7
EC-specific promoter in zebrafish, revealing that intracellular vacuoles were
observed and participate in the lumen formation process of intersegmental vessels
during vascular development [62, 104]. Additional work in vivo reveals the clear
presence of intracellular vacuoles as a major determinant of EC lumen formation in
other species, including mouse and quail [36, 110].
Both Cdc42 and Rac1 are required for intracellular vacuole formation, as well as
EC lumen and tube formation [8, 30, 67]. We have performed these experiments
using either dominant-negative mutants of Cdc42 and Rac1 or specific siRNAs to
these GTPases. Developing multicellular lumenal structures then interconnect into
more extensive networks over time (Fig. 1.1b, middle and lower panels). Using an
angiogenic sprouting model [9, 68], time-lapse images are shown which reveal how
invading ECs interact, develop intracellular vacuoles, and migrate toward each other
to form multicellular lumen and tube structures over time (Fig. 1.1c).
After much effort over many years, we have elucidated the molecular requirements and signaling pathways that underlie the ability of human ECs to form lumen
and tube structures in 3D matrices [8, 25, 27, 36, 60, 63, 64, 79, 84, 87, 89, 93, 95].
Most of our studies have focused on collagen matrices, and, thus, a major requirement for these events is the α2β1 integrin, a collagen-binding integrin [28] (Fig. 1.2).
Blocking antibodies directed to α2β1 markedly block lumen formation, as do α2
integrin subunit siRNAs. Interestingly, blocking antibodies directed to many other
integrin subunits, including α5β1, a fibronectin receptor, have no effect in this system. Also, considerable work has shown that α2β1 integrin is important for vascularization events in vivo and in both developmental and postnatal life contexts [85,
88]. In contrast, when our studies utilized fibrin matrices, we identified that both
αvβ3 and α5β1 were required for EC lumen and tube formation [11], while α2β1
was not shown to be involved. Interestingly, the first fibrin system that we developed was performed in the presence of serum. More recently, we developed a
serum-free defined system in fibrin matrices, where fibronectin was added in with
the matrix when it is polymerized [91]. Under these conditions, α5β1 was involved
in the tube formation process, not αvβ3. This result suggests that serum-derived
vitronectin might have been the reason why αvβ3 played a role in this first system
[11]. Nonetheless, both of these integrins have been shown to be involved during
vessel formation during development and in postnatal mice [57, 97]. Thus, an
important point is that the in vitro models by our laboratory and others have very
accurately predicted in vivo findings made by other groups. Another key point is
that the in vitro model systems demonstrated first that multiple integrin chains
could be utilized by ECs to control tube morphogenesis and second that there is
little evidence to suggest that any particular integrin is unique or special in this
property to regulate the morphogenic cascade necessary to form new blood vessels.
The role of particular integrins in morphogenesis appears to be directly linked to
the ECM environment (and the predominant ECM components) that are in contact
with the ECs.
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…
EC-specific promoter in zebrafish, revealing that intracellular vacuoles were
observed and participate in the lumen formation process of intersegmental vessels
during vascular development [62, 104]. Additional work in vivo reveals the clear
presence of intracellular vacuoles as a major determinant of EC lumen formation in
other species, including mouse and quail [36, 110].
Both Cdc42 and Rac1 are required for intracellular vacuole formation, as well as
EC lumen and tube formation [8, 30, 67]. We have performed these experiments
using either dominant-negative mutants of Cdc42 and Rac1 or specific siRNAs to
these GTPases. Developing multicellular lumenal structures then interconnect into
more extensive networks over time (Fig. 1.1b, middle and lower panels). Using an
angiogenic sprouting model [9, 68], time-lapse images are shown which reveal how
invading ECs interact, develop intracellular vacuoles, and migrate toward each other
to form multicellular lumen and tube structures over time (Fig. 1.1c).
After much effort over many years, we have elucidated the molecular requirements and signaling pathways that underlie the ability of human ECs to form lumen
and tube structures in 3D matrices [8, 25, 27, 36, 60, 63, 64, 79, 84, 87, 89, 93, 95].
Most of our studies have focused on collagen matrices, and, thus, a major requirement for these events is the α2β1 integrin, a collagen-binding integrin [28] (Fig. 1.2).
Blocking antibodies directed to α2β1 markedly block lumen formation, as do α2
integrin subunit siRNAs. Interestingly, blocking antibodies directed to many other
integrin subunits, including α5β1, a fibronectin receptor, have no effect in this system. Also, considerable work has shown that α2β1 integrin is important for vascularization events in vivo and in both developmental and postnatal life contexts [85,
88]. In contrast, when our studies utilized fibrin matrices, we identified that both
αvβ3 and α5β1 were required for EC lumen and tube formation [11], while α2β1
was not shown to be involved. Interestingly, the first fibrin system that we developed was performed in the presence of serum. More recently, we developed a
serum-free defined system in fibrin matrices, where fibronectin was added in with
the matrix when it is polymerized [91]. Under these conditions, α5β1 was involved
in the tube formation process, not αvβ3. This result suggests that serum-derived
vitronectin might have been the reason why αvβ3 played a role in this first system
[11]. Nonetheless, both of these integrins have been shown to be involved during
vessel formation during development and in postnatal mice [57, 97]. Thus, an
important point is that the in vitro models by our laboratory and others have very
accurately predicted in vivo findings made by other groups. Another key point is
that the in vitro model systems demonstrated first that multiple integrin chains
could be utilized by ECs to control tube morphogenesis and second that there is
little evidence to suggest that any particular integrin is unique or special in this
property to regulate the morphogenic cascade necessary to form new blood vessels.
The role of particular integrins in morphogenesis appears to be directly linked to
the ECM environment (and the predominant ECM components) that are in contact
with the ECs.
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…