This study determines that microvascular EC response to VEGF-A meets established criteria for a morphogenic response (Ashe and Briscoe, 2006
; Gurdon and Bourillot, 2001
; Ibanes and Belmonte, 2008
; Jaeger et al., 2008
) and provides insight into how concentration-dependent patterns of target gene expression are established. In vitro the full range of response is achieved over a 50-fold difference in VEGF-A with linear VEGFR2 autophosphorylation interrogated across PLCγ/p44/p42MAPK phosphorylation and activation of target genes EGR-1, EGR3 and NR4A1. Within the same VEGF-A range, concentration-dependent information was not transduced through the AKT signal pathway. It is possible that the AKT pathway may transmit concentration-dependent information at for instance very low VEGF or at later times. However these data suggest that within the dynamic VEGF-A range for VEGFR2 phosphorylation, there is bifurcation of concentration-dependent and concentration-independent information at the level of signal kinase activation. Further analysis is required to understand how crosstalk between signal pathways influences concentration-dependent output. Induction of transcriptional effectors also acts a node for differential transmittance of dose-dependent information, as some but not all of VEGF-A and MAPK activated immediate early genes analyzed had induction profiles proportional to VEGF-A concentration.
Both in vitro and in vivo analyses shown here support the cell autonomous response of microvascular EC, another criteria for morphogenic response. Within the microvascular network of the developing mouse lung, individual cells were activated as indicated by phosphorylated MAPK or EGR-1 induction but neighboring cells, presumably experiencing similar VEGF-A concentration, were not activated. Unequal response of EC receiving seemingly similar positional information is also seen in the influence of VEGF-A on specification of tip and stalk phenotypes by adjacent EC (Gerhardt et al., 2003
). The differential activation of jagged and DII4 in the Notch signaling pathway specifies migration versus proliferation and determination of tip versus stalk phenotype (Benedito et al., 2009
; Gerhardt et al., 2003
). Ruhrberg, Gerhardt et al. (2002)
report that VEGF-A gradient position determines branch point location as the microvascular network is refined. Arterial differentiation is induced through the PLCγ/MAPK and Notch signaling pathways (Siekmann et al., 2008
) and our model predicts that induction of arterial differentiation via PLCγ/MAPK is dependent on position of EC within the gradient.
Several factors are likely to contribute to dissimilar response of microvascular EC at the same position within a VEGF-A gradient field. Variable expression of VEGFR2 binding partners including VEGFR1 and neuropilin-1 (NRP-1) or of the soluble, ligand-sequestering form of VEGFR1 (sFLT-1) by EC within the microvascular network could contribute to the response of individual cells. Kappas et al (Kappas et al., 2008
) propose that local discontinuous expression of sFLT-1 regulates VEGF-A signal amplitude and creates a mosaic of expression. NRP-1 as a coreceptor enhances VEGF-A165 binding to VEGFR2 (Soker et al., 1998
). Variable expression of NRP-1 could account for differential activation of EC within a vascular bed. Also, the microvasculature is actively expanding throughout lung organogenesis (Schachtner, 2000
). Differential response to VEGF-A by EC within the population may be cell cycle dependent, with the response of new daughter cells dependent on the VEGF-A concentration zone into which they replicate. Finally, receptor desensitization due to decreasing cell surface receptor and diminished VEGFR2 synthesis could account for differences in cellular response with time.
Cellular interpretation of morphogens is specified by both spatial and temporal input (Ashe and Briscoe, 2006
; Kholodenko, 2006
). The temporal activation profile of VEGFR2 autophosphorylation and PLCγ/MAPK activation at low, mid and high VEGF-A showed that EC sense duration of signal and concentration differently. This is unlike systems where duration is sensed in a manner similar to signal strength, for example digit specification by Shh (Harfe et al., 2004
). VEGFR2 autophosphorylation and signal kinase activation were very rapid, detected in 30 sec, but extending the time of exposure at low concentration did not generate a response equivalent to short exposure at high concentration. Further, induction of immediate early target gene EGR-1 required only a short pulse of VEGFR2 activation suggesting that initial and not sustained receptor occupancy determines response. Moreover, analysis of very long-term exposure demonstrated down regulation of cell surface VEGFR2, suggesting a mechanism for desensitization. This was confirmed by diminished receptor autophosphorylation and increased threshold for activation of PLCγ and MAPK. This data predicts that first exposure and not long-term exposure sets the critical parameters for response.
While the essential role of VEGFs and their receptors is well appreciated, many important questions about transcriptional mechanisms downstream remain unanswered. In this study we report that VEGF gradient derived positional information is transduced through specific signal kinase pathways. Analysis of a limited set of immediate early genes () suggested how positional and temporal information within a VEGF-A gradient field is transduced through combinatorial changes in transcription effectors that could influence microvascular EC fate decisions. Recent theoretical approaches have modeled the distribution and availability of VEGF-A in tissues accounting for low affinity binding of heparin-binding VEGF-A isoforms including VEGF-A165 to extracellular matrix (Ibanes and Belmonte, 2008
; Mac Gabhann and Popel, 2004
; Mac Gabhann and Popel, 2007
), making approximations of positional concentrations possible. Receptor activation analysis () demonstrate the dynamic range for EC response to be between 0.10 and 2.0nM VEGF-A. Using a gradient field of 55um (Ruhrberg et al., 2002
) and the theoretical approach of MacGabban and Popel (Mac Gabhann and Popel, 2007
), we calculated that to achieve a gradient from 2.0nM to 0.10nM would require a decrease in VEGF-A of 25% with each 5μm, . The result is an uneven slope of VEGF-A distribution, another characteristic of morphogen fields (Ashe and Briscoe, 2006
; Gurdon and Bourillot, 2001
). This approach predicts cells less than 15μm from the VEGF-A source would be in a high concentration zone (> 1.0nM) while cells further than 40μm from the source would be in a zone of low concentration (<0.25nM). Our data () predicts that within the zone closest to the epithelial source of VEGF-A (<15um), EC response would be initially determined by expression of high levels of EGR-3, NR4A1, NR4A2 and DSCR1. In the zone 40 μm or more from the source, DSCR1 transcripts would predominant. At exposure continues EGR1 transcripts predominate in high and mid concentration zones (<40μm). By 24hr, only EGR1 and EGR3 would have significant influence on cellular response. While these results suggest how EC transduce positional information, system-wide computational analyses are needed to fully elucidate cell-context dependent effects of signal strength and duration of signal on EC response to a VEGF-A gradient field.
Model of VEGF-A gradient to estimate positional concentration