Fluid Shear Stress-Induced Alignment of Cultured Vascular Smooth Muscle Cells

[+] Author and Article Information
Ann A. Lee, Dionne A. Graham, Sheila Dela Cruz, Anthony Ratcliffe

Advanced Tissue Sciences, Inc., La Jolla, CA 92037

William J. Karlon

Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093

J Biomech Eng 124(1), 37-43 (Oct 02, 2001) (7 pages) doi:10.1115/1.1427697 History: Received April 09, 2000; Revised October 02, 2001
Copyright © 2002 by ASME
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Fluid shear stress induces alignment in canine vascular SMC. Cells were cultured on fibronectin-coated glass slides under static conditions (A) or exposed to 20 dyn/cm2 shear stress for 48 hours (B). Fluorescent visualization of F-actin in cells cultured under static (C) or flow conditions (D). Arrows indicate the direction of fluid flow. An intensity gradient imaging technique was used to quantify the distribution and angular standard deviation of mean cell orientations for static (E) and flow (F) conditions.
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Vascular SMC alignment is dependent on the magnitude of and exposure time to applied shear stress. Canine SMC were subjected to fluid shear stresses up to 20 dyn/cm2 for 0, 24, and 48 hours. Histograms of mean cell orientation angle are shown for 10, 15 and 20 dyn/cm2 . Mean cell angle measurements range from 0 to 180 deg, with 0 deg defined as the direction of fluid flow.
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Summary of time- and magnitude-dependence of shear stress-induced SMC alignment. Cells subjected to 20 dyn/cm2 aligned more rapidly and to a higher degree than those cultured under lower flow conditions (*p=0.002 at 24 hours, compared with static control; at 48 hours, **p<0.0001, p=0.001, p=0.005, compared with 0, 10, and 15 dyn/cm2 , respectively). At 48 hours, alignment of SMC subjected to 10 and 15 dyn/cm2 were also significantly different compared with static control (p<0.05 for both conditions). Cells cultured in static and very low flow conditions (0 and 1 dyn/cm2 ) did not align in any preferred direction and remained randomly distributed throughout the time course.
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Vascular SMC initiate the alignment process within hours of the onset of fluid flow. Images of cells subjected to fluid shear stress of 20 dyn/cm2 were acquired by time-lapse video over a 24-hour period. Images were digitized to quantify the increasing degree of cellular alignment, measured as the decreasing angular standard deviation of mean cell orientation.
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Inhibition of intracellular calcium by quin-2 AM prevents the alignment of vascular SMC under fluid flow. After pretreatment with 10 μM quin-2 AM for 1 hour in static culture, cells were cultured under several conditions for 48 hours: “Static+Vehicle” (a), “Flow + Vehicle” (b), and “Flow+Quin-2 AM” (c). In the flow conditions, SMC were subjected to 20 dyn/cm2 shear stress with vehicle or 1 μM quin-2 AM in the circulating medium. Arrows indicate the direction of fluid flow.
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Image analysis of the effects of quin-2 AM on flow-induced SMC alignment. Treatment with quin-2 AM was shown to significantly block flow-induced alignment, as measured by the angular standard deviation of mean cell angles (*p=0.007, compared with “Flow+Vehicle”; **p=0.001, compared with “Flow”).
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Flow-induced SMC alignment was attenuated by disrupting actin filaments and microtubules. To examine the role of actin filaments in cell alignment, 40 nM cytochalasin D was used to pre-treat the cells for 1 hour (top row). Cells were subsequently cultured as static controls (a) or subjected to 20 dyn/cm2 for 48 hours with vehicle (b) or with 40 nM cytochalasin D in the circulating medium for 48 hours (c). To disrupt microtubules, cells were pretreated with 3 μM nocodazole for 1 hour (bottom row). Static controls (d) were compared with cells subjected to fluid flow for 48 hours under circulating DMSO vehicle (e) or nocodazole treatment (f).




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