Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division

Abstract

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 die of vascular overgrowth, and we are interested in how flt-1 normally prevents this outcome. Our results support a model whereby aberrant endothelial cell division is the cellular mechanism resulting in vascular overgrowth, and they suggest that VEGF-dependent endothelial cell division is normally finely modulated by flt-1 to produce blood vessels. Flt-1 ؊/؊ embryonic stem cell cultures had a 2-fold increase in endothelial cells by day 8, and the endothelial cell mitotic index was significantly elevated before day 8. . Some of the mitogenic signals that promote division of endothelial cells and their precursors are known, but how these signals are modulated to initiate cell divisions only when and where they are needed is not known in detail. After blood vessels initially form, maturation and remodeling steps involve the recruitment of ancillary cells, such as smooth muscle and pericytes. These cells and the extracellular matrix that is also produced can negatively modulate endothelial cell division. [4][5][6][7][8] However, modulators of endothelial cell mitogenesis at the earliest stages of blood vessel formation have not been identified.The vascular endothelial growth factor (VEGF) signaling pathway is clearly critical to both early endothelial cell division and morphogenesis, and its regulation is complex (reviewed in Ferrara & Davis-Smyth 9 and Neufeld et al 10 ). Mouse embryos lacking even one copy of the VEGF gene die in utero with severe vascular defects, and vascular development in differentiating embryonic stem (ES) cells is compromised in VEGF-A ϩ/Ϫ and VEGF-A Ϫ/Ϫ ES cells in a dose-dependent manner. [11][12][13] Moreover, even modestly elevated levels of VEGF lead to vascular abnormalities, 14 and large doses of VEGF invariably severely compromise both vascular development and neovascularization in adult organisms. [15][16][17] These findings suggest that VEGF signaling must be precisely controlled during vascularization to result in proper vessels. The location and duration of VEGF expression provide the first level of control, [18][19][20][21] but other components of the pathway are likely to be involved in fine-tuning the signal.Two high-affinity receptors, flk-1 and flt-1, participate in VEGF signal transduction and are candidates to be involved in fine-tuning mechanisms. Both receptors are membrane-spanning receptor tyrosine kinases that bind VEGF with high affinity, 22-26 but their effects on VEGF signaling are very different. Mice or ES cells lacking flk-1 have little or no blood vessel formation, suggesting that many downstream effects of VEGF on endothelial cells are mediated through flk-1. 27,28 Specifically, numerous studies show that VEGF signaling through flk-1 produces a strong mitogenic signal for endothelial cells. [29][30][31][32] In contrast, VEGF binding to flt-1 does not produce a strong mitogenic signal, and flt-1 Ϫ/Ϫ mice die at mid-gestation with vascular overgrowth and disorganization. 23,29,33 This ph...

Antitumour efficacy of VEGFR2 tyrosine kinase inhibitor correlates with expression of VEGF and its receptor VEGFR2 in tumour models

“…Conversely, others have proposed that high level of VEGF receptor expression by tumour cells can also negatively modulate VEGF signalling by sequestering VEGF, which otherwise will bind to the receptors expressed by vascular endothelial cells (Hiratsuka et al, 1998;Kearney et al, 2002). Vascular endothelial growth factor receptors are in most cases specifically expressed on vascular endothelial cells, but certain tumour cells also express VEGFR2 (Masood et al, 1997;Dias et al, 2000;Masood et al, 2001;Podar et al, 2001;Strizzi et al, 2001;Jackson et al, 2002;Nakopoulou et al, 2002).…”

“…Vascular endothelial growth factor receptors are in most cases specifically expressed on vascular endothelial cells, but certain tumour cells also express VEGFR2 (Masood et al, 1997;Dias et al, 2000;Masood et al, 2001;Podar et al, 2001;Strizzi et al, 2001;Jackson et al, 2002;Nakopoulou et al, 2002). The expression of VEGFR2 by tumour cells may be a mechanism to become less dependent on VEGF signalling for tumour angiogenesis as well as to negatively regulate the VEGF expression (Hiratsuka et al, 1998;Kearney et al, 2002). Consistent with this hypothesis, the two tumours PC3 and A375P, which express very high levels of human tumour VEGFR2 as determined by Western blots and mRNA analysis also express very low levels of VEGF (Figure 2 and data not shown).…”

Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation

“…Activation of VEGFR-2 positively regulates angiogenesis (Breier 2000;Yancopoulos et al, 2000;Gille et al, 2001) and it is highly autophosphorylated in response to ligand stimulation (Waltenberger et al, 1994;Shalaby et al, 1995;Rahimi et al, 2000). VEGFR-1 is poorly autophosphorylated in response to ligand stimulation in endothelial cells and appears to regulate angiogenesis negatively (Waltenberger et al, 1994;Fong et al, 1999;Rahimi et al, 2000;Kearney et al, 2002). Despite its poor autophosphorylation, VEGFR-1 can heterodimerize with VEGFR-2 and transphosphorylates VEGFR-2 (Rahimi et al, 2000;Autiero et al, 2003).…”

“…Selective activation of VEGFR-1 in endothelial cells is not associated with endothelial cell proliferation (De Vries et al, 1992;Waltenberger et al, 1994;Rahimi et al, 2000;Kearney et al, 2002). To test whether the Ctail-swapped VEGFR-1 is able to simulate biological responses, we subjected cells either expressing CTR or CTR/cFLK-1 to proliferation assay.…”

“…Homozygous VEGFR-2 null mice exhibited early embryonic lethality due to the absence of endothelial cells (Shalaby et al, 1995). In contrast, targeted deletion of VEGFR-1 resulted in early embryonic lethality due to overgrowth of endothelial cells, suggesting a negative role for VEGFR-1 in angiogenesis (Fong and Rossant, 1995;Fong et al, 1999;Kearney et al, 2002). The molecular mechanisms that account for these differences between the two highly related receptors are not fully understood.…”