n pixel squared. For the evaluation of the cell area, a mean value of the 5 data set was calculated. Materials and Methods Cell Culture Primary cultures of normal human bronchial smooth muscle cells from multiple donors were obtained from Cambrex Co.. The cells were maintained in culture medium containing 5% fetal bovine serum, human recombinant epidermal growth factor, insulin, human recombinant fibroblast growth factor, gentamycin and amphotericin B in an atmosphere of 5% CO2 and 95% air at 37uC. The cells retain expression of smooth muscle marker proteins such as a-smooth muscle actin, smooth muscle myosin heavy chain, and calponin. Immunofluorescent Staining The cells 21358117” subjected to cyclic stretch or static condition on the silicone chamber were fixed with 4% formaldehyde in phosphatebuffered saline for 1 h at room temperature, and permeabilized with 0.25% Triton X-100 containing 0.5% bovine serum albumin for 1 h. For the detection of microtubules, cells were incubated with a mouse monoclonal anti-a-tubulin antibody for 8 h and then with 
an FITC-conjugated anti-mouse ” secondary antibody for 1 h at room temperature for immunodetection. F-actin was stained with rhodamine-phalloidin for 1 h at room temperature. Nuclei were stained with the DNA binding dye, 4,6-diamino-2-phenylindole . The immunofluorescently stained cells were then visualized by fluorescence microscopy with an imaging-system. Application of Uniaxial Cyclic Stretch The cells at the 47th passage were removed from the dish with 0.01% EDTA-0.02% trypsin and transferred to a 4 cm2 silicone chamber coated with type I collagen at a density of 2.06104 cells/cm2. A uniaxial sinusoidal stretch of 20% in strain at 30 cycle/min was applied using stretching apparatus driven by a computer-controlled stepping motor in an atmosphere of 5% CO2 and 95% air at 37uC as described previously. Briefly, one end of the chamber was attached to a fixed frame, while the other end was attached to a movable frame. Other two sides were free to move. The movable frame was connected to a motor driven shaft whose amplitude and frequency of stretch was controlled by a programmable microcomputer. Twelve hours prior to stretching, cells were brought to a quiescent state by incubation in order Relebactam Dulbecco’s modified Eagle’s medium /F-12 culture medium with 0.03% FBS. The silicon chamber had a 200-mM thick transparent bottom and the side walls were 4 mm thick to reduce compression of the chamber perpendicular to the direction of the stretch. The relative elongation of the silicone membrane was uniform across the whole membrane area, and lateral contraction did not exceed 3% at 20% stretch. The cells incubated under a static condition on the silicone chamber were used as a time-matched control. To determine whether microtubules play a role in the cyclic stretchinduced cell reorientation, effects of inhibitors of microtubule polymerization, nocodazole and colchicine, or a microtubule stabilizer paclitaxel were examined. Either drug was applied to the cell culture medium 30-min prior to the application of cyclic stretch. Model Development Previous studies have established that when ASM cells are subjected to cyclic uniaxial stretching, the actin stress fibers in the direction of stretch dissemble and reassemble in a new direction which is almost perpendicular to the direction of stretching. To understand how the dynamics of microtubules fit into this picture, we developed a model where microtubules and actin stress fibers
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