CSB331H1 Lecture Notes - Lecture 18: Rho-Associated Protein Kinase, Syndecan, Platelet-Derived Growth Factor

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4 Apr 2012
Lecture 18 Notes March 30, 2011
Text taken verbatim from cited articles with minor modifications
Angiogenesis and endothelial cell migration
Eiken and Adams (2010) Dynamics of endothelial cell behavior in sprouting angiogenesis Current
Opinion in Cell Biology 22:617-625
Endothelial cells (ECs) form two extensive tubular networks, the blood vasculature and the lymphatic
vasculature, that transport a large variety of molecular and cellular cargo. The formation and function of
these vessels supports the growth of organs during development and early postnatal life, but also helps to
defend diseases, stabilize the body temperature and maintain homeostatic balance in the adult. Blood
vessels form a closed tubular system for blood circulation, which consists of hierarchically organized
arteries, capillaries and veins. In contrast, lymphatic vessels are blind-ending tubules that transport
protein-rich fluid (lymph) and immune cells unidirectionally from distal tissues into the venous branch of
the circulation. All vessels consist of interconnected, tube-forming ECs. However, ECs are highly
heterogeneous in terms of functional properties and gene expression profiles, which depend on their
differentiation and proliferation status, identity as blood vascular or lymphatic endothelium, location
within a specific part of the vasculature, and organ-specific specialization. In addition, mural cells
(smooth muscle cells and pericytes) are attached to the abluminal (basal) surface of certain vessels and
help, for example, to stabilize the vessel wall and regulate blood pressure. Whereas vasculogenesis, the de
novo formation of vessels from mesoderm-derived endothelial precursor cells (angioblasts), is responsible
for the formation of the first, primitive blood vessels in the embryo, physiological and pathological blood
vessel growth in later life is predominantly, Most of our knowledge about the morphological processes and
molecular regulation of angiogenesis results from studies in developing zebrafish embryos or the
vascularization of the retina in the postnatal mouse. Its great experimental accessibility and the possibility
of life imaging are main advantages of the zebrafish system. Key benefits of the mouse retina model are
the progressive expansion of the growing retinal blood vessels from the center to the periphery, their initial
organization as a flat two-dimensional network, the spatial separation of growing vs. maturing areas, and
the relatively easy identification of sprouts and tip cells (Figure 1). Growth of the vascular network is
highly dynamic and requires a multitude of individual processes such as the proliferation of ECs, their
directional migration, the establishment of appropriate connections, tube formation (tubulogenesis),
remodeling and pruning, specification into arteries, veins and capillaries, and the recruitment of mural
cells. Sprouting and the emergence of tip cells are early steps in this complex program. Tip cells possess
many long and motile filopodia that extend towards the source of pro-angiogenic growth factors and
respond to other positive or negative guidance cues to enable directional and prevent unorganized and
random vessel growth (Figures 1 and 2).
VEGF family growth factors — master regulators of angiogenesis
A key molecule for the initiation and direction of sprouting is vascular endothelial growth factor A
(VEGF-A). In the postnatal mouse retina, VEGF-A is secreted by astrocytes in the avascular region, which
leads to the formation of a VEGF gradient. This role of VEGF-A as a directional, chemoattractive cue for
angiogenic sprouting has been also observed in the embryonic spinal cord of the mouse or during the
vessel growth in the developing zebrafish. A second VEGF family member, the growth factor VEGF-C, is
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also strong promoter of angiogenic growth in the retina, in the zebrafish embryo and in tumors. While
VEGF-A binds to and signals though the receptor tyrosine kinase (RTK) VEGFR2/KDR/Flk1, the effects
of VEGF-C are mainly mediated by the receptor VEGFR3/Flt4. Both VEGFR2 and VEGFR3 are strongly
expressed in the angiogenic endothelium. High levels of VEGFR3 in lymphatic ECs are the basis for the
important role of VEGF-C as a promoter of lymphangiogenesis. Another related RTK, VEGFR1/Flt1,
which has high affinity for VEGF-A but weak tyrosine kinase activity and also exists in a secreted,
catalytically inactive form, acts as a negative regulator of angiogenesis. Soluble VEGFR1 expression in
the vicinity of emerging sprouts, presumably in stalk cells, appears to enhance the steepness of the VEGF-
A signaling gradient and has been found to control the guidance of tip cells and filopodia.
Tip and stalk cells — angiogenic ECs are not all alike
When vessels are exposed to pro-angiogenic signals such as VEGF, only a fraction of the ECs acquire the
tip cell phenotype, whereas others form the stalk of the vascular sprout or stay behind to maintain the
integrity and perfusion of the growing vascular bed. This decision is strongly controlled by the Notch
pathway, a key regulator of cell fate, differentiation and patterning processes in a large variety of tissues
and organisms. Tip cells contain relatively high levels of Delta-like 4 (Dll4), a transmembrane protein and
Notch ligand, whereas stalk cells preferentially express a different ligand, namely Jagged-1. Activation of
Notch signaling by Dll4 is thought to occur predominantly in stalk ECs and leads to the downregulation of
VEGFR2 and VEGFR3 expression in these cells. Accordingly, the blockade of Notch signaling or
insufficient expression of the Notch ligand Delta-like 4 (DLL4) increases the number of tip-like cells and
EC proliferation in the postnatal mouse retina and in sprouting intersegmental vessels in zebrafish.
Likewise, disruption of Dll4–Notch interactions in experimental tumors causes extensive endothelial
sprouting. On the basis of these results, it is currently believed that cells at the angiogenic front compete
with each other through Notch signaling. In response to VEGF, all cells upregulate Dll4 and signal to their
neighbors. Factors such as high exposure to Dll4-expressing cells in their vicinity or limited access to
VEGF might favor stalk cell behavior. Conversely, cells with higher levels of Dll4, low Notch activity
and stronger VEGF receptor transcription are thought to convert into tip cells (Figure 2). Mediated by
lateral inhibition effects, Notch signaling presumably becomes increasingly directional (between signal-
sending tip and signal-receiving stalk cells) so that differences
in EC behavior and gene expression get enhanced further (Figure 2). The ligand Jagged1 is a pro-
angiogenic regulator that antagonizes Dll4–Notch signaling and thereby positively controls the number of
sprouts and tips. Postranslational modification of Notch receptors by Fringe family glycosyltransferases,
which enhances Delta-mediated Notch activation but strongly weakens the signaling capability of Jagged1,
are critical for the opposing roles of the two ligands. Jagged1 and Dll4 not only maintain differential
Notch signaling among tip and stalk cells, they also control the levels of VEGF receptor expression and
thereby EC growth and proliferation throughout the angiogenic vasculature. The phenotypic specialization
of ECs as tip or stalk cells is presumably very transient. Tips form connections with other sprouts or
nearby capillaries and thereby get incorporated into the endothelial lining of new vessels. Conversely,
local changes in VEGF and Notch signaling can trigger the conversion of stalk cells into tips and induce a
new round of sprouting (Figure 4). The Notch pathway can act in a very transient, oscillating modus,
which might well promote the dynamic interconversion of tip and stalk cells. Thus, tip and stalk cell
determination is
probably transient, reversible and depends on the balance between pro-angiogenic factors (like VEGF and
Jagged1) and signals suppressing EC sprouting and proliferation (e.g., Dll4–Notch activity).
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Meet the leaders — similarities between tip cells and axonal growth cones
The filopodia-extending and motile endothelial tip cells share many structural and functional features with
growth cones found at the distal end of extending axons in the nervous system. Both are responsible for
the formation of new connections and respond to similar cues that they encounter in their vicinity. While
vascular sprouts or axonal growth are attracted by guidance cues, they are blocked by repulsive signals
leading to either growth inhibition or changes in the direction of growth. Eph/ephrin molecules, which
provide mostly repulsive signals for growing nerve cells, are one such example. The receptor EphB4 and
its ligand ephrin-B2 are critical regulators of vascular morphogenesis in the early mouse embryo.
EphB4/ephrin-B2 signaling also controls vein and artery segregation in the developing zebrafish, where
highly migratory EphB4-positive cells leave a common precursor vessel to form the cardinal vein, whereas
ephrin-B2-expressing cells form the dorsal aorta. Recent work has linked ephrin-B2 to motile and invasive
tip cell behavior as well as to VEGF receptor signaling. ECs lacking or expressing a mutant form ephrin-
B2 fail to internalize VEGF receptors in response to growth factor stimulation, which also compromises
the activation of downstream signaling pathways. In addition, ephrin-B2 also regulates the recruitment of
mural cells to endothelial tubes.
Matrix reloaded — reorganizing the subendothelial basement membrane
Endothelial sprouting is just an early step in the formation of new vessels. To generate new vessels,
endothelial sprouts need to connect to other sprouts or nearby vessels, a process termed anastomosis, and
assemble new lumen-containing tubules (Figure 4). Extracellular matrix signals play important roles in
these processes. Binding of VEGF to heparan sulfate proteoglycans and thereby physical linkage to matrix
scaffolds is essential for the generation of VEGF gradients, which, in turn, are critical for filopodia
extension and the directional growth of endothelial sprouts. As blood vessels are surrounded by a layer of
sub-endothelial basement membrane, sprouting requires the local breakdown and/or reorganization of this
matrix. This process is presumably mediated by matrix metalloproteinases, like MT1-MMP, and ADAM
family disintegrin and metalloproteases (Figure 4). However, proteolytic processing can also affect VEGF,
trigger its release from the extracellular matrix and thereby impair the guided growth of vessel sprouts.
When conditions do not favor the formation of new endothelial tubules, like the presence of repulsive
signals or inhibition of VEGF, the retraction of vessels sprouts leaves behind empty matrix sleeves (Figure
4). Cell–matrix interactions can also help to stabilize the vasculature and thereby counteract the angiogenic
activation of vessels. In line with these findings, the formation of the sub-endothelial basement membrane
is likely to play a key role in the establishment of stable and mature blood vessels.
Sticking together — dynamics of adhesive
EC–cell interactions
As outlined above, the behavior of individual cells and, as a consequence, intercellular interactions are
highly dynamic in the growing vasculature. Since ECs are connected to each other through junctional
protein complexes, these structures need to be modulated in a highly dynamic fashion during processes
such as sprouting, proliferation or tubulogenesis. VE-cadherin, a junctional transmembrane protein that
mediates adhesive, homophilic interactions between adjacent ECs, is a critical regulator of vascular
integrity and permeability. Like for other cadherin family proteins, binding to cytoplasmic catenin proteins
and linkage to the actin cytoskeleton are major effects downstream of VE-cadherin. Recent evidence
suggests that VE-cadherin interactions inhibit angiogenic growth. In cultured ECs, the protein limits
proliferation by contact inhibition, which suppresses VEGFR2-induced pro-mitogenic signals. Consistent
with this finding, blocking VE-cadherin function or expression in endothelial 3D cultures enhances
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