Volume 94, Issue 4, Pages 1497-1507 (February 2008) Role of Cyclic Strain Frequency in Regulating the Alignment of Vascular Smooth Muscle Cells In Vitro  Bo Liu, Ming-Juan Qu, Kai-Rong Qin, He Li, Zhen-Kun Li, Bao-Rong Shen, Zong-Lai Jiang  Biophysical Journal  Volume 94, Issue 4, Pages 1497-1507 (February 2008) DOI: 10.1529/biophysj.106.098574 Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 1 Change of SMC alignment after stretching at a variety of frequencies. SMCs were cultured on collagen I-coated elastic membranes and subjected to cyclic stretch with frequencies 0.5, 1.0, 1.5, and 2.0Hz, respectively, for 24h. Photographs were taken under microscopy, and their middle vertical lines were fixed consistently with the radial direction of the culture membrane. SMCs without stretch (S group) or statically stretched without variation in frequency (C group) were also observed. Bar, 200μm, and the arrow indicates the radial direction. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 2 Change of SMC orientation after stretching at a variety of frequencies. The angle of SMCs (0–90°) was divided into six angle regions, each covering 15°. The percentages of SMCs within the C and 1.5-Hz groups were calculated after stretching for 24h (A). The AOA (B) and the percentage of SMCs from 75° to 90° (C) were contrasted between groups. Results are mean±SD. *p<0.05; **p<0.01 versus the C group; +p<0.05; ++p<0.01 versus the 0.5-Hz group, n=4. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 3 Time courses of AIE change with different frequencies of cyclic stretch from the experimental and mathematic models. (A) SMCs were stretched for 3, 6, 12, 24, 36, and 48h with frequencies of 0.5, 1.0, 1.5, and 2.0Hz, respectively, and the C group was also observed at the same time points. Based on the data of the percentage of cells in each angle interval, the AIE was calculated and contrasted. The time courses of AIE were fitted with a logistic curve, and the frequency and each parameter were then fitted again using the power function or binomial equation. The lines are fitted curves, and spots represent the corresponding experimental data; n=4. (B). Prediction and experimental verification of AIE. The AIE under the cyclic stretch frequency of 1.2Hz were predicted, and for experimental verification, SMCs were stretched with a frequency of 1.2Hz for 3, 6, and 24h, respectively. The dotted lines are fitted curves for each experimental group, the solid line is a predicted curve for 1.2Hz, and spots represent experimental data; n=7. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 4 Activation of integrin-β1 in SMCs under different frequencies of cyclic strain analyzed by flow cytometry. (A) SMCs were analyzed by flow cytometry after stretched for 1, 6, and 12h with frequencies of 0.5, 1.25, and 2.0Hz, respectively. Results are mean±SD. *p<0.05, **p<0.01 versus C group of the same time point; +p<0.05 versus 0.5-Hz group of the same time point; #p<0.05 versus 1.25-Hz group of the same time point; n=4. (B) Activation of integrin-β1 in SMCs stretched for 1h without cytochalasin D after pretreatment with cytochalasin D for 1h. Results are mean±SD. #p<0.05, ##p<0.01 versus the untreated group of the same time point; n=4. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 5 Change of AIE of SMCs under cyclic strain after pretreatment with specific inhibitors. (A) SMCs were stretched after pretreatment with different inhibitors for 1h and stretched at 1.25Hz for 12h with specific inhibitors in the medium. (B) Change of AIE of SMCs stretched at different frequencies for 6h without cytochalasin D in the medium after pretreatment with cytochalasin D for 1h, n=4. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 6 Change of filament orientation after cyclic stretching. (A) Control group, which was statically stretched without frequency for 12h. (B) SMCs stretched at the frequency of 1.25Hz for 6h. (C) SMCs stretched for 12h at the frequency of 1.25Hz after pretreatment with cytochalasin D for 1h. (D) SMCs were stretched for 12h at the frequency of 1.25Hz with cytochalasin D in the medium throughout. Stained with rhodamine phalloidin. Bar, 100μm, and the arrow indicates the radial direction of the culture membrane. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 7 F/G-actin ratio changed with different frequencies of cyclic strain while disturbed by cytochalasin D. SMCs were stretched for 1, 6, and 12h (A) or stretched for the same time after pretreatment with cytochalasin D for 1h (B). G-actin in cytosol protein and F-actin in cytoskeleton protein were immunoblotted with anti-β-actin antibody. Each column represents the mean±SD. *p<0.05; **p<0.01 versus the C group at the same time point; +p<0.05; ++p<0.01 versus the 0.5-Hz group at the same time point; n=5. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 8 Change of phospho-p38 MAPK (P-p38) induction and F/G-actin ratio of SMCs stretched after having been inhibited with anti-integrin-β1 blocking antibody. (A) Western blotting result from P-p38 after stretching for 1h with anti-integrin-β1 blocking antibody, and total-p38 MAPK (T-p38) served as loading control. (B) The F/G-actin ratio of SMCs after stretching for 12h with anti-integrin-β1 blocking antibody. Each column represents the mean±SD. +p<0.05; ++p<0.01 versus the C group; #p<0.05; ##p<0.01 versus the 1.25Hz group; *p<0.05 versus the untreated group at the same frequency; n=5. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions

Figure 9 Immunocytochemistry result of actin and integrin-β1. (A) C group, in which SMCs were stretched without frequency. (B) SMCs were stretched at 1.25Hz for 6h. (C) SMCs were stretched at 1.25Hz for 12h. (D) SMCs were stretched at 1.25Hz for 6h after pretreatment with cytochalasin D for 1h. The cells were fixed, and actin filament was stained with red rhodamine-phalloidin, and integrin-β1 was stained with an anti-integrin-β1 antibody and green FITC-IgG, respectively. Photographs were taken with the fluorescence microscope. Bar, 100μm, and the arrow indicates the radial direction. Biophysical Journal 2008 94, 1497-1507DOI: (10.1529/biophysj.106.098574) Copyright © 2008 The Biophysical Society Terms and Conditions