1 Department of Medical Biochemistry, Göteborg University, PO Box 440, SE
405 30 Göteborg, Sweden
2 Department of Genetics and Pathology, Rudbeck Laboratory, SE 751 85 Uppsala,
Sweden
3 Department of Experimental Pathology, Lund University, SE 221 85 Lund,
Sweden
4 Max Planck Institute for Biochemistry, Department of Molecular Medicine, Am
Klopferspitz 18a, D-82152 Martinsried, Germany
* Author for correspondence (e-mail: christer.betsholtz{at}medkem.gu.se)
Accepted 17 December 2003
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SUMMARY |
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Key words: PDGFB, Endothelium, Cre, loxP, Pericytes, Microaneurysm
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Introduction |
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VSMCs and PCs express PDGFRß, and therefore probably constitute a
primary target for PDGFB in the embryo
(Holmgren et al., 1991;
Lindahl et al., 1997
).
However, the critical source of PDGFB in the process of VSMC/PC recruitment is
not firmly established. Endothelial cells have been shown to express PDGFB in
vitro (Jaye et al., 1985
) and
in vivo (Hellström et al.,
1999
; Lindahl et al.,
1997
), suggesting that PC recruitment is controlled by short-range
PDGFB/PDGFRß signaling between the endothelium and the PCs. However,
several other cell types express PDGFB, for example megakaryocytes/platelets
and monocytes/macrophages (Heldin and
Westermark, 1999
). These sources are implicated in inflammation
and wound healing, but may contribute also to vascular development. PDGFB is
also expressed by neurons (Sasahara et
al., 1991
). It is not known whether any of the extra-endothelial
sources of PDGFB may have more long-range paracrine, or endocrine, effects on
VSMC and PC growth and vascular recruitment.
Pdgfb and Pdgfrb null mutant mouse embryos die
perinatally. Death is probably connected to microvascular dysfunction,
manifested as widespread microvascular hemorrhage and generalized edema
(Levéen et al., 1994;
Soriano, 1994
). The
PC-deficient mutant microvessels of PDGFB- and PDGFRß-deficient embryos
show endothelial cell hyperplasia, hypervariable diameter, abundant
microaneurysms, abnormal endothelial ultrastructure and signs of increased
permeability (Hellström et al.,
2001
). In addition to these widespread signs of microvascular
dysfunction, certain organs show rather specific defects, in particular the
kidney, heart and placenta (Levéen
et al., 1994
; Ohlsson et al.,
1999
; Soriano,
1994
). In kidneys, the glomeruli are malformed as a consequence of
deficient recruitment of mesangial cells
(Levéen et al., 1994
;
Soriano, 1994
). Like PCs,
mesangial cells express PDGFRß and may be attracted into the developing
glomerular tuft by PDGFB released from endothelial cells
(Lindahl et al., 1998
). Other
studies, however, have localized glomerular PDGFB expression to podocytes
(Alpers et al., 1992
) and to
the mesangial cells themselves (Floege et
al., 1992
). Thus, the critical source of PDGFB in glomerulogenesis
remains unclear.
To what extent the heart and placenta abnormalities of Pdgfb and
Pdgfrb mutants are secondary to microvessel dysfunction is also not
established. VSMC and PC recruitment is defective in both organs
(Lindahl et al., 1997;
Ohlsson et al., 1999
), and
hence these organ defects may be secondary to microvascular dysfunction;
however, other functions for PDGFB/PDGFRß have also been suggested at
these sites. Cardiomyocytes express PDGFRß transiently during early
development (Soriano, 1994
),
and chimeric analysis has indicated that PDGFRß may have a
cell-autonomous role in these cells as well as in other muscle lineage cell
types (Crosby et al., 1998
).
Placenta trophoblasts express PDGFB and PDGFRß
(Goustin et al., 1985
), and
Pdgfb or Pdgfrb null mice show a reduction in trophoblast
numbers (Ohlsson et al.,
1999
), suggesting that the proliferation of these cells is under
autocrine control.
In order to directly test the relative importance of various cellular
sources of PDGFB in embryonic development, we have taken advantage of the
Cre-lox system for conditional gene inactivation
(Gu et al., 1994;
Rajewsky et al., 1996
). We
generated mice carrying a Pdgfb allele flanked by loxP sites
(Enge et al., 2002
) and
crossed these with transgenic mice that express Cre under the vascular
endothelial specific Tie1 promoter
(Gustafsson et al., 2001
). The
resulting offspring showed compromised PC recruitment to retinal blood vessels
and developed a diabetic retinopathy-like condition
(Enge et al., 2002
). In the
present study, we compare the endothelium-specific Pdgfb knockout
with Pdgfb null mice in order to clarify the role of endothelial
PDGFB expression during development. Interestingly, we found that
endothelium-specific knockouts and complete Pdgfb null embryos
developed a similar set of defects in glomeruli, microvessels, heart and
placenta. However, in contrast to the null mutants, most endothelium-specific
knockouts survived into adulthood with persistent microvascular pathology.
Thus, these mice allowed us to unambiguously identify endothelial-derived
PDGFB as source for PC recruitment and to address the role of PCs in the adult
microvasculature.
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Materials and methods |
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Histological analysis
Tissues were fixed in 4% paraformaldehyde (PFA), paraffin embedded and
sectioned at 4-5 µm. Sections were stained with Hematoxylin and Erythrosin
(H/E) according to standard protocols, with -smooth muscle actin (SMA)
as previously described (Hellström et
al., 1999
), and with Periodic Acid Schiff (PAS), Hotchkiss McManus
modified (Bio-optica 130802). Staining of whole tissues and sections for
ß-galactosidase were performed as described
(Hogan et al., 1994
), and
sections were counterstained with Erythrosin. Images were captured using a
Nikon Eclipse E1000 microscope. Vibratome brain sections (200 µm thick)
were stained with isolectin (Bandeiraea simplicifolia, Sigma L-2140)
and GFAP labeling was achieved using a polyclonal rabbit antibody (1:75, Dako
Z 0334) as described (Enge et al.,
2002
). Confocal images were captured using a Leica TCS NT
microscope system. All images were processed using Adobe Photoshop
software.
Glomerulus morphometry
The maximum capillary diameter was measured in individual glomeruli on
H/E-stained sections (as illustrated in
Fig. 3). Mature glomeruli were
selected at random from different kidney areas of embryonic day (E) 18.5 and
postnatal day (P) 21 mice. In P21 mice, only glomeruli in the deep
peripapillary region of the cortex were selected for analysis in order to
compare glomeruli of a similar size and developmental age. The number of
glomeruli analyzed ranged from 13 to 22 per animal at E18.5, and from 19 to 42
in the adult animals. Images were captured using a Nikon Eclipse E1000
microscope equipped with a Nikon Plan Fluor 60X lens. The Easy Image
Measurement 2000 software was used to measure the actual capillary diameter,
giving each animal its own range of values. P values were calculated
using a t-test, two-sample unequal variance/two-tailed distribution.
Graphs were produced using Microsoft Excel software.
|
PDGFB protein measurements
Glomeruli were isolated as described
(Takemoto et al., 2002), and
frozen and thawed three times in lysis buffer [60% Acetonitrile, 1% Trifluoric
Acid (TFA) 1% Tween]. PDGFB concentrations were then analyzed by homogenous
proximity ligation as described
(Fredriksson et al., 2002
).
Prior to analysis the samples were diluted in 100 mM Tris (pH 8.0). After
dilution, 1 µl of each sample was analyzed in triplicate. The analyses were
carried out on 22 mice belonging to two age groups: 6 months and >1 year.
P values were calculated using a t-test, two-sample unequal
variance/two-tailed distribution. Graphs were produced using Microsoft Excel
software.
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Results |
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Renal abnormalities in endothelium-specific Pdgfb mutants
Endothelial cells in the developing kidney glomerular tuft express Tie1
(Lindahl et al., 1998). To
confirm that the Tie1 promoter directs Cre expression to the renal
endothelium, we crossed Pdgfb mutants with the R26R reporter strain
(Soriano, 1999
) and identified
Cre-activity using ß-gal staining
(Fig. 2). In the kidney,
abundant ß-gal staining was seen in the glomeruli, as well as in the
tubular interstitium (Fig. 2A). At higher magnification, it was evident that the ß-gal staining was
confined to endothelial cells within the glomerular
(Fig. 2B,C) and interstitial
capillaries (Fig. 2D,E), and
that most but not all endothelial cells were ß-gal positive
(Fig. 2E). Based on these
observations, we conclude that the Tie1-Cre transgene is expressed in the
vascular endothelial cell population in the kidney. The proportion of
ß-gal-positive versus negative endothelial cells in the kidney (>70%)
is comparable to the ratio of recombined versus unrecombined Pdgfb
flox alleles in brain capillaries reported previously
(Enge et al., 2002
),
suggesting that the R26R and Pdgfb flox alleles recombine with
similar efficiency in the presence of Cre. To confirm that recombination at
the Pdgfb locus led to attenuated PDGFB protein levels in the
glomerulus, we applied a sensitive and specific method based on PDGFB-binding
aptamers and proximity-dependent DNA ligation
(Fredriksson et al., 2002
).
Using this method, we could demonstrate a reduction in PDGFB protein levels in
glomerular preparations from lox/ mice in comparison with controls (see
below Fig. 4J,K).
|
|
The postnatal renal phenotype was analyzed using H/E-stained paraffin
sections of kidneys. Interestingly, the mesangial deficiency was largely
corrected in 3-week-old animals (Fig.
4A,B, and data not shown). However, all lox/ animals showed
glomerular dilation at this age in comparison with controls, both measured as
an increased glomerulus diameter (Fig.
4A,B) and as an increased diameter of the individual capillary
loops (Fig. 4C). By 6 months of
age the picture was further normalized and no morphological signs of increased
glomerular pathology were apparent in lox/ mice up to 21 months of age
(Fig. 4D-G). Increased
mesangial matrix accumulation has been observed in mutants of PDGFRß
signaling (Klinghoffer et al.,
2001), and in PDGFB retention
(Lindblom et al., 2003
)
mutants, but was not seen in the PDGFB lox/ glomeruli stained by
periodic acid-Schiff's (PAS) reagent (Fig.
4D-G). However, lox/ mice older than 12 months developed
mild but significant increases in albumin content in the urine
(Fig. 4I), suggesting that
lox/ glomerular filtration become abnormal at older age. The glomerular
PDGFB protein content was lower in lox/ mice of both age groups
although statistically significant differences were obtained only in mice
older than 12 months (Fig.
4J,K).
Cardiac and placenta abnormalities in endothelium-specific Pdgfb mutants
Both cardiac and placenta abnormalities have been reported in
Pdgfb and Pdgfrb mutant embryos at late gestation
(Hellström et al., 1999;
Levéen et al., 1994
;
Ohlsson et al., 1999
;
Soriano, 1994
). The cardiac
abnormalities include dilation, myocardial hypotrophy with thinning of the
myocardial wall, myocardial hypertrabeculation and septal abnormalities
(Hellström et al., 1999
;
Levéen et al., 1994
;
Soriano, 1994
) (M.
Hellström and M. Kalén, unpublished). When compared with
Pdgfb/, +/+ and various other controls
(flox/+, flox/, lox/+), we found that E18.5 lox/ embryos showed
similar heart abnormalities as the same age / embryos
(Fig. 5A-D). We also analyzed
the heart morphology at three different postnatal ages (1 month, 6 months and
>1 year). Like the kidney glomeruli, the heart phenotype normalized
histologically with age. At 1 month of age, the myocardium of lox/ mice
was already of normal thickness, and no statistically significant deviations
were noticed at older ages (data not shown). To address the cause of the
embryonic myocardial hypotrophy, we analyzed cardiac Cre expression in
Tie1-Cre/R26R mice. Expression was evident in the endocardium
(Fig. 2I), interstitial
capillaries (Fig. 2H), valvular
mesenchyme (Fig. 2F,G) and
endothelium of the large arteries (Fig.
2F,L). A chimeric situation was evident, with areas of
ß-gal-positive as well as negative cells
(Fig. 2J,K arrowheads). The
lacZ expression sites in the postnatal heart were compatible with an
early endocardial/endothelial expression, including the valvular mesenchyme,
which is derived from the cardiac cushion. The cardiac cushion arises through
mesenchymal transdifferentiation of endocardial cells, and has previously been
shown to express lacZ in Tie1-Cre/R26R crosses
(Gustafsson et al., 2001
).
|
Endothelium-specific Pdgfb deletion leads to impaired PC recruitment to brain microvessels
To allow for visualization of PC recruitment to brain microvessels, we
crossed the Tie1-Cre/Pdgfb/flox alleles onto the XlacZ4
transgenic background in which lacZ expression is restricted to
vascular smooth muscle cells and PCs from late gestation onwards
(Abramsson et al., 2002;
Klinghoffer et al., 2001
;
Tidhar et al., 2001
).
Whole-mount ß-gal-staining of E15.5 brains allowed for quantification of
PC density in various brain regions following dissection. Using this method,
we observed a significant reduction in PC density in lox/ embryos. We
compared the PC densities in the midbrain of control,
Pdgfb/ and lox/ embryos
(Fig. 6). Vascularization of
this region follows a stereotypical pattern: blood vessels enter the brain
tissue perpendicular to the surface (radial branches) and ramify to form a
capillary plexus in the subventricular zone. XlacZ4-positive PCs were
found in association with the radial vessels, as well as with the vessels of
the subventricular plexus, in both mutants and controls, however the densities
differed markedly. The lox/ embryos showed intermediate densities
between those of the control and / situations [70-90% reduction
the two lox/ embryos shown (Fig.
6C,D,G,H,K,L), compared with >95% reduction in the
Pdgfb/ embryo
(Fig. 6B,F,J)]. Both the
lox/ and Pdgfb/ mutants showed
irregular capillary diameter, with focal distensions (microaneurysms) and
hemorrhages (Fig. 6B-D,G,H). A
noticeable variation in PC coverage between neighboring capillaries was seen
in lox/ midbrain (Fig.
6K). Even in the lox/ embryo with a 90% overall reduction
in XlacZ4-positive PCs, individual capillaries were found with
seemingly normal PC coverage and distribution
(Fig. 6L). This result would be
expected from a chimeric situation in which most capillaries are composed of
PDGFB-negative endothelial cells, whereas individual capillary segments may be
composed of mostly unrecombined cells retaining PDGFB expression, which hence
stimulate local recruitment of PCs.
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Discussion |
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The importance of endothelium-derived PDGFB in PC, VSMC and mesangial
recruitment suggests that PDGFB acts mainly through a short-range paracrine
route. The inability of hematopoietic cell-derived PDGFB to compensate for the
lack of endothelial PDGFB may seem surprising, given the presence of platelets
and monocytes two cell types with proven capacity to produce and
release high levels of PDGFB in developing microvessels. However,
these cells probably release PDGFB mainly in conjunction with thrombus
formation, blood clotting and inflammatory conditions
(Heldin and Westermark, 1999).
An important role for hematopoietic PDGFB in vascular turnover and homeostasis
is also unlikely given the lack of vascular pathology in mouse radiation
chimeras substituted with PDGFB-negative bone marrow
(Kaminski et al., 2001
). We
have also failed to see any effects on vascular development by
macrophage-specific knockout of PDGFB (M.E., unpublished). Likewise, neuronal
PDGFB expression, which appears at detectable levels at late gestation, and
increased postnatally (Sasahara et al.,
1992
; Sasahara et al.,
1991
), is not able to compensate for the loss of endothelial PDGFB
in the CNS vasculature. A neuron-specific knockout of PDGFB also lacked
detectable vascular as well as neuronal and glial phenotypic abnormalities
(Enge et al., 2003
). In
summary, therefore, the only source of PDGFB with a clear developmental
function detected so far is the vascular endothelium.
Role of endothelial PDGFB in glomerular development and postnatal function
Both genetic approaches and the use of pharmacological inhibitors have
defined a crucial role for PDGFB and PDGFRß in mesangial cell recruitment
into developing glomeruli (Levéen
et al., 1994; Lindahl et al.,
1998
; Sano et al.,
2002
; Soriano,
1994
). Our present data identify the glomerular endothelium as a
critical PDGFB source in this process. To our surprise, the severe reduction
in mesangial cells observed at late embryonic age was largely corrected
postnatally. By 3 weeks of postnatal age, the glomeruli appeared to have
acquired a normal number of mesangial cells, although a mild increase in
capillary diameter (and total glomerular diameter) persisted. The reason for
this recovery is unclear. One possibility is that PDGFB regulates the rate of
mesangial cell recruitment, but is not exclusively involved in the process. In
such a scenario, PDGFB would be expected to cause a delay rather than an
irreversible block in mesangial cell recruitment. Alternatively, other sources
of PDGFB (such as macrophages or podocytes) could substitute for the loss of
endothelial PDGFB, which could potentially be tested by generating
combinations of, for exampl, endothelial, podocytic and hematopoietic
knockouts. Such crosses are ongoing in our laboratory. The partial deletion of
the Pdgfb gene could also provide an explanation, as a few remaining
PDGFB-producing endothelial cells in each glomerulus might be enough for
delayed recruitment. The recently discovered novel PDGFRß agonists PDGFC
and PDGFD (Bergsten et al.,
2001
; Gilbertson et al.,
2001
; LaRochelle et al.,
2001
; Li et al.,
2000
) also raise the possibility that other PDGF ligands could
replace PDGFB as the PDGFRß ligand in glomerulogenesis. So far we have
been unable to detect PDGFD expression at the mRNA level in developing
glomeruli. Although PDGFC is primarily a PDGFR
ligand, it might mediate
PDGFRß signaling through PDGFRß/
heterodimers
(Gilbertson et al., 2001
).
PDGFC is expressed by metanephric mesenchyme in the developing kidney
(Ding et al., 2000
;
Li et al., 2000
), and both
PDGFR
and PDGFRß are expressed by developing mesangial cells
(Eitner et al., 2002
;
Floege et al., 1997
;
Lindahl et al., 1998
;
Matsumoto et al., 2002
).
Finally it is possible that factors other than PDGFs may play a role in
mesangial cell recruitment.
The albuminuria observed in lox/ mice older than 12 months suggests
that endothelium-derived PDGFB has a role in the maintenance of glomerulus
function. PDGFB is upregulated in glomeruli in conjunction with injury,
suggesting a protective role that may be operational also in the normal aging
glomerulus. One would expect such a role to be mediated by the mesangial cells
(as these are the only glomerular cells known to harbor PDGF receptors).
However, the albuminuria implicates a problem with the podocytes, the
endothelial cells or the intervening glomerular basement membrane. Therefore,
the age-associated glomerular pathology in the lox/ mice probably
reflects that mesangial cells influence the function of other glomerular cell
types. Interestingly, the lox/ albuminuria was not accompanied by signs
of morphological glomerular abnormalities. Glomerular extracellular matrix
accumulation (glomerulosclerosis) has previously been reported in PDGFRß
signaling mutants, in which the intracellular domain was replaced with that of
PDGFR (Klinghoffer et al.,
2001
), and in PDGFB retention-motif knockouts, in which the
C-terminal heparan sulphate proteoglycan-binding motif was deleted
(Lindblom et al., 2003
). In
the latter case, albuminuria preceded glomerulosclerosis, which might suggest
that glomerulosclerosis would develop also in the lox/ mice if they
lived long enough.
Why do Pdgfb null mice die and endothelium-specific Pdgfb knockout mice survive?
Considering the extensive PC loss, the prevalent microvascular bleedings,
and the glomerular, cardiac and placental changes, it was a surprise to find
that the majority of the PDGFB lox/ mice survive birth and reach
reproductive age without overt problems, in sharp contrast to the null mutants
that invariably die before or at birth. The likely explanation for this,
supported by both genetic and morphological data, is that the
endothelium-specific Pdgfb knockout is incomplete, i.e. all
endothelial cells do not recombine their Pdgfb flox allele, resulting
in a chimeric vasculature with regard to endothelial PDGFB production. Genetic
analysis of freshly isolated microvascular fragments demonstrated that the
ratio between Pdgfb flox and lox alleles in the endothelium
varied between individual mice, but that most of them show more than 70%
recombination, and some more than 90%
(Enge et al., 2002). This
corresponds well with the data obtained by crossing Tie1-Cre with ROSA26R
mice, which resulted in Cre-activated lacZ expression in 50-90% of
the endothelial cells depending on the site analyzed
(Gustafsson et al., 2001
)
(this study). This correlates with the actual PC loss seen in Pdgfb
lox/ brain microvessels, which is in the order of 50-90% in different
individuals (Enge et al.,
2002
), compared with 95-98% in the complete knockout (this study).
The existence of parallel midbrain radial vessels devoid of, or normally
invested by, PCs in the endothelium-restricted knockouts also strongly
suggests that the vascular bed is chimeric with regard to endothelial PDGFB
expression and recruitment of PCs. It appears from these observations that
mice can tolerate up to 90% PC loss without severe organ dysfunction, but die
when the loss is more extensive. We have observed a small number of dead
newborn lox/ pups, which may represent rare individuals in which the
recombination efficiency led to higher than 90% PC loss (data not shown).
Based on our previous experience with inter-individual variation in the
Tie1-Cre-mediated recombination efficiency, we assume that the perinatal
lethal cases represent situations in which recombination is near complete. Our
data do not formally rule out the possibility that the perinatal death of
Pdgfb null embryos reflects the loss of non-endothelial expression of
PDGFB, but because selective knockout of any of the other major sources of
PDGFB (neurons or hematopoietic cells) leads to viable mice without any
obvious developmental or pathological defects, we consider this possibility
unlikely.
Diabetic microangiopathy-like condition in endothelium-specific Pdgfb knockout mice
Although the lox/ mice survive, they show several pathological
changes associated with microvascular dysfunction in the CNS, such as
microaneurysm formation, microhemorrhage, and associated astro- and
microgliosis. It is unclear whether these defects solely reflect the
persistence of the developmental defects, or whether they also develop as a
result of a need for continuous endothelial PDGFB expression in the postnatal
animal. Irrespective of the exact cause of the microvascular defects in the
adult mice, the Pdgfb lox/ mouse provides a model for
microvascular changes that bears striking resemblance to diabetic
microangiopathy (Cogan et al.,
1961; Engerman,
1989
; Kuwabara and Cogan,
1963
). As the microvascular pathology of Pdgfb
lox/ mice involves PCs and mesangial cells, which are both affected in
diabetic microangiopathy (Frank,
1991
; Wada et al.,
2002
), these mice may provide a useful model for some of the later
steps in this microvascular complication.
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ACKNOWLEDGMENTS |
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