0
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
Article
References
Figures
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.
FIGURES IN THIS ARTICLE
<>
Copyright © 2013 by ASME
Your Session has timed out. Please sign back in to continue.

References

1
Paszek, M. J., and Weaver, V. M., 2004, “The Tension Mounts: Mechanics Meets Morphogenesis and Malignancy,” J. Mammary Gland Biol. Neoplasia, 9(4), pp. 325–342. [CrossRef] [PubMed]
2
Paszek, M. J., Zahir, N., Johnson, K. R., Lakins, J. N., Rozenberg, G. I., Gefen, A., Reinhart-King, C. A., Margulies, S. S., Dembo, M., Boettiger, D., Hammer, D. A., and Weaver, V. M., 2005, “Tensional Homeostasis and the Malignant Phenotype,” Cancer Cells, 8(3), pp. 241–254. [CrossRef]
3
Sieminski, A. L., Hebbel, R. P., and Gooch, K. J., 2004, “The Relative Magnitudes of Endothelial Force Generation and Matrix Stiffness Modulate Capillary Morphogenesis In Vitro,” Exp. Cell Res., 297(2), pp. 574–584. [CrossRef] [PubMed]
4
Kniazeva, E., Weidling, J. W., Singh, R., Botvinick, E. L., Digman, M. A., Gratton, E., and Putnam, A. J., 2012, “Quantification of Local Matrix Deformations and Mechanical Properties During Capillary Morphogenesis in 3d,” Integr. Biol. (Camb.), 4(4), pp. 431–439. [CrossRef] [PubMed]
5
Peyton, S. R., Ghajar, C. M., Khatiwala, C. B., and Putnam, A. J., 2007, “The Emergence of ECM Mechanics and Cytoskeletal Tension as Important Regulators of Cell Function,” Cell Biochem. Biophys., 47(2), pp. 300–320. [CrossRef] [PubMed]
6
Engler, A. J., Sweeney, H. L., Discher, D. E., and Schwarzbauer, J. E., 2007, “Extracellular Matrix Elasticity Directs Stem Cell Differentiation,” J. Musculoskeletal and Neuronal Interact., 7(4), p. 335.
7
Califano, J. P. and Reinhart-King, C. A., 2010, “Exogenous and Endogenous Force Regulation of Endothelial Cell Behavior,” J. Biomech., 43(1), pp. 79–86. [CrossRef] [PubMed]
8
Sieminski, A. L., Hebbel, R. P., and Gooch, K. J., 2005, “Improved Microvascular Network In Vitro by Human Blood Outgrowth Endothelial Cells Relative to Vessel-Derived Endothelial Cells,” Tissue Eng., 11(9–10), pp. 1332–1345. [CrossRef] [PubMed]
9
Ghajar, C. M., Chen, X., Harris, J. W., Suresh, V., Hughes, C. C., Jeon, N. L., Putnam, A. J., and George, S. C., 2008, “The Effect of Matrix Density on the Regulation of 3-D Capillary Morphogenesis,” Biophys. J., 94(5), pp. 1930–1941. [CrossRef] [PubMed]
10
Rao, R. R., Peterson, A. W., Ceccarelli, J., Putnam, A. J., and Stegemann, J. P., 2012, “Matrix Composition Regulates Three-Dimensional Network Formation by Endothelial Cells and Mesenchymal Stem Cells in Collagen/Fibrin Materials,” Angiogenesis, 15(2), pp. 253–264. [CrossRef] [PubMed]
11
Vernon, R. B., Angello, J. C., Iruela-Arispe, M. L., Lane, T. F., and Sage, E. H., 1992, “Reorganization of Basement Membrane Matrices by Cellular Traction Promotes the Formation of Cellular Networks In Vitro,” Lab. Invest., 66(5), pp. 536–547. [PubMed]
12
Kanzawa, S., Endo, H., and Shioya, N., 1993, “Improved In Vitro Angiogenesis Model by Collagen Density Reduction and the Use of Type III Collagen,” Ann. Plast. Surg., 30(3), pp. 244–251. [CrossRef] [PubMed]
13
Nehls, V., and Herrmann, R., 1996, “The Configuration of Fibrin Clots Determines Capillary Morphogenesis and Endothelial Cell Migration,” Microvasc. Res., 51(3), pp. 347–364. [CrossRef] [PubMed]
14
Sieminski, A. L., Was, A. S., Kim, G., Gong, H., and Kamm, R. D., 2007, “The Stiffness of Three-Dimensional Ionic Self-Assembling Peptide Gels Affects the Extent of Capillary-Like Network Formation,” Cell Biochem. Biophys., 49(2), pp. 73–83. [CrossRef] [PubMed]
15
Vernon, R. B., Lara, S. L., Drake, C. J., Iruela-Arispe, M. L., Angello, J. C., Little, C. D., Wight, T. N., and Sage, E. H., 1995, “Organized Type I Collagen Influences Endothelial Patterns During”Spontaneous Angiogenesis In Vitro”: Planar Cultures as Models of Vascular Development,” In Vitro Cell Dev. Biol.: Anim., 31(2), pp. 120–131. [CrossRef] [PubMed]
16
Reinhart-King, C. A., Dembo, M., and Hammer, D. A., 2008, “Cell-Cell Mechanical Communication Through Compliant Substrates,” Biophys. J., 95(12), pp. 6044–6051. [CrossRef] [PubMed]
17
Korff, T., and Augustin, H. G., 1999, “Tensional Forces in Fibrillar Extracellular Matrices Control Directional Capillary Sprouting,” J. Cell Sci., 112, pp. 3249–3258. [PubMed]
18
Lee, P. F., Yeh, A. T., and Bayless, K. J., 2009, “Nonlinear Optical Microscopy Reveals Invading Endothelial Cells Anisotropically Alter Three-Dimensional Collagen Matrices,” Exp. Cell Res., 315(3), pp. 396–410. [CrossRef] [PubMed]
19
Ceccarelli, J., Cheng, A., and Putnam, A. J., 2012, “Mechanical Strain Controls Endothelial Patterning During Angiogenic Sprouting,” Cell Mol. Bioeng., 5(4), pp. 463–473. [CrossRef]
20
Pizzo, A. M., Kokini, K., Vaughn, L. C., Waisner, B. Z., and Voytik-Harbin, S. L., 2005, “Extracellular Matrix (ECM) Microstructural Composition Regulates Local Cell-ECM Biomechanics and Fundamental Fibroblast Behavior: A Multidimensional Perspective,” J. Appl. Physiol., 98(5), pp. 1909–1921. [CrossRef] [PubMed]
21
Stevenson, M. D., Sieminsk, A. L., Mcleod, C. M., Byfield, F. J., Barocas, V. H., and Gooch, K. J., 2010, “Pericellular Conditions Regulate Extent of Cell-Mediated Compaction of Collagen Gels,” Biophys. J., 98, pp. 1–10. [CrossRef] [PubMed]
22
Reinhardt, J. W., Krakauer, D. A., and Gooch, K. J., “Complex Matrix Remodeling and Durotaxis Can Emerge from Simple Rules for Cell-Matrix Interaction in Agent-Based Models,” ASME J. Biomech. Eng., 135(7), p. 071003. [CrossRef]
23
Dickinson, R. B., Guido, S., and Tranquillo, R. T., 1994, “Biased Cell-Migration of Fibroblasts Exhibiting Contact Guidance in Oriented Collagen Gels,” Ann. Biomed. Eng., 22(4), pp. 342–356. [CrossRef] [PubMed]
24
Raeber, G. P., Lutolf, M. P., and Hubbell, J. A., 2008, “Part II: Fibroblasts Preferentially Migrate in the Direction of Principal Strain,” Biomech. Model. Mechanobiol., 7(3), pp. 215–225. [CrossRef] [PubMed]
25
Deroanne, C. F., Lapiere, C. M., and Nusgens, B. V., 2001, “In Vitro Tubulogenesis of Endothelial Cells by Relaxation of the Coupling Extracellular Matrix-Cytoskeleton,” Cardiovasc. Res., 49(3), pp. 647–658. [CrossRef] [PubMed]
26
Sieminski, A. L., and Gooch, K. J., 2002, “Systemic Delivery of HGH Using Genetically Modified Tissue-Engineered Microvascular Networks: Prolonged Delivery and Endothelial Survival With Inclusion of Non-Endothelial Cells,” Tissue Eng., 8(6), pp. 1057–1069. [CrossRef] [PubMed]
27
Sieminski, A. L., and Gooch, K. J., 2004, “Salmon Fibrin Supports an Increased Number of Sprouts and Decreased Degradation While Maintaining Sprout Length Relative to Human Fibrin in an In Vitro Angiogenesis Model,” J. Biomater. Sci. Polym. Ed., 15(2), pp. 237–242. [CrossRef] [PubMed]
28
Anderson, C. R., Ponce, A. M., and Price, R. J., 2004, “Immunohistochemical Identification of an Extracellular Matrix Scaffold That Microguides Capillary Sprouting in vivo,” J. Histochem. Cytochem., 52(8), pp. 1063–1072. [CrossRef] [PubMed]
29
Anghelina, M., Krishnan, P., Moldovan, L., and Moldovan, N. I., 2006, “Monocytes/Macrophages Cooperate With Progenitor Cells During Neovascularization and Tissue Repair: Conversion of Cell Columns Into Fibrovascular Bundles,” Am. J. Pathol., 168(2), pp. 529–541. [CrossRef] [PubMed]
30
Moon, J. J., Saik, J. E., Poche, R. A., Leslie-Barbick, J. E., Lee, S. H., Smith, A. A., Dickinson, M. E., and West, J. L., 2010, “Biomimetic Hydrogels With Pro-Angiogenic Properties,” Biomaterials, 31(14), pp. 3840–3847. [CrossRef] [PubMed]
31
Leslie-Barbick, J. E., Moon, J. J., and West, J. L., 2009, “Covalently-Immobilized Vascular Endothelial Growth Factor Promotes Endothelial Cell Tubulogenesis in Poly(Ethylene Glycol) Diacrylate Hydrogels,” J. Biomater. Sci. Polym. Ed., 20(12), pp. 1763–1779. [CrossRef] [PubMed]
32
Ma, X., Schickel, M., Stevenson, M. D., Sarang-Sieminski, A. L., Gooch, K. J., Ghadiali, S. N., and Hart, m R. T., 2013, “Fibers in the Extracellular Matrix Enable Long-Range Stress Transmission between Cells,” Biophys. J. (in press).

Figures

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

Tables

Errata

Discussions

Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging and repositioning the boxes below.

Related Journal Articles
Related eBook Content
Bacteriophage T4 Tail Fibers as a Basis for Structured Assemblies
Tail fiber function and structure
Bacteriophage T4 Tail Fibers as a Basis for Structured Assemblies> Chapter 2
Tail fiber production and purification
Bacteriophage T4 Tail Fibers as a Basis for Structured Assemblies> Chapter 3
Tail fiber modifications
Bacteriophage T4 Tail Fibers as a Basis for Structured Assemblies> Chapter 4
Conclusions
Bacteriophage T4 Tail Fibers as a Basis for Structured Assemblies> Chapter 5
Topic Collections

Sorry! You do not have access to this content. For assistance or to subscribe, please contact us:

  • TELEPHONE: 1-800-843-2763 (Toll-free in the USA)
  • EMAIL: asmedigitalcollection@asme.org
Sign In