15
expression of wild-type MT1-MMP without its cytoplasmic tail markedly stimulates the rate and extent of EC lumen formation compared to full-length wild-type
MT1- MMP expression [83]. A number of studies suggest that its cytoplasmic tail
plays a role in endocytic recycling, and thus, deleting the tail increases cell surface
expression, which in our case leads to additional increases in EC lumen formation
events. Overall, these results demonstrate that MT1-MMP is a major regulator of
EC lumen and tube formation and that it works closely in conjunction with the α2β1
integrin as well as Cdc42, Rac1, and other small GTPases to control this process.
1.3.8 MT1-MMP-Dependent EC Lumen and Tube Formation
Leads to the Formation of a Network of Physical Spaces
Within the ECM Termed Vascular Guidance Tunnels
During the course of the above studies, we made the novel observation that during
EC lumen and tube formation, ECs are also creating a network of physical spaces
that we term vascular guidance tunnels. These form as a result of MT1-MMPmediated proteolysis of collagen matrices [95]. In every instance examined, there is
a direct relationship between EC tube formation and the formation of vascular guidance tunnels [95]. Tunnels were first detected by staining the collagen type I matrix
with a monoclonal antibody that recognizes native type I collagen and not denatured
collagen (which is generated at 37 °C when it is cut with mammalian collagenases).
The lumen and tube formation creates an extensive interconnecting network of
these tunnel spaces within the 3D collagen matrices [95]. To further prove that these
represent physical spaces in the ECM, they were microinjected with silicone oil
[95]. Dramatic filling of networks was demonstrated showing that EC tube formation leads to the formation of interconnecting vascular guidance tunnel spaces. We
further showed that the ECs produced twice as many tunnel spaces than were occupied by EC-lined tubes [95], raising the interesting possibility that vessel remodeling could occur through these pre-formed physical tunnel spaces. Although
MT1-MMP was required for the formation of vascular guidance tunnel formation,
once they were formed, blockade of MT1-MMP did not affect the ability of ECs to
migrate within the spaces [95]. Thus, EC migration events which are necessary for
EC tube formation are completely inhibited in 3D collagen matrices, if MT1-MMP
is blocked from the beginning of culture. However, once vascular guidance tunnels
have formed through MT1-MMP-mediated events, ECs are then able to migrate
within these physical spaces in an MMP-independent manner [95]. Thus, vascular
guidance tunnel spaces are similar to 2D matrix surfaces where EC motility is
insensitive to MT1-MMP inhibition (siRNA or inhibitors) [95]. We also observed
that while the creation of vascular guidance tunnels by ECs requires the α2β1 integrin, a native collagen-binding integrin, the motility of ECs within MT1-MMPgenerated tunnels was not sensitive to inhibition with anti-α2 integrin subunit
blocking antibodies. In contrast, EC motility was blocked using anti-αv subunit
blocking antibodies [95] which are known to bind matricryptic RGD sites that are
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…
expression of wild-type MT1-MMP without its cytoplasmic tail markedly stimulates the rate and extent of EC lumen formation compared to full-length wild-type
MT1- MMP expression [83]. A number of studies suggest that its cytoplasmic tail
plays a role in endocytic recycling, and thus, deleting the tail increases cell surface
expression, which in our case leads to additional increases in EC lumen formation
events. Overall, these results demonstrate that MT1-MMP is a major regulator of
EC lumen and tube formation and that it works closely in conjunction with the α2β1
integrin as well as Cdc42, Rac1, and other small GTPases to control this process.
1.3.8 MT1-MMP-Dependent EC Lumen and Tube Formation
Leads to the Formation of a Network of Physical Spaces
Within the ECM Termed Vascular Guidance Tunnels
During the course of the above studies, we made the novel observation that during
EC lumen and tube formation, ECs are also creating a network of physical spaces
that we term vascular guidance tunnels. These form as a result of MT1-MMPmediated proteolysis of collagen matrices [95]. In every instance examined, there is
a direct relationship between EC tube formation and the formation of vascular guidance tunnels [95]. Tunnels were first detected by staining the collagen type I matrix
with a monoclonal antibody that recognizes native type I collagen and not denatured
collagen (which is generated at 37 °C when it is cut with mammalian collagenases).
The lumen and tube formation creates an extensive interconnecting network of
these tunnel spaces within the 3D collagen matrices [95]. To further prove that these
represent physical spaces in the ECM, they were microinjected with silicone oil
[95]. Dramatic filling of networks was demonstrated showing that EC tube formation leads to the formation of interconnecting vascular guidance tunnel spaces. We
further showed that the ECs produced twice as many tunnel spaces than were occupied by EC-lined tubes [95], raising the interesting possibility that vessel remodeling could occur through these pre-formed physical tunnel spaces. Although
MT1-MMP was required for the formation of vascular guidance tunnel formation,
once they were formed, blockade of MT1-MMP did not affect the ability of ECs to
migrate within the spaces [95]. Thus, EC migration events which are necessary for
EC tube formation are completely inhibited in 3D collagen matrices, if MT1-MMP
is blocked from the beginning of culture. However, once vascular guidance tunnels
have formed through MT1-MMP-mediated events, ECs are then able to migrate
within these physical spaces in an MMP-independent manner [95]. Thus, vascular
guidance tunnel spaces are similar to 2D matrix surfaces where EC motility is
insensitive to MT1-MMP inhibition (siRNA or inhibitors) [95]. We also observed
that while the creation of vascular guidance tunnels by ECs requires the α2β1 integrin, a native collagen-binding integrin, the motility of ECs within MT1-MMPgenerated tunnels was not sensitive to inhibition with anti-α2 integrin subunit
blocking antibodies. In contrast, EC motility was blocked using anti-αv subunit
blocking antibodies [95] which are known to bind matricryptic RGD sites that are
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…