27
proteins. Although the EC apical membrane domain has been difficult to define in
molecular terms, the clear evidence of EC polarization in our coculture model is the
deposition of basement membrane matrices specifically to the abluminal surface
and the prior recruitment of pericytes to these same abluminal membranes [94].
1.3.18 Specific Upregulation of EC and Pericyte Integrins
Recognizing Basement Membrane Matrices
During EC-Pericyte Tube Co-Assembly in 3D Collagen
Matrices
Again, using our coculture system with human ECs and bovine pericytes, we
assessed how EC vs. pericyte integrins were regulated during this process. We
assessed mRNA levels and performed function blocking experiments with antiintegrin monoclonal antibodies. As discussed above, blocking antibodies to the
α5β1 integrin had function blocking effects that selectively occurred in the
EC-pericyte cocultures but not the EC-only cultures [94]. Interestingly, the EC α5
integrin subunit was induced at the mRNA level in EC-pericyte cocultures, but not
in EC-only cultures where it was downregulated. An important theme which
emerged from these studies is that integrins which recognize the newly remodeled
ECM assembling between ECs and pericytes were induced, while others that recognized collagen type I matrices, such as the α2 integrin subunit from ECs, were
downregulated [94] (Fig. 1.5e). Thus, as basement membranes assemble around EC
tubes, the direct interaction of ECs with collagen type I decreases, while their contact with basement membrane matrices increases. Concomitantly, we observed
increases in the expression of integrin α5, α3, and α6 from ECs, which can recognize fibronectin, nidogens, and laminin isoforms, while α5, α3, α6, and α1 integrin
subunits were increased, from pericytes which recognize fibronectin, nidogens,
laminin isoforms, and collagen type IV [94]. We also observed functional effects of
these integrins, since blocking antibodies to the α5, α3, α6, and α1 integrin all
caused abnormalities in the tube maturation process, by significantly increasing
tube width [94]. None of these antibodies have any influence on EC-only cultures,
which are solely dependent on the collagen-binding integrin, α2β1 [28, 94]. This
data strongly indicates that the purpose of the multiple β1 integrins, on the EC cell
surface, is to recognize key ECM components they encounter at different stages of
the tube morphogenic and maturation process. When they are exposed to collagen
type I matrices, which serves as a strong agonist for tubulogenesis, they utilize
collagen-binding integrins such as α2β1. However, when EC-lined tubes attract
pericytes, ECM remodeling occurs that induces deposition of basement membrane
matrices that are recognized by different sets of integrins such as α5β1 (a fibronectin
receptor), α3β1 (a nidogen and laminin isoform receptor), α6β1 (a laminin isoform
receptor), and α1β1 (a collagen type IV, collagen type I, and laminin receptor) [94].
Interestingly, α1β1 appears to be predominantly pericyte-derived during the
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…
proteins. Although the EC apical membrane domain has been difficult to define in
molecular terms, the clear evidence of EC polarization in our coculture model is the
deposition of basement membrane matrices specifically to the abluminal surface
and the prior recruitment of pericytes to these same abluminal membranes [94].
1.3.18 Specific Upregulation of EC and Pericyte Integrins
Recognizing Basement Membrane Matrices
During EC-Pericyte Tube Co-Assembly in 3D Collagen
Matrices
Again, using our coculture system with human ECs and bovine pericytes, we
assessed how EC vs. pericyte integrins were regulated during this process. We
assessed mRNA levels and performed function blocking experiments with antiintegrin monoclonal antibodies. As discussed above, blocking antibodies to the
α5β1 integrin had function blocking effects that selectively occurred in the
EC-pericyte cocultures but not the EC-only cultures [94]. Interestingly, the EC α5
integrin subunit was induced at the mRNA level in EC-pericyte cocultures, but not
in EC-only cultures where it was downregulated. An important theme which
emerged from these studies is that integrins which recognize the newly remodeled
ECM assembling between ECs and pericytes were induced, while others that recognized collagen type I matrices, such as the α2 integrin subunit from ECs, were
downregulated [94] (Fig. 1.5e). Thus, as basement membranes assemble around EC
tubes, the direct interaction of ECs with collagen type I decreases, while their contact with basement membrane matrices increases. Concomitantly, we observed
increases in the expression of integrin α5, α3, and α6 from ECs, which can recognize fibronectin, nidogens, and laminin isoforms, while α5, α3, α6, and α1 integrin
subunits were increased, from pericytes which recognize fibronectin, nidogens,
laminin isoforms, and collagen type IV [94]. We also observed functional effects of
these integrins, since blocking antibodies to the α5, α3, α6, and α1 integrin all
caused abnormalities in the tube maturation process, by significantly increasing
tube width [94]. None of these antibodies have any influence on EC-only cultures,
which are solely dependent on the collagen-binding integrin, α2β1 [28, 94]. This
data strongly indicates that the purpose of the multiple β1 integrins, on the EC cell
surface, is to recognize key ECM components they encounter at different stages of
the tube morphogenic and maturation process. When they are exposed to collagen
type I matrices, which serves as a strong agonist for tubulogenesis, they utilize
collagen-binding integrins such as α2β1. However, when EC-lined tubes attract
pericytes, ECM remodeling occurs that induces deposition of basement membrane
matrices that are recognized by different sets of integrins such as α5β1 (a fibronectin
receptor), α3β1 (a nidogen and laminin isoform receptor), α6β1 (a laminin isoform
receptor), and α1β1 (a collagen type IV, collagen type I, and laminin receptor) [94].
Interestingly, α1β1 appears to be predominantly pericyte-derived during the
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…