28
EC-pericyte tube co-assembly process. Thus, the effects of blocking antibodies that
have been observed may be due to an inhibitory influence on pericyte recognition of
basement membrane matrices during these events [94]. EC-dependence on α2β1,
which is continuously observed over time in EC-only cultures is lost with time in
EC-pericyte cocultures, as basement membrane matrix assembly occurs and exposure of ECs to collagen type I is strongly diminished. In conclusion, our findings
show that EC-pericyte interactions control vascular basement membrane matrix
assembly and that concomitant changes in EC and pericyte integrins occurs to recognize this newly remodeled matrix to facilitate further tube maturation and stabilization events.
1.3.19 Defining the Critical Growth Factors that Control
Human Vascular Tube Morphogenesis and Pericyte
Recruitment to EC-Lined Tubes
A central question in vascular biology is to elucidate the nature of the growth factors
that are necessary to assemble the developing vasculature. Considerable data, particularly obtained from knockout mice or zebrafish, suggested a major role for
VEGF [1]. Using serum-free defined models of vascular tube morphogenesis, we
have assessed a role for VEGF and FGF-2, singly or in combination, in directly
regulating EC tube assembly. Neither factor (alone or in combination) (i.e.,
VEGF+FGF) was able to support EC tubulogenesis [93]. Using a broad screen for
growth factors, peptides, and other small molecules in a 96-well plate microassay
format, we identified a single combination of five growth factors that leads to human
EC lumen and tube network assembly. The five Factors are stem cell factor (SCF),
interleukin-3 (IL-3), stromal-derived factor-1 alpha (SDF-1α), FGF-2, and insulin
[93] (Fig. 1.3). Detailed screening of hundreds of additional combinations of molecules failed to identify any other mixture that supported this process [18]. Adding
pericytes to ECs does not substitute for the Factor requirements [93]. Importantly,
admixing pericytes with ECs under these Factor-driven defined conditions leads to
EC tubulogenesis and marked pericyte recruitment as well as proliferation [93]. Of
great interest is that pericytes proliferate, but ECs do not. In addition, this stimulated
EC-pericyte tube co-assembly results in capillary maturation events, including
basement membrane matrix assembly [93, 94]. Pericyte recruitment and basement
membrane formation lead to narrower tube diameters, compared to EC-only cultures [94]. Using this Factor-driven system, we identified a key role for EC-derived
PDGF-BB and HB-EGF, in pericyte recruitment and proliferation, during
EC-pericyte tube co-assembly [96]. Disruption of recruitment using antagonists of
PDGF-BB and HB-EGF (using blocking antibodies or receptor traps) leads to wider
tubes, fewer pericytes, and markedly reduced basement membrane deposition [96].
Overall, these data strongly indicate that we have identified a key combination of
five Factors that are necessary to stimulate the process of human capillary tube
G. E. Davis
EC-pericyte tube co-assembly process. Thus, the effects of blocking antibodies that
have been observed may be due to an inhibitory influence on pericyte recognition of
basement membrane matrices during these events [94]. EC-dependence on α2β1,
which is continuously observed over time in EC-only cultures is lost with time in
EC-pericyte cocultures, as basement membrane matrix assembly occurs and exposure of ECs to collagen type I is strongly diminished. In conclusion, our findings
show that EC-pericyte interactions control vascular basement membrane matrix
assembly and that concomitant changes in EC and pericyte integrins occurs to recognize this newly remodeled matrix to facilitate further tube maturation and stabilization events.
1.3.19 Defining the Critical Growth Factors that Control
Human Vascular Tube Morphogenesis and Pericyte
Recruitment to EC-Lined Tubes
A central question in vascular biology is to elucidate the nature of the growth factors
that are necessary to assemble the developing vasculature. Considerable data, particularly obtained from knockout mice or zebrafish, suggested a major role for
VEGF [1]. Using serum-free defined models of vascular tube morphogenesis, we
have assessed a role for VEGF and FGF-2, singly or in combination, in directly
regulating EC tube assembly. Neither factor (alone or in combination) (i.e.,
VEGF+FGF) was able to support EC tubulogenesis [93]. Using a broad screen for
growth factors, peptides, and other small molecules in a 96-well plate microassay
format, we identified a single combination of five growth factors that leads to human
EC lumen and tube network assembly. The five Factors are stem cell factor (SCF),
interleukin-3 (IL-3), stromal-derived factor-1 alpha (SDF-1α), FGF-2, and insulin
[93] (Fig. 1.3). Detailed screening of hundreds of additional combinations of molecules failed to identify any other mixture that supported this process [18]. Adding
pericytes to ECs does not substitute for the Factor requirements [93]. Importantly,
admixing pericytes with ECs under these Factor-driven defined conditions leads to
EC tubulogenesis and marked pericyte recruitment as well as proliferation [93]. Of
great interest is that pericytes proliferate, but ECs do not. In addition, this stimulated
EC-pericyte tube co-assembly results in capillary maturation events, including
basement membrane matrix assembly [93, 94]. Pericyte recruitment and basement
membrane formation lead to narrower tube diameters, compared to EC-only cultures [94]. Using this Factor-driven system, we identified a key role for EC-derived
PDGF-BB and HB-EGF, in pericyte recruitment and proliferation, during
EC-pericyte tube co-assembly [96]. Disruption of recruitment using antagonists of
PDGF-BB and HB-EGF (using blocking antibodies or receptor traps) leads to wider
tubes, fewer pericytes, and markedly reduced basement membrane deposition [96].
Overall, these data strongly indicate that we have identified a key combination of
five Factors that are necessary to stimulate the process of human capillary tube
G. E. Davis