Blood vessel formation
Scanning electron micrograph of an isolated capillary
Which genes are expressed during endothelial cell sprouting and tube formation?
There are two recognized mechanisms for formation of new blood vessels, these are vasculogenesis -- the formation of vessels de novo through aggregation of cells derived from endothelial precursors, and angiogenesis -- the growth of new vessels from pre-existing vessels. Vasculogenesis is particularly critical during development of the embryo. Angiogenesis, while important in development, also occurs in the adult during, for example, wound healing and ovulation. New blood vessel growth is also a critical phase of solid tumor growth -- without a new blood supply tumors cannot grow more than about 1-2mm in diameter. We have used differential cDNA screening and microarrays to identify genes involved in sprouting and tube formation, both critical processes in the development of a blood vessel.
We have isolated several candidate genes including NrCAM (a "neural" cell adhesion molecule), beta Ig-h3 (an extracellular matrix protein) and ESM-1 (a secreted molecule of unkown function) (Aitkenhead et al. 2002)
We have also found upregulation of the bHLH transcription factor HESR1 in EC undergoing tube formation and have shown that the gene acts as a switch to control EC phenotype. Expression of HESR1 downregulates the growth factor receptor VEGFR2, thus promoting a more quiescent EC phenotype (Henderson et al 2001).
We have gone on to show that HESR1 is a downstream effector of notch, a transmembrane receptor that in many systems controls cell differentiation. Our data suggest that Notch/HESR1 may be critical regulators of the angiogenic phenotype (Taylor et al 2002).
HESR1 appears to act through multiple weak interactions with SP-1 and other factors on promoters driven by initiator elements. A TATA box can override the inhibitory effects of HESR1 on transcription (Holderfield et al 2006).
We have developed an in vitro angiogenesis assay that closely models many of the aspects of in vivo angiogenesis, including sprouting, tube formation, branching and anastomosis (see pictures below and: Nakatsu, Sainson, Aoto et al; Nakatsu and Hughes, 2008). Using this assay we have defined an important role for VEGF in determining blood vessel diameter. Its effects appear to be largely through MAPK-mediated cell proliferation (Nakatsu, Sainson, Pérez del Pulgar et al 2005).
We have also established a role for cell-autonomous notch signaling in EC in the control of vessel branching and setting of vessel diameter (Sainson et al) and have reviewed the literature on crosstalk between notch, VEGF and TGFbeta (Holderfield and Hughes, 2008).
Recently we described how TNF can coordinate angiogenesis with the resolution of inflammation during wound healing by blocking EC migration and proliferation downstream of VEGF, while simultaneously priming EC for sprouting by inducing a "tip cell" phenotype. TNF induces the notch ligand jagged-1 in tip cells through an NFkB-dependent mechanism, and also induces the known tip cell-enriched genes PDGFB and VEGFR2 (Sainson et al 2008).
We are currently identifying stromal cell-derived factors that regulate EC sprouting and tube formation (Hughes, 2008).
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| Sprouting and branching | Anastomosis and network formation | |||||||||
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| Vessels have clear lumens surrounded by endothelial cells | Capillary-like vessel stained to show nuclei (blue) and tubulin (green) | |||||||||
Click here for two movies showing capillary growth in vitro
References
Henderson AM, Wang S-J, Taylor AC, Aitkenhead M and Hughes CCW 2001 The bHLH transcription factor HESR1 regulates endothelial cell tube formation. J. Biol. Chem. 276: 6169-6176 PubMed
Aitkenhead M, Wang S-J, Mestas J, Heard C and Hughes CCW. 2002. Identification of endothelial cell genes expressed in an in vitro model of angiogenesis: upregulation of ESM-1, beta-ig-h3 and NrCAM. Microvascular Research. 63: 159-171 PubMed
Taylor KT and Hughes CCW. 2002. Notch activation during endothelial cell tube formation in vitro targets the basic HLH transcription factor HESR1 and downregulates VEGFR-2/KDR expression. Microvascular Research. 64: 372-383 PubMed
Nakatsu MN, Sainson RCA, Aoto JN, Taylor KL, Aitkenhead M, Pérez del Pulgar S, Carpenter PM, and Hughes CCW. 2003 Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1 Microvascular Research. 66: 102-112 PubMed
Nakatsu MN, Sainson RCA, Pérez del Pulgar S, Aoto JN, Aitkenhead M, Taylor KL, Carpenter PM and Hughes CCW. 2003 VEGF121 and VEGF165 regulate blood vessel diameter through VEGFR-2 in an in vitro angiogenesis model. Laboratory Investigation 83: 1873-1885 PubMed.
Sainson RCA, Aoto J, Nakatsu MN, Holderfield M, Conn E, Koller E, and Hughes CCW. 2005 Re-iterative Notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis FASEB J 19: 1027-9 PubMed
Holderfield MT, Henderson Anderson AM, Kokubo H, Chin MT, Johnson RL, and Hughes CCW. 2006 HESR1/CHF2 suppresses VEGFR2 transcription independent of binding to E-boxes. Biol. Biochem. Res. Comm. 346: 637-648 PubMed
Nakatsu MN, Davis J, and Hughes CCW 2007 An optimized fibrin gel bead assay for the study of angiogenesis in vitro. J. Vis. Exp. (JoVE) 3: (Apr) doi:10.3791/186 PubMed
Sainson RCA, Johnston DA, Chu HC, Holderfield MT, Nakatsu MN, Crampton SP, Davis J, Conn E, and Hughes CCW. 2008 TNF Primes Endothelial Cells for Angiogenic Sprouting by Inducing a Tip Cell Phenotype. Blood 111: 4997-5007 PubMed.
Holderfield MT and Hughes CCW. 2007 Crosstalk between VEGF, notch, and TGFbeta in vascular morphogenesis. Circ. Res 102: 637-52 PubMed
Nakatsu MN and Hughes CCW 2008. An optimized three-dimensional in vitro model for the analysis of angiogenesis. Methods in Enzymology 443: 65-82 PubMed
Hughes CCW 2008. Endothelial-stromal interactions in angiogenesis. Curr. Opinion Hematol. 15: 204-209. PubMed