|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 30 July 2008
doi: 10.1242/dev.017863
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-targeted deletion of bone morphogenetic protein receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis
1 Cardiopulmonary Research Program, Vera Moulton Wall Center for Pulmonary
Vascular Disease, Stanford University School of Medicine, Stanford,
California, CA XXXXX?, USA.
2 Department of Pediatrics, Stanford University School of Medicine, Stanford,
California, CA XXXXX?, USA.
3 Department of Medicine, Stanford University School of Medicine, Stanford,
California, CA XXXXX?, USA.
4 Molecular Developmental Biology Group, Laboratory of Reproductive and
Developmental Toxicology, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina, NC XXXXX?, USA.
* Author for correspondence (e-mail: marlener{at}stanford.edu)
Accepted 2 July 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
-Cre mice.
SM22-Cre;R26R;Bmpr1aflox/flox mice died
soon after embryonic day 11 (E11) with massive vascular and pericardial
hemorrhage and impaired brain development. At E10.5,
SM22-Cre;R26R;Bmpr1aflox/flox embryos
showed thinning of the myocardium associated with reduced cell proliferation.
These embryos also had severe dilatation of the aorta and large vessels with
impaired investment of SMCs that was also related to reduced proliferation.
SM22-Cre;R26R;Bmpr1aflox/flox mice showed
collapsed telencephalon in association with impaired clearing of brain
microvessels in areas where reduced apoptosis was observed. Transcript and
protein levels of matrix metalloproteinase (MMP) 2 and 9 were reduced in E9.5
and E10.5 SM22-Cre;R26R;Bmpr1aflox/flox
embryos, respectively. Knock-down of BMPR1A by RNA interference in
human pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated
serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA
interference of MMP2 or MMP9 recapitulated these
abnormalities, supporting a functional interaction between BMP signaling and
MMP expression. In human brain microvascular pericytes, knock-down of
BMPR1A reduced MMP2 activity and knock-down of either BMPR1A
or MMP2 caused resistance to apoptosis. Thus, loss of
Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for
vascular dilatation and persistence of brain microvessels, leading to the
impaired organogenesis documented in the brain.
Key words: Bmpr1a (Alk3), Vasculogenesis, Heart development, Craniofacial development, Matrix metalloproteinase (metallopeptidase), MMP2, MMP9, Smooth muscle cell proliferation, Pericyte apoptosis, SM22 (transgelin, Tagln), Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Mehra and Wrana, 2002).
Heteromeric complexes form between BMPR1 and BMPR2
(Gilboa et al., 2000).
Aberrant BMP signaling has been linked to pulmonary arterial hypertension
(PAH). Various germline mutations in BMPR2 have been identified in
familial and even sporadic forms of the disease
(Deng et al., 2000;
Lane et al., 2000;
Thomson et al., 2001).
Moreover, independent of a mutation, expression of BMPR2
(Atkinson et al., 2002) and
BMPR1A (Du et al.,
2003) is reduced in lungs of PAH patients.
PAH is a potentially fatal disease
(Abenhaim et al., 1996)
characterized by both obliteration of proximal pulmonary arteries resulting
from vascular smooth muscle cell (VSMC) proliferation and migration
(Jeffery and Morrell, 2002),
and loss of distal arteries associated with endothelial cell (EC)
(Campbell et al., 2001) and
pericyte apoptosis (Zhao et al.,
2003). These pathological features account for the progressive
increase in pulmonary vascular resistance culminating in right-side heart
failure (Humbert et al., 2004;
Rubin, 1997).
Mice homozygous null for Bmpr2
(Beppu et al., 2000),
Bmpr1a (Mishina et al.,
1995), the ligand Bmp4
(Winnier et al., 1995) and the
effector Smad4 (Sirard et al.,
1998) die early in embryonic life owing to a lack of mesodermal
induction. In mice with Flk1-targeted deletion of Bmpr1a
(Flk1-Cre;Bmpr1aflox/flox) (Flk1 is also known
Kdr - Mouse Genome Informatics)
(Park et al., 2006), lethality
occurs between E10.5 and E11.5, in association with massive abdominal
hemorrhage. These mice exhibit dilatation of large vessels owing to poor
recruitment of VSMCs around the EC layer, but it is not clear whether the
vascular phenotype is due to Bmpr1a-deficient ECs or SMCs
(Park et al., 2006).
In this study, we determined whether VSMC deletion of Bmpr1a could
cause abnormalities in vasculogenesis that might explain a propensity to PAH.
We bred mice expressing floxed Bmpr1a and ROSA26 with
SM22-Cre mice [SM22 is also known as
transgelin (Tagln) - Mouse Genome Informatics]. Progeny homozygous
for deletion of Bmpr1a,
SM22-Cre;R26R;Bmpr1aflox/flox, died soon after
E11 with massive vascular and pericardial hemorrhage. These mice had a thin
ventricular wall and aneurysmal dilatation of large vessels associated with
reduced myocyte proliferation related to decreased MMP9 and MMP2 activities.
Defective brain development documented in the
SM22-Cre;R26R;Bmpr1aflox/flox mice was
associated with impaired clearing of brain microvessels related to a
resistance of pericytes to apoptosis and decreased levels of MMP2.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
-Cre;R26R;Bmpr1aflox/flox mice-Cre mice with mice homozygous for
the floxed Bmpr1a gene (Mishina
et al., 2002) and the Cre reporter gene ROSA26
(R26R) (Soriano,
1999) (Bmpr1aflox/flox;R26R+/+). F2
breeding was then realized by backcrossing F1 mice
(SM22-Cre;R26R+/-;Bmpr1aflox/+)
with the Bmpr1aflox/flox;R26R+/+ mice to
produce mice that were
SM22-Cre;R26R;Bmpr1aflox/flox
(flox/flox). All studies were performed under a protocol approved by
the Animal Care Committee at Stanford University in accordance with the
guidelines of the American Physiological Society.
Genotyping
PCR was used to amplify Cre
(Saam and Gordon, 1999),
R26R (Soriano, 1999),
the floxed Bmpr1a gene (Mishina
et al., 2002), and the Bmpr1a gene with an exon 2
deletion (Mishina et al.,
2002), using DNA extracted from mouse embryonic yolk sacs.
Preparation of embryos for histological analyses
Isolated E9.5-11 mouse embryos were fixed with formalin or 4%
paraformaldehyde (PFA) in phosphate-buffered saline (PBS), embedded in
paraffin and cut transversely (7 µm).
Histology and immunostaining
Paraffin sections of brains, hearts and dorsal aortae of formalin-fixed
embryos were stained with Hematoxylin and Eosin (H&E) to assess the
phenotype resulting from deletion of Bmpr1a. To assess apoptosis, we
performed the TUNEL assay using the ApopTag Peroxidase In Situ Oligo Ligation
Apoptosis Detection Kit (Chemicon International, Temecula, CA). Sections were
counterstained with Methyl Green (Vector Labs, Burlingame, CA).
To assess alpha smooth muscle actin (SM-actin) or the proliferating
cell nuclear antigen (PCNA), formalin-fixed tissue sections were incubated
with either mouse anti-SM-actin antibody (1:200, Sigma-Aldrich, St
Louis, MO) or with biotinylated mouse anti-PCNA antibody (1:100, Zymed, South
San Francisco, CA). For SM-actin staining, sections were then incubated
with goat anti-mouse-biotinylated antibody (1:500, Jackson ImmunoResearch,
West Grove, PA). For both SM-actin and PCNA staining, sections were
incubated with streptavidin-horseradish peroxidase (HRP)-conjugated antibody
(1:500, Jackson ImmunoResearch). Brown immunoreactivity was observed by
subjecting the sections to diaminobenzidine substrate (DAB; Vector Labs).
Sections stained with antibodies to SM-actin and PCNA were
counterstained with Hematoxylin and Methyl Green, respectively.
To assess apoptosis in brain pericytes, TUNEL assay using the ApopTag Red In Situ Apoptosis Detection Kit (Chemicon) was followed by immunostaining for the pericyte marker NG2 (CSPG4 - Mouse Genome Informatics) (primary antibody, 1:100, Chemicon) on formalin fixed-head sections.
Expression of MMP2 and MMP9 in aortic walls and heart was analyzed in tissue sections of PFA-fixed embryos incubated with either an anti-MMP2 (Ab-4) mouse mAb (75-7F7) or an anti-MMP9 (Ab-3) mouse mAb (56-2A4) (1:100, Calbiochem, EMD Biosciences, San Diego, CA) followed by Alexa Fluor 488 goat anti-mouse IgG (H+L, 1:200, Molecular Probes, Invitrogen, Carlsbad, CA).
To assess BMP10 signaling in embryo hearts, PFA-fixed tissue sections were incubated with a p57KIP2 (CDKN1C - Mouse Genome Informatics) primary antibody (clone 57P06, 1:100, Neomarkers, Fremont, CA) followed by a biotinylated rabbit anti-mouse secondary antibody (1:250, BMK-2202, MOM Kit, Vector Labs) and ABC Reagent (PK6100, ABC Elite Kit, Vector Labs). Sections were then subjected to DAB+ (DAKO, Carpinteria, CA) and counterstained with Hematoxylin.
Whole-mount lacZ staining
E8.5-10.5 PFA-fixed mouse embryos were stained with 0.7 mg/ml X-Gal for
assessment under the microscope or were sectioned and counterstained with
Nuclear Fast Red (Vector Labs).
Whole-mount PECAM staining
PFA-fixed E10.5 mouse embryos were incubated with PECAM antibody [1:100,
rat anti-mouse CD31 (PECAM1), clone MEC13.3, BD Pharmingen, BD Biosciences,
San Jose, CA] followed by HRP-conjugated goat anti-rat IgG (1:500, Jackson
ImmunoResearch). Embryos were then subjected to DAB substrate (Vector Labs),
cleared (benzyl alcohol/benzyl benzoate) for better visualization of the
vascular tree and then assessed under the microscope or sectioned and
counterstained with Methyl Green (Vector Labs) for histological analysis.
Primary cell cultures and RNA interference (RNAi)
Adult human pulmonary artery smooth muscle cells (HPASMCs) and human brain
vascular pericytes (HBVPs) were cultured as previously described
(El-Bizri et al., 2008). Cells
were transiently transfected with control, human BMPR1A, MMP9 or
MMP2 siRNA (Dharmacon, Lafayatte, CO) in Opti-MEM I (Gibco,
Invitrogen) using Lipofectamine 2000 (Invitrogen). `Starvation media' (media
supplemented with 0.1% FBS) were added 6 hours later for a total of 48
hours.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from whole E9.5 mouse embryos or from HPASMCs or
HBVPs and then reverse transcribed as previously described
(El-Bizri et al., 2008). Gene
expression levels were quantified using preverified Assays-on-Demand TaqMan
primer/probe sets (Applied Biosystems, Foster City, CA) and normalized to 18S
RNA and B2M for murine and human samples, respectively, using the
comparative delta-CT method.
Cell proliferation (MTT) and apoptosis (caspase 3 and 7 activity)
Forty-eight hours following transfection in 0.1% FBS, HPASMCs were exposed
to 10% FBS for 72 hours, and cell growth was assessed by the MTT Cell
Proliferation Assay (American Type Culture Collection, Manassas, VA) and by
cell counts. Transfected HBVPs were kept under serum-free conditions for an
additional 24 hours, after which apoptosis was assessed by measuring caspase 3
and 7 activity using the Caspase 3/Caspase 7 Luminescent Assay Kit
(Caspase-Glo, Promega, Madison, WI), and proliferation was assessed by the MTT
assay.
Cell migration assay (Boyden Chamber)
Migration was assessed using a modified Boyden Chamber (BD Falcon, BD
Biosciences) as previously described
(Leung et al., 2004).
SiControl- and SiBMPR1A-transfected HPASMCs were stimulated to migrate for 6
hours in 0.1% FBS for baseline measurements and in response to 10% FBS or
PDGF-BB (20 ng/ml) (R&D Systems, Minneapolis, MN) as chemoattractants in
the lower compartments of the chambers.
Gelatin zymography
Conditioned media collected from the upper compartments of the Boyden
Chambers to evaluate production of MMPs in HPASMCs migrating in response to
0.1% FBS, 10% FBS or PDGF-BB (20 ng/ml) and from HBVPs after 48 hours of serum
starvation as well as extracts of individual mouse embryos were used for
gelatin zymography. The supernatants were subjected to electrophoresis in an
8% SDS-PAGE gel co-polymerized with gelatin (1 mg/ml, Sigma-Aldrich)
(Cann et al., 2008). The
gelatinolytic activities were detected as transparent bands against the
background of Coomassie Brilliant Blue-stained gelatin and quantified using
ImageJ.
Statistical analysis
Values for each determination are expressed as mean±s.e.m. For
comparisons between two groups, statistical significance was determined using
the unpaired two-tailed t-test. For comparisons of multiple groups,
one-way analysis of variance (ANOVA) followed by Bonferroni's
multiple-comparison test was carried out. The number of mouse embryos or
samples used in each experiment is provided in the figure legends.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
-targeted deletion of Bmpr1a; Roelen et al.,
1997). To assess the profile of Cre activity reflecting areas of
Bmpr1a deletion, we performed whole-mount lacZ staining on
SM22-Cre;R26R;Bmpr1aflox/+ mouse embryos
at E8.5-10.5 (Fig. 1B). Cre
activity was evident in the heart from E8.5 by blue lacZ staining
(data not shown), and in the heart and vasculature at E9.25
(Fig. 1Ba). At E10.5, smaller
intersomitic vascular branches and somitic myotomes showed positive
lacZ staining (Fig.
1Bb).
|
-Cre;R26R;Bmpr1aflox/flox mice
appeared normal until E9.5 (Fig.
1C, compare b with a). By E10.5, they were somewhat smaller than
wild-type (WT) (Fig. 1C,
compare d with c), and at E11 the mice showed massive perivascular and
pericardial hemorrhage (Fig.
1C, compare f with e) and died soon after. Following the breeding
strategy described in the Materials and methods, the expected frequency of the
flox/flox genotype was 25%. This frequency was observed by genotyping
embryos at different ages up to E11-11.5; the frequency was 10% at E12.5, and
there were no fetuses with this genotype by E18.0 or in postnatal mice
assessed after weaning (Fig.
1D).
Cardiac defect in SM22-Cre;R26R;Bmpr1aflox/flox embryos: thinning of the myocardium associated with reduced proliferation
Cre activity was confined to atrial and ventricular myocytes, with no
expression in the endocardium of E10.5 flox/flox embryos as assessed
by whole-mount lacZ staining (Fig.
2A,B). Myocardial deletion by SM22-Cre is
consistent with transient expression of SM22 in the developing heart
(Li et al., 1996;
Umans et al., 2007).
To assess the sequelae of loss of Bmpr1a in cardiomyocytes on
embryonic cardiac development, histological analysis of heart sections of
viable E10.5-11
SM22-Cre;R26R;Bmpr1aflox/flox and
age-matched littermate control embryos (WT) was carried out to show the four
chambers and outflow tract anatomy at multiple levels. We noted thinning of
the ventricular wall in the flox/flox versus WT hearts
(Fig. 2, compare D with C),
quantified as a 
35% reduction in the number of ventricular cells per
heart section (P<0.05) (Fig.
2E). The cardiac phenotype was not due to enhanced apoptosis as
only the occasional TUNEL-positive cell was seen
(Fig. 2F,G), but rather was
associated with attenuated cell proliferation. There was a reduction in the
percentage of PCNA-positive cells over the total number of ventricular cells
in heart sections of the flox/flox
(Fig. 2I) versus WT
(Fig. 2H) (P<0.05)
at E9.5 that persisted at E10.5-11 (Fig.
2J). As Bmp10-deficient embryos show thinning of the
myocardium associated with decreased cell proliferation and ectopic expression
of p57KIP2 (Chen et al.,
2004), we assessed the expression of p57KIP2 by immunofluorescence
to address the possibility that a deletion of Bmpr1a in the heart
might lead to a defect in BMP10 signaling. Our results showed no difference in
p57KIP2 immunoreactivity between mutant
(Fig. 2L) and WT hearts
(Fig. 2K).
Vascular defect in SM22-Cre;R26R;Bmpr1aflox/flox embryos: dilatation of large vessels associated with reduced proliferation of vascular smooth muscle cells
To characterize the vascular defect resulting in perivascular hemorrhage
and lethality in flox/flox embryos, we performed whole-mount PECAM
staining on embryos at E10.5. Gross morphological examination revealed massive
dilatation of the large vessels appreciated in the dorsal aortae, mesenteric
(Fig. 3Ab,d,f) and cranial
vessels (not shown) of the mutants versus WT littermates
(Fig. 3Aa,c,e). There were more
ramifications or interconnections in the interlimb vessels of the
flox/flox (Fig. 3Af)
versus WT (Fig. 3Ae) embryos.
H&E-stained transverse sections of E10.5 embryos showed dilated aortae
(Fig. 3Ah) in
flox/flox embryos relative to WT controls
(Fig. 3Ag).
|
-Cre-expressing lacZ-positive cells in
the dilated aortic wall of the
SM22-Cre;R26R;Bmpr1aflox/flox mutants
(Fig. 3Aj) as compared with WT
(Fig. 3Ai), where strong
lacZ staining was evident. The lacZ-positive cells were
identified as being of smooth muscle lineage by immunoperoxidase staining
using an antibody for SM-actin. There were also fewer surrounding
mesenchymal cells expressing SM-actin in
SM22-Cre;R26R;Bmpr1aflox/flox
(Fig. 3Bb) versus WT
(Fig. 3Ba) embryos. TUNEL
staining on sections of aorta revealed only occasional positive mesenchymal
cells (Fig. 3Bc,d, arrows).
Instead, the decreased number of SM-actin-positive perivascular cells
was consistent with reduced proliferation as assessed by PCNA staining
(Fig. 3B, compare f with e,
arrows). Quantitative analysis revealed a 53% reduction in the percentage
of PCNA-positive SMCs forming the vessel wall
(Fig. 3Bg)
(P<0.05). PECAM staining of sections did not reveal a difference
in the number of ECs surrounding the dilated vessels, but the cells appeared
`stretched' (data not shown).
Defective brain development of SM22-Cre;R26R;Bmpr1aflox/flox embryos associated with impaired clearing of small vessels
Cre activity was seen in the forebrain of an E10.5 WT embryo by whole-mount
lacZ staining (Fig.
4A). To characterize and better visualize any brain development
abnormality, we examined heads of embryos incubated with ethidium bromide
under UV light. Compared with WT (Fig.
4Ba,c), flox/flox mutant embryos
(Fig. 4Bb,d) showed brain
compression and collapse of telencephalic vesicles. These defects were
apparent in H&E-stained transverse sections of the heads at multiple
levels (Fig. 4Bf,h,j,l). To
determine how loss of Bmpr1a in SM22-expressing cells could
impair brain development, we performed whole-mount PECAM staining on embryos
at E9.5 and E10.5. We observed similar brain vessel distribution in the WT and
flox/flox mutants at E9.5 (data not shown); however, at E10.5, we
noted evidence of clearing of telancephalic vessels in the WT
(Fig. 4Ca,c) but not the
mutants (Fig. 4Cb,d).
Transverse sections of the brains stained for PECAM at the level of the
nasal-mandibular processes showed histologic evidence of clearing of vessels
in the WT heads (Fig. 4Ce),
whereas flox/flox mutant heads
(Fig. 4Cf) showed persistent
vessels (brown). To determine whether the clearing of vessels is related to
apoptosis, the TUNEL assay was performed on brain sections. TUNEL-positive
cells were plentiful in the WT (Fig.
4Cg) but were almost absent from the flox/flox mutant
heads (Fig. 4Ch). Quantitative
analysis showed a 62% reduction in the percentage of TUNEL-positive cells
over the total number of cells in the flox/flox group
(Fig. 4Ck)
(P<0.05).
Since pericytes express SM22
(Ding et al., 2004), we
speculated that loss of Bmpr1a in these cells led to resistance to
apoptosis and reduced clearing of brain microvessels. We therefore performed a
fluorescent TUNEL assay followed by fluorescent immunostaining for NG2, a
pericyte marker. Reduced apoptosis was associated with an increased number of
pericytes in the mutant (Fig.
4Cj) versus WT (Fig.
4Ci) brains. Because co-localization of the TUNEL and NG2 staining
was not observed in the WT brain (Fig.
4Ci), we could not confirm ongoing apoptosis of pericytes,
suggesting that this occurred before E10.5. Persistence of brain microvessels
was not due to enhanced cell growth, as PCNA immunoreactivity showed no
difference between E10.5 WT and flox/flox mutants
(Fig. 4Cl).
|
-targeted deletion of Bmpr1a; Palosaari et
al., 2003) and MMPs can regulate cell survival
(Jones et al., 1997) and
induce proliferation of VSMCs (Zempo et
al., 1994). Quantitative RT-PCR was applied to embryonic extracts
to assess differential expression of MMPs and other extracellular matrix genes
that could be modulated by loss of Bmpr1a and might account for these
altered vascular cell phenotypes. Many of the genes that we assessed are
modified in other embryonic mouse models of vascular dilatation
(Oh et al., 2000). We also
assessed the transcript levels of genes implicated in vasculo/angiogenesis -
vascular endothelial growth factor (Vegf; also known as
Vegfa) and the angiopoietins (Angpt1, Angpt2) - and of the
phosphatase and tensin homolog gene (Pten), a known gene downstream
of BMP signaling implicated in juvenile polyposis
(He et al., 2004) that might
also impact cell growth (Beck and
Carethers, 2007). RNA was extracted from E9.5 embryos, preceding
the appearance of the phenotype in
SM22-Cre;R26R;Bmpr1aflox/flox mice. We
found a significant decrease in the mRNA expression of Mmp9
(P<0.05) and Mmp2 (P<0.05), and trends toward
reduced expression of tenascin C (Tnc), fibronectin, connective
tissue growth factor (Ctgf) and urokinase plasminogen activator
(uPA; Plau - Mouse Genome Informatics) were observed
(Fig. 5A). No differences in
tissue plasminogen activator (tPA; Plat)
(Fig. 5A), Angpt1, Angpt2,
Vegf (data not shown) and Pten
(Fig. 5A) mRNA levels were
noted between the WT and mutants.
This decrease in Mmp2 and Mmp9 transcripts was associated
with a decrease, although not statistically significant, in the pro (40%) and
active (30%) forms of MMP2 in
SM22-Cre;R26R;Bmpr1aflox/flox mutants
versus WT, as assessed by gelatin zymography on mouse embryos (data not
shown).
To determine whether the decrease in mRNA levels of Mmp9 and Mmp2 in whole E9.5 mutant mouse embryos is translated into reduced protein expression at a later age, we performed immunostaining of MMP9 and MMP2 at E10.5. We found abundant MMP9 and, to a greater extent, MMP2, in the aortic walls of WT embryos (Fig. 5Ba,c) and only weak immunoreactivity in the mutants (Fig. 5Bb,d). However, a low and diffuse immunostaining was noted in the heart and brains of WT and mutants (data not shown).
|
Consistent with our hypothesis and our findings in the mouse embryo, we
showed that RNAi-mediated reduction in mRNA of BMPR1A (by 66%),
MMP9 (to undetectable levels) or MMP2 (by >80%), resulted
in a 35-40% reduction in HPASMC proliferation in response to 10% FBS as
assessed by the MTT assay (P<0.001,
Fig. 6B) and cell counts (data
not shown). Since MMP9 and MMP2 levels increase in migrating SMCs
(Bendeck et al., 2002;
Franco et al., 2006;
Kuzuya et al., 2003;
Mason et al., 1999), we
determined whether the chemotactic migratory behavior of SMCs was impaired by
loss of BMPR1A, in association with reduced MMP9 and/or MMP2. A
deficiency in SMC migration could also account for the lack of SMC investment
of the aneurysmally dilated vessels in flox/flox embryos. We serum
starved HPASMCs in 0.1% FBS for 48 hours and then assessed their response to a
6-hour treatment with PDGF-BB (20 ng/ml) using a modified Boyden Chamber
assay. The MMP9 and MMP2 activities in SiControl HPASMCs, as assessed by
gelatin zymography, were repressed in SiBMPR1A-treated cells
(P<0.01 for MMP9 and P<0.001 for proMMP2 and MMP2)
(Fig. 7A). Although basal
levels of migration were increased in SiBMPR1A-transfected HPASMCs
(P<0.001), these cells did not significantly migrate in response
to PDGF-BB when compared with SiControl HPASMCs (P<0.05)
(Fig. 7B).
|
|
). Using
RNAi under conditions of serum starvation (0.1% FBS) for 48 hours, we showed a
53% reduction in BMPR1A transcript levels in HBVPs. To induce
apoptosis, cells were serum deprived for an additional 24 hours, after which a
reduction in both pro and active forms of MMP2 was demonstrated by gelatin
zymography (P<0.05) (Fig.
8A). We then used RNAi to reduce mRNA levels of MMP2 in
HBVPs and confirmed the decrease in pro and active forms of MMP2 by gelatin
zymography (P<0.001) (Fig.
8B). A control experiment showing the sensitivity of gel
zymography to detect gelatinase activity in a dose-dependent manner is
provided in Fig. S1 (see supplementary material). Transfecting HBVPs with
SiBMPR1A, or with SiMMP2, induced resistance to apoptosis when compared with
SiControl HBVPs (P<0.001), as assessed by caspase 3 and 7
activities (Fig. 8C), without
affecting cell proliferation as assessed by the MTT assay (data not shown).
Therefore, lack of MMP2 in pericytes could account for the resistance
to apoptosis seen in highly vascularized areas of flox/flox mutant
brains.
|
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
-Cre;R26R;Bmpr1aflox/flox mouse
embryos was characterized by thinning of the ventricular wall and was
attributed to reduced cell proliferation evident at E9.5. Bmp10-null
mice also develop hearts with hypoplastic walls owing to reduced proliferation
of cardiac myocytes at E9.0-9.5 (Chen et
al., 2004). However, our results showed that BMP10 signaling in
E10.5 SM22-Cre;R26R;Bmpr1aflox/flox heart
sections was not affected by Bmpr1a deletion.
It is interesting that in the mouse in which Bmpr1a was deleted
following activation of the cardiac myocyte-specific promoter alpha myosin
heavy chain (MHC-Cre;Bmpr1aflox/flox)
(Gaussin et al., 2002), the
ventricular thinning that took place at a later time point (E11.5-12.5) was
attributed to enhanced apoptosis. Ventricular thinning was also seen at
E11.5-12.5 in mice lacking Bmpr1a in cardiac progenitors
(Islet1-Cre;Bmpr1a nulls) (Yang
et al., 2006), or at E11.5 with cardiac-specific ablation of
Smad4 (Song et al.,
2007), and both were associated with attenuated proliferation and
enhanced apoptosis of the ventricular septal myocytes. This suggests that
differences in the timing of promoter activation and Bmpr1a deletion
in cardiomyocytes might dictate whether the thinning of the ventricular wall
will be the result of apoptosis and/or reduced proliferation.
Bmpr1a and vasculogenesis
We cannot exclude the possibility that the myocardial thinning is secondary
to a hemodynamic abnormality caused by the vascular phenotype observed in the
SM22-Cre;R26R;Bmpr1aflox/flox embryos, and
characterized by aneurysmal dilatation of the dorsal aorta and other large
vessels. Dilatation of the aorta was observed in embryos with
Flk1-targeted deletion of Bmpr1a
(Flk1-Cre;Bmpr1aflox/flox)
(Park et al., 2006) and in
embryos null for Alk1 (Acvrl1 - Mouse Genome Informatics)
(Oh et al., 2000) or
Smad5 (Yang et al.,
1999). In those models, the dilatation was attributed to a
paracrine effect of Bmpr1a-deficient ECs repressing the recruitment
of VSMCs or pericytes, as observed in mice lacking PDGF-BB or PDFG-Rβ
(Hellstrom et al., 1999;
Lindahl et al., 1997). Other
possibilities suggested include poor transdifferentiation of ECs into SMCs, or
a defect in SMC growth affecting vessel maturation and integrity
(Park et al., 2006). The third
explanation fits best with the further delineation of the phenotype of
SM22-Cre;R26R; Bmpr1aflox/flox embryos
that we carried out.
|
-Cre;R26R;Bmpr1aflox/flox embryos by
whole-mount PECAM immunostaining (data not shown) that might explain the
vascular defect through a non-cell-autonomous contribution. In addition, we
did not observe upregulation of angiogenic factors, such as of Angpt1
and Angpt2 as described in Alk1-null embryos
(Oh et al., 2000), or of
Vegf as observed in both Flk1-Cre;Bmpr1aflox/flox
(Park et al., 2006) and
Alk1 nulls (Oh et al.,
2000). It follows that there was no concomitant angiogenic defect
in SM22-Cre;R26R;Bmpr1aflox/flox mutant
embryos, such as the impaired yolk sac vascular remodeling seen in the
Flk1-Cre;Bmpr1aflox/flox
(Park et al., 2006) and the
Alk1-null (Oh et al.,
2000) embryos. Since Flk1 is a mesodermal marker and
Alk1 is mostly expressed in ECs, the angiogenic defect in the yolk
sac is likely to be due to the loss of Bmpr1a in ECs, a feature we
reproduced by ablating Bmpr1a using Tie2-Cre (our
unpublished observations) (Tie2 is also known as Tek). In
contrast to other mice models of aneurysmal vascular dilatation, the
SM22-Cre;R26R;Bmpr1aflox/flox mice did not
exhibit an increase in expression of proteases such as uPA and
tPA (Oh et al., 2000;
Park et al., 2006). In
contrast to the Smad5-null embryos with dilated aorta, the
SM22-Cre;R26R; Bmpr1aflox/flox embryos did
not show apoptosis in VSMCs or in neighboring mesenchymal cells
(Yang et al., 1999).
When we assessed gene expression of extracellular matrix proteins and
proteinases previously implicated in VSMC proliferation and migration, a
consistent reduction in the expression of Mmp9 and Mmp2,
genes downstream of BMP signaling in other cell types
(Mishina et al., 2004;
Palosaari et al., 2003), was
observed. A direct association between reduced BMPR1A and impaired
production of MMP9 and MMP2 was then demonstrated in cultured human VSMCs, in
which knock-down of BMPR1A by RNAi attenuated MMP9 and MMP2
activities. The role of both MMP9 and MMP2 in VSMC proliferation and migration
is well documented (Bendeck et al.,
2002; Franco et al.,
2006; Kuzuya et al.,
2003; Mason et al.,
1999). Expression of Pten, downstream of Bmpr1a
and implicated in juvenile polyposis and affecting cell growth, was not
modified in the mutants.
Our observations linking reduced MMP9 and MMP2 to aneurysmal dilatation
might seem at odds with clinical studies in human tissue in which increased
MMP2 and especially MMP9 are observed in abdominal aortic aneurysm
(Goodall et al., 2001;
Thompson et al., 1995).
Moreover, reduction of MMP9 activity by Doxycycline protects against
experimentally induced aortic aneurysm
(Kaito et al., 2003), as does
local expression of TIMP1, an inhibitor of MMP9 activity
(Allaire et al., 1998;
McMillan et al., 1995). In
addition, mice that are null for Mmp9 are resistant to
elastase-induced aortic aneurysms (Pyo et
al., 2000). It therefore appears that during vascular development,
a reduction in both MMP9 and MMP2 in SMCs is required to produce aneurysmal
dilatation, as a result of reduced proliferation and perhaps migration of
SMCs. It is interesting that the Mmp2/9 double nulls
(Lambert et al., 2003) do not
recapitulate our phenotype. This could reflect compensatory induction of other
MMPs in response to a global, rather than a tissue-specific, deletion.
Alternatively, the mixed background of the flox/flox mutants compared
with the C57BL/6J background of the Mmp2/9 double nulls might account
for the difference in the phenotype.
Bmpr1a expression in pericytes mediates vessel regression during brain development
SM22-Cre;R26R;Bmpr1aflox/flox mutants
showed severe brain asymmetry and collapse of telencephalic vesicles. A
vascular defect produced by impaired BMPR1A signaling that has not previously
been described might explain these abnormalities.
Regression of vessels is crucial in triggering mesenchymal condensation
culminating in chondrogenesis and skeletogenesis
(Yin and Pacifici, 2001). As
MMP2 activity is linked to retinal pericyte apoptosis in diabetic retinopathy
(Yang et al., 2007), we
reasoned that suppression of MMP2 resulting from lack of BMPR1A signaling
might make pericytes resistant to apoptosis, preventing EC apoptosis and
microvessel clearing, and subsequently leading to defective brain development.
Indeed, we showed that lack of BMPR1A or MMP2 by RNAi
renders pericytes in culture resistant to apoptosis.
The same phenomenon might explain the enhanced ramification of the
interlimb vessels seen in the flox/flox mutants, suggesting that
vascular deletion of Bmpr1a might impair organogenesis of other
tissues not investigated here. In the rat aortic model of angiogenesis, MMP9
and MMP2 expression and activity not only increased during the angiogenic
growth phase of microvessels, but also remained elevated and were necessary
for microvessel regression (Zhu et al.,
2000). Consistent with this, maximal MMP2 activity is observed in
the late corpus luteum concomitant with vessel regression
(Duncan et al., 1998). The
deletion of Bmpr1a in brain cells
(Hebert et al., 2002) did not
recapitulate the phenotype, further indicating the importance of the
vasculature in this cell-autonomous mechanism.
The discrepancy between the phenotypes resulting from loss of
BMPR1A in VSMCs and pericytes might be related to the fact that they
have different basement membranes (Meyrick
and Reid, 1979) and hence could exhibit different effects
resulting from reduced MMPs (Fig.
9).
It is worth mentioning that our findings did not recapitulate any aspects
of juvenile polyposis (JP), a condition associated with mutations in
BMPR1A. However, the site of pathology in JP, the villus, forms after
E15.5 (Batts et al., 2006) and
SM22-Cre;R26R;Bmpr1aflox/flox embryos die
several days earlier.
Clinical significance
Our study is the first to show that both MMP9 and MMP2 are developmentally
regulated by expression of Bmpr1a and that attenuation in their
levels could reduce the proliferation of SMCs leading to aneurysmal dilatation
of large vessels. These observations could also explain the reduced cell
proliferation that leads to thinning of the ventricular wall. Our findings
linking repression of Bmpr1a-mediated MMP2 activity to reduced
apoptosis of pericytes, point to a feature not only of developmental
importance in clearing of microvessels, but potentially to a mechanism that
might help in preserving or regenerating microvessels in disease. In our
recent studies (El-Bizri et al.,
2008), in which patchy deletion of Bmpr1a was induced in
VSMCs, mice were actually protected against both the excessive muscularization
and loss of distal vessels associated with chronic hypoxia-induced PAH.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2981/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Abenhaim, L., Moride, Y., Brenot, F., Rich, S., Benichou, J.,
Kurz, X., Higenbottam, T., Oakley, C., Wouters, E., Aubier, M. et al.
(1996). Appetite-suppressant drugs and the risk of primary
pulmonary hypertension. International Primary Pulmonary Hypertension Study
Group. New Engl. J. Med.
335,609
-616.
Allaire, E., Forough, R., Clowes, M., Starcher, B. and Clowes,
A. W. (1998). Local overexpression of TIMP-1 prevents aortic
aneurysm degeneration and rupture in a rat model. J. Clin.
Invest. 102,1413
-1420.[Medline]
Atkinson, C., Stewart, S., Upton, P. D., Machado, R., Thomson,
J. R., Trembath, R. C. and Morrell, N. W. (2002). Primary
pulmonary hypertension is associated with reduced pulmonary vascular
expression of type II bone morphogenetic protein receptor.
Circulation 105,1672
-1678.
Batts, L. E., Polk, D. B., Dubois, R. N. and Kulessa, H.
(2006). Bmp signaling is required for intestinal growth and
morphogenesis. Dev. Dyn.
235,1563
-1570.[CrossRef][Medline]
Beck, S. E. and Carethers, J. M. (2007). BMP
suppresses PTEN expression via RAS/ERK signaling. Cancer Biol.
Ther. 6,1313
-1317.[Medline]
Bendeck, M. P., Conte, M., Zhang, M., Nili, N., Strauss, B. H.
and Farwell, S. M. (2002). Doxycycline modulates smooth
muscle cell growth, migration, and matrix remodeling after arterial injury.
Am. J. Pathol. 160,1089
-1095.
Beppu, H., Kawabata, M., Hamamoto, T., Chytil, A., Minowa, O.,
Noda, T. and Miyazono, K. (2000). BMP type II receptor is
required for gastrulation and early development of mouse embryos.
Dev. Biol. 221,249
-258.[CrossRef][Medline]
Campbell, A. I., Zhao, Y., Sandhu, R. and Stewart, D. J.
(2001). Cell-based gene transfer of vascular endothelial growth
factor attenuates monocrotaline-induced pulmonary hypertension.
Circulation 104,2242
-2248.
Cann, G. M., Guignabert, C., Ying, L., Deshpande, N., Bekker, J.
M., Wang, L., Zhou, B. and Rabinovitch, M. (2008).
Developmental expression of LC3alpha and beta: absence of fibronectin or
autophagy phenotype in LC3beta knockout mice. Dev.
Dyn. 237,187
-195.[CrossRef][Medline]
Chen, H., Shi, S., Acosta, L., Li, W., Lu, J., Bao, S., Chen,
Z., Yang, Z., Schneider, M. D., Chien, K. R. et al. (2004).
BMP10 is essential for maintaining cardiac growth during murine cardiogenesis.
Development 131,2219
-2231.
de Caestecker, M. (2004). The transforming
growth factor-beta superfamily of receptors. Cytokine Growth Factor
Rev. 15,1
-11.[CrossRef][Medline]
Deng, Z., Morse, J. H., Slager, S. L., Cuervo, N., Moore, K. J.,
Venetos, G., Kalachikov, S., Cayanis, E., Fischer, S. G., Barst, R. J. et
al. (2000). Familial primary pulmonary hypertension (gene
PPH1) is caused by mutations in the bone morphogenetic protein receptor-II
gene. Am. J. Hum. Genet.
67,737
-744.[CrossRef][Medline]
Ding, R., Darland, D. C., Parmacek, M. S. and D'Amore, P. A.
(2004). Endothelial-mesenchymal interactions in vitro reveal
molecular mechanisms of smooth muscle/pericyte differentiation.
Stem Cells Dev. 13,509
-520.[Medline]
Du, L., Sullivan, C. C., Chu, D., Cho, A. J., Kido, M., Wolf, P.
L., Yuan, J. X., Deutsch, R., Jamieson, S. W. and Thistlethwaite, P. A.
(2003). Signaling molecules in nonfamilial pulmonary
hypertension. New Engl. J. Med.
348,500
-509.
Duncan, W. C., McNeilly, A. S. and Illingworth, P. J.
(1998). The effect of luteal "rescue" on the
expression and localization of matrix metalloproteinases and their tissue
inhibitors in the human corpus luteum. J. Clin. Endocrinol.
Metab. 83,2470
-2478.
El-Bizri, N., Wang, L., Merklinger, S. L., Guignabert, C.,
Desai, T., Urashima, T., Sheikh, A. Y., Knutsen, R. H., Mecham, R. P.,
Mishina, Y. et al. (2008). Smooth muscle protein
22alpha-mediated patchy deletion of Bmpr1a impairs cardiac contractility but
protects against pulmonary vascular remodeling. Circ.
Res. 102,380
-388.
Franco, C., Ho, B., Mulholland, D., Hou, G., Islam, M.,
Donaldson, K. and Bendeck, M. P. (2006). Doxycycline alters
vascular smooth muscle cell adhesion, migration, and reorganization of
fibrillar collagen matrices. Am. J. Pathol.
168,1697
-1709.
Gaussin, V., Van de Putte, T., Mishina, Y., Hanks, M. C.,
Zwijsen, A., Huylebroeck, D., Behringer, R. R. and Schneider, M. D.
(2002). Endocardial cushion and myocardial defects after cardiac
myocyte-specific conditional deletion of the bone morphogenetic protein
receptor ALK3. Proc. Natl. Acad. Sci. USA
99,2878
-2883.
Gilboa, L., Nohe, A., Geissendorfer, T., Sebald, W., Henis, Y.
I. and Knaus, P. (2000). Bone morphogenetic protein receptor
complexes on the surface of live cells: a new oligomerization mode for
serine/threonine kinase receptors. Mol. Biol. Cell
11,1023
-1035.
Goodall, S., Crowther, M., Hemingway, D. M., Bell, P. R. and
Thompson, M. M. (2001). Ubiquitous elevation of matrix
metalloproteinase-2 expression in the vasculature of patients with abdominal
aneurysms. Circulation
104,304
-309.
He, X. C., Zhang, J., Tong, W. G., Tawfik, O., Ross, J.,
Scoville, D. H., Tian, Q., Zeng, X., He, X., Wiedemann, L. M. et al.
(2004). BMP signaling inhibits intestinal stem cell self-renewal
through suppression of Wnt-beta-catenin signaling. Nat.
Genet. 36,1117
-1121.[CrossRef][Medline]
Hebert, J. M., Mishina, Y. and McConnell, S. K.
(2002). BMP signaling is required locally to pattern the dorsal
telencephalic midline. Neuron
35,1029
-1041.[CrossRef][Medline]
Hellstrom, M., Kalen, M., Lindahl, P., Abramsson, A. and
Betsholtz, C. (1999). Role of PDGF-B and PDGFR-beta in
recruitment of vascular smooth muscle cells and pericytes during embryonic
blood vessel formation in the mouse. Development
126,3047
-3055.[Abstract]
Humbert, M., Morrell, N. W., Archer, S. L., Stenmark, K. R.,
MacLean, M. R., Lang, I. M., Christman, B. W., Weir, E. K., Eickelberg, O.,
Voelkel, N. F. et al. (2004). Cellular and molecular
pathobiology of pulmonary arterial hypertension. J. Am. Coll.
Cardiol. 43,13S
-24S.
Jeffery, T. K. and Morrell, N. W. (2002).
Molecular and cellular basis of pulmonary vascular remodeling in pulmonary
hypertension. Prog. Cardiovasc. Dis.
45,173
-202.[CrossRef][Medline]
Jones, P. L., Crack, J. and Rabinovitch, M.
(1997). Regulation of tenascin-C, a vascular smooth muscle cell
survival factor that interacts with the alpha v beta 3 integrin to promote
epidermal growth factor receptor phosphorylation and growth. J.
Cell Biol. 139,279
-293.
Kaito, K., Urayama, H. and Watanabe, G. (2003).
Doxycycline treatment in a model of early abdominal aortic aneurysm.
Surg. Today 33,426
-433.[CrossRef][Medline]
Kuzuya, M., Kanda, S., Sasaki, T., Tamaya-Mori, N., Cheng, X.
W., Itoh, T., Itohara, S. and Iguchi, A. (2003). Deficiency
of gelatinase a suppresses smooth muscle cell invasion and development of
experimental intimal hyperplasia. Circulation
108,1375
-1381.
Lambert, V., Wielockx, B., Munaut, C., Galopin, C., Jost, M.,
Itoh, T., Werb, Z., Baker, A., Libert, C., Krell, H. W. et al.
(2003). MMP-2 and MMP-9 synergize in promoting choroidal
neovascularization. FASEB J.
17,2290
-2292.
Lane, K. B., Machado, R. D., Pauciulo, M. W., Thomson, J. R.,
Phillips, J. A., 3rd, Loyd, J. E., Nichols, W. C. and Trembath, R. C.
(2000). Heterozygous germline mutations in BMPR2, encoding a
TGF-beta receptor, cause familial primary pulmonary hypertension. The
International PPH Consortium. Nat. Genet.
26, 81-84.[CrossRef][Medline]
Leung, W. C., Lawrie, A., Demaries, S., Massaeli, H., Burry, A.,
Yablonsky, S., Sarjeant, J. M., Fera, E., Rassart, E., Pickering, J. G. et
al. (2004). Apolipoprotein D and platelet-derived growth
factor-BB synergism mediates vascular smooth muscle cell migration.
Circ. Res. 95,179
-186.
Li, L., Miano, J. M., Cserjesi, P. and Olson, E. N.
(1996). SM22 alpha, a marker of adult smooth muscle, is expressed
in multiple myogenic lineages during embryogenesis. Circ.
Res. 78,188
-195.
Lindahl, P., Johansson, B. R., Leveen, P. and Betsholtz, C.
(1997). Pericyte loss and microaneurysm formation in
PDGF-B-deficient mice. Science
277,242
-245.
Mason, D. P., Kenagy, R. D., Hasenstab, D., Bowen-Pope, D. F.,
Seifert, R. A., Coats, S., Hawkins, S. M. and Clowes, A. W.
(1999). Matrix metalloproteinase-9 overexpression enhances
vascular smooth muscle cell migration and alters remodeling in the injured rat
carotid artery. Circ. Res.
85,1179
-1185.
McMillan, W. D., Patterson, B. K., Keen, R. R., Shively, V. P.,
Cipollone, M. and Pearce, W. H. (1995). In situ localization
and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in
aneurysmal, occlusive, and normal aorta. Arterioscler. Thromb.
Vasc. Biol. 15,1139
-1144.
Mehra, A. and Wrana, J. L. (2002). TGF-beta and
the Smad signal transduction pathway. Biochem. Cell
Biol. 80,605
-622.[CrossRef][Medline]
Meyrick, B. and Reid, L. (1979).
Ultrastructural features of the distended pulmonary arteries of the normal
rat. Anat. Rec. 193,71
-97.[CrossRef][Medline]
Mishina, Y., Suzuki, A., Ueno, N. and Behringer, R. R.
(1995). Bmpr encodes a type I bone morphogenetic protein receptor
that is essential for gastrulation during mouse embryogenesis.
Genes Dev. 9,3027
-3037.
Mishina, Y., Hanks, M. C., Miura, S., Tallquist, M. D. and
Behringer, R. R. (2002). Generation of Bmpr/Alk3 conditional
knockout mice. Genesis
32, 69-72.[CrossRef][Medline]
Mishina, Y., Starbuck, M. W., Gentile, M. A., Fukuda, T.,
Kasparcova, V., Seedor, J. G., Hanks, M. C., Amling, M., Pinero, G. J.,
Harada, S. et al. (2004). Bone morphogenetic protein type IA
receptor signaling regulates postnatal osteoblast function and bone
remodeling. J. Biol. Chem.
279,27560
-27566.
Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe,
P. K., Li, L., Miyazono, K., ten Dijke, P., Kim, S. et al.
(2000). Activin receptor-like kinase 1 modulates transforming
growth factor-beta 1 signaling in the regulation of angiogenesis.
Proc. Natl. Acad. Sci. USA
97,2626
-2631.
Palosaari, H., Pennington, C. J., Larmas, M., Edwards, D. R.,
Tjaderhane, L. and Salo, T. (2003). Expression profile of
matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in mature human
odontoblasts and pulp tissue. Eur. J. Oral Sci.
111,117
-127.[CrossRef][Medline]
Park, C., Lavine, K., Mishina, Y., Deng, C. X., Ornitz, D. M.
and Choi, K. (2006). Bone morphogenetic protein receptor 1A
signaling is dispensable for hematopoietic development but essential for
vessel and atrioventricular endocardial cushion formation.
Development 133,3473
-3484.
Pyo, R., Lee, J. K., Shipley, J. M., Curci, J. A., Mao, D.,
Ziporin, S. J., Ennis, T. L., Shapiro, S. D., Senior, R. M. and Thompson, R.
W. (2000). Targeted gene disruption of matrix
metalloproteinase-9 (gelatinase B) suppresses development of experimental
abdominal aortic aneurysms. J. Clin. Invest.
105,1641
-1649.[Medline]
Roelen, B. A., Goumans, M. J., van Rooijen, M. A. and Mummery,
C. L. (1997). Differential expression of BMP receptors in
early mouse development. Int. J. Dev. Biol.
41,541
-549.[Medline]
Rubin, L. J. (1997). Primary pulmonary
hypertension. New Engl. J. Med.
336,111
-117.
Saam, J. R. and Gordon, J. I. (1999). Inducible
gene knockouts in the small intestinal and colonic epithelium. J.
Biol. Chem. 274,38071
-38082.
Sirard, C., de la Pompa, J. L., Elia, A., Itie, A., Mirtsos, C.,
Cheung, A., Hahn, S., Wakeham, A., Schwartz, L., Kern, S. E. et al.
(1998). The tumor suppressor gene Smad4/Dpc4 is required for
gastrulation and later for anterior development of the mouse embryo.
Genes Dev. 12,107
-119.
Song, L., Yan, W., Chen, X., Deng, C. X., Wang, Q. and Jiao,
K. (2007). Myocardial smad4 is essential for cardiogenesis in
mouse embryos. Circ. Res.
101,277
-285.
Soriano, P. (1999). Generalized lacZ expression
with the ROSA26 Cre reporter strain. Nat. Genet.
21, 70-71.[CrossRef][Medline]
Thompson, R. W., Holmes, D. R., Mertens, R. A., Liao, S.,
Botney, M. D., Mecham, R. P., Welgus, H. G. and Parks, W. C.
(1995). Production and localization of 92-kilodalton gelatinase
in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by
aneurysm-infiltrating macrophages. J. Clin. Invest.
96,318
-326.[Medline]
Thomson, J., Machado, R., Pauciulo, M., Morgan, N., Yacoub, M.,
Corris, P., McNeil, K., Loyd, J., Nichols, W. and Trembath, R.
(2001). Familial and sporadic primary pulmonary hypertension is
caused by BMPR2 gene mutations resulting in haploinsufficiency of the bone
morphogenetic protein type II receptor. J. Heart Lung
Transplant. 20,149
.[Medline]
Umans, L., Cox, L., Tjwa, M., Bito, V., Vermeire, L., Laperre,
K., Sipido, K., Moons, L., Huylebroeck, D. and Zwijsen, A.
(2007). Inactivation of Smad5 in endothelial cells and smooth
muscle cells demonstrates that Smad5 is required for cardiac homeostasis.
Am. J. Pathol. 170,1460
-1472.
Winnier, G., Blessing, M., Labosky, P. A. and Hogan, B. L.
(1995). Bone morphogenetic protein-4 is required for mesoderm
formation and patterning in the mouse. Genes Dev.
9,2105
-2116.
Yang, L., Cai, C. L., Lin, L., Qyang, Y., Chung, C., Monteiro,
R. M., Mummery, C. L., Fishman, G. I., Cogen, A. and Evans, S.
(2006). Isl1Cre reveals a common Bmp pathway in heart and limb
development. Development
133,1575
-1585.
Yang, R., Liu, H., Williams, I. and Chaqour, B.
(2007). Matrix metalloproteinase-2 expression and apoptogenic
activity in retinal pericytes: implications in diabetic retinopathy.
Ann. N. Y. Acad. Sci.
1103,196
-201.[CrossRef][Medline]
Yang, X., Castilla, L. H., Xu, X., Li, C., Gotay, J., Weinstein,
M., Liu, P. P. and Deng, C. X. (1999). Angiogenesis defects
and mesenchymal apoptosis in mice lacking SMAD5.
Development 126,1571
-1580.[Abstract]
Yin, M. and Pacifici, M. (2001). Vascular
regression is required for mesenchymal condensation and chondrogenesis in the
developing limb. Dev. Dyn.
222,522
-533.[CrossRef][Medline]
Zempo, N., Kenagy, R. D., Au, Y. P., Bendeck, M., Clowes, M. M.,
Reidy, M. A. and Clowes, A. W. (1994). Matrix
metalloproteinases of vascular wall cells are increased in balloon-injured rat
carotid artery. J. Vasc. Surg.
20,209
-217.[Medline]
Zhao, Y. D., Campbell, A. I., Robb, M., Ng, D. and Stewart, D.
J. (2003). Protective role of angiopoietin-1 in experimental
pulmonary hypertension. Circ. Res.
92,984
-991.
Zhu, W. H., Guo, X., Villaschi, S. and Francesco Nicosia, R.
(2000). Regulation of vascular growth and regression by matrix
metalloproteinases in the rat aorta model of angiogenesis. Lab.
Invest. 80,545
-555.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
