103
ECM degradation is accompanied by ECM production and the secretion of cells.
Once the quiescent state of the ECs composing the blood vessel walls is perturbed
and angiogenesis is induced, ECs start to proliferate and invade the neighboring
ECM by using proteinases. At the same time, they start to remodel the existing ECM
by synthesizing new ECM. In healing wounds, ECs produce transitional ECM proteins, including fibrinogen and fibronectin, and temporarily deposit them in the
ECM in order to provide available ligands during vessel growth [38]. Moreover,
ECs also produce such matricellular proteins as tenascin C and SPARC in the ECM
to mediate angiogenesis [38]. Clearly, the new ECM synthesis of cells is crucial for
angiogenesis, and hypoxia, through HIF1α, has been shown to regulate the expressions of many different types of ECM proteins [167]. For example, many in vivo
and in vitro studies have shown that hypoxia enhanced the synthesis of collagen, the
most abundant protein in mammalian tissues [22, 103, 217].
Moreover, proliferation of the cells during angiogenesis/vasculogenesis in 3D
scaffolds is regulated by basic fibroblast growth factor (bFGF) and VEGF, which are
known to be hypoxia-dependent proteins [177]. In most studies of the vascularization of 3D scaffolds, both GFs are broadly used as soluble factors that supplement
cell growth medium to induce proliferation and migration [127, 193]. In addition,
Shen et al. demonstrated that immobilization of VEGF into a 3D collagen scaffold
promotes EC viability, proliferation, and vascularization [198]. VEGF has been
shown to promote blood vessel formation, not only by inducing cellular proliferation and migration but also by directly regulating elongation and capillary networking in 3D ECM constructs deprived of O 2 and nutrients [93]. Helminger et al. used
a sandwich system to seed HUVECs inside a collagen gel [93]. The transfer of O 2
and nutrients was accomplished with only simple diffusion through the edges of the
collagen, so that O 2 and nutrient levels decreased toward the center. They found that,
in a short time period (about nine hours), VEGF intensity increased in the interior
regions deprived of O 2 , which correlates well with cell elongation and branching.
VEGF promoted capillary networking independently of proliferation, highlighting
the role of autocrine VEGF in the reorganization of vascular networks in hypoxic
regions of solid tumors. Another study focusing on quantitative measurements of O 2
gradients in 3D collagen also showed that increased VEGF concentrations correlated well to decreasing O 2 levels throughout the 3D constructs during a ten day
period of cultivation [36].
A few studies considering the induction of angiogenesis by hypoxia in 3D scaffolds have emphasized that lowering the O 2 tension in 3D gels improved cellular
branching and tube formation of ECs [170, 177]. As made abundantly clear throughout this chapter, both O 2 and matrix mechanics act as potent upstream regulators of
a variety of signaling pathways that contribute significantly to the regulation of
vascular differentiation as well as morphogenesis. To better understand these
processes, engineers and biologists together have built an impressive library of
materials and assays to examine an array of signaling cascades guiding the formation of blood vessels. There are several interesting platforms that can be used to
study the effect of hypoxia and O 2 gradients on cellular function, including hydrogels (Fig. 4.6) [143, 144, 173, 187] and microfabrication or microfluidic devices [2,
4 Hypoxia and Matrix Manipulation for Vascular Engineering
ECM degradation is accompanied by ECM production and the secretion of cells.
Once the quiescent state of the ECs composing the blood vessel walls is perturbed
and angiogenesis is induced, ECs start to proliferate and invade the neighboring
ECM by using proteinases. At the same time, they start to remodel the existing ECM
by synthesizing new ECM. In healing wounds, ECs produce transitional ECM proteins, including fibrinogen and fibronectin, and temporarily deposit them in the
ECM in order to provide available ligands during vessel growth [38]. Moreover,
ECs also produce such matricellular proteins as tenascin C and SPARC in the ECM
to mediate angiogenesis [38]. Clearly, the new ECM synthesis of cells is crucial for
angiogenesis, and hypoxia, through HIF1α, has been shown to regulate the expressions of many different types of ECM proteins [167]. For example, many in vivo
and in vitro studies have shown that hypoxia enhanced the synthesis of collagen, the
most abundant protein in mammalian tissues [22, 103, 217].
Moreover, proliferation of the cells during angiogenesis/vasculogenesis in 3D
scaffolds is regulated by basic fibroblast growth factor (bFGF) and VEGF, which are
known to be hypoxia-dependent proteins [177]. In most studies of the vascularization of 3D scaffolds, both GFs are broadly used as soluble factors that supplement
cell growth medium to induce proliferation and migration [127, 193]. In addition,
Shen et al. demonstrated that immobilization of VEGF into a 3D collagen scaffold
promotes EC viability, proliferation, and vascularization [198]. VEGF has been
shown to promote blood vessel formation, not only by inducing cellular proliferation and migration but also by directly regulating elongation and capillary networking in 3D ECM constructs deprived of O 2 and nutrients [93]. Helminger et al. used
a sandwich system to seed HUVECs inside a collagen gel [93]. The transfer of O 2
and nutrients was accomplished with only simple diffusion through the edges of the
collagen, so that O 2 and nutrient levels decreased toward the center. They found that,
in a short time period (about nine hours), VEGF intensity increased in the interior
regions deprived of O 2 , which correlates well with cell elongation and branching.
VEGF promoted capillary networking independently of proliferation, highlighting
the role of autocrine VEGF in the reorganization of vascular networks in hypoxic
regions of solid tumors. Another study focusing on quantitative measurements of O 2
gradients in 3D collagen also showed that increased VEGF concentrations correlated well to decreasing O 2 levels throughout the 3D constructs during a ten day
period of cultivation [36].
A few studies considering the induction of angiogenesis by hypoxia in 3D scaffolds have emphasized that lowering the O 2 tension in 3D gels improved cellular
branching and tube formation of ECs [170, 177]. As made abundantly clear throughout this chapter, both O 2 and matrix mechanics act as potent upstream regulators of
a variety of signaling pathways that contribute significantly to the regulation of
vascular differentiation as well as morphogenesis. To better understand these
processes, engineers and biologists together have built an impressive library of
materials and assays to examine an array of signaling cascades guiding the formation of blood vessels. There are several interesting platforms that can be used to
study the effect of hypoxia and O 2 gradients on cellular function, including hydrogels (Fig. 4.6) [143, 144, 173, 187] and microfabrication or microfluidic devices [2,
4 Hypoxia and Matrix Manipulation for Vascular Engineering