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Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis

Richard C. A. Sainson^*, Jason Aoto^*, Martin N. Nakatsu^*, Matthew Holderfield^*, Erin Conn^*, Erich Koller and Christopher C. W. Hughes^*,1

^* Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine California, USA; and
Isis Pharmaceuticals, Inc. Carlsbad, California, USA

¹ Correspondence: Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine CA 92612, USA. E-mail: cchughes{at}uci.edu

SPECIFIC AIMS

The requirement for notch signaling during vascular development is well documented but poorly understood. We used a 3-dimensional (3D) in vitro assay that models all of the early stages of angiogenesis, including sprouting, migration, proliferation, alignment, tube formation, and branching, to determine whether cell-autonomous use of notch was required for proper endothelial cell (EC) function during each of these stages.

PRINCIPAL FINDINGS

1. Notch regulates tubule diameter through suppression of cell proliferation
We have previously shown by an in vitro angiogenesis assay that VEGF signaling regulates tubule diameter through induction of EC proliferation. Moreover, we have shown that notch signaling induces the bHLH transcription factor HESR1, which then down-regulates expression of the VEGF receptor VEGFR2. These findings led us to propose a feedback mechanism for regulation of vessel diameter involving VEGF, VEGFR2, notch, and HESR1. To test this hypothesis, we blocked notch signaling using an inhibitor of the notch processing enzyme -secretase (GSI). After 8 days, control and GSI-treated cultures showed extensive capillary-like tubule formation. In the GSI-treated cultures, however, the average sprout diameter was increased by 60% (n=3, P<0.01). We did not observe a statistically significant increase in sprout length. Nuclear staining with DAPI revealed a 4-fold increase (n=3, P<0.01) in the number of cells present in sprouts cultured with GSI. Using immunofluorescent detection of BrdU incorporation, we demonstrated that the increase was largely the result of increased cell proliferation. The negative regulation of cell proliferation by notch signaling was further demonstrated using other specific inhibitors of this pathway, including a dominant-negative, secreted form of notch1, Notch1-EC(EGF11/12), and notch1 and notch4 antisense. Thus, cell-autonomous notch signaling limits tube diameter by suppressing EC proliferation, as hypothesized. Notch and VEGF act antagonistically in this 3D in vitro system as part of a negative feedback loop that fine-tunes EC proliferation and thus vessel diameter.

2. Notch suppresses branching at the tip of developing sprouts
Angiogenic capillary sprouts are composed of EC of two distinct phenotypes: the trunk and tip cells. Trunk cells proliferate and contribute to lumen formation whereas the lead, or "tip" cell, does not proliferate or form a lumen. The tip cell is, however, highly motile and extends numerous processes into the surrounding matrix. When cultures were treated with GSI, we noted a dramatic increase in vascular arborization due to enhanced secondary branching (Fig. 1 A). Time-lapse video microscopy demonstrated that secondary branches were mainly generated from bifurcation of the vessel tip (Fig. 1B ). When the number of vessel tips was quantified, a 4-fold increase was observed as early as day 6 (n=3, P<0.01) in GSI-supplemented cultures relative to control (Fig. 1C ). Intriguingly, proliferating cells were often found at the tip of spouts in GSI-treated cultures but rarely in controls (Fig. 1D ). These data suggest that bifurcation of the tip correlates with deregulated cell proliferation.

View larger version (104K): [in this window] [in a new window]   Figure 1. Notch inhibition induces extensive branching at the tip of growing vessels. A) EC were coated onto dextran beads and embedded in fibrin gels as described previously. Circles outline bifurcations at the tip of growing vessels in the presence of GSI. B) Time-lapse images of sprouting branches from a single culture supplemented with GSI. Images were acquired over 72 h starting 4 days after plating. Vessel branching occurs at the tip of growing vessels (white arrowheads) and within the trunk of the vessels (black arrowhead). Scale bar: 100 µm. C) Quantification of cultures established as in panel A, *P < 0.01. D) BrdU staining (green) of GSI-treated cultures showing proliferation of tip cells. Cell nuclei were counterstained with DAPI (blue). Arrowheads denote proliferating tip cells.

We confirmed these findings using notch1EC-(EGF11/12) and notch 1 and notch 4 antisense. Previous studies have demonstrated that a 17-mer peptide corresponding to the delta/serrate/lag-2 domain of Jagged-1 can activate notch signaling, and we confirmed this in EC. Activation of notch signaling with this peptide inhibited sprouting by up to 80% at day 6 (n=3, P<0.05). These data demonstrate that notch regulates the phenotype of tip cells, normally acting to suppress proliferation and bifurcation.

3. Notch signaling does not regulate lumen formation or apical-basal polarity
We took advantage of the fact that our assay induces clear lumen formation to study the function of notch during tubulogenesis (Fig. 2 A). In this assay, lumen formation is initiated by cell hollowing and vacuole fusion; later, however, cord hollowing prevails, leading to mature sprouts composed of a single layer of EC, connected by adherens junctions and surrounding an intercellular lumen. In vivo studies have suggested that impaired notch signaling correlates with severe defects in lumen formation. Using 2-photon imaging and semi-thin sections of plastic-embedded 3D cultures, we demonstrated that GSI-treated sprouts did indeed have lumens, but these were often collapsed and flattened in morphology (Fig. 2A ).

View larger version (51K): [in this window] [in a new window]   Figure 2. Inhibition of notch signaling does not block lumen formation or establishment of apical-basal polarity. A) Top two panels and bottom panel show phase contrast images of sprouts in fibrin gels grown in the presence of DMSO or GSI. In the upper middle two panels, semi-thin cross sections of vessels are shown demonstrating the presence of lumens in treated and untreated cultures. Scale bars: 50 µm. Lower middle panel demonstrates the presence of a lumen surrounded by tubulin-positive EC (green); nuclei are counterstained with DAPI (blue). B) Two-photon fluorescence images of cultures treated with DMSO or GSI demonstrate correct apical-basal polarization for both conditions. Vessels were stained for the basally expressed ECM protein collagen IV (green), F-actin (red), and nuclear DNA (DAPI, blue). Scale bar: 100 µm. C) Two-photon fluorescence images showing polarized collagen IV staining (green) on the basal surfaces of vessels. Cells were counterstained with DAPI (blue) and ß-catenin (red). Note the lack of collagen IV staining around the emerging EC sprout. Scale bar: 10 µm.

The establishment of cell polarity is a critical early stage in the formation of tubular structures. The establishment of apical-basal polarity in epithelial cells correlates with a mesenchyme-to-epithelial transition, whereas its loss correlates with an epithelial-to-mesenchyme transition (EMT). Recent studies have suggested that angiogenesis may require such plasticity in EC, and it has been suggested that notch signaling may play a role. Immunofluorescence staining for collagen IV, a major component of basement membranes, demonstrated that inhibition of notch signaling does not affect the basal localization of this extracellular protein (Fig. 2B ). Where a secondary sprout is emerging from a parent vessel, staining for collagen IV is dramatically reduced, consistent with the absence of apical-basal polarity in tip cells (Fig. 2C ). Taken together, theseexperiments demonstrate that notch signaling is not required for lumen formation, nor is it essential for establishment of EC apical-basal polarity during tubulogenesis.

CONCLUSIONS AND SIGNIFICANCE

These studies confirm that notch plays an important cell-autonomous role in EC tubulogenesis. While sprouting and lumen formation can occur in the absence of notch signaling, the subsequent patterning of the vessel, including the setting of diameter and number of branches, appears to require a functional notch pathway. In the regulation of diameter, notch works antagonistically with VEGF in a negative feedback loop that limits EC proliferation; in the absence of notch, VEGF-driven proliferation is unchecked and vessel diameter increases as a consequence. Consistent with this interpretation, we previously showed that a high concentration of VEGF, even in the presence of functional notch signaling, increases vessel diameter. A second related function of notch is in the regulation of branching. Normally, notch acts to block division of tip cells, but when signaling is inhibited, tip cells divide and both daughter cells take on a tip cell phenotype, leading to bifurcation of the vessel. A number of studies have implicated notch in transitions between epithelial and mesenchymal phenotypes. We find that notch signaling is not necessary for establishment of polarity or for lumen formation; however the expression of some genes associated with EMT, such as snail and c-met, do appear to be regulated by notch. In conclusion, notch is used cell autonomously by EC to regulate vessel patterning.

View larger version (40K): [in this window] [in a new window]   Figure 3. Schematic model. Notch signaling regulates EC proliferation and vessel morphology in a cell-autonomous manner. During normal angiogenesis, activation of the notch pathway antagonizes VEGF signaling by repressing EC proliferation and branching. Notch signaling is required to establish and/or maintain phenotypically distinct tip and trunk cells (see text). Inhibition of the notch pathway increases proliferation of cells (white nuclei) located at the trunk and the tip of the vessels, which correlates with an increase in vessel diameter and extensive arborization of vessels. Also note that notch signaling is not necessary to establish apical-basal polarity or for lumen formation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3172fje; doi: 10.1096/fj.04-3172fje

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