Endothelial Extracellular Matrix

George E. Davis and Donald R.
Senger
Vascular Morphogenesis and Neovessel Stabilization
Endothelial Extracellular Matrix : Biosynthesis, Remodeling, and Functions During
Print ISSN: 0009-7330. Online ISSN: 1524-4571
Copyright 2005 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Circulation Research
doi: 10.1161/01.RES.0000191547.64391.e3
2005;97:1093-1107 Circ Res.
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This Review is part of a thematic series on Vascular Cell Diversity, which includes the following articles:
Heart Valve Development: Endothelial Cell Signaling and Differentiation
Molecular Determinants of Vascular Smooth Muscle Cell Diversity
Endothelial/Pericyte Interactions
Endothelial Extracellular Matrix: Biosynthesis, Remodeling, and Functions During Vascular Morphogenesis and
Neovessel Stabilization
Joyce Bischoff, Guest Editor
Endothelial Extracellular Matrix
Biosynthesis, Remodeling, and Functions During Vascular Morphogenesis
and Neovessel Stabilization
George E. Davis, Donald R. Senger
AbstractThe extracellular matrix (ECM) is critical for all aspects of vascular biology. In concert with supporting
cells, endothelial cells (ECs) assemble a laminin-rich basement membrane matrix that provides structural and
organizational stability. During the onset of angiogenesis, this basement membrane matrix is degraded by
proteinases, among which membrane-type matrix metalloproteinases (MT-MMPs) are particularly significant. As
angiogenesis proceeds, ECM serves essential functions in supporting key signaling events involved in regulating
EC migration, invasion, proliferation, and survival. Moreover, the provisional ECM serves as a pliable scaffold
wherein mechanical guidance forces are established among distal ECs, thereby providing organizational cues in the
absence of cellcell contact. Finally, through specific integrin-dependent signal transduction pathways, ECM
controls the EC cytoskeleton to orchestrate the complex process of vascular morphogenesis by which proliferating
ECs organize into multicellular tubes with functional lumens. Thus, the composition of ECM and therefore the
regulation of ECM degradation and remodeling serves pivotally in the control of lumen and tube formation and,
finally, neovessel stability and maturation. (Circ Res. 2005;97:1093-1107.)
Key Words: extracellular matrix

endothelial cells

angiogenesis

vascular morphogenesis
vessel stabilization
T
he extracellular matrix (ECM) provides critical support
for vascular endothelium. Primarily through adhesive
interactions with integrins on the endothelial cell (EC) sur-
face, ECM provides a scaffold essential for maintaining the
organization of vascular ECs into blood vessels. In addition,
EC adhesion to ECM is required for EC proliferation,
migration, morphogenesis, survival, and ultimately blood
vessel stabilization, all of which are critical for neovascular-
ization. The specific mechanisms through which ECM sup-
ports EC functions are complex and involve both external
structural support and regulation of multiple signaling path-
ways within the cell, including signaling pathways that
control apoptosis, proliferation, the cytoskeleton, and cell
shape. Thus, through both mechanical and signaling func-
Original received June 23, 2005; revision received October 3, 2005; accepted October 11, 2005.
From the Department of Pathology (G.E.D.), Texas A&M University System Health Science Center, College Station; and Department of Pathology
(D.R.S.), Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass.
Correspondence to Donald R. Senger, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Research North,
99 Brookline Ave, Boston, MA 02215. E-mail dsenger@bidmc.harvard.edu
2005 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000191547.64391.e3
1093
Review
tions, the ECM affects many fundamental aspects of EC
biology. Moreover, the diversity of ECM components in the
EC microenvironment and the diversity of mechanisms for
controlling the synthesis and degradation of ECM have
suggested an intricate level of complexity sufficient for ECM
to exert significant and precise control over many aspects of
neovascularization and blood vessel maturation.
The purpose of this review is to provide an overview of the
importance of ECM for the biology of vascular ECs, and it is
divided into 4 parts: (1) ECM Function in Cellular Morphogen-
esis and Signaling; (2) ECM Function in EC Lumen Formation
and the Switch to Vessel Maturation; (3) Endothelial ECM:
Remodeling; and (4) Endothelial ECM Biosynthesis, Assembly,
and Structural Functions: Critical Role for EC Basement Mem-
brane Matrix in Vessel Stabilization. Because of our own
specific research interests, we have focused particularly on the
importance of ECMfor vascular morphogenesis, ie, the complex
process through which proliferating ECs organize into new
blood vessels with functional lumens.
ECM Function in EC Signaling
and Morphogenesis
Although much emphasis has been placed on the role of
angiogenic cytokines such as vascular endothelial growth
factor (VEGF) in EC migration, survival, and proliferation,
considerable evidence indicates that ECM is equally or more
important. Moreover, in most cases, cytokine function is
entirely dependent on EC adhesion to ECM. There is a
growing body of evidence that ECM also drives capillary
morphogenesis through sustained signaling, resulting in per-
sistent EC cytoskeletal reorganization and changes in cell
shape. Also, ECM scaffolds may function during angiogen-
esis to immobilize angiogenic cytokines and thereby coordi-
nate signals transduced through both growth factor and ECM
receptors, as addressed in the section Endothelial ECM
Biosynthesis, Assembly, and Structural Functions: Critical
Role for EC Basement Membrane Matrix in Vessel Stabili-
zation. In this section, we focus attention on how ECM
regulates signaling pathways to control EC proliferation,
survival, and capillary morphogenesis.
Adhesion to ECM Regulates EC Migration,
Proliferation, and Survival
Vascular ECs require adhesion to ECM for migration, and EC
migration is important for angiogenesis, particularly during
sprouting of new blood vessels from the existing vasculature.
1
Although gradients of cytokines or other agonists are required
to drive chemotactic migration, such directed motility is
absolutely dependent on EC adhesion to ECM. Moreover,
evidence from in vitro experiments indicate that many of the
interstitial and provisional ECM components that are encoun-
tered during sprouting angiogenesis, including interstitial
fibrin and collagen I, are capable of supporting chemotactic
migration.
2,3
In addition, gradients of immobilized ECM
components can by themselves drive haptotactic migration in
vitro, and this type of motility is not dependent on cyto-
kines.
3,4
Although the significance of such haptotactic migra-
tion has not been established in vivo, it seems plausible that
the high concentrations of interstitial collagen encountered by
ECs during the sprouting phase of angiogenesis may drive
outward migration, in part, through haptotaxis. Indeed, inter-
stitial collagen is highly effective at promoting haptotactic
migration in vitro.
3
Thus, sprouting ECs may migrate in
response to both chemotactic gradients of angiogenic cyto-
kine and haptotactic gradients of ECM. Regardless, any and
all such motility-promoting stimuli are dependent on EC
adhesion to ECM.
One of the most fundamental functions provided by ECM
involves support of EC proliferation and survival. Both
proliferation and survival are highly dependent on adhesion
to ECM through cell-surface integrins.
58
In particular, acti-
vation of the p44/p42 (Erk1/Erk2) mitogen-activated protein
kinase (MAPK) signal transduction pathway in ECs is critical
for EC proliferation and angiogenesis.
912
EC anchorage to
ECM through integrins is necessary for efficient MAPK
activation by cytokines.
13,14
Also, the expression and activi-
ties of cyclin-dependent kinases, which are required for cell
cycle progression and therefore for cell proliferation, are
dependent on EC adhesion to ECM.
1517
Thus, failure of ECs
to attach to ECM results in cessation of cell proliferation
through multiple mechanisms. Adhesion to ECM is similarly
important for EC survival.
6
In particular, adhesion-dependent
activation of the MAPK pathway, which, as noted above, is
required for EC proliferation, functions critically in EC
survival.
1820
By controlling the transcription of key signaling
molecules that regulate caspase-mediated apoptosis, the
MAPK pathway suppresses cell death.
19
Thus, apoptosis is
induced and proliferation ceases when ECs are prevented
from attaching to ECM, indicating that ECM is pivotal for the
most fundamental aspects of EC biology.
Although the importance of ECM for EC migration,
proliferation, and survival is well established, the relative
importance of specific ECM components in supporting these
processes is less understood and often difficult to ascertain
because of functional overlap. For example, cytokine activa-
tion of the MAPK pathway in microvascular ECs and
proliferation of microvascular ECs are similarly supported by
attachment to either collagen I or vitronectin.
20
Moreover, a
variety of ECM components provide sufficient support for
EC migration,
2,3,21
although not with equal potency.
21
There
is also evidence that components of ECM exhibit maximal
activity in combination with each other and thereby may
function cooperatively.
20
ECM Provides Guidance Cues That Support
Capillary Morphogenesis
During angiogenesis, proliferating and migrating ECs orga-
nize to form new 3D capillary networks. This process has
been studied extensively in the embryo, thus establishing that
an early stage of capillary morphogenesis involves transition
of endothelial precursor cells to a spindle-shaped morpholo-
gy.
22
Coincident with this spindle-shape transition, EC pre-
cursors align and connect into solid, multicellular, precapil-
lary cord-like structures that form an integrated polygonal
network.
23,24
Solid precapillary cord-like structures have also
been identified during angiogenesis in the adult.
25
During
maturation, solid vascular cords form hollow lumens for the
transport of blood, and ECs are sequestered from the inter-
1094 Circulation Research November 25, 2005
stitial matrix through establishment of a continuous basal
lamina.
22,25
During vascular morphogenesis, the ECM serves as a 3D
malleable scaffold in which individual ECs and clusters of
ECs can transduce mechanical forces to other ECs at a
considerable distance. Thus, by generating mechanical, con-
tractile forces within ECM, ECs are able to establish tension-
based guidance pathways that allow them to form intercon-
nected cords. These guidance pathways provide a mechanism
for ECs to organize into large multicellular structures at a
distance without the initial requirement of cell:cell
contact.
24,26
ECM Signaling Controls EC Morphogenesis
In addition to serving as an adhesive support in which EC
mechanical forces can be transduced to promote multicellular
organization, ECM exerts important signaling functions di-
rectly on ECs to regulate cell shape and contractility. It has
long been recognized that 3D interstitial collagen type I
provokes ECs in culture to undergo marked shape changes
that closely imitate precapillary cord formation observed
during embryonic vasculogenesis and adult angiogenesis.
Within hours after addition of collagen I to confluent EC
cultures, the cells partially retract and exhibit a spindle-
shaped morphology, coincident with realignment to form
solid cords organized in a polygonal pattern
2731
(see Figure
1). Subsequently, over the course of several days, these
structures mature to form tubes with hollow lumens through
a process involving development and coalescence of intracel-
lular vacuoles.
26
In sharp contrast to ECs, fibroblasts do not
respond to collagen I with changes in cell shape or alignment
into cords, suggesting a special connection between collagen
I signaling and EC morphogenesis.
32
In support of the importance of interstitial collagens in
driving ECs to form precapillary cords, there is much evi-
dence that interactions between interstitial collagens and ECs
are highly relevant in vivo. During sprouting angiogenesis,
ECs within existing blood vessels degrade basement mem-
brane
33
and migrate and proliferate within connective tissue
abundant in interstitial collagens.
34
Furthermore, the key
angiogenic cytokine VEGF, which induces sprouting angio-
genesis also induces microvascular ECs to express integrins
1
and
2
1
,
35
which are the key interstitial collagen
receptors on microvascular ECs. Moreover, antagonism of
these integrins inhibits dermal and tumor angiogenesis in
vivo.
3,35
Also, analyses of genes expressed in human tumor
endothelium have demonstrated that ECs isolated from tu-
mors express 10-fold more transcripts encoding interstitial
collagens type I and III than ECs isolated from corresponding
control tissue, indicating that tumor ECs express their own
interstitial collagens,
36
thus suggesting that interstitial colla-
gen expression by tumor ECs may be conducive for angio-
genesis. In support of this hypothesis, expression of collagen
I by isolated EC clones in vitro correlates with spontaneous
multicellular organization of these ECs into cords.
37,38
Fi-
nally, neovascularization can be inhibited in animal models
Figure 1. Inuence of collagen type I and
laminin-1 signaling in ECs on capillary
morphogenesis in vitro. In this schematic
diagram, collagen type I, but not
laminin-1, activates Src and Rho and
suppresses Rac and PKA through
1
integrins. This results in induction of
actin stress bers, disruption of
VE-cadherin, and formation of precapil-
lary cords. In contrast, laminin-1 induces
Rac and PKA and suppresses Rho and,
therefore, does not provoke morphogen-
esis (top). These marked distinctions in
signaling by collagen type I (re) and
laminin-1 (ice) suggest a mechanism
through which degradation of basement
membrane and exposure of activated
and proliferating ECs to collagen type I
initiates morphogenesis of new capillary
sprouts (bottom). n/c indicates no
change. This is a modied version of a
previously published gure
40
and is
reprinted by permission of the Federation
of American Societies for Experimental
Biology 2004.
Davis and Senger ECM in Vessel Morphogenesis and Stabilization 1095
both by proline analogues that interfere with collagen triple-
helix assembly and by -aminopropionitrile, which inhibits
collagen cross-linking,
39
indicating that collagens play a
crucial role in angiogenesis.
Recently, the mechanisms through which collagen I selec-
tively provokes EC formation of cord-like structures that
closely imitate precapillary cords in vivo have begun to be
identified. First, collagen I-ligation of integrins
1
1
and
2
1
in microvascular ECs in vitro suppresses cAMP and thereby
suppresses the activity of cAMP-dependent protein kinase A
(PKA). Suppression of PKA activity leads to a marked
induction of actin polymerization that contributes to the
formation of prominent stress fibers and EC contractility.
Suppression of PKA and induction of actin polymerization
closely parallel cord formation in vitro, consistent with
functional significance. In contrast to collagen I, laminin-1
neither suppresses cAMP nor PKA activity nor induces actin
polymerization, indicating specificity among matrix proteins
regarding this signaling pathway.
32
Consistent with the im-
portance of this pathway for formation of precapillary cords,
laminin-1 also failed to induce changes in cell shape. More-
over, in contrast to ECs, fibroblasts did not respond to
collagen I with changes in cAMP or actin polymerization,
consistent with the failure of collagen I to provoke cord
formation by fibroblasts. Pharmacological elevation of cAMP
blocks collagen-induced actin polymerization and formation
of cords by ECs; conversely, pharmacological suppression of
either cAMP or PKA induces actin polymerization. Thus,
collagen Iinduced suppression of cAMP-dependent PKA
and induction of actin polymerization is an important mech-
anism through which collagen I drives EC organization into
multicellular precapillary cords.
32
In addition to suppressing cAMP-dependent PKA, collagen
I stimulation of microvascular ECs in vitro induces activation
of Src kinase and the GTPase Rho, also through interactions
with
1
integrins.
40
Similar to suppression of PKA, activation
of Src and Rho parallels cord formation. The Src inhibitor
PP2, dominant-negative Src, the Rho inhibitor exoenzyme C3
transferase, and dominant-negative RhoA each individually
inhibit collagen I induction of actin stress fibers that mediate
cell retraction, and each inhibit capillary morphogenesis.
40,41
Thus, collagen I induces actin stress fibers through activation
of Src and Rho,
40
and such activation likely complements
collagen Iinduced actin polymerization, as mediated by
PKA suppression (see above), to achieve the robust cytoskel-
etal contractility that drives cord assembly. Importantly, the
significance of actin stress fibers and EC contractility for
angiogenesis in vivo has been established in a mouse model
of VEGF-driven neovascularization. Specifically, retrovirus-
mediated transduction of ECs with a dominant-negative
RhoA mutant that blocks EC contractility and cord formation
in vitro markedly suppressed organization of proliferating
ECs into new blood vessels in vivo.
41
Activation of Src by collagen I not only contributes to the
formation of actin stress fibers but also disrupts vascular
endothelial (VE)-cadherin from intercellular junctions. Both
the Src inhibitor PP-2 and dominant-negative Src preserve
VE-cadherin localization to regions of cellcell contact; in
the absence of such inhibitors, VE-cadherin is disrupted by
collagen I. Also, an active Src mutant disrupted VE-cadherin
and cellcell contacts similarly to collagen I. In sharp
contrast to collagen I, laminin-1, which, as noted above, does
not induce capillary morphogenesis, also does not induce
activation of Src or Rho. Rather, laminin-1 induces persistent
activation of the GTPase Rac.
40
Thus, in addition to suppres-
sion of PKA activity, activation of Src and Rho is also a key
mechanism through which collagen I provokes capillary
morphogenesis of microvascular ECs. Moreover, activation
of p38 MAPK also has been implicated as important to the
mechanism by which collagen I drives ECs to form cords,
42
although the precise contribution of such activation to mor-
phogenesis is unclear.
Finally, an intriguing aspect of differences observed between
collagen I and laminin-1 signaling in microvascular ECs is that
they suggest a model whereby interstitial collagen and basement
membrane laminin differentially regulate various stages of
angiogenesis. As summarized in Figure 1 (top), interstitial
collagen I activation of both Src and Rho and suppression of
PKA promotes formation of prominent actin stress fibers that
mediate EC retraction and capillary morphogenesis. In addition,
collagen I activation of Src disrupts VE-cadherin from cell
junctions and promotes disruption of cellcell contacts, thus
facilitating multicellular reorganization. In sharp contrast to
collagen I, basement membrane laminin-1 does not provoke EC
morphogenesis or induce activation of Src or Rho or suppression
of PKA. Rather, laminin-1 induces persistent activation of the
GTPase Rac, whereas collagen I suppresses Rac activity after a
transient increase.
40
The potential significance of such distinc-
tions in ECM signaling is summarized in Figure 1 (top). During
the sprouting and proliferative stages of angiogenesis the
laminin-rich basal lamina is degraded, resulting in reduction of
EClaminin interactions. Thus, the available in vitro data predict
that loss of laminin substratum results in reduced Rac activity
and therefore a loss of Rac function in supporting integrity of
cellcell junctions during this phase. Moreover, on degradation
of basement membrane, sprouting ECs are exposed to underly-
ing interstitial collagens and begin to invade it, resulting in
activation of Src and Rho, suppression of PKA, and initiation of
capillary morphogenesis. Subsequently, as the newly formed
capillary sprouts mature into new vessels with mature lumens,
the intact basement membrane is reestablished. The continuous
basement membrane sequesters ECs from interstitial collagens
and thereby reestablishes normal activation levels for Rac, Rho,
Src, and PKA. Thus, the in vitro evidence supports a model in
which the laminin-rich basement membrane serves not only to
maintain the integrity of the mature endothelium but also to
sequester and thereby insulate ECs from interstitial collagens. In
contrast, degradation of basement membrane exposes ECs to
interstitial collagens and activates signaling pathways that drive
cytoskeletal reorganization and sprouting morphogenesis.
ECM Function in EC Lumen Formation and
the Switch to Vessel Maturation
ECM Stimulates EC Lumen Formation and
Tubular Morphogenesis Through Integrins
As discussed above, considerable data suggest that EC
integrin interactions with interstitial matrix proteins (ie,
interstitial collagens and fibrin/fibronectin) are key receptors in
stimulating EC tubular morphogenesis as well as concurrent EC
activation (Figures 1 and 2).
32,40,4345
The major integrins re-
sponsible for these interactions are
2
1
,
1
1
, and
v
3
,
5
1
,
which are collagen and fibrin/fibronectin receptors, respective-
ly
3,26,35,4446
(Figure 2). ECtubular morphogenesis is initiated by
the matrix-integrin-cytoskeletal signaling axis,
44
which causes
simultaneous sprouting (ie, cord formation) and vacuole and
lumen formation events that eventually lead to interconnecting
networks of EC-lined tubes (Figure 3). These events depend on
integrin interactions with collagen or fibrin matrices through
their respective integrins, as indicated above.
Figure 2. Role of integrins and ECM in
the control of EC morphogenesis and
stabilization. This schematic diagram
depicts the hypothesis that interstitial
collagens and provisional plasma-derived
bronectin/brin matrices stimulate EC
tubular morphogenic events, whereas
laminin-rich matrices lead to EC differen-
tiation and stabilization events. We pro-
pose a re and ice model whereby EC
contacts with interstitial collagens or
brin matrices lead to EC activation/mor-
phogenesis (ie, re), and the accumula-
tion of basement membrane matrices
around developing tubes leads to EC
differentiation and stabilization (ie, ice).
Furthermore, we hypothesize that these
distinct stages and steps in the EC tubu-
lar morphogenic and stabilization cas-
cade are mediated by overlapping sub-
sets of integrins and their associated
signaling molecules (eg, tetraspanins).
The subsets of integrins may act
together in clustered signaling com-
plexes to provide unique signals that
characterize either EC activation/mor-
phogenesis or EC
differentiation/stabilization.
Figure 3. Fundamental contribution of the ECMintegrincytoskeletal signaling axis to the assembly of EC cords as well as formation
of lumens and tubular networks during vasculogenesis and angiogenesis. In this schematic, 2 models of EC morphogenesis are illus-
trated. a, Conuent human EC monolayers were overlaid with a type I collagen gel and allowed to undergo cord formation, illustrating
how collagenous ECM markedly converts an EC monolayer to an interconnecting network of cords. This process is dependent on the
1
and
2
1
integrins and the small GTPase RhoA. Right (b), Human ECs are suspended as individual cells in 3D collagen matrices
and are xed, stained, and photographed at 8 and 48 hours of culture, illustrating intracellular vacuoles and early lumen formation at 8
hours and interconnecting networks of EC-lined tubes at 48 hours. Bar50 m. The 3 panels on the far right (c) are cross-sections
from plastic-embedded collagen gels revealing EC intracellular vacuoles (top and middle right) or EC lumens (bottom right).
Bar25 m. The vacuole and lumen formation process shown is completely dependent on the
2
1
integrin and the Cdc42 and Rac1
small GTPases.
Much work indicates that lumen formation during capillary
morphogenesis in 3D collagen matrices depends on the
formation and coalescence of integrin-dependent pinocytic
intracellular vacuoles.
26,4447
These intracellular vacuoles
fuse together to form intracellular lumens.
44,45,4752
These
compartments then fuse through exocytic events to create
interconnections between adjacent ECs to form continuous
luminal structures.
44,45
This intracellular lumen formation
process is a highly efficient mechanism to rapidly create
ECM-free space
26,44,45
and begin the establishment of apical-
basal polarization. Intracellular vacuole and lumen formation
requires the activity of MT-MMPs on the EC surface (see the
section Endothelial ECM: Remodeling).
The integrin-dependent pinocytic process that regulates
intracellular vacuole and lumen formation, which resembles
in many respects the process of macropinocytosis, also
depends on Rho GTPases, which are downstream of integrin
ECM interactions.
45,47
In particular, Cdc42 and Rac1 are
required for intracellular vacuole and lumen formation to
occur. We observed that dominant-negative forms of Cdc42
and Rac1 markedly block these events,
47
whereas RhoA
blockade did not regulate lumen formation in our 3D collagen
morphogenesis model.
47
Furthermore, we demonstrated that
green fluorescent protein fusion proteins with Cdc42 or Rac1
allowed for the targeting of these constructs to intracellular
vacuole membranes during the lumen formation process.
45,47
Thus, integrinECM interactions regulate Cdc42 and Rac1
activation and vacuole membrane targeting to control the
proper cytoskeletal rearrangements and vacuole targeting
mechanisms to properly orient the position of the developing
lumen.
Potential Key Roles for Integrin Signaling to
Control the Switch Between EC Tubular
Morphogenesis Versus EC Tube Stabilization
In contrast to the provisional matrix-binding integrins dis-
cussed above, basement membrane laminin-binding integrins
such as
6
1
and
3
1
may be more important for the process
of tube stabilization (see Figure 2). As illustrated in Figure 1,
the marked distinctions in signaling by collagen type I
(fire) and laminin-1 (ice) suggest a mechanism through
which degradation of basement membrane and exposure of
activated and proliferating ECs to collagen type I initiates
morphogenesis of new capillary sprouts. Conversely, they
suggest that laminins serve a maturation function. Integrin
1
has been reported to regulate cord formation in planar
angiogenesis systems in which ECs are placed on the surface
of basement membrane matrix gels.
24,53,54
Therefore, it is
important to consider that multiple integrins may act together
in regulating vascular morphogenesis (ie,
2
1
and
1
1
in
collagen matrices or
5
1
or
v
3
in fibrin matrices)
44,45
and
vascular stabilization (possibly
6
1
or
3
1
in combination
with
2
1
or
1
1
) to thereby create unique combinations of
signals (see Figures 1 and 2). The ability of
3
1
and
6
1
to
interact with components of the tetraspanin web
55
makes
them particularly attractive as key regulators of the switch
from EC activation/morphogenesis to EC stabilization/quies-
cence. Also,
3
1
is intriguing because of its known affinity
for tissue inhibitor of metalloproteinase (TIMP)-2, an angio-
genic inhibitor,
56
as well as other molecules that may regulate
the EC proteolytic machinery (eg, extracellular MMP inducer
and urokinase-type plasminogen activator receptor)
5760
that
control morphogenesis or regression (see the section Endo-
thelial ECM: Remodeling). Furthermore,
3
1
may partici-
pate in early EC tubular morphogenic events along with
integrins such as
2
1
and
1
1
(Figure 2). For example, the
tetraspanin, CD151, which interacts strongly with
3
1
, has
been shown to stimulate cell motility and invasion,
55
which
are critical for tubular morphogenesis. Although
3
1
shows
very low affinity for laminin-1, it shows strong affinity for the
epithelial laminin, laminin-5,
61
and laminin-10, with detect-
able but variable binding to laminin-8, depending on the cell
type.
62,63
Integrin
6
1
appears to have a broader specificity
for different laminin isoforms.
62,64
Thus, these receptors
interact with the key laminins in EC basement membrane
matrices and signals derived from these individual integrins
or in combination with other laminin-binding receptors (ie,
1
,
2
1
, dystroglycan, sulfatides)
64,65
may act to suppress
the EC tubular morphogenic program by blocking sprouting
and vacuole and lumen-formation events. In addition, these
receptors may suppress EC proliferation and signaling path-
ways that regulate EC activation such as the Ras-MAPK and
nuclear factor B pathways,
66,67
thereby promoting EC qui-
escence. Following establishment of the EC basement mem-
brane, signals from
6
1
and
3
1
may also control EC gene
expression, which regulates EC quiescence.
Thus, signaling events downstream of laminin-binding
integrins are likely crucial to the establishment of EC tube
stabilization through their interactions with the EC basement
membrane. The components of this basement membrane
matrix including laminin-10 and laminin-8 are likely to
interact with both the EC as well as its supporting cells such
as pericytes, which are known to be required for vascular
stabilization. These integrinlaminin interactions appear to
tip the balance toward stabilization and, furthermore, proba-
bly actively inhibit sprouting morphogenesis (Figures 1 and
2). Further work is required to define the key molecular
interactions between EC or pericyte integrins and ECM,
together with elucidation of key downstream signaling path-
ways. Such interactions and signaling pathways very likely
regulate the critical balance between sprouting morphogene-
sis and vessel stabilization. A better understanding of these
fundamental issues will hopefully lead to more sophisticated
approaches for regulating neovascularization in clinical
settings.
Endothelial ECM: Remodeling
Critical Role for Membrane-Type Matrix
Metalloproteinases in EC Tubular Morphogenesis
in Three-Dimensional Extracellular Matrices
The ability of ECs and their supporting cellular elements to
directly modify (ie, remodel) their immediate ECM environ-
ment is critically important for forming tubular networks,
stabilizing these networks, and inducing regression of these
networks. Many studies indicate that ECs are highly proteo-
lytic during the formation or regression of vascular net-
works.
44,68,69
A fundamental goal in vascular biology is to
understand which proteinases are relevant and to identify
their substrate targets at various stages of neovascularization.
Steps in which proteolysis appears likely to be critical during
EC morphogenesis include (1) the initiation of angiogenesis
whereby basement membrane breakdown occurs, (2) invasion
into collagen type I or fibrin matrices, and (3) lumen
formation.
44,45
Considerable data now suggest that MT-
MMPs are the critical regulators of EC invasion into 3D
collagen or fibrin matrices.
44,68,7077
In addition, we have
shown that that MT-MMPs are important regulators of lumen
formation in 3D extracellular matrices.
70
Moreover, the pro-
teinase inhibitors TIMP-2, TIMP-3, and TIMP-4 all inhibit
EC vacuole and lumen formation, whereas TIMP-1 does not
(W.B. Saunders and G.E. Davis, manuscript in preparation).
MT-MMPs are blocked by TIMP-2, -3, and -4 but not by
TIMP-1.
78
In support of these conclusions, small-interfering
RNA knockdown of MT1-MMP also markedly inhibits EC
vacuole and lumen formation (W.B. Saunders and G.E.
Davis, manuscript in preparation).
In contrast to MT-MMPs, many soluble MMPs have not
been found to be involved in these invasive or morphogenic
events, despite the fact that some of these enzymes are
induced during EC tubular morphogenesis (ie, MMP-1 or
MMP-9).
44,71,79,80
A few studies indicate a role for MMP-9
during retinal angiogenic responses in vivo,
81
and MMP-9 has
been reported to liberate VEGF from ECM sites.
82
In con-
trast, several studies using in vitro approaches have failed to
reveal a role for MMP-9 in tubular morphogenesis in 3D
collagen matrices.
71,80
Thus, the induction of an MMP during
morphogenesis does not necessarily mean that it plays a role
during the tube-formation process. However, it remains
possible that the influence of a particular MMP may depend
on the vascular bed analyzed or on a particular type of EC. In
other studies, we have recently shown that induction of both
MMP-1 and MMP-10 during tubular morphogenesis appears
to be primarily associated with regression of tubular net-
works
80
and not the formation of these networks (see below).
Thus, some soluble MMPs appear to be produced to control
the process of tube regression rather than morphogenesis (see
below).
44,79,80,83
Also, some of the complexity and confusion
related to the influence of MMP inhibitors on wound- or
tumor-induced angiogenesis
84,85
may directly relate to the
different functional roles of specific MMPs in controlling
tubular morphogenesis versus regression. An MMP inhibitor
that blocks regression may actually promote angiogenesis by
facilitating the maintenance and survival of neovessels.
ECM Degradation As a Regulatory Signal for
Initiating Vascular Morphogenesis and Controlling
Vessel Stabilization
The enzymes that control basement membrane degradation
and the initiation of angiogenesis have yet to be defined,
including the relative contributions of membrane-associated
versus soluble MMPs or other classes of enzymes such as
serine proteinases. Various MMPs are known to degrade
basement membrane matrix components, and these include
MT-MMPs, MMP-3, MMP-10, and MMP-9.
44,68,69,75
Perhaps
a combination of these enzymes controls the invasion of ECs
through basement membrane during the sprouting phase of
angiogenesis. In addition to the function of MMPs or other
proteases in degrading basement membrane matrix, another
possible function of proteinases during sprouting angiogene-
sis may be to inactivate a constitutive inhibitor of EC
sprouting morphogenesis. Such an inhibitor may be present
within basement membranes to prevent sprouting in a quies-
cent vessel and may be produced by supporting cells such as
pericytes.
44,86
One such inhibitor may be the heparan sulfate
binding proteinase inhibitor TIMP-3
78
(ie, allowing it to
interact directly with the basement membrane proteoglycan,
perlecan), which is highly expressed by pericytes
87
and
induced by EC-pericyte interactions (W.B. Saunders et al,
manuscript in preparation ). We have previously shown that
TIMP-3 is able to markedly inhibit EC invasion and tube
formation in 3D collagen or fibrin matrices.
70
It has been
reported that serine proteinases can inactivate TIMP-1,
88
suggesting the possibility that a function of serine or other
proteinases may be to inactivate TIMPs that control vascular
stabilization. Another function of TIMPs and TIMP-3, in
particular, is to decrease stimuli that activate ECs. Delivery of
TIMP-3 is known to markedly suppress various membrane-
associated MMPs including MT-MMPs as well as various
disintegrins and metalloproteinases such as ADAM17,
ADAM10, and ADAM15.
78,8992
This class of proteinases
controls ECM degradation as well as the release of EC
activators such as membrane-associated growth factors that
interact with epidermal growth factor receptors (eg, heparin-
binding epidermal growth factor).
8991
The integrity of ECEC junctions
9396
is also key for
maintaining a stabilized state. Membrane-associated protein-
ases that are known to facilitate disassembly of junctional
contacts include ADAM-15 (which is concentrated in junc-
tions of both endothelial and epithelial cells) and ADAM-
10,
96,97
MT-MMPs,
98
and soluble proteinases such as MMP-3
or MMP-7.
99
Because ADAMs and MT-MMPs are known to
control proteolysis of cellcell junctional adhesion mole-
cules, blockade of these proteinases with TIMP-3 (which,
uniquely among the TIMPs, blocks both ADAMs proteinases
and MT-MMPs)
78
may strongly promote cellcell junction
formation and stability. Junctional stability may also enhance
basement membrane matrix stability by facilitating the con-
tinued deposition of basement membrane components in a
polarized basal direction.
100,101
The combination of ECEC
junctional stability (with appropriate apical-basal polarity),
and continuous basement membrane matrix deposition is
likely required for EC tube stabilization and a quiescent
phenotype.
Soluble MMPs Such As MMP-1 and MMP-10 Play
a Role As Vascular Regression Factors
Whereas considerable data now suggests that MT-MMPs
control EC tube morphogenesis in 3D extracellular matri-
ces,
44,7073,102
other MMPs such as MMP-1 and MMP-10
(stromelysin-2) appear to control the process of regression
rather than morphogenesis.
79,80
Blockade of these MMPs with
proteinase inhibitors such as TIMP-1 and
2
-macroglobulin,
blocking antibodies, and small-interfering RNA knockdown
does not influence tube formation. In contrast, these reagents
markedly inhibit EC tube regression events in 3D collagen
matrices.
79
We have found that multiple serine proteases such
as plasmin and plasma kallikrein induce activation of both
MMP-1 and MMP-10 proenzymes to control regression.
80
Plasmin and MMP-10 (as well as previous reports with
MMP-3) synergistically activate proMMP-1, creating a
superactive enzyme that is approximately 10-fold more active
than MMP-1 activated with plasmin alone.
103,104
Thus, the
ability of ECs to coinduce MMP-1 and MMP-10 during tube
morphogenesis establishes the conditions necessary to regu-
late tube regression when a serine proteaseactivating stim-
ulus is supplied.
44,79,80
Endothelial ECM Biosynthesis, Assembly, and
Structural Functions: Critical Role for EC
Basement Membrane Matrix in
Vessel Stabilization
EC Synthesis and Deposition of Basement
Membrane Matrix
ECs exposed to blood flow are typically associated with
continuous basement membrane matrix structures. It has
largely been assumed that ECs are primarily responsible for
the synthesis and deposition of these ECM components.
However, little is known about how basement membrane
assembly is regulated by ECs or whether the ECs require
supporting cells such as pericytes
105109
or astrocytes
110
to
properly assemble these structures. The mechanisms that
regulate basement membrane matrix assembly by ECs and its
supporting elements remain a critical question in vascular
biology. Nevertheless, much has been learned over the past
decade or so concerning the molecular control of basement
membrane matrix assembly during development with a pri-
mary focus on how epithelia and muscle cells assemble
basement membranes.
64,65,101,111114
A major message from
these studies is that laminins are the primary determinants of
basement membrane assembly and that other basement mem-
brane components such as collagen type IV variants,
115
perlecan (basement membrane heparan sulfate proteoglycan-
2), nidogens, and collagen type XVIII are accessory compo-
nents.
64,101,114
Collagen type IV deletion results in embryonic
lethality when defects arise in cells in contact with basement
membranes that are placed under mechanical stress such as in
the heart and blood vessels.
116
EC basement membranes are
thought to contain predominantly the collagen type IV iso-
form [
1
]
2
1
.
111
Consistent with the data from collagen type
IV knockout mice, knockout of the collagen-specific chaper-
one heat shock protein (Hsp) 47 also resulted in a similar
lethal vascular phenotype.
117,118
Hsp47 controls the assembly
of triple-helical collagens
115
and without this chaperone,
117,118
the triple-helical regions of collagen type IV as well as other
collagens are unstable and rapidly degraded by enzymes such
as serine proteinases (which are normally unable to proteo-
lyze these triple helices). Thus, in Hsp47 knockout mouse,
marked abnormalities were observed in both interstitial col-
lagen matrices and basement membrane matrices. Recent
studies reveal an accumulation of collagen type IV in the
endoplasmic reticulum of Hsp47 knockout cells that leads to
cellular apoptosis.
117
Similar vascular defects, but of lesser
severity to those of the collagen type IV null mice, were
observed in perlecan-null mice, suggesting that perlecan also
plays a key role in basement membrane function in mechan-
ically stressed cells.
119
Thus, both collagen type IV and
perlecan, although not absolutely required for basement
membrane formation, are necessary for stability of basement
membranes under conditions of mechanical stress, and both
genes are required for embryonic survival during develop-
ment.
116,119
In mice lacking these genes, the continuous
laminin-containing basement membrane matrix forms nor-
mally but then over time becomes unstable and begins to
develop discontinuities and physical breaks.
116,119
Although
the mechanisms underlying such instability of basement
membrane laminins are not entirely clear, the absence of
collagen type IV or perlecan may render laminins more
susceptible to proteolysis. A summary of the vascular pheno-
types of mice with null mutations for various basement
membrane matrix components is presented in Table I in the
online data supplement available at http://circres.ahajournals.
org.
Role for Laminin-8 and Laminin-10 in EC
Tubular Morphogenesis and Stabilization
Laminins are a family of proteins containing three subunits
arranged in different combinations. These molecules are
discussed in detail in several excellent recent reviews.
64,101,114
Some laminin isoforms are thought to be particularly adept at
assembling through polymerization reactions to form base-
ment membranes containing laminin-1 (
1
1
)
112,113,120,121
(embryonic basement membranes), laminin-2 (
2
1
) (skel-
etal muscle basement membranes),
122,123
and laminin-10
(adult and vascular basement membranes) (
5
1
).
123125
EC
basement membranes are thought to primarily consist of the
laminin-8
123,126
and laminin-10 isoforms,
123,127,128
with some
additional contribution of laminin-9 (
4
1
) or laminin-11
(
5
1
). The
2
subunit of laminin creates isoforms contain-
ing forms of laminin (s-laminin) found to be concentrated in
synaptic contacts at neuromuscular junctions.
123,129
Laminin-10 is an abundant and broadly expressed laminin in
adult tissues.
64,123
This latter laminin variant is a major
laminin synthesized by ECs and appears to be the major
self-polymerizing laminin that is likely responsible for EC
basement membrane assembly,
64,101,114,127,130
particularly in
adult tissues. During embryonic development, it appears that
laminin-8 (
4
1
) is the major laminin produced by ECs, and
it predominates in vascular basement membranes over
laminin-10 at this time.
124,126,127,131
However, laminin-10
does appear to overlap with laminin-8 expression in some
vascular beds during development.
123,127
Laminin-8 lacks globular domains on its short arms, which
are thought to be critical for self-polymerization reactions to
form basement membranes. Although laminin-8 can interact
with other basement membrane components, it may not be
capable of itself assembling the actual basement membrane
structure.
64,124
Knockout of the laminin
4
chain results in the
appearance of hemorrhages around many vessels in embryos,
but, despite this, many animals survive beyond embryonic
development and eventually normalize because of the appear-
ance of laminin-10 in vascular basement membranes within
the first 2 to 3 weeks of postnatal age (see online Table I).
126
The
4
laminin chain knockout animals show poorly
formed vascular basement membranes during development
and shortly after birth show observable discontinuities in
basement membrane structure, and, despite this, most
animals survive with a relatively normal appearing vascu-
lature. These knockout animals show increased angiogenic
responses following application of angiogenic cytokines or
the implantation of tumor cells.
132
This response may
reveal that the EC/pericyte-derived basement membrane
matrix is a signal for vascular quiescence, and stabilization
and it may be that a decrease in this quiescence signal
(perhaps laminin-8, -10, or both) can facilitate the activa-
tion of ECs or initiation of neovascularization during
angiogenic responses (see section ECM Function in EC
Lumen Formation and the section Switch to Vessel Mat-
uration and section ECM Function in EC Lumen Forma-
tion and the Switch to Vessel Maturation). Similar results
have recently been obtained using the aortic ring EC
sprouting assay from collagen type XVIII knockout mice,
whereby increased sprouting is observed compared with
wild-type mice.
133
However, these mice are, for the most
part, normal with respect to their vasculature (except in the
developing eye).
134
These results may be attributable to the
inhibitory activity of the NC1 domain fragment of collagen
type XVIII, endostatin, which, like many NC1 domain
fragments, is able to block angiogenic responses.
135137
The knockout of the laminin
5
subunit results in an
embryonic lethal phenotype that does not progress beyond
embryonic day 16.5.
127
The lethality appears to relate to
abnormalities of the placental as well as fetal vasculature
(online Table I). In these animals, reduced complexity in
the branching patterns of these vascular beds was ob-
served, and existing vessels had larger lumen diameters.
127
Similarly, angiogenic vessels from laminin
4
knockout
vessels also had markedly increased lumen diameters.
126
Thus, a function of basement membrane matrix through
laminins may be to inhibit tubular morphogenesis by
interfering with lumen expansion. Normally, some fetal
vessels are juxtaposed with trophoblasts (eg, in the pla-
cental labyrinth) that adhere to the EC basement mem-
brane, and these were clearly abnormal in the laminin
5
knockout mice.
127
A similar defect occurs in the develop-
ing kidney glomerulus in these mice such that there are
marked abnormalities of mesangial cellEC basement
membrane interactions causing an absence of capillary
loop formation
130
(a key morphological change required
for glomerular development). The related nature of mes-
angialEC interactions with pericyteEC interactions
138,139
are such that molecules such as laminin-10 may represent
key regulators of this interaction that when absent may
cause the observed microvascular abnormalities and insta-
bility in the knockout animals. Thus, laminins mediate
interactions of ECs with basement membranes as well as
adjacent supporting cells such as pericytes to facilitate
vessel stabilization.
An interesting concept is that there may be intermediate
stages of basement membrane assembly (ie, provisional
basement membranes) and that these may be critical during
processes such as EC sprouting and tubular morphogenesis.
Early work suggested the concept that fibronectin may
provide such provisional matrix signals for vascular morpho-
genesis while laminins are deposited later for stabilization.
140
Other work suggests the possibility that laminin-8 and colla-
gen type IV might participate in providing a provisional ECM
scaffold during tubular morphogenesis before the develop-
ment of a mature basement membrane containing laminin-10
(ie, leading to tube stabilization).
64,126
In support of these
concepts, antagonism of the
4
laminin subunit with blocking
antibodies resulted in inhibition of EC morphogenic re-
sponses and EC survival in various in vitro models,
141,142
and,
in our model of EC tubular morphogenesis in 3D collagen
matrices, the
1
and
4
laminin subunit genes were induced,
whereas the
5
laminin subunit gene was expressed in a stable
fashion.
143
In addition, a laminin switch is known to occur
during kidney development where the laminin-1 isoform
(embryonic development) is eventually replaced with
laminin-10 (adult).
130
Thus, the EC basement membrane
matrix may undergo regulated changes in laminins during the
transition from morphogenesis to stabilization.
A related question involves the extent to which ECs are
able to assemble basement membrane matrices in a provi-
sional matrix such as fibrin.
46,144147
Fibrin and fibronectin
are recruited together from plasma through increased vascular
permeability following tissue injury to modify the local ECM
that regulates vascular morphogenesis.
43
Recent studies have
revealed that fibrin-induced EC tube morphogenesis is mark-
edly enhanced by the presence of fibroblasts
145
that similarly
may contribute interstitial matrix proteins or missing compo-
nents of basement membrane matrix such as nidogen-1.
148
Potential Role for Endothelial Cell Supporting
Cells in the Regulation of Basement Membrane
Matrix Assembly and Stabilization
Another important question is the extent to which ECs
themselves can produce functional basement membranes
versus the necessity of these cells to interact with support-
ing cells such as pericytes to regulate basement membrane
matrix synthesis or assembly.
149,150
Supporting cells such
as pericytes may contribute molecules such as nidogen-1,
which, along with perlecan, can facilitate the assembly of
a stabilized basement membrane containing laminin-10
(which self-assembles), collagen type IV, perlecan, and
nidogens 1 and 2.
64
Nidogen-2 has been found to be
present in higher amounts in EC basement mem-
branes,
64,151,152
and it is induced during EC tubular mor-
phogenesis,
153
although it is expressed at much lower
levels compared with nidogen-1 basement membranes
throughout most tissues.
151,154
It is clear in many instances that epithelialmesenchymal
interactions regulate basement membrane assembly by cells
such as epithelium.
148,155160
This is particularly apparent in
the skin whereby keratinocytes are not able to assemble a
basement membrane structure (as determined with electron
microscopy and immunofluorescent staining) unless underly-
ing fibroblasts are present. Fibroblasts have been shown to be
a producer of nidogens
148
that control interactions of laminins
with collagen type IV isoforms in different basement mem-
branes.
64
In our system of EC tube morphogenesis in 3D type
I collagen matrices over 3 to 5 days,
26,143
we have never
observed the development of complete basement membrane
structures with EC cultures alone (electron microscopic
analysis). If pericytes are required for EC basement mem-
brane assembly, this would provide explanation for why these
supporting cells are required for vascular stabiliza-
tion.
86,105,107,108,149,150,161
As such, EC basement membrane
matrix assembly may closely resemble the parallel process of
epithelial basement membrane assembly where mesenchymal
cells are necessary. Further support for this possibility comes
from findings that tubular morphogenesis and stabilization of
endothelial versus epithelial structures appear highly relat-
ed.
44,48,162164
Furthermore, in some situations, ECs them-
selves have been shown to produce ECM proteins that are
typically classified as interstitial matrix proteins such as
collagen type I, collagen type VI, and fibronectin.
36,38,165
A
major step in embryonic development involves epithelial
mesenchymal transition, and ECs can undergo a similar
transition, for example, during cardiac valve development.
166
In contrast, many differential gene array experiments using
ECs undergoing morphogenesis in 3D matrices have revealed
a much more prominent induction of basement membrane
matrixrelated genes (ie, epithelial-like genes).
143,153
Yet
another question relates to EC production of collagens that
bridge EC basement membranes to the collagen type I
interstitial matrix. Such proteins include collagen type VI,
which through its NC1 domains can bind both collagen type
IV and type I,
115,167,168
as well as possibly collagen type
VIII.
169
ECs are capable of synthesizing collagen type VI,
although it is possible that pericytes or other supporting cells
such as fibroblasts may contribute collagen type VI to
facilitate the linkage of EC basement membranes to intersti-
tial matrices.
Limited information is available concerning the ability
of perivascular cells such as pericytes to produce ECM
components. It is clear that pericytes synthesize basement
membrane matrix proteins, proteoglycans, such as decorin,
biglycan, versican, and aggrecan, and fibronectin and
various collagens.
105,170175
A key regulator of ECM syn-
thesis in this and other contexts is transforming growth
factor-, particularly in concert with connective tissue
growth factor.
176178
PericyteEC interactions are known
to markedly induce transforming growth factor- activa-
tion,
179182
which ultimately results in increased ECM
synthesis, deposition, and decreased ECM turnover
through secretion of proteinase inhibitors (ie, plasminogen
activator inhibitor-1 and TIMP-1). Major questions re-
maining include whether pericytes are required for EC
basement membrane synthesis and assembly or whether
they are particularly involved in stabilization of these
structures.
Immobilization of Angiogenic Cytokines by
ECM Scaffolds
Finally, an interesting concept in EC/ECM biology involves
immobilization of angiogenic cytokines on ECM scaffolds.
Such immobilization may provide important cues for direc-
tional growth of EC sprouts during early morphogenesis. In
addition, such growth factorrich ECM scaffolds may pro-
vide the means for ECs to respond simultaneously to growth
factor receptor and integrin cosignaling pathways (eg,
through clustering of receptors). Most angiogenic cytokines
have affinity for heparin and heparan sulfate and thereby
become anchored to heparan sulfate proteoglycans (ie, syn-
decans, perlecan, versican) either on the surface of ECs or
within the surrounding ECM.
86,111,183
Recent work suggests
that MMPs and serine proteinases such as plasmin can
liberate functional VEGF-165 from heparan sulfatecontain-
ing ECM.
184,185
Also, relevant angiogenic cytokines may be
capable of directly binding to angiogenesis-promoting ECM
scaffolds such as collagen type I and fibrin/fibronectin
matrices.
186
In support of this possibility are studies showing
that VEGF can bind directly to fibronectin
187
and that
hepatocyte growth factor and keratinocyte growth factor each
can bind directly to collagen type I and remain function-
al.
188,189
Also, the ECM protein thrombospondin-1,
190
which
in some cases inhibits angiogenesis,
191
binds various angio-
genic cytokines such as fibroblast growth factor-2, VEGF,
and hepatocyte growth factor
192
and thereby may prevent
their binding to proangiogenic ECM. Thus, angiogenic cyto-
kines show direct binding affinities for angiogenesis-
promoting ECM scaffolds that stimulate the process of
vascular morphogenesis.
Acknowledgments
This work was supported by NIH grants HL59373 and HL79460 (to
G.E.D.), NIH grant CA77357 (to D.R.S.), and grants from the V.
Kann Rasmussen Foundation (to D.R.S.).
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Online Table 1. Vascular defects in mouse knockouts of ECM basement membrane matrix genes
Basement membrane
component
Laminin 4 chain
(laminin-8)
Laminin 5 chain
(laminin-10)
Collagen type IV,
alpha 1/2 chain
Perlecan
Nidogens 1 and 2
Collagen type XVIII
Mouse knockout phenotype
Mild, early postnatal lethality, newborns lethargic,
pale and icteric due to anemia and hemorrhage,
survivors improve and have normal life span. Corneal
angiogenesis assays with FGF-2 reveal early, more
intense sprouting and grossly distorted vasculature with
apparent hemorrhages and dilated vessels.
Specific vascular phenotypic
defects/ comments
Diffuse hemorrhagic phenotype from smaller vessels, particularly
associated with parturition; Discontinous basement membrane in
capillaries with reduction in collagen type IV and nidogen staining
but normal levels of perlecan. Adult null mice correct these defects
and have normal levels of collagen type IV, nidogen, perlecan and
express laminin-10 in capillary basement membranes.
References
Embryonic lethality between E13.5 and 16.5 with limb,
neural tube and placental defects. Placental labyrinth is
malformed which primarily consists of interacting trophoblasts
and endothelial cells with intervening basement membranes.
At E13.5-16.5, vessels were present in placental labryrinth, but
the branching complexity was markedly reduced and vessel
diameters were signficantlyincreased. In normal embryos,
trophoblasts and ECs are separated only by a basement membrane
while in the mutant, only the ECs remained adherent to the
basement membrane creating a cell-free space.
Embryonic lethality between E10.5-E11.5; basement
membrane laminin and nidogen-1 were detected underlying
epithelia and endothelia, although showed weaker and more
patchy staining. Areas of basement membrane discontinuity
and fragmentation were apparent. Excessive amounts of
maternal blood was present in the yolk sac cavity indicative
of hemorrhage; evidence of pericardial bleeding.
Impairment of the placental labyrinth was apparent as its
thickness was reduced. At the time of embryonic death, dilated
blood vessels were observed. Overall, vascular development
appeared relatively normal with reduced capillary density
sprouting into the neural layer. However, hemorrhage was
observed in the heart and arteries with excessive yolk sac
hemorrhage indicative of abnormalities in EC basement
membrane contacts or stability.
Embryonic lethality between E10-E12 as well as perinatally.
The lethality occurs due to hemopericardiumand cardiac
arrest. There is no evidence for placental or vascular
defects.
There are few apparent vascular developmental defects
except for some microaneurysms that form in several tissues
Including lung, skin and brain.
Viable mice, some seizure-like symptoms with loss of muscle
control in hindlimbs, no discernable vascular phenotype.
Overall, normal basement membrane assembly and
immunostaining of major basement membrane components
Minimal to no vascular abnormalities, some structural
abnormalities in basement membrane matrix of brain
capillaries. Nidogen 2 appears to be expressed in higher
amounts in EC basement membranes compared to other
basement membranes. Both null phenotypes suggest
probable compensation.
Viable mice with eye abnormalities. Overall, vascular
basement membranes and vascular development (with the
exception of the eye) appear normal.
Delayed regression of hyaloid vessels in the vitreous;
abnormal outgrowth of retinal vessels which may be
secondary to the lack of regression and/or alterations
in VEGF expression through changes in retinal hypoxia.
Thyboll et al, 2001
Miner et al., 1998
Poschl et al., 2004
Costell et al., 1999
Miosge et al., 2002;
Murshedet al., 2000
Fukai et al., 2002
126
127
116
119
151, 154
134

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