Microfluidic Devices for Quantifying the Role of Soluble Gradients in Early Angiogenesis - Mechanical and Chemical Signaling in Angiogenesis - page 52

proliferation [ 49 , 50 ]. These identities are fluid and result from dynamic VEGF
receptor-2 (VEGFR2) and Notch signaling [ 51 ]. VEGFR2, when potentiated by
VEGF, signals for migration and matrix degradation. Delta-like ligand-4, which is
up-regulated by VEGFR2 signaling, is a cell-displayed Notch ligand that triggers
cell proliferation and expression of VEGF receptor-1 (VEGFR1) in neighboring
cells, i.e., in the stalk [ 52 ]. These proliferating cells ignore the VEGF gradient as
VEGFR1 sequesters VEGF from migration-inducing VEGFR2 [ 53 ]. Stalk cells
eventually form a perfusible lumen by merging pinocytic vacuoles [ 54 ]. This
division of labor, with the proliferating stalk cells and migratory tip cells, means
that gradients not only regulate path-finding but also stalk morphology. Steeper
VEGF gradients increase the velocity of the tip cell as it travels through the ECM
by statistically increasing the directional bias of the cell's random walk. The stalk
cells, meanwhile, proliferate along the path of least resistance, which is the trail
blazed by the tip cells though the mechanically cell-restrictive ECM. It has been
hypothesized that faster migration results in thinner sprouts; the proliferation of the
stalk can only marginally keep up with the tip [ 55 ]. Slower migration, i.e., shal-
lower gradients, results in thicker spouts, since cells must proliferate outward if
there is insufficient space along the forward path. Through these mechanisms,
VEGF gradients instruct sprouts to grow in the appropriate direction and with the
appropriate morphology.
Although VEGF is the primary soluble factor that initiates sprouting angio-
genesis, gradients of other factors play crucial roles in maturation of nascent
sprouts. Tip cells secrete angiopoietin-2 (Ang-2) [ 56 ], destabilizing nearby mature
vessels and potentially eliciting a sympathy sprout with which to anastomose.
Ang-2 is sensed by the receptor ''tyrosine kinase with immunoglobulin-like and
EGF-like domains-2'' (TIE-2) [ 57 ], which triggers directed release of proteases
such as MMP-9 [ 58 ]. Digestion of the local ECM results in increased interstitial
flow and a gradient of liberated VEGF, potentially leading to sympathetic
sprouting. Merger of the initial sprout with a perfusable network is essential to
flow, without which nascent vessels revert [ 59 ]. Meanwhile, up-regulated
expression of autocrine angiopoietin-1 (Ang-1), a TIE-2 antagonist, protects the
proliferating stalk cells from Ang-2-mediated destabilization [ 60 ]. As an example
of another maturation process, pericytes, cells that reside in the perivascular niche
surrounding the endothelial sprout, detect soluble gradients and chemotax to
nascent blood vessels where they deposit basal lamina [ 61 ] and help to re-establish
quiescence [ 62 ]. Gradients of platelet-derived growth factor (PDGF), which induce
directionally-biased clustering of PDGF receptors, can induce chemotaxis of
pericytes to nascent vessels [ 63 ]. Chemotactic PDGF gradients enable recruitment
and proliferation of pericytes from distant tissues, including bone marrow [ 64 ]. In
summary, while VEGF gradients are sufficient to induce the beginning stages of
sprouting morphogenesis, soluble gradients of other factors are required for the
maturation of nascent blood vessels.