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Journal of Biomechanical Engineering | Volume 135 | Issue 7 | research-article
Research Papers

Microscopic Matrix Remodeling Precedes Endothelial Morphological Changes During Capillary Morphogenesis

[+] Author and Article Information
Claire McLeod

Franklin W. Olin College of Engineering,
Needham, MA 02492;
Department of Bioengineering,
University of Pennsylvania,
Philadelphia, PA 19014

John Higgins, Alisha L. Sarang-Sieminski

Franklin W. Olin College of Engineering,
Needham, MA 02492

Yekaterina Miroshnikova

Franklin W. Olin College of Engineering,
Needham, MA 02492;
Department of Surgery,
Center for Bioengineering and Tissue Regeneration,
University of California,
San Francisco,
San Francisco, CA 94143

Rachel Liu

Franklin W. Olin College of Engineering,
Needham, MA 02492;
Department of Bioengineering,
University of Pennsylvania,
Philadelphia, PA 19014

Contributed by the Bioengineering Division of ASME for publication in the JOURNAL OF BIOMECHANICAL ENGINEERING. Manuscript received October 1, 2012; final manuscript received February 25, 2013; accepted manuscript posted March 8, 2013; published online June 11, 2013. Assoc. Editor: Edward Sander.

J Biomech Eng 135(7), 071002 (Jun 11, 2013) (7 pages) Paper No: BIO-12-1453; doi: 10.1115/1.4023984 History: Received October 01, 2012; Revised February 25, 2013; Accepted March 08, 2013
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Citing Articles

The formation of microvascular networks (MVNs) is influenced by many aspects of the microenvironment, including soluble and insoluble biochemical factors and the biophysical properties of the surrounding matrix. It has also become clear that a dynamic and reciprocal interaction between the matrix and cells influences cell behavior. In particular, local matrix remodeling may play a role in driving cellular behaviors, such as MVN formation. In order to explore the role of matrix remodeling, an in vitro model of MVN formation involving suspending human umbilical vein endothelial cells within collagen hydrogels was used. The resulting cell and matrix morphology were microscopically observed and quantitative metrics of MVN formation and collagen gathering were applied to the resulting images. The macroscopic compaction of collagen gels correlates with the extent of MVN formation in gels of different stiffness values, with compaction preceding elongation leading to MVN formation. Furthermore, the microscopic analysis of collagen between cells at early timepoints demonstrates the alignment and gathering of collagen between individual adjacent cells. The results presented are consistent with the hypothesis that endothelial cells need to gather and align collagen between them as an early step in MVN formation.
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Grahic Jump Location
Fig. 1

Schematic of hypothesized role of matrix gathering and alignment between cells. Cells exert forces on (a) the matrix, and both (b) sense the matrix properties, and (c) remodel the matrix. Cells interact with each other and move directionally (d) as a precursor to MVN formation.

+Schematic of hypothesized role of matrix gathering and alignment between cells. Cells exert forces on (a) the matrix, and both (b) sense the matrix properties, and (c) remodel the matrix. Cells interact with each other and move directionally (d) as a precursor to MVN formation.
View Large  |  Save Figure  |  Download Slide (.ppt)
Schematic of hypothesized role of matrix gathering and alignment between cells. Cells exert forces on (a) the matrix, and both (b) sense the matrix properties, and (c) remodel the matrix. Cells interact with each other and move directionally (d) as a precursor to MVN formation.
Grahic Jump Location
Fig. 2

MVN formation and macroscopic gel compaction as functions of collagen concentration and time. HUVECs cultured in various collagen concentrations (n = 6) for 48 h show (a) decreased MVN formation and decreasing compaction ((b) size) with increasing initial collagen concentration. (c) Plotting the final size against the extent of the MVN formation shows an inverse linear relationship (R2 = 0.99) between these two readouts. HUVECs cultured in 2 mg/ml collagen and fixed at various timepoints show increased elongation and MVN formation ((d) n = 9) and decreasing gel size ((e) n = 5) with time. The number of cells per structure also increases with time ((d) n = 3). (f) Plotting size against the extent of the MVN formation for the timecourse shows a nearly linear relationship (R2 = 0.94) with a slight concave-up trend. (d) Plotting size against cells/structure shows a highly concave up trend. Error bars represent standard deviations and the lines are connecting points to illustrate trends except for linear fit in (c).

+MVN formation and macroscopic gel compaction as functions of collagen concentration and time. HUVECs cultured in various collagen concentrations (n = 6) for 48 h show (a) decreased MVN formation and decreasing compaction ((b) size) with increasing initial collagen concentration. (c) Plotting the final size against the extent of the MVN formation shows an inverse linear relationship (R2 = 0.99) between these two readouts. HUVECs cultured in 2 mg/ml collagen and fixed at various timepoints show increased elongation and MVN formation ((d) n = 9) and decreasing gel size ((e) n = 5) with time. The number of cells per structure also increases with time ((d) n = 3). (f) Plotting size against the extent of the MVN formation for the timecourse shows a nearly linear relationship (R2 = 0.94) with a slight concave-up trend. (d) Plotting size against cells/structure shows a highly concave up trend. Error bars represent standard deviations and the lines are connecting points to illustrate trends except for linear fit in (c).
View Large  |  Save Figure  |  Download Slide (.ppt)
MVN formation and macroscopic gel compaction as functions of collagen concentration and time. HUVECs cultured in various collagen concentrations (n = 6) for 48 h show (a) decreased MVN formation and decreasing compaction ((b) size) with increasing initial collagen concentration. (c) Plotting the final size against the extent of the MVN formation shows an inverse linear relationship (R2 = 0.99) between these two readouts. HUVECs cultured in 2 mg/ml collagen and fixed at various timepoints show increased elongation and MVN formation ((d) n = 9) and decreasing gel size ((e) n = 5) with time. The number of cells per structure also increases with time ((d) n = 3). (f) Plotting size against the extent of the MVN formation for the timecourse shows a nearly linear relationship (R2 = 0.94) with a slight concave-up trend. (d) Plotting size against cells/structure shows a highly concave up trend. Error bars represent standard deviations and the lines are connecting points to illustrate trends except for linear fit in (c).
Grahic Jump Location
Fig. 3

Cell morphology timecourse. HUVECs cultured in 2 mg/ml collagen were fixed, stained with Alexa 633 phalloidin, and imaged after (a) 0, (b) 4, (c) 8, (d) 16, (e) 24, or (f) 48 h of culture. The scale bar (a) is 100 μm for all images.

+Cell morphology timecourse. HUVECs cultured in 2 mg/ml collagen were fixed, stained with Alexa 633 phalloidin, and imaged after (a) 0, (b) 4, (c) 8, (d) 16, (e) 24, or (f) 48 h of culture. The scale bar (a) is 100 μm for all images.
View Large  |  Save Figure  |  Download Slide (.ppt)
Cell morphology timecourse. HUVECs cultured in 2 mg/ml collagen were fixed, stained with Alexa 633 phalloidin, and imaged after (a) 0, (b) 4, (c) 8, (d) 16, (e) 24, or (f) 48 h of culture. The scale bar (a) is 100 μm for all images.
Grahic Jump Location
Fig. 4

Microscopic timecourse of collagen remodeling. HUVECs cultured in 2 mg/ml collagen were fixed, stained with phalloidin and DAPI, and imaged after (a) 0, (b) 4, (c) 8, (d) 16, (e) 24, or (f) 48 h of culture. Upper panels show overlay of collagen (green) with cells (red/blue). Insets show cells only. The scale bar (a) is 25 μm for all images.

+Microscopic timecourse of collagen remodeling. HUVECs cultured in 2 mg/ml collagen were fixed, stained with phalloidin and DAPI, and imaged after (a) 0, (b) 4, (c) 8, (d) 16, (e) 24, or (f) 48 h of culture. Upper panels show overlay of collagen (green) with cells (red/blue). Insets show cells only. The scale bar (a) is 25 μm for all images.
View Large  |  Save Figure  |  Download Slide (.ppt)
Microscopic timecourse of collagen remodeling. HUVECs cultured in 2 mg/ml collagen were fixed, stained with phalloidin and DAPI, and imaged after (a) 0, (b) 4, (c) 8, (d) 16, (e) 24, or (f) 48 h of culture. Upper panels show overlay of collagen (green) with cells (red/blue). Insets show cells only. The scale bar (a) is 25 μm for all images.
Grahic Jump Location
Fig. 5

Regional quantification of collagen concentration. Confocal stacks of cells and collagen underwent image processing to identify regions of depleted, normal, and compacted collagen along with the cellular regions. Individual images from confocal stacks from (a) 0, (b) 4, and (c) 16 h are shown. Different regions of collagen and cells, by percent, were determined for different timepoints (d).

+Regional quantification of collagen concentration. Confocal stacks of cells and collagen underwent image processing to identify regions of depleted, normal, and compacted collagen along with the cellular regions. Individual images from confocal stacks from (a) 0, (b) 4, and (c) 16 h are shown. Different regions of collagen and cells, by percent, were determined for different timepoints (d).
View Large  |  Save Figure  |  Download Slide (.ppt)
Regional quantification of collagen concentration. Confocal stacks of cells and collagen underwent image processing to identify regions of depleted, normal, and compacted collagen along with the cellular regions. Individual images from confocal stacks from (a) 0, (b) 4, and (c) 16 h are shown. Different regions of collagen and cells, by percent, were determined for different timepoints (d).
Grahic Jump Location
Fig. 6

Gathering between cells. Collagen intensity was quantified between pairs of HUVECs cultured in collagen gels for 4 h as a function of the cell-cell distance and collagen concentration for 1.5, 2, and 3.25 mg/ml collagen

+Gathering between cells. Collagen intensity was quantified between pairs of HUVECs cultured in collagen gels for 4 h as a function of the cell-cell distance and collagen concentration for 1.5, 2, and 3.25 mg/ml collagen
View Large  |  Save Figure  |  Download Slide (.ppt)
Gathering between cells. Collagen intensity was quantified between pairs of HUVECs cultured in collagen gels for 4 h as a function of the cell-cell distance and collagen concentration for 1.5, 2, and 3.25 mg/ml collagen

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