1. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
Subcellular ECM fibers aligned in response to CS on native but not glycated collagen. (a) Representative fibronectin immunofluorescent images. PAEC cultured on PDMS membranes were exposed to static culture or 12 h of CS. Subcellular ECM was isolated, labeled for fibronectin (green), and imaged by confocal microscopy. Alternatively, PAEC were fixed and labeled for actin with rhodamine phalloidin (red) and nuclei with bis-benzamide (blue). Scale bar = 20 μm. (b) Fibronectin fiber and actin alignment was quantified by edge detection [35]. Fibers oriented between 80 deg and 90 deg to the stretch direction were considered aligned. **p < 0.001.

2. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
Collagen I decreased with CS, while fibronectin, collagen IV and vitronectin did not change. All matrix protein levels were similar in cells on native (NC) or glycated (GC) collagen. After stretching, cells were lysed and collected with the ECM. Collagen I, collagen IV, fibronectin, and vitronectin were measured in lysates by Western blot. The protein levels in CS samples were normalized to the respective static sample.

3. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
Glycated collagen decreased endothelial cell MMP-2 activity in static culture and in response to CS. (a) PAEC were cultured on native or glycated collagen coated substrates. After 48 h, conditioned medium samples were taken and immediately analyzed by gelatin zymography. (b) PAEC were cyclically stretched for 0 and 24 h on native and glycated coated substrates and then conditioned medium samples were collected and analyzed by gelatin zymography. #p < 0.05, *p < 0.01, and **p < 0.001.

4. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
MMP protein levels changed in response to CS in cells on native but not glycated collagen. Cells were cyclically stretched for 0 to 12 h and then cell lysates were used to quantify MMP-2, TIMP-2, and MT1-MMP protein levels by ELISA or Western blot. (a) MMP-2 increased in a time-dependent manner in cells on native collagen exposed to CS, but MMP-2 levels did not change significantly in cells on glycated collagen. (b) TIMP-2 decreased in cells on native collagen but was unchanged in cells on glycated collagen in response to CS. (c) MT1-MMP only increased with CS in cells on native collagen. #p < 0.05 and **p < 0.001.

5. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
Jnk and p38 phosphorylation were decreased in response to CS in cells on glycated collagen, whereas Erk phosphorylation was unchanged. After 0–12 h of CS, cell lysates were collected and analyzed for Jnk (a), p38 (b), and Erk (c) activation by Western blot. Phosphorylated samples were normalized to the corresponding total protein. *p < 0.01 and **p < 0.001.

6. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
Inhibition of MMP-2 activation did not prevent subcellular fibronectin alignment in response to CS. Endothelial cells incubated with or without human TIMP-2 for 40 h prior to CS both aligned their fibronectin. (a) Representative fibronectin immunofluorescent images. PAEC cultured on native collagen coated PDMS membranes in the presence (200 ng/ml) or absence of TIMP-2 were exposed to static culture or 12 h CS. Subcellular ECM was isolated, labeled for fibronectin (green), and imaged by confocal microscopy. Scale bar = 20 μm. (b) Fibronectin fiber alignment was quantified by edge detection [35]. Fibers oriented between 80 deg and 90 deg to the stretch direction were considered aligned. *p < 0.01.

7. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
Inhibition of actin alignment in endothelial cells on native collagen disrupted subcellular ECM alignment. (a) Representative fibronectin and actin immunofluorescent images. PAEC cultured on PDMS membranes were incubated with or without cytochalasin D for 30 min prior to applying 12 h of CS. After stretching, PAEC were fixed with paraformaldehyde and labeled for actin with rhodamine phalloidin (red) and nuclei with bis-benzamide (blue). Subcellular ECM was isolated and labeled for fibronectin (green). All samples were imaged by confocal microscopy. (b) Actin and fibronectin fiber alignment were quantified by edge detection. Fibers oriented between 80 deg and 90 deg to the stretch direction were considered aligned. *p < 0.01.

8. Date of download: 11/2/2017
Copyright © ASME. All rights reserved.
From: Glycated Collagen Decreased Endothelial Cell Fibronectin Alignment in Response to Cyclic Stretch Via Interruption of Actin Alignment
J Biomech Eng. 2014;136(10): doi: /
Figure Legend:
A model for endothelial cell basement remodeling in response to CS on native and glycated collagen. In cells on native collagen, CS activates FAK and induces actin alignment via Rho activation. Actin alignment applies tension to the matrix, causing subcellular ECM fiber alignment. FAK also phosphorylates the MAP kinase protein Jnk. Active Jnk increases MMP-2 protein levels while also increasing MT1-MMP and decreasing TIMP-1. This ratio of MMP proteins activates MMP-2. In cells on glycated collagen, CS does not activate FAK or align actin, which inhibits both subcellular matrix protein alignment and MMP-2 activity. Thus, glycated collagen prevents cells exposed to CS from remodeling the subcellular ECM by inhibiting actin tension.