Statement on animal welfare
Fischer 344 rat strains (male, 7-9 wk-old) were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and housed in a conventional animal facility at Harvard School of Public Health (Boston, MA). All experimental protocols conducted on animals were performed in accordance with the standards established by the US Animal Welfare Acts, as well as the Policy and Procedures Manual of the Harvard University School of Public Health Animal Care and Use Committee.
Isometric force measurements
As described previously by us and others [14, 24], bovine tracheal strips and rat tracheal rings (i.e. transverse rings, 1.0 mm in width) were prepared and mounted in organ bath containing a bicarbonate buffer. Tissue strips/rings were tied with surgical silk and attached at one end to a force transducer (Kent Scientific, Litchfield, CT). The other end of tissue strips/rings were connected to a length manipulator. Tissue strips/rings were progressively stretched to a total force of ~10 g and then released to a passive force of ~0.5 g. Subsequently, the isometric force in response to a contracting agonist acetylcholine was determined until a consistent maximal force was produced. Here we used sub-maximally activated tissue strips/rings (~80% of the maximal contraction with 3 μM acetylcholine) and used 5% w/v cyclodextrin as a vehicle for the delivery of compounds. For each pre-contracted tissue, compounds were added directly to the organ bath. To ensure the viability of the tissue, the isometric force in response to 110 mM KCl (with equimolar replacement of NaCl in bicarbonate buffer) was measured after each experiment.
Cell isolation and culture
Smooth muscle (i.e. vascular and airway) cells were isolated from either the aorta or the trachealis of the highly inbred Fischer 344 rat strains (male, 7-9 wk-old) as described previously [15, 25]. Human ASM cells were isolated, characterized and provided by Dr. Reynold A. Panettieri, Jr. (University of Pennsylvania). Cells were grown until confluence at 37°C in humidified air containing 5% CO2 and passaged with 0.25% trypsin-0.02% EDTA solution every 10-14 days. ASM cells in culture were elongated and spindle shaped, grew with the typical hill-and-valley appearance, and showed positive staining for the smooth muscle-specific protein α-actin and calponin. In the present study, we used cells in passages 3-7. Unless otherwise specified, serum-deprived post-confluent cells were plated at 30,000 cells/cm2 on plastic wells (96-well Removawell, Immunlon II: Dynetech) previously coated with type I collagen (Vitrogen 100; Cohesion, Palo Alto, CA) at 500 ng/cm2. Cells were maintained in serum-free media for 24 h at 37°C in humidified air containing 5% CO2. These conditions have been optimized for seeding cultured cells on collagen matrix and for assessing their mechanical properties [25–31].
Magnetic twisting cytometry (MTC)
Stiffness of the adherent ASM cell was measured as described by us in detail elsewhere [25, 29, 32]. In brief, an RGD-coated ferrimagnetic microbead (4.5 μm in diameter) bound to the surface of the cell was magnetized horizontally and then twisted in a vertically aligned homogenous magnetic field that varied sinusoidally in time. The sinusoidal twisting magnetic field causes both a rotation and a pivoting displacement of the bead: as the bead moves, the cell develops internal stresses which in turn resist bead motions . Lateral bead displacements in response to the resulting oscillatory torque were detected optically (with a spatial resolution of ~5 nm), and the ratio of specific torque to bead displacements was computed and expressed here as the cell stiffness in units of Pascal per nm (Pa/nm).
For each individual cell, stiffness was measured continuously for the duration of 600 s (Additional file 1, Figure S1): baseline stiffness was measured for the first 0-60 s and stiffness changes in response to compounds were measured up to the indicated time (60-600 s). In general, changes in cell stiffness approached a steady-state level within 600 s. In the present study, we reported this steady-state cell stiffness (540-600 s) upon treatment with various compounds. Moreover, to adjust for cell-to-cell and day-to-day variability in baseline stiffness, we normalized stiffness changes to respective baseline stiffness of an individual ASM cell.
Fourier transform traction microscopy (FTTM)
The contractile stress arising at the interface between each adherent cell and its substrate was measured with traction microscopy [25, 27]. Cells were plated sparsely on elastic gel blocks (Young's modulus of 8 kPa with a Poisson's ratio of 0.48), and allowed to adhere and stabilize for 24 h. For each adherent cell, the traction field was computed using Fourier transform traction cytometry as described previously [33, 34]. The computed traction field was used to obtain the net contractile moment, which is a scalar measure of the cell's contractile strength . The net contractile moment is expressed in units of pico-Newton meters (pNm).
Protein expression/phosphorylation detection
The expression of HSP20, cofilin, and phosphorylated cofilin was detected as previously described [19, 35]. For each well of confluent ASM cells (on 6-well plates), total cell protein was quantified by the Bradford method (using Bio-Rad dye reagent, Richmond, CA), and equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked and then probed with primary antibodies to HSP20, cofilin or phosphorylated cofilin. Immunoreactive proteins were detected with appropriate secondary antibodies and visualized by light emission on film with enhanced chemiluminescent substrate (Cell Signaling, Danvers, MA).
Surface plasmon resonance (SPR) assay
All SPR experiments were performed on a BIAcore 3000 instrument. Phosphorylated HSP20 (pHSP20) peptide was immobilized to one flow cell of a CM5 chip (BIAcore) via a standard amino coupling procedure. The other three flow cells contained immobilized unphosphorylated HSP20 peptide (HSP20), a phosphorylated peptide containing a scrambled sequence of the pHSP20 peptide, and an empty surface blocked with ethanolamine, respectively. The 5 different 14-3-3 isoforms (β, ζ, η, ε and ϒ), expressed and purified from E. coli (described in detail below), were injected separately at equal concentrations in HBS (HEPES Buffered Saline, pH 7.4) with a flow rate of 20 μl/min across the pHSP20 and control surfaces. The dissociation was monitored for ca. 12 min in a HBS flow. Between injections, the surfaces were regenerated with a 30s pulse of 10 mM NaOH. The signal obtained from the HSP20 peptide surface were subtracted from that of the pHSP20 peptide surface.
Fluorescence polarization (FP) assay
The 58,019 structurally diverse chemical compounds were obtained from ChemBridge (San Diego, CA) and ChemDiv (San Diego, CA). 8-mer peptides containing the recognition motif for 14-3-3 proteins were synthesized and purified via HPLC to > 95% purity, and their size confirmed by mass spectrometry (BioSynthesis, Inc., Lewisville, TX). The sequences of 8-mer peptides used were: 1) fluorophore-pHSP20 (6-FAM-WLRRApSAP); 2) positive control (WLRRApSAP); and 3) negative control (WLRRASAP).
The 247-amino acid 14-3-3γ coding region was cloned as a fusion with an N-terminal GST-His tag using the vector pDEST15 (Life Technologies) with expression under the control of the T7 promoter. BL21 (DE3) competent cells were transformed with pDEST15- GST-His14-3-3γ. Transformed bacteria were inoculated in 100 mL of LB media containing ampicillin at 10 μg/mL and grown overnight at 37°C. The overnight culture was diluted 1:50 in 4 L of fresh LB with the same concentration of antibiotic as described above. These cells were allowed to grow at 37°C for approximately 2-3 h, until the optical density at 600 nm reached 0.4 to 0.8. Induction was started by addition of IPTG at a final concentration of 0.1 mM, followed by incubation at 30°C for 5 h. Cells were harvested by centrifuge at 5000 rpm for 30 min. The cell pellet was resuspended, sonicated and centrifuged, and the soluble protein was subjected to two-step GST-His tag affinity purification according to manufacturer's instructions (Sigma-Aldrich Inc., St. Louis, MO; Qiagen Inc., Valencia, CA). Fractions containing GST-His-14-3-3γ (determined through SDS-PAGE) were pooled, and the protein concentration measured using the Bradford protein assay (Bio-rad, Hercules, CA). GST-His-14-3-3γ purity was assessed by SDS-PAGE and Coomassie Blue staining. This method was also used to prepare the other 14-3-3 isoforms used in the Surface Plasmon Resonance (SPR) experiments.
For the FP assay, we used 384-well microplates (low-volume, flat-bottom, black plates; Greiner-Bio-One North America Inc., Monroe, NC). First, GST-His-14-3-3γ and FAM-pHSP20 were added to the wells at final concentrations of 1 μM and 2 nM, respectively, in a final reaction buffer of 1X HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% (v/v) polysorbate 20, 3 mM EDTA, 10 mM MgCl2). Compounds or negative/positive controls were then added at final concentrations of 10 μM and 1 μM, respectively. After 4 h incubation at room temperature, the FP was read using Perkin-Elmer Fusion Universal Microplate Analyzer (Perkin-Elmer, Shelton, CT) using 485 nm excitation (light-emitting diode) and 515 nm emission (20 nm bandwidth) settings. Compounds eliciting a variation of FP greater than 20% were flagged as optically active. After initial screening, flagged compounds were verified for inhibition in a concentration-responsive manner in order to establish their IC50 concentrations. All FP reactions were assayed in triplicate for each compound.
For the comparisons among treatments, we used two sample t-test, the Analysis of Variance (ANOVA) with adjusting for multiple comparisons by applying the Tukey's method, or the Wilcoxon test depending on the distribution of data. To satisfy the distributional assumptions associated with ANOVA, cell stiffness data were converted to log scale prior to analyses. All analyses were performed in SAS V.9.1, and the 2-sided P-values less than 0.05 were considered significant.