Microfluidic Devices for Quantifying the Role of Soluble Gradients in Early Angiogenesis - Mechanical and Chemical Signaling in Angiogenesis - page 60

Fig. 10 GPR124 is a receptor that mediates endothelial cell chemotaxis in response to paracine
factors secreted by the embryonic forebrain. a GPR124-overexpressing bEND3 cells (GPR124+)
migrate within a gradient of conditioned medium from cultured E12.5 forebrain cortical neurons.
Migration is random under b an equivalent hindbrain gradient, c a gradient from HEK293T
conditioned medium, d a uniform concentration of forebrain-conditioned medium. e, f Directional
bias is ablated by treatment with shRNA against GPR124. g Chemotaxis is preserved under
sequestration of VEGF by a recombinant soluble VEGFR1 ectodomain (400 ng/mL). Originally
published in [ 72 ]
in studies of matrix remodeling during sprouting morphogenesis, as the proteolytic
degradation of existing matrix and the deposition of new matrix are both expected
to be intimately connected to interstitial flow.
Although VEGF is the most commonly studied angiogenic cue, gradients of
other factors also play a role in endothelial cell chemotaxis and sprouting mor-
phogenesis. Recent microfluidic experiments identified the G-protein coupled
receptor 124 (GPR124) as a critical regulator of organ-specific angiogenesis in the
central nervous system [ 72 ]. GPR124 is an endothelial receptor of previously
unknown function that induces chemotaxis of brain-derived endothelial cells
through a non-VEGF pathway. Expression of GPR124 caused cells from the brain
vascular endothelial cell line bEND3 to chemotax along a gradient of paracrine
secretions from cortical neurons of the murine embryonic forebrain (Fig. 10 a). In
contrast, a gradient of hindbrain-derived secretions did not elicit chemotaxis
(Fig. 10 b). As controls for specificity, a gradient of conditioned medium from the
293T cell line (Fig. 10 c) and a uniform concentration profile of forebrain-condi-
tioned medium (Fig. 10 d) did not induce chemotaxis. Similarly, knocking down
GPR124 through use of small hairpin RNA (shRNA) prevented chemotaxis
(Fig. 10 e, f). Blocking the VEGF activity through inhibition with a soluble
receptor did not inhibit forebrain-specific chemotactic signaling (Fig. 10 g). These
results corroborate data from GPR124-knockout mice, which are deficient in the
embryonic development of forebrain vasculature [ 72 ]. Together, these data support
the hypothesis that GPR124 is a critical mediator of brain-specific endothelial cell
chemotaxis and that the GPR124 ligand is secreted by forebrain cortical neurons.
To further demonstrate that the GPR124 ligand promotes stable sprout for-
mation, sprouting morphogenesis assays were performed in a microfluidic device