Chapter 5 In Vitro Three Dimensional Collagen Matrix Models of Endothelial Lumen Formation During Vasculogenesis and Angiogenesis

Koh, Wonshill; Stratman, Amber N.; Sacharidou, Anastasia; Davis, George E.

doi:10.1016/s0076-6879(08)02005-3

Cited by 203 publications

(243 citation statements)

References 52 publications

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“…Angiogenic potential of ECs, therefore, is typically assayed in vitro by quantifying endothelial networks that form either on 2D or within 3D gels. 20,24 The advantage of the 3D model is that it allows generation of capillary lumens that resemble capillaries that form in vivo. This method, therefore, has been widely used to elucidate the signaling pathways that control EC morphogenesis and lumen formation.…”

Section: Discussionmentioning

confidence: 99%

Oxidized Low‐Density Lipoprotein Alters the Effect of Matrix Stiffness on the Formation of Endothelial Networks and Capillary Lumens

et al. 2013

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Formation of new blood vessels is essential for vascular repair and remodeling, and it is known that biomechanical properties of extracellular matrix play a major role in this process. Our earlier studies have also shown that exposing endothelial cells to oxidized modification of low-density lipoproteins (oxLDL) increases endothelial stiffness and facilitates their ability to form cellular networks, suggesting that it facilitates endothelial angiogenic potential. The goal of this study, therefore, was to test the interrelationship between matrix stiffness and oxLDL in the regulation of angiogenesis. Our results show that, as expected, an increase in matrix stiffness inhibited endothelial network formation and that exposure to oxLDL significantly facilitated this process. We also show, however, that oxLDL-induced facilitation of endothelial networks was observed only in stiff (3 mg/mL) but not in soft (1 mg/mL) collagen gels, resulting in blunting the effect of matrix stiffness. Also unexpectedly, we show that an increase in matrix stiffness results in a significant increase in the number of capillary lumens that are formed by single cells or pairs of cells, suggesting that while endothelial connectivity is impaired, formation of single-cell lumens is facilitated. oxLDL facilitates lumen formation, but this effect is also matrix dependent and is observed only in soft gels and not in stiff gels. Finally, an increase in both matrix stiffness and oxLDL exposure results in changes in capillary morphology, with the formation of larger capillary lumens. Overall, our study suggests that oxLDL plays an important role in formation of new capillaries and their morphology and that this effect is critically dependent on the extracellular environment's compliance, thereby underlining the importance of the interdependence of these parameters.

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Section: Discussionmentioning

confidence: 99%

Oxidized Low‐Density Lipoprotein Alters the Effect of Matrix Stiffness on the Formation of Endothelial Networks and Capillary Lumens

et al. 2013

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“…HUVECs resuspended in 200 l medium 199 (1ϫ) were then mixed with the gel solution for a final concentration of 2 million cells/ml. We transferred an appropriate volume of gel solution into either 24-or 96-well plates to obtain a gel thickness of 1.7 mm [as used in other studies (24)] and incubated the gels for 30 min at 37°C. Finally, the EGM, supplemented with 50 ng/ml VEGF, was added at a ratio of 1.78 ml media to 10 3 cells (24).…”

Section: Methodsmentioning

confidence: 99%

“…In recent decades, our understanding of the molecular regulation underlying vascular development has vastly expanded, primarily because of newly available, well-defined in vitro models. One common model is the culturing of ECs in gels made of the extracellular matrix component collagen, which has enabled us to investigate the effects of many factors on tubulogenesis (24,38,39).The availability of oxygen (O 2 ) is a critical parameter affecting how vascular cells form tubes. In vitro models have demonstrated that hypoxic conditions enhance tube formation, as do hypoxic regions formed by the O 2 consumption of the cells in threedimensional (3D) structures (3,19,32).…”

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Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

Abaci

Truitt

Tan

et al. 2011

American Journal of Physiology-Cell Physiology

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The availability of oxygen (O2) is a critical parameter affecting vascular tube formation. In this study, we hypothesize that dissolved oxygen (DO) levels in collagen gels change during the three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs) in atmospheric conditions and that such changes affect the kinetics of tube formation through the production of reactive oxygen species (ROS). We demonstrate a decrease in O2 tension during 3D cultures of HUVECs. Noninvasive measurements of DO levels during culture under atmospheric conditions revealed a profound decrease that reached as low as 2% O2 at the end of 24 h. After media replacement, DO levels rose rapidly and equilibrated at ϳ15% O 2, creating a reoxygenated environment. To accurately estimate DO gradients in 3D collagen gels, we developed a 3D mathematical model and determined the Michaelis-Menten parameters, V max and K m, of HUVECs in collagen gels. We detected an increase in ROS levels throughout the culture period. Using diphenyliodonium to inhibit ROS production resulted in the complete inhibition of tube formation. Interference RNA studies further showed that hypoxiainducible factors (HIFs)-1␣ and -2␣ are not involved in the formation of 3D tubes in collagen gels. We conclude that ROS affect the tubulogenesis process through HIF␣-independent pathways, where the levels of ROS are influenced by the uncontrolled variations in DO levels. This study is the first demonstration of the critical and unexpected role of O 2 during 3D in vitro culture models of tubulogenesis in atmospheric conditions. microvasculature; reactive oxygen species; hypoxia-inducible factors AN UNDERSTANDING OF THE MECHANISMS controlling how blood vessels form from endothelial cells (ECs) is crucial for developmental biology and vascular tissue engineering. In recent decades, our understanding of the molecular regulation underlying vascular development has vastly expanded, primarily because of newly available, well-defined in vitro models. One common model is the culturing of ECs in gels made of the extracellular matrix component collagen, which has enabled us to investigate the effects of many factors on tubulogenesis (24,38,39).The availability of oxygen (O 2 ) is a critical parameter affecting how vascular cells form tubes. In vitro models have demonstrated that hypoxic conditions enhance tube formation, as do hypoxic regions formed by the O 2 consumption of the cells in threedimensional (3D) structures (3,19,32). Research has shown that the rate of O 2 consumption of cells follows the Michaelis-Menten kinetics and depends on the cell type, cell density (15), and dissolved oxygen (DO) levels (1). Variations in DO levels strongly affect angiogenesis (19, 48) through different vascular cell responses, such as viability, differentiation, migration, and remodeling of the extracellular matrix (4, 27, 40).Hypoxia-inducible factors (HIFs) and reactive oxygen species (ROS) are among the effectors responsible for cellular responses to changing DO levels. HIF1␣ and HI...

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“…Ex vivo assays have been developed to mimic human vessel formation either in fibrin or collagen gels [28][29][30][31][32][33][34][35][36][37]. These have combined endothelial cells or ECFCs with mural cells (eg, lung fibroblasts, dermal fibroblasts, retinal pericytes, or mesenchymal stromal cells) and specialized endothelial growth medium containing cytokines [eg, VEGF and fibroblast growth factor-2 (FGF-2)].…”

Section: Introductionmentioning

confidence: 99%

The Hematopoietic Chemokine CXCL12 Promotes Integration of Human Endothelial Colony Forming Cell–Derived Cells into Immature Vessel Networks

Newey

Tsaknakis

Khoo

et al. 2014

Stem Cells and Development

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Chapter 5 In Vitro Three Dimensional Collagen Matrix Models of Endothelial Lumen Formation During Vasculogenesis and Angiogenesis

Cited by 203 publications

References 52 publications

Oxidized Low‐Density Lipoprotein Alters the Effect of Matrix Stiffness on the Formation of Endothelial Networks and Capillary Lumens

Oxidized Low‐Density Lipoprotein Alters the Effect of Matrix Stiffness on the Formation of Endothelial Networks and Capillary Lumens

Unforeseen decreases in dissolved oxygen levels affect tube formation kinetics in collagen gels

The Hematopoietic Chemokine CXCL12 Promotes Integration of Human Endothelial Colony Forming Cell–Derived Cells into Immature Vessel Networks

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