Matrix stiffening in the formation of blood vessels

MINI REVIEW

Matrix stiffening in the formation of blood vessels

Danielle J. LaValley and Cynthia A. Reinhart-King*

Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA

Received: 21 June 2014; Revised: 1 September 2014; Accepted: 3 September 2014; Published: 15 October 2014

Abstract

Angiogenesis, the process where new blood vessels form from existing vasculature, is essential for the successful integration of most tissue-engineered constructs and is dysregulated in many diseases, including cancer. To be functional, the newly formed vasculature must have similar structure and integrity as existing blood vessels, both of which are dependent upon mechanical and chemical cues from the surrounding extracellular matrix (ECM). ECM stiffness has emerged as a critical extracellular parameter that can modulate capillary network formation and barrier integrity. Moreover, matrix stiffness can alter how endothelial cells respond to soluble, angiogenic factors released by stromal cells, such as vascular endothelial growth factor (VEGF). In this review, we will discuss how matrix stiffness can affect the formation and structure of angiogenic vessels, and we will highlight the role of this work in the development of therapeutics to treat angiogenesis in cancer. Knowledge of the governing parameters for vessel formation is critical to the intelligent design of materials made to foster blood vessel growth for tissue-engineering applications and pharmaceuticals designed to intervene with newly formed vasculature in diseased tissue.

Keywords: angiogenesis; extracellular matrix stiffness; capillary-like networks; endothelial cells; cancer

In context

New blood vessels develop from existing ones in a process called angiogenesis. Angiogenesis is critical for wound healing and most tissue-engineering applications, where a blood supply is necessary for the viability of the new tissue. In tissue-engineered constructs for wound repair, for example, angiogenesis needs to occur for proper skin regeneration. Angiogenesis also occurs during cancer, where newly formed vessels feed a growing tumor. These same vessels are also used as one of the primary routes to deliver chemotherapeutics into the tumor. Therefore, there is a clinical need to understand and control angiogenesis to foster tissue regrowth and facilitate the delivery of drugs into tumors. For newly formed vessels to function properly, they must match the growth and integrity of native vessels to form a fully functional vasculature. Numerous factors dictate vessel formation, including the properties of the extracellular matrix (ECM). The ECM surrounds cells within tissues and provides growth factors and other nutrients to the cells. It also provides the cells with mechanical and chemical cues that can affect and control their behavior. Endothelial cells (ECs), which comprise the inner lining of blood vessels, interact with their ECM, and the nature of these interactions can determine how many new vessels form, the shape of those vessels, and whether those vessels are structurally stable. Many studies have focused on how various chemicals in the ECM affect angiogenesis, but recently, mechanical cues have emerged as important features of tissues that affect the nature of blood vessel formation. In this review, we discuss the role of tissue stiffness as a major determinant of angiogenesis. New biomaterials are emerging where stiffness is tuned in order to control and study angiogenesis. This work lays an important foundation to understand how to foster angiogenesis for tissue-engineering applications or modulate the structure of growing vessels to treat cancer.

*Correspondence to: Cynthia A. Reinhart-King, Department of Biomedical Engineering, Cornell University, 302 Weill Hall, Ithaca, NY 14853, USA, Email: cak57@cornell.edu

Advances in Regenerative Biology 2014. © 2014 Danielle J. LaValley and Cynthia A. Reinhart-King. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 Unported (CC BY 4.0) License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.

Citation: Advances in Regenerative Biology 2014, 1: 25247 - http://dx.doi.org/10.3402/arb.v1.25247

 

Endothelial cells (ECs) maintain vascular homeostasis and, upon injury, participate in vascular repair and regeneration (1). The ECM serves as a reservoir of matrix proteins and as a structural scaffold to support EC organization and stabilization during blood vessel formation (2). However, it also serves a mechanical function, as the stiffness of the matrix can dictate the nature and extent of angiogenesis. Understanding the role of matrix mechanics in angiogenesis is critical to the intelligent design of scaffolds tailored to foster angiogenesis and to the treatment of cancer, where uncontrolled angiogenesis contributes to tumor pathology.

2D and 3D regulation of vascular growth and integrity by matrix stiffness

Cells are capable of sensing and responding to changes in matrix stiffness (3), which, in turn, regulates angiogenesis. Significant work has been performed using deformable 2D substrates to show that ECs form capillary-like networks on compliant substrates, but not on stiffer matrices (2, 47). Using planar substrates of tunable stiffness, several groups, including our own, have observed that ECs form capillary networks on compliant matrices independently of exogenously applied growth factors (Fig. 1) (2, 46). Cells are thought to sense one another and communicate mechanically through the ECM, facilitating the formation of cell–cell junctions during tissue formation (8). Compliant substrates induce enhanced EC elongation and alignment (2, 4), whereas increasing 2D matrix stiffness decreases network formation (4, 5) and sprouting (7). Conversely, decreasing stiffness results in decreased matrix metalloproteinase (MMP) expression and increased network formation (1). Furthermore, more EC proliferation was observed on stiffer matrices than on more compliant substrates (9). Overall, data in 2D suggests that compliant matrices foster the self-assembly and growth of vascular networks.

Fig 1

Fig. 1.   (a, b) ECs grown on 2D polyacrylamide hydrogels. Capillary-like networks form on soft matrices (a, 1 kPa), but not on stiff substrates (b, 10 kPa). Scale is 200 µm. (c, d) Multicellular spheroids embedded into 3D collagen gels. EC sprouting increases with stiffness from 175 Pa (c) to 515 Pa (d). Scale is 200 µm. Reprinted with permission from Elsevier (14).

Although most studies report that elevated matrix stiffness on 2D substrates inhibits network formation (2, 47), results differ in 3D matrices. Increased matrix density, which results in increased stiffness, is reported to reduce neovascularization and capillary network formation (10, 11), resulting in shorter, thicker, slower growing sprouts, fewer branch points, and reduced network connectivity (11, 12), but more stable lumen formation (12). Importantly, there exists a collagen density above which vessels no longer form (12). It is crucial to note, however, that in these studies, stiffness is regulated by density.

Stiff, 3D, collagen matrices can also be fabricated by cross-linking collagen gels, in which case, the collagen density and gel pore size remain constant. For example, Yamamura et al. reported thicker, deeper capillary networks on rigid 3D collagen gels and the formation of large, multicellular lumens (13). In a 3D spheroid model, cell spreading, the number and length of EC sprouts, and overall angiogenic outgrowth increased as a function of stiffness in 3D when stiffness was modulated by collagen glycation (Fig. 1) (14). However, separate experiments have shown that stiffening collagen delays EC sprouting and increases resistance to matrix degradation during remodeling (15).

It is hypothesized that, in 3D, matrix stiffness alters EC force generation that is critical for early sprouting morphogenesis (16, 17) and is required for later maintenance stages as well (17). Additionally, stiffness is thought to regulate MMP production as it has been shown that increasing stiffness results in increased MMP expression and decreased microvascular density (18). Together, these data underscore the need to parse apart the effects of density from stiffness in both 2D and 3D (Table 1).


Table 1.  A summary of the effects of matrix stiffening on angiogenesis in two-dimensional (2D) and three-dimensional (3D) models
Parameter 2D 3D
Network formation Decreases with increasing stiffness (1, 4, 5); networks present on compliant matrices but not on stiff substrates (2, 47) Decreases with increasing ECM density (1012, 15); thicker, deeper networks form with increasing stiffness (13)
EC elongation and sprouting EC elongation decreases with increasing stiffness (2, 4) Sprouting and outgrowth increase with increasing glycation of ECM proteins (14)
Lumen formation Larger lumens formed on stiffer gels (16) Forms more stable lumens with increased stiffness (12); large lumens observed on rigid gels (13)
MMP expression Decreases with decreasing stiffness, resulting in increased EC elongation (1) Increases with increasing stiffness, resulting in decreased vessel density (18)
EC proliferation Increases with increasing stiffness (9) No change in proliferation with increasing collagen glycation (14)

To better mimic the in vivo microenvironment, microfabrication approaches have been used to introduce complex topographies into gels of tunable stiffness to investigate the combined effects of stiffness and topography on angiogenesis. Sun et al. showed that ECs formed denser, shorter cord networks near a physical boundary compared to regions without boundaries (6). In our own work, we have demonstrated that topography in compliant or stiff gels can induce EC alignment (19). Collectively, these microfabricated systems create more complex, realistic geometries than those found in typical 2D assays without the density and stiffness issues that are at play in 3D matrices.

To complement these experimental approaches, computational modeling has been used to predict the effects of ECM stiffness on angiogenesis (11, 20). Computationally, increasing matrix density and network connectivity were shown to decrease sprout extension speed and alter morphology (20, 21). The models can predict the optimal protein density to maximize sprout extension speed (20) and simulate individual ECs forming sprouts (21).

Stromal cell synergies with the mechanical microenvironment

Stromal cells interact with ECs, and their interaction is vital for vascularization (22, 23). Fibroblasts can stimulate angiogenesis both chemically, with the release of angiogenic cues, and mechanically, by matrix disruption leading to ECM deposition and remodeling (22). Interestingly, increased ECM density hinders diffusion of proangiogenic macromolecules through the ECM and reduces network formation, but adding fibroblasts atop the EC-embedded 3D gels resulted in the robust formation of capillary networks regardless of collagen density (10).

Cell response to chemical factors secreted by stromal cells has also been shown to be mediated by matrix stiffness. High levels of VEGF are needed for tubulogenesis initiation (1), and VEGF-mediated sprout formation is dependent upon ECM protein concentration (12). Interestingly, established networks on compliant matrices are disrupted with the addition of growth factors like VEGF (5, 7). The addition of VEGF leads to coordinated EC migration and proliferation, resulting in stable sprout formation at intermediate matrix density (12) and rigidity (7), with uncoordinated migration at lower densities and no elongation at higher densities (12). No network formation was reported on extremely stiff gels, even with the addition of VEGF and other growth factors (5).

Matrix stiffness in tumor angiogenesis

Angiogenesis is highly upregulated in cancer, but the tumor vasculature is far more dense, tortuous, and permeable than healthy vasculature (24, 25). The disorganized tumor network results in non-uniform perfusion and chaotic blood flow within tumors (24). Much of this disorganization in vessel architecture is attributed to an overproduction of proangiogenic factors, which does not allow newly formed vessels to mature and stabilize (25). Collectively, the abnormalities in tumor vasculature can inhibit effective chemotherapeutic delivery (24, 25).

In addition to upregulation of proangiogenic factors in the tumor microenvironment, matrix stiffness is also elevated in tumors. Increased stiffness is due to increased matrix deposition and increased matrix cross-linking via the action of enzymes like lysyl oxidase. Stiffness has been shown to influence numerous aspects of tumor angiogenesis, including capillary sprouting (26, 27) and malignancy (28). In normoxic conditions, increased stiffness reduces EC sprouting and invasion (26), whereas under hypoxic conditions, there is increased sprouting compared to normoxia regardless of stiffness (26). Studies within the vascular biology community have suggested that increased matrix stiffness can also disrupt vascular integrity by increasing EC contractility and disrupting EC–EC adhesion (29, 30). These data suggest that the increased permeability found in the tumor microenvironment may also be due to changes in matrix mechanics.

Concluding thoughts

As more sophisticated models are developed that more accurately recapitulate the physiological microenvironment, we may better understand the signal transduction pathways that lead to altered angiogenic signaling in response to changes in the mechanical properties of a matrix. Targeting these pathways may help circumvent some of the problems associated with angiogenesis that occur in current regenerative medicine approaches and tumor growth.

Acknowledgements

This work was supported in part by a National Science Foundation (NSF) CAREER Award (1055502), by the Cornell Center on the Microenvironment & Metastasis through Award Number U54CA143876 from the National Cancer Institute, and by the National Heart, Lung and Blood Institute (HL097296) to CAR.

Conflict of interest and funding

The authors have not received any funding or benefits from industry or elsewhere to conduct this study.

References

  1. Hanjaya-Putra D, Yee J, Ceci D, Truitt R, Yee D, Gerecht S. Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells. J Cell Mol Med. 2010; 14: 2436–47. PubMed Abstract | Publisher Full Text
  2. Dickinson LE, Rand DR, Tsao J, Eberle W, Gerecht S. Endothelial cell responses to micropillar substrates of varying dimensions and stiffness. J Biomed Mater Res A. 2012; 100: 1457–66. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  3. Discher DE, Janmey P, Wang Y-L. Tissue cells feel and respond to the stiffness of their substrate. Science. 2005; 310: 1139–43. PubMed Abstract | Publisher Full Text
  4. Califano JP, Reinhart-King CA. A balance of substrate mechanics and matrix chemistry regulates endothelial cell network assembly. Cell Mol Bioeng. 2008; 1: 122–32. Publisher Full Text
  5. Saunders RL, Hammer DA. Assembly of human umbilical vein endothelial cells on compliant hydrogels. Cell Mol Bioeng. 2011; 3: 60–7. Publisher Full Text
  6. Sun J, Jamilpour N, Wang F-Y, Wong PK. Geometric control of capillary architecture via cell-matrix mechanical interactions. Biomaterials. 2014; 35: 3273–80. PubMed Abstract | Publisher Full Text
  7. Wu Y, Al-Ameen MA, Ghosh G. Integrated effects of matrix mechanics and vascular endothelial growth factor (VEGF) on capillary sprouting. Ann Biomed Eng. 2014; 42: 1024–36. PubMed Abstract | Publisher Full Text
  8. Reinhart-King CA, Dembo M, Hammer DA. Cell-cell mechanical communication through compliant substrates. Biophys J. 2008; 95: 6044–51. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  9. Yeh Y-T, Hur SS, Chang J, Wang K-C, Chiu J-J, Li Y-S, et al. Matrix stiffness regulates endothelial cell proliferation through septin 9. PLoS One. 2012; 7: e46889. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  10. Ghajar CM, Chen X, Harris JW, Suresh V, Hughes CCW, Jeon NL, et al. The effect of matrix density on the regulation of 3-D capillary morphogenesis. Biophys J. 2008; 94: 1930–41. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  11. Edgar LT, Underwood CJ, Guilkey JE, Hoying JB, Weiss JA. Extracellular matrix density regulates the rate of neovessel growth and branching in sprouting angiogenesis. PLoS One. 2014; 9: e85178. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  12. Shamloo A, Heilshorn SC. Matrix density mediates polarization and lumen formation of endothelial sprouts in VEGF gradients. Lab Chip. 2010; 10: 3061–8. PubMed Abstract | Publisher Full Text
  13. Yamamura N, Sudo R, Ikeda M, Tanishita K. Effects of the mechanical properties of collagen gel on the in vitro formation of microvessel networks by endothelial cells. Tissue Eng. 2007; 13: 1443–53. PubMed Abstract | Publisher Full Text
  14. Mason BN, Starchenko A, Williams RM, Bonassar LJ, Reinhart-King CA. Tuning three-dimensional collagen matrix stiffness independently of collagen concentration modulates endothelial cell behavior. Acta Biomater. 2013; 9: 4635–44. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  15. Francis-Sedlak ME, Moya ML, Huang J-J, Lucas SA, Chandrasekharan N, Larson JC, et al. Collagen glycation alters neovascularization in vitro and in vivo. Microvasc Res. 2010; 80: 3–9. PubMed Abstract | Publisher Full Text
  16. Sieminski AL, Hebbel RP, Gooch KJ. The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro. Exp Cell Res. 2004; 297: 574–84. PubMed Abstract | Publisher Full Text
  17. Kniazeva E, Putnam AJ. Endothelial cell traction and ECM density influence both capillary morphogenesis and maintenance in 3-D. Am J Physiol – Cell Physiol. 2009; 297: 179–87. Publisher Full Text
  18. Chung AWY, Yang HHC, Sigrist MK, Brin G, Chum E, Gourlay WA, et al. Matrix metalloproteinase-2 and -9 exacerbate arterial stiffening and angiogenesis in diabetes and chronic kidney disease. Cardiovasc Res. 2009; 84: 494–504. PubMed Abstract | Publisher Full Text
  19. Charest JM, Califano JP, Carey SP, Reinhart-King CA. Fabrication of substrates with defined mechanical properties and topographical features for the study of cell migration. Macromol Biosci. 2012; 12: 12–20. PubMed Abstract | Publisher Full Text
  20. Bauer AL, Jackson TL, Jiang Y. Topography of extracellular matrix mediates vascular morphogenesis and migration speeds in angiogenesis. PLoS Comput Biol. 2009; 5: e1000445. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  21. Bauer AL, Jackson TL, Jiang Y. A cell-based model exhibiting branching and anastomosis during tumor-induced angiogenesis. Biophys J. 2007; 92: 3105–21. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  22. Hurley JR, Balaji S, Narmoneva DA. Complex temporal regulation of capillary morphogenesis by fibroblasts. Am J Physiol – Cell Physiol. 2010; 299: 444–53. Publisher Full Text
  23. Rao RR, Peterson AW, Ceccarelli J, Putnam AJ, Stegemann JP. Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials. Angiogenesis. 2012; 15: 253–64. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  24. Munn LL. Aberrant vascular architecture in tumors and its importance in drug-based therapies. Drug Discov Today. 2003; 8: 396–403. PubMed Abstract | Publisher Full Text
  25. Chauhan VP, Stylianopoulos T, Boucher Y, Jain RK. Delivery of molecular and nanoscale medicine to tumors: transport barriers and strategies. Annu Rev Chem Biomol Eng. 2011; 2: 281–98. PubMed Abstract | Publisher Full Text
  26. Shen Y, Abaci HE, Krupski Y, Weng L, Burdick JA, Gerecht S. Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting. Biomater Sci. 2014; 2: 655–65. PubMed Abstract | Publisher Full Text
  27. Ghosh K, Thodeti CK, Dudley AC, Mammoto A, Klagsbrun M, Ingber DE. Tumor-derived endothelial cells exhibit aberrant Rho-mediated mechanosensing and abnormal angiogenesis in vitro. Proc Natl Acad Sci. 2008; 105: 11305–10. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
  28. Liang Y, Jeong J, DeVolder RJ, Cha C, Wang F, Tong YW, et al. A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity. Biomaterials. 2011; 32: 9308–15. PubMed Abstract | Publisher Full Text
  29. Krishnan R, Klumpers DD, Park CY, Rajendran K, Trepat X, Van Bezu J, et al. Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces. Am J Physiol – Cell Physiol. 2011; 300: 146–54. Publisher Full Text
  30. Huynh J, Nishimura N, Rana K, Peloquin JM, Califano JP, Montague CR, et al. Age-related intimal stiffening enhances endothelial permeability and leukocyte transmigration. Sci Transl Med. 2011; 3: 112ra122. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
About The Authors

Danielle J. LaValley

United States

Cynthia A. Reinhart-King
Associate Professor Department of Biomedical Engineering Cornell University
United States

Associate Professor

Department of Biomedical Engineering