1 Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department
of Surgery, University of California, San Francisco, CA 94143-0507, USA
2 Veterans Administration Medical Research Center, Surgical Services (112G), San
Francisco, CA 94121, USA
3 Department of Cell Biology and Anatomy, University of Arizona College of
Medicine, Tucson, AZ 85724, USA
Author for correspondence (e-mail:
rongw{at}itsa.ucsf.edu)
Accepted 12 May 2005
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SUMMARY |
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Key words: Angiogenesis, Space of Disse, Sinusoids, Vascular development
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Introduction |
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Proper formation of the intricate sinusoidal vascular network provides all
hepatocytes with direct access to the blood plasma and efficient lipoprotein
metabolism. A cooperative effort between hepatocytes and endothelial cells
(ECs) during liver organogenesis may be required for the proper development of
SECs and the space of Disse. In support of this, Matsumoto et al. recently
showed that ECs are required for liver bud emergence into the septum
transversum mesenchyme at embryonic day 9.5 (E9.5), and that ECs delimit the
extent of hepatic cell migration
(Matsumoto et al., 2001).
These interactions suggest that there are specific signals that are
communicated between ECs and endoderm that stimulate liver organogenesis.
Morphological studies have highlighted the importance of the space of Disse,
and SEC and hepatocyte structural features, such as fenestrations and
microvilli, respectively, for effective lipoprotein-remnant passage from the
blood to hepatocytes (Fraser et al.,
1995
). It is therefore likely that there are communication signals
between these two cell lineages that are responsible for establishing the
structural characteristics of these cell types. We have chosen to undertake a
genetic approach to test the hypothesis that disruption of the liver
vasculature would affect the ability of the liver to properly function in
lipoprotein homeostasis.
Vascular endothelial growth factor (VEGF) is a potent angiogenic regulator
required for embryonic development. Mice heterozygous for the VEGF allele
(VEGF+/) are embryonic lethal by E12
(Carmeliet et al., 1996;
Ferrara et al., 1996
).
VEGF+/ and VEGF/ embryos display
severely abnormal vasculature and organ development. In addition to being
required for embryonic development, VEGF is also required for continued
development in neonatal mice (Gerber et
al., 1999
). In the liver, hepatocytes of
VEGF/ mice have an abnormal morphology and an
underdeveloped sinusoidal network. Additionally, VEGF inactivation leads to a
significant increase in EC apoptosis in neonatal mice, suggesting a role for
VEGF in EC survival (Gerber et al.,
1999
).
Communication between hepatocytes and ECs through VEGF signaling has been
suggested by the expression patterns of VEGF by hepatocytes
(Mochida et al., 1998;
Yamane et al., 1994
), and of
its tyrosine kinase receptors VEGFR1 (FLT1) and VEGFR2 (FLK1/KDR) by ECs
(de Vries et al., 1992
;
Quinn et al., 1993
).
Differential signaling of VEGF through its receptors highlights the
intricacies of this communication system in the liver. Signaling through
VEGFR1 induces SECs to release various cytokines that stimulate hepatocyte
proliferation; signaling through VEGFR2 stimulates SEC proliferation
(LeCouter et al., 2003
).
During regeneration of the adult liver following partial hepatectomy,
expression of both VEGF and its receptors is upregulated
(Mochida et al., 1998
;
Ross et al., 2001
;
Sato et al., 2001
;
Shimizu et al., 2001
). Adult
rats undergoing partial hepatectomy concomitant with VEGF administration
showed a significant increase in both SEC and hepatocyte proliferation
(Taniguchi et al., 2001
).
Conversely, rats treated with an anti-VEGF antibody following partial
hepatectomy displayed significantly reduced proliferation of SECs and
hepatocytes (Taniguchi et al.,
2001
).
In this study, we hypothesized that VEGF signaling plays an important role in the development of the space of Disse and SEC structure during liver organogenesis. We chose to downregulate VEGF activity by using a liver-specific, conditional VEGF-knockdown system. We show here that downregulation of VEGF signaling does not cause detectable gross abnormalities in liver development, but that it selectively abrogates hepatic sinusoidal structure, resulting in incomplete lining of the sinusoidal lumen, decreased network complexity, lack of fenestrae, impaired lipoprotein uptake and decreased hepatocellular lipid content. These results demonstrate that VEGF signaling is both important for SEC development and required for lipoprotein uptake in the liver.
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Materials and methods |
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Western blot
Western-blot analyses were performed with a mouse anti-HA antibody (clone
12CA5, Exalpha Biologicals) or anti-albumin antibody (Accurate Chemical and
Scientific Corporation) at 20 µg/ml in blocking solution.
VEGF ELISA assay
VEGF protein concentration was determined using the Quantikine Mouse VEGF
Immunoassay (R&D Systems) according to the manufacturer's recommended
protocol.
Histology and immunohistochemistry
Tissues were fixed in 4% paraformadehyde (PFA)/phosphate-buffered saline
(PBS) for 24 to 48 hours and processed according to standard histology
procedures. Paraffin sections were analyzed for glycogen storage using the
Periodic Acid-Schiff (PAS) Staining System (Sigma). Proliferating cell nuclear
antigen (PCNA) staining was performed using the Zymed Kit (Zymed
Laboratories). Apoptosis was analyzed using a terminal deoxynucleotidyl
transferase biotin-dUTP nick-end labeling (TUNEL)-staining kit (Roche).
Immunohistochemistry was performed as previously described
(Wang et al., 2001). Rabbit
anti-VEGFR2 antibody (TO14) (Brekken et
al., 1998
) was used at a final concentration of 4 µg/ml. Rat
anti-CD31 antibody (clone MEC 13.3, PharMingen) was used at 5 µg/ml. Rabbit
anti-murine apolipoprotein (APO) E and anti-murine APOB antibodies
(Raffai and Weisgraber, 2002
)
were used at 1:1000 dilution. Signals were amplified with Vectastain Elite ABC
kits (Vector Laboratories). Signals were visualized using the DAB peroxidase
substrate kit (Vector Laboratories); sections were counterstained with
Hematoxylin.
Vascular perfusion
Using a 26-gauge needle, anesthetized newborn pups were perfused by
injection of 10 ml of PBS, followed by 5 ml of 1% PFA into the left ventricle
of their hearts.
Preparation of DiI-labeled murine lipoproteins and lipoprotein-uptake experiment
Murine remnant lipoproteins were prepared from plasma of
Apoeh/hLdlr/ mice
deficient in the low-density lipoprotein receptor as previously described
(Raffai, 2003; Raffai and Weisgraber,
2002). Remnant lipoproteins in this mouse model are composed of
APOB48 and APOB100-containing lipoproteins, both enriched with APOE (R.L.R.,
unpublished). Purified remnants were dialyzed against PBS (pH 7.2) and labeled
with the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI)
(Molecular Probes), as described (Pitas, 1981), and were adjusted to a
concentration of 0.5 mg/ml protein. DiI-labeled remnant lipoproteins (30
µl) were injected into the hearts of anesthetized newborn pups. After 10
minutes of circulation, the livers were harvested and fixed in 4% PFA/PBS at
4°C overnight, equilibrated in 30% sucrose overnight, and embedded in
optimal cutting temperature (OCT). Frozen liver sections (10 µm) were
mounted with VECTASHIELD mounting medium (Vector Laboratories).
ß-Galactosidase staining
Mouse embryos were fixed in PBS with 0.2% glutaraldehyde, 5 mM EGTA, 1 mM
MgCl2 for 1-3 hours at 4°C, then rinsed in PBS. For whole-mount
staining, embryos were incubated overnight at room temperature in PBS
containing 1 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium
ferricyanide, 2 mM MgCl2 and 0.02% NP40. After staining, samples
were washed in PBS, fixed in 4% PFA and stored in 2% PFA. For thick sections
(100-200 µm), fixed tissues were embedded in 4% low-melting agarose/PBS,
sectioned using a vibratome (VT1000S, Leica) and stained as described
above.
Oil Red O staining
Tissues were fixed in 4% PFA/PBS at 4°C overnight, equilibrated in 30%
sucrose overnight, and embedded in OCT. Frozen liver sections were stained
with Oil Red O (Sigma) for 10 minutes and counterstained with Hematoxylin.
Electron microscopy
For transmission electron microscopy (TEM), anesthetized newborn pups were
perfused with 0.1 M sodium cacodylate (pH 7.4) and then fixative solution (2%
glutaraldehyde, 1% PFA, 0.1 M sodium cacodylate pH 7.4). The liver was
harvested and fixed at 4°C for 1-2 hours, dissected into 1-2 mm sections
and fixed for an additional hour at 4°C. Standard procedures for TEM were
then performed.
Routine methods were used to prepare liver specimens for scanning electron
microscopy (SEM) (McCuskey,
1986). Livers were fixed by perfusion with 0.1 M cacodylate
buffer, followed by 1.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4).
Pieces of fixed livers were dehydrated in a graded ethanol series,
critical-point dried, fractured, sputter-coated with 10 nm gold and examined
using the XL35 scanning electron microscope (Philips Electronic
Instruments).
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Results |
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To determine the onset and tissue specificity of VEGFR2T expression, the
LAP-tTA mice were bred with a lacZ reporter line,
TRE-lacZ (Redfern et al.,
1999). ß-Galactosidase activity was observed specifically in
the livers of double-transgenic (LAP-tTA/TRE-lacZ) embryos as early
as E10.5 (Fig. 1C). At E11.5, a
liver-specific, speckled lacZ expression pattern was observed
(Fig. 1D). These results
indicate that tTA is expressed in a subset of hepatocytes early after the
initial onset of liver organogenesis (E9.5)
(Matsumoto et al., 2001
).
|
VEGFR2T-expressing newborn mice exhibit a dark-red liver phenotype
We next focused on the phenotypic analysis of newborn mice that expressed
VEGFR2T throughout embryonic development. Analysis of the progeny of two
TRE-VEGFR2T transgenic lines revealed that 26-38% of
double-transgenic newborns were stillborn, and 30-63% were jaundiced
(Table 1). By comparison,
control LAP-tTA littermates never exhibited signs of jaundice and all
were born alive. While the body weight and liver:body weight ratios of both
LAP-tTA and LAP-tTA/TRE-VEGFR2T newborns were comparable
(Fig. 2B,C), 100% of
double-transgenic livers were dark-red in appearance, in contrast to the pink
color of normal control livers (Fig.
2A). No detectable abnormalities were observed in all other organs
examined. Given the 100% penetrance of the dark-red liver phenotype, this was
the most striking gross phenotype of the double-transgenic mice.
|
Because our transgene was expressed in hepatocytes, we investigated whether
it had induced any direct effects in hepatocytes that may have been
responsible for the observed phenotype. Both liver albumin
(Fig. 2D) and glycogen levels
(data not shown) were similar in double-transgenic and control livers. Serum
bilirubin levels were comparable in mutants and controls (data not shown).
Furthermore, no visual differences were observed between mutant and control
hepatocytes that were isolated from adult livers and cultured in vitro (data
not shown). Recent studies have shown that VEGF signaling through EC-specific
receptors can regulate hepatocyte proliferation during adult liver
regeneration (LeCouter et al.,
2003). We therefore evaluated hepatocyte proliferation in
VEGFR2T-expressing mice. Newborn liver sections stained with PCNA showed no
significant change in hepatocyte proliferation in double-transgenic livers
(Fig. 2E), demonstrating that
hepatocyte growth is normal in these mice. Hepatocyte cell death, as
determined by TUNEL assay, was also not significantly different in livers of
control and mutant mice (Fig.
2F). Thus, downregulation of VEGF activity did not result in any
detectable alteration of the function of hepatocytes.
|
|
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|
VEGF is required for vascular development throughout liver organogenesis
Because a disrupted vasculature and impaired lipid uptake are both evident
in newborn transgenic mice, we wanted to identify the developmental stage at
which these abnormalities manifested themselves. To visualize the liver
vasculature throughout embryonic development, we used a
VEGFR2lacZ reporter allele
(Shalaby et al., 1995) to
observe blood vessel ECs. At E11.5, the vasculature in the mutant and control
livers was comparable, having formed the primary plexus from which the mature
vasculature would arise (Fig.
6A,B). In control livers at E12.5, a blood vessel network was
apparent (Fig. 6C). However, in
the E12.5 mutant livers, the vasculature appeared to be less organized and the
vessels were dilated (Fig. 6D).
At E13.5, major blood vessels had formed in both the control and mutant
livers, but the microvascular network in the mutant livers appeared to be more
disorganized, with fewer branches than the control livers
(Fig. 6E,F). At the newborn
stage, very little microvascular network was evident in the mutant livers
(Fig. 6H), supporting the
immunohistochemical observations (Fig.
3F).
To further assess the temporal requirement for VEGF in this process, we used the Tet-regulatable feature of our transgenic system. Pregnant mothers were fed with Tet-supplemented drinking water during the initial stages of pregnancy, and Tet was removed at specific time points during gestation (Table 2). Owing to the time required for the complete metabolism of Tet, there is a 3-4 day lag between the time when it is removed from the diet and the time when the transgene (VEGFR2T) is expressed (data not shown). When VEGFR2T expression was turned on as late as E15.5-E16.5, 100% of newborn mutant mice exhibited a dark-red liver color, suggesting that hepatic lipid uptake was impaired in these mice. Anti-CD31 immunohistochemical staining showed a disrupted sinusoidal network in these mutant mice (Fig. 6J; Table 2), when compared with control mice (Fig. 6I), similar to the vascular defects observed in mice that expressed VEGFR2T throughout development (Fig. 3F). This result suggests that as little as 3-4 days of expression of VEGFR2T can sufficiently deplete VEGF in the liver, and the phenotypic observations are consistent with a role for VEGF in modulating the sinusoidal network development and SEC fenestrations necessary for proper lipid homeostasis. When VEGFR2T expression is turned on at E16.5-E17.5, newborn mutant mice exhibited a light-red colored liver and a disrupted vasculature. This intermediary phenotype suggests that only 2-3 days may not be sufficient to turn on VEGFR2T expression and to cause full-capacity depletion of VEGF, suggesting either a dose-dependent role of VEGF in SEC development or that the vasculature is fully developed by this stage and is no longer responsive to VEGF signaling.
|
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Discussion |
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Liver-specific VEGF depletion results in defective lipid uptake in hepatocytes
A defect in lipid homeostasis in VEGFR2T-expressing mice was first proposed
based on the gross phenotypical observations that their livers displayed a
dark-red color (Fig. 2A). In
the normal fetal liver, one function of the hepatocyte is to store and process
lipids. These vast fat stores give the liver a lighter-colored appearance;
reduced lipid stores may, therefore, result in a darker-colored liver
phenotype. Several lines of evidence supported this hypothesis. First,
histological analysis of the liver with Oil Red O staining revealed markedly
decreased intracellular lipid droplets in mutant livers
(Fig. 4). Second, TEM confirmed
a decrease in hepatocellular lipid droplets in mutant livers
(Fig. 5B,D). Third, the absence
of robust APOE localization in the sinusoids in mutant livers
(Fig. 4E,F) demonstrated an
aberrant space of Disse, which is normally enriched with APOE
(Mahley and Ji, 1999) and
functions to allow efficient remnant-lipoprotein clearance
(Raffai and Weisgraber,
2002
).
The most direct line of evidence demonstrating that aberrant hepatic-lipid
homeostasis in mutant mice was due to defective hepatic-lipoprotein uptake
came from a remnant lipoprotein-clearance study using fluorescently labeled,
murine remnant lipoproteins. In this experiment, control livers rapidly (in 10
minutes) took up the injected lipoproteins, which diffused broadly into the
hepatic layers; whereas, in the mutant livers, the labeled lipoproteins
remained mostly restricted to the sinusoids
(Fig. 4C,D). This pattern of
hepatic lipoprotein uptake is strikingly similar to what Ji et al. reported in
their classic experiments using intravenous heparinase to inhibit remnant
lipoprotein uptake in liver (Ji et al.,
1995).
Defects in hepatocyte morphology, consisting of abnormal cell shape and reduced numbers of microvilli (Fig. 5C-F) also contributed to reduced lipid homeostasis. Hepatocyte microvilli normally extend into the space of Disse, where they are bathed in extra-sinusoidal serum. The microvilli act to increase the surface area of the hepatocyte, allowing a more efficient passage of lipoproteins from the space of Disse. The observed reduction in hepatocyte microvilli in the livers of VEGFR2T-expressing mice may also explain the reduced uptake of lipoprotein remnants observed in mutant mice. It is unclear, however, why the number of microvilli is reduced. It could simply be a consequence of having a disrupted space of Disse in the mutant livers, or it might suggest that further communication between SECs and hepatocytes is necessary to stimulate microvilli projections. A caveat to our experimental design is that the overexpression of VEGFR2T on hepatocyte surface membranes may have a direct effect on hepatocytes, either on their ability to form microvilli or on the assembly of lipoprotein receptors within these villi. However, we have not detected any other abnormalities in the hepatocytes by other methods. Furthermore, in a similar transgenic system, in which the Met receptor was overexpressed in mouse hepatocytes to induce tumor formation, LAP-tTA/TRE-hMET mice did not exhibit the same defect (R.W., unpublished). This suggests that the observed defects in lipid homeostasis in our mice are specific to the VEGFR2T transgene and its depletion of VEGF signaling, and are not a consequence of non-specific receptor overexpression on hepatocyte membranes.
VEGF is required for the fenestrations in SECs that are required for lipoprotein passage
SEM revealed a lack of fenestrations in the SECs of the mutant livers,
suggesting that VEGF signaling is crucial in the morphogenesis of SECs. SEC
fenestrations function as a sieve to allow small lipoprotein remnants to pass
out of the sinusoids and into the space of Disse, where they can be
endocytosed by receptor-mediated mechanisms on the surface of hepatocytes. A
lack of fenestrae would inhibit lipoprotein remnant exit from the sinusoids,
thus reducing uptake by the liver and resulting in reduced lipid content in
hepatocytes.
VEGF has previously been suggested to play a role in maintaining the
integrity of endothelial fenestrations, on the basis of observations that VEGF
is continuously expressed by epithelial cells associated with fenestrated
endothelium, in organs such as the kidney and choroid plexus
(Breier et al., 1992;
Esser et al., 1998
).
Additionally, ectopic expression of VEGF has been shown to induce
fenestrations in normally unfenestrated ECs
(Roberts and Palade, 1995
).
Gerber and colleagues observed a loss of endothelial fenestrations in the
glomeruli of the kidney in mice treated with a dominant-negative soluble
VEGF-sequestering receptor, further supporting a role for VEGF in endothelial
fenestrae development (Gerber et al.,
1999
). It has also recently been shown that VEGF signaling during
development is necessary for establishing endothelial fenestrae in pancreatic
islets (Lammert et al., 2003
).
Our results represent the first demonstration of an essential role for VEGF
signaling in establishing and maintaining the fenestrations in the SECs of the
developing liver necessary for a functional space of Disse.
VEGF depletion impairs microvascular network formation throughout embryogenesis
In addition to structural defects in SEC, the vascular network is also
disrupted, even collapsed, in VEGFR2T-expressing liver, as shown by both
immunostaining and electron miscroscopy analyses. A disrupted vasculature
would also reduce access of hepatocytes to the sinusoids, which would impair
the process of lipoprotein passage from the blood, as we observed. A defective
vasculature resulting in impaired blood flow through the liver was also
evident by the accumulation of red blood cells. It is possible that these
accumulated red blood cells contribute to the color phenotype of the mutant
liver, in addition to the decreased hepatic lipid stores. In contrast to the
vascular system, the lymphatic system appears to be unaffected. Although
VEGFR2 (and VEGFR2T as well) can bind to the lymphatic growth factors VEGFC
and VEGFD, lymphatic endothelium-specific anti-LYVE1 staining did not reveal
any apparent differences between controls and mutants, suggesting that the
impairment of microvascular networks may not be secondary to the depletion, if
any, of VEGFC and VEGFD. Thus, VEGFR2T could specifically target VEGF
signaling in the liver.
Further analysis of the vasculature during embryonic development, using a
VEGFR2lacZ reporter allele, showed normal capillary plexus
formation at E11.5 (Fig. 6A,B).
Expression of the tTA was observed as early as E10.5
(Fig. 1C). It is possible that
VEGFR2T expression may not have been induced sufficiently early to affect
initial capillary plexus formation. Alternatively, but less likely, VEGF
signaling in the liver may not be required during this initial phase of
vascular development. However, remodeling of the vasculature after this stage
was significantly impaired in VEGFR2T-expressing mice. Although major blood
vessels formed in the mutant livers at E13.5
(Fig. 6F), the microvascular
network was generally characterized by fewer branches and thicker and more
disorganized vessels at E12.5 and E13.5
(Fig. 6D,F) than in control
livers. Very little sinusoidal vasculature remained at the newborn stage
(Fig. 6J). A similarly
compromised sinusoidal vasculature was observed when VEGFR2T-expression was
suppressed during development until E15.5-E16.5
(Fig. 6H,
Table 2), supporting a role for
VEGF signaling in the developing sinusoidal vasculature, and that this
signaling is required throughout organogenesis, beginning as early as E12.5 or
as late as E16.5. The partial phenotypes we observed in mice that expressed
VEGFR2T at E16.5-E17.5 indicate that sinusoidal vascular development is
regulated by VEGF in a dose- and/or time-dependent manner. While the embryonic
requirement for VEGF in the context of the whole organism has long been known
(Carmeliet et al., 1996;
Ferrara et al., 1996
), our
findings represent the first demonstration of a requirement for VEGF signaling
for the vascular development in a single organ, the liver.
VEGFR2T represents the most potent system for functionally depleting VEGF in a tissue-specific manner
In this study, we chose to use a membrane-bound VEGF-sequestering model
system to functionally deplete VEGF in the developing liver. VEGFR2T is
membrane bound, and therefore does not circulate to other organs systems,
making it specific to the liver when expressed under the control of the LAP
promoter. Overexpression of this allele allows for the sufficient
sequestration of secreted VEGF, even if VEGFR2T is not expressed by all
hepatocytes within the liver. The Tet-regulation feature of our VEGFR2T system
is essential, not only because it allows for temporal control of transgene
expression, but also because it allows for transgene suppression in breeding
stocks and control animals, which may be necessary when studying otherwise
lethal phenotypes. Combination of this regulation system with a liver-specific
promoter further strengthens the effectiveness of this model system in
dissecting the role of VEGF signaling and angiogenesis in liver development,
hepatic regeneration, and liver tumor progression. Additionally, this unique
and powerful system can be adapted for studying the effects of numerous other
transgenes in many other organ systems.
It should be noted, however, that other systems exist which may prove
useful for such analysis. One possible approach to deplete VEGF signaling
would be to use a soluble VEGF receptor system, such as VEGF-Trap
(Holash et al., 2002), which
would effectively sequester secreted VEGF and could eliminate any potential
non-specific defects caused by overexpressing a membrane-bound receptor.
However, such a soluble receptor system cannot be restricted specifically to
the liver, and therefore would be likely to cause significant defects in other
organ systems, as has been observed with other soluble VEGF receptors in
newborn mice (Gerber et al.,
1999
).
Cre/lox-mediated VEGF knockout is advantageous in that it can also eliminate potential artifacts due to overexpression of membrane-bound receptors, and it can be tissue specific, thereby reducing the occurrence of potentially confounding whole-embryo defects that a soluble receptor may cause. We performed such an experiment in our LAP-tTA/TRE-Cre/VEGFfloxed/floxed mice, in which Cre expression (and thus VEGF deletion) was hepatocyte specific. However, we observed only a 40% reduction in liver VEGF levels (data not shown). Although these mice did have a slightly underdeveloped sinusoidal vasculature, this defect was not as severe as that observed in VEGFR2T-expressing mice, and there was no observable reduction in hepatocellular lipid stores, as determined by Oil Red O analysis and liver color observations (data not shown). The incomplete knockout of VEGF in this system is likely to be a result of two phenomena. First, LAP-tTA (and hence Cre) expression is limited to only a subset of hepatocytes (Fig. 1D and data not shown). Therefore, only a fraction of VEGF alleles are deleted. Second, although hepatocytes are the primary VEGF-expressing cells in the liver, VEGF is also expressed by other cell types in the liver and can arrive via the circulatory system from other organs. Thus, even VEGF gene deletion in all hepatocytes will not eliminate all sources of circulating VEGF in the liver and would not achieve the sequestration effects of VEGFR2T.
Significance
Morphological evidence has long suggested a strong link between ECs and
hepatocytes in the process of lipid uptake by the liver. Our results present
the first genetic evidence for the existence of a signaling mechanism derived
from ECs responsible for lipid uptake by the liver. We have shown that VEGF
signaling in the liver is essential for the development of the functional
sinusoidal vasculature required for efficient plasma lipoprotein uptake.
Depletion of VEGF in the liver results in a disrupted vascular network, a lack
of SEC fenestrations and a non-functional space of Disse.
The role of VEGF in microvascular network and SEC structural development
has important pathological implications. Impaired remnant lipoprotein uptake
and clearance from plasma by the liver results in premature atherosclerosis
and cardiovascular disease (Fraser et al.,
1995; Mahley and Ji,
1999
). In addition to the defects associated with lipoprotein
uptake, defenestration itself is linked to other pathologies, including
cirrhosis and cancer (Mori, 1993). Although not necessarily implicated as a
cause of these diseases, defenestration of the endothelium occurs in both of
these situations. If decreased SEC porosity impairs the transport and/or
metabolism of metabolites or other nutrients necessary for normal liver
function and development, defenestration could be a factor in the development
of these, and other, pathologies.
The demonstration of a crucial role for VEGF signaling in the regulation of
SEC fenestration and lipid uptake by the liver is of particular significance
given the tremendous progress in the development of anti-VEGF therapies,
including the recently approved cancer therapeutic drug Avastin (Genentech)
(Muhsin et al., 2004). It is
conceivable that continued research and drug development based on VEGF
function may clarify the cellular mechanisms of plasma lipid metabolism, and
provide the treatment of cardiovascular disease caused by abnormal lipoprotein
metabolism and lipid uptake in the liver.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Auvation Limited, Aberdeen Science and Technology Park,
Balgownie Drive, Bridge of Don, Aberdeen AB22 8GU, UK
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