102
results in the collapsing and deformation of tubes [195]. The critical issues to consider to prevent cellular necrosis during 3D vascular cell cultures are the permeability of the ECM material to O 2 and glucose, the cell seeding density, and the thickness
of the gel. Thus, cell seeding density is constrained by an upper limit, above which
the cells undergo necrosis due to nutrient deprivation, and a lower limit, below
which the cells cannot assemble sufficiently to form tube-like structures. Both limits
depend on the equilibrium O 2 levels in the environment.
Cells may also die as a result of apoptosis after their encapsulation in the gel.
Interestingly, some groups have demonstrated that programmed cellular death is
necessary for angiogenesis/vasculogenesis [195]. Segura et al., having studied tube
formation of ECs in both 2D Matrigel and 3D collagen, concluded that a considerable number of cells undergo apoptosis at the initial stages of cultivation and that,
once angiogenesis is induced and tube formation has started, no further apoptosis
occurs throughout the process. Inhibition of proapoptotic proteins has been shown
to correlate with defective tube formations, suggesting that apoptosis is important
for avoiding imperfections during blood vessel growth. Hypoxia, as already discussed, induces angiogenic responses and also regulates proapoptotic gene expressions. Thus, spatial variations in O 2 levels may alter the apoptotic responses in the
gel and therefore regulate vascular tube morphogenesis.
MMPs are promoted by integrin-ligand interactions between cells and the ECM,
leading to the degradation of the ECM and facilitating the migration of the cells
[81]. It is hypothesized that ECM fragmentation, orchestrated by the secretion of
MMPs, can mediate caspase activity through the rebinding of ECM protein fragments to unligated integrins, namely, death receptors [38]. Therefore, the survival of
ECs depends on the balance between cell survival promoters, such as FAK, Src, and
Raf, and cellular apoptosis promoters, such as caspase 8 and caspase 3. Hypoxia
may again play a critical role here, affecting both sides of the equilibrium, by upregulating MMPs and VEGF at the same time [21, 93]. Hypoxia, accompanied by
nonuniform distribution of O 2 throughout the gel, can result in spatial differences of
cellular viability, which may subsequently disrupt vascular networking.
Overall, blood vessel growth requires remodeling of the ECM, which is based on
two distinct mechanisms: (1) degradation of the ECM by secreted proteases, and (2)
production of new ECM to support the invading vasculature. Many studies have
shown that hypoxia can regulate the degradation, maintenance, and synthesis of the
ECM [61, 179]. ECM degradation is important for cellular migration into and blood
vessel invasion of tissue. MMPs, as mentioned above, are a major family of proteinases that participate in the degradation of the ECM during angiogenesis. In particular,
MMP-2 and MMP-9, both members of the gelatinase subgroup of MMPs, have been
shown to contribute to the process of angiogenesis [85]. MMP-2 secreted by the cells
is activated through membrane MT1-MMPs where the activation can be avoided in
the presence of tissue inhibitor of MMP-2 (TIMP-2) at high levels [99]. Furthermore,
hypoxia was shown to influence the expression of MMP-2, as well as of MT1-MMP
and TIMP-2, in ECs [21]. Lahat’s group demonstrated the upregulation of MMP-2
expression in hypoxic (0.3% O 2 ) cultures of HUVECs, whereas MT1-MMP and
TIMP-2 are downregulated, enhancing migration and tube formation [21].
M. R. Blatchley et al.
results in the collapsing and deformation of tubes [195]. The critical issues to consider to prevent cellular necrosis during 3D vascular cell cultures are the permeability of the ECM material to O 2 and glucose, the cell seeding density, and the thickness
of the gel. Thus, cell seeding density is constrained by an upper limit, above which
the cells undergo necrosis due to nutrient deprivation, and a lower limit, below
which the cells cannot assemble sufficiently to form tube-like structures. Both limits
depend on the equilibrium O 2 levels in the environment.
Cells may also die as a result of apoptosis after their encapsulation in the gel.
Interestingly, some groups have demonstrated that programmed cellular death is
necessary for angiogenesis/vasculogenesis [195]. Segura et al., having studied tube
formation of ECs in both 2D Matrigel and 3D collagen, concluded that a considerable number of cells undergo apoptosis at the initial stages of cultivation and that,
once angiogenesis is induced and tube formation has started, no further apoptosis
occurs throughout the process. Inhibition of proapoptotic proteins has been shown
to correlate with defective tube formations, suggesting that apoptosis is important
for avoiding imperfections during blood vessel growth. Hypoxia, as already discussed, induces angiogenic responses and also regulates proapoptotic gene expressions. Thus, spatial variations in O 2 levels may alter the apoptotic responses in the
gel and therefore regulate vascular tube morphogenesis.
MMPs are promoted by integrin-ligand interactions between cells and the ECM,
leading to the degradation of the ECM and facilitating the migration of the cells
[81]. It is hypothesized that ECM fragmentation, orchestrated by the secretion of
MMPs, can mediate caspase activity through the rebinding of ECM protein fragments to unligated integrins, namely, death receptors [38]. Therefore, the survival of
ECs depends on the balance between cell survival promoters, such as FAK, Src, and
Raf, and cellular apoptosis promoters, such as caspase 8 and caspase 3. Hypoxia
may again play a critical role here, affecting both sides of the equilibrium, by upregulating MMPs and VEGF at the same time [21, 93]. Hypoxia, accompanied by
nonuniform distribution of O 2 throughout the gel, can result in spatial differences of
cellular viability, which may subsequently disrupt vascular networking.
Overall, blood vessel growth requires remodeling of the ECM, which is based on
two distinct mechanisms: (1) degradation of the ECM by secreted proteases, and (2)
production of new ECM to support the invading vasculature. Many studies have
shown that hypoxia can regulate the degradation, maintenance, and synthesis of the
ECM [61, 179]. ECM degradation is important for cellular migration into and blood
vessel invasion of tissue. MMPs, as mentioned above, are a major family of proteinases that participate in the degradation of the ECM during angiogenesis. In particular,
MMP-2 and MMP-9, both members of the gelatinase subgroup of MMPs, have been
shown to contribute to the process of angiogenesis [85]. MMP-2 secreted by the cells
is activated through membrane MT1-MMPs where the activation can be avoided in
the presence of tissue inhibitor of MMP-2 (TIMP-2) at high levels [99]. Furthermore,
hypoxia was shown to influence the expression of MMP-2, as well as of MT1-MMP
and TIMP-2, in ECs [21]. Lahat’s group demonstrated the upregulation of MMP-2
expression in hypoxic (0.3% O 2 ) cultures of HUVECs, whereas MT1-MMP and
TIMP-2 are downregulated, enhancing migration and tube formation [21].
M. R. Blatchley et al.