Mol. Hum. Reprod. Advance Access originally published online on February 25, 2005
Molecular Human Reproduction 2005 11(4):253-260; doi:10.1093/molehr/gah159
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In utero angiopoietin-2 gene delivery remodels placental blood vessel phenotype: a murine model for studying placental angiogenesis
1Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, 2UCSF Comprehensive Cancer Center, University of California and 3Department of Pathology, University of California, San Francisco, CA, USA
4 To whom correspondence should be addressed at: Box 0556, 505 Parnassus, Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA 94143-0556, USA. E-mail: jaffer{at}obgyn.ucsf.edu
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Abstract |
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Angiopoietin (Ang)-2, the natural antagonist of the Ang1/Tie2 receptor is a complex regulator of blood vessel plasticity that plays a pivotal role in both vessel sprouting [in the presence of vascular endothelial growth factor (VEGF)-A] and vessel regression (in the absence of VEGF-A). Based on the spatial and temporal expression of Ang2 throughout human gestation, we recently suggested that the Ang2 may play a pivotal role in placental angiogenesis. Further, to examine this tenet we have developed a novel murine model system in which in utero Ang2 gene delivery via a non-replicating adenoviral expression vector has the potential to manipulate the blood vessel phenotype in vivo during pregnancy. Ang2 overexpression selectively and rapidly remodels the labyrinth perivascular extracellular matrix, subsequently promoting plasticity of the maternal and fetal vessels, which appear honeycombed due to a 2-fold increase in blood vessel luminal area. High levels of Ang2 impair endothelial cell adhesiveness, leading to vascular leakiness with perivascular oedema, which increases placental weight. These observations suggest that the Ang2 overexpression may play a key role in placental vascular remodelling. Furthermore, we suggest a novel new model to study the pathobiology of placental vascularization and the effect of placental blood vessels on fetal phenotype.
Key words: angiogenesis/angiopoietin-2/placenta
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Introduction |
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Successful pregnancy requires the development of a complex maternal and fetal vascular network to support the increasing oxygen and metabolic demands of the growing fetus. Human placental vascular development is characterized by vasculogenesis, followed by transformation to branching and then non-branching angiogenesis (Geva et al., 2002). Elucidating the molecular mechanisms that control placental blood vessel development is critical to the understanding of the pathophysiology of conditions associated with chorionic villous vasculopathy, e.g. intrauterine growth restriction (IUGR). Numerous approaches have been used to investigate placental function. The inability to access placentas from ongoing human pregnancies to study primary trophoblast cells in vitro and the paucity of the appropriate animal models have severely hampered progress in understanding the molecular mechanisms underlying abnormal placental vascularization (hypervascularization and hypermaturation) and poor obstetric outcome. These shortcomings have also precluded earlier diagnosis and possible development of the effective intrauterine therapeutic options. Furthermore, the availability of genetic, cellular and molecular tools permits application of reductionist approaches to explore spatially and temporally different genes and the ligands that they encode that might influence components of human placental angiogenesis in ongoing human pregnancy.
The process of placental vasculogenesis/angiogenesis requires tight orchestration between vascular endothelial cell-specific ligands and their tyrosine kinase receptors. These include the vascular endothelial growth factor (VEGF) family members, including placental growth factor and the angiopoietins (Ang's) primarily Ang1 and Ang2 (Geva et al., 2002). Lack of appropriate function of one or more of these molecules and/or their receptors may adversely affect pregnancy outcome (Sato et al., 1995; Carmeliet et al., 1996
; Ferrara et al., 1996; Suri et al., 1996; Maisonpierre et al., 1997).
VEGF-A is a potent mitogen and survival factor for endothelial cells that has been shown to initiate vasculogenesis and angiogenesis by inducing the endothelial cell proliferation, migration and sprouting, as well as by promoting endothelial cell formation of tubule-like structures (Ferrara et al., 2004). The Ang play a critical role in angiogenesis by blood vessel stabilization, maturation and regression (Yancopoulos et al., 2000). Ang1 is expressed in a wide variety of tissues, while Ang2 is required for normal adult vascular remodelling, particularly in components of the female reproductive tract: the placenta, ovary and uterus (Suri et al., 1996; Maisonpierre et al., 1997). Previous descriptive studies suggested that the opposing actions of Ang1 and Ang2 on the activation state of the Tie2 tyrosine kinase receptor might regulate the plasticity of endothelial cells (Yancopoulos et al., 2000). Ang1 plays a role in endothelial cell maturation and vascular stabilization, promoting endothelial cell survival and endothelial integrity (Sato et al., 1995; Suri et al., 1996; Asahara et al., 1998; Witzenbichler et al., 1998; Yancopoulos et al., 2000) while Ang2 has the opposite effect, promoting blood vessel destabilization and regression in the absence of VEGF-A (Maisonpierre et al., 1997; Yancopoulos et al., 2000; Carlson et al., 2001; Gale et al., 2002; Lobov et al., 2002). An Ang2 negative signal initially causes endothelial cells to loosen, reducing endothelial cell contacts with the matrix and dissociating periendothelial support cells and basal lamina (Carlson et al., 2001; Gale et al., 2002; Lobov et al., 2002). The characterization of Ang1 and Ang2 as agonist and antagonist, respectively, was based on the inability of Ang2 to cause Tie2 receptor phosphorylation in endothelial cells (Maisonpierre et al., 1997; Yancopoulos et al., 2000). However, several studies of Ang2 function have suggested a more complex situation. Ang2 seems to activate Tie2 on some cells, while blocking Tie2 receptor phosphorylation on others (Gale et al., 2002; Lobov et al., 2002). Furthermore, a Tie2-independent mechanism has been suggested recently based upon Ang2 modulation of endothelial cell adhesion via integrins (Carlson et al., 2001).
Based on spatial and temporal expression of Ang2 throughout human gestation, we recently suggested that the Ang2 may play a pivotal role in human placental angiogenesis (Geva et al., 2002). To examine this issue further, we have developed a novel in vivo model system. Our study demonstrates that in utero Ang2 gene delivery via a non-replicating adenoviral expression vector promotes vascular plasticity by remodelling the extracellular matrix (ECM) and increasing blood vessel luminal area. Ang2 overexpression selectively and rapidly remodels the labyrinth perivascular ECM, subsequently promoting plasticity of maternal and fetal vessels, which appeared honeycombed due to increasing blood vessel diameter. Higher levels of Ang2 impair endothelial cell adhesiveness, leading to vascular leakiness and perivascular oedema, which increases the placental weight. Based upon these studies, we suggest a new model for studying the pathobiology of placental vascularization and the effect of placental blood vessels on the fetal phenotype.
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Materials and methods |
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Adenoviral production
Recombinant adenovirus expressing Ang2 (Ad-Ang2) was constructed using the Adeno-QuestTM system (Quantum Biotechnology Inc., Montreal, Canada). Full-length cDNA encoding human Ang2 was cloned into shuttle vector pQBI-AdCMV5GFP (Thurston et al., 2000).
Animals
Animal procedures were carried out with approval from the Committee on Animal Research, UCSF. C57BL/6 mice (7–8 weeks; Charles River Laboratories, Inc., Wilmington, MA, USA) were mated and vaginal plugs were used to determine gestation day 0. A midline abdominal incision was made in anaesthetized (isoflurane) embryonic day (E) 14 mice (for explanation see below) and the gravid uterus delivered though the incision. Ang2 (4 x 106 plaque-forming units) (n=37 mice) and green fluorescent protein (GFP) (n=31 mice) adenovirus were injected into the placental labyrinth using a Hamilton syringe (Reno, NV, USA). Untreated mice (n=37) and saline-injected mice (n=16) served as additional controls. The uterus was reinserted into the abdominal cavity that was closed in two layers. Mice were killed by cervical dislocation on E17. Tissues used for RNA and protein analyses were dissected and frozen immediately in liquid nitrogen and stored at –80 °C (see below). Tissues used for immunohistochemistry were fixed by immersion for 24 h in 10% paraformaldehyde, embedded in paraffin, and 5 µm sections were stained.
Human placentas
Human placental tissues were obtained from first (n=7), second (n=7) and third (n=8) trimester normotensive pregnancies (Geva et al., 2002). Placental tissues from the first and second trimesters were obtained after elective pregnancy termination (7–24 weeks' gestation). Terminations were performed by dilation and evacuation, and gestational age was determined by fetal foot length. Tissues from third trimester placentas were obtained after vaginal or caesarean deliveries (25–41 weeks' gestation). This study was approved by the UCSF Committee on Human Research.
RNA and protein extraction
Total RNA was prepared from snap-frozen placental tissue samples by homogenization in TRIzol reagent (Life Technologies, Rockville, MD, USA). RNA was quantified by absorbance at 260 nm, analysed by electrophoresis on a 1% agarose gel and stored at –80 °C. Proteins were extracted from snap-frozen tissue samples by homogenization in radioimmune precipitation (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 5 mM benzamidine, 50 mM sodium fluoride, 1 mM sodium orthoanadate, and CompleteTM tablet protease inhibitors)/10% NP40 buffer (Roche, Indianapolis, IN, USA). Protein levels were assayed using a BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA) and were quantified by absorbance at 595 nm using a DU 640 spectrophotometer (Beckman Coulter, Palo Alto, CA, USA) and stored at –80 °C.
Quantitative mRNA expression
Reverse transcriptase reactions were performed by heating a 15 µl reaction mixture containing 2 µg total RNA and 0.5 µg oligodeoxythymidine (Life Technologies, Inc.) at 70 °C for 10 min. After cooling, 25 U rRNAasin ribonuclease inhibitor (Life Technologies, Inc., Gaithersburg, MD, USA) and 200 U Moloney murine leukaemia virus (MMLV) ribonuclease reverse transcriptase (Promega Corp., Madison, WI, USA) were added in a final 25 µl reaction mixture containing 10 mM deoxy-NTP (Life Technologies, Inc.) and 5 µl MMLV reaction buffer (Promega Corp.), incubated for 1 h at 42 °C and then heated for 15 min at 70 °C. Relative expression levels were measured using the 5' fluorogenic nuclease assay in quantitative PCR, using the 5' nuclease assay on the ABI PRISM 7700 (Applied Biosystems, Foster City, CA, USA). For each gene, PCR was conducted in triplicate with 50 µl reaction volumes of 1x PCR buffer A (Applied Biosystems), 5.5 mM MgCl2, 0.4 µM of each primer, 200 µM each deoxy-NTP, 100 nM probe and 0.025 U/µl Taq Gold (Applied Biosystems). In each experiment, a large master mix of the above components was made for each cDNA and aliquoted into each optical reaction tube. Each primer/probe set (5–10 µl) was then added, and PCR conducted using the following cycle parameters: 95 °C for 15 min and 40 cycles at 95 °C for 20 s, 60 °C for 1 min. Analysis was carried out using the sequence detection software supplied with the ABI PRISM 7700. This software calculates the threshold cycle (Ct) for each reaction and this Ct is used to quantify the amount of starting template in the reaction. The Ct values for each set of three reactions were averaged for all subsequent calculations. A difference in Ct values (
Ct) was calculated for each gene by taking the mean Ct of triplicates and subtracting the mean Ct of the control gene triplicates for each cDNA sample at the same concentration (Geva et al., 2002). The primers chosen using Primer Express Software (Applied Biosystems) for sense and antisense, respectively, were: human Ang2 cDNA (GenBank accession no. AF004327
[GenBank]
) 5'-CAGATTTTGGACCAGACCAGTG-3' and 5'-ACTGTATGTTGGATGATGTGCTTG-3' and human ß-glucuronidase (GUS) (GenBank accession no. NM000181) 5'-CTCATTTGGAATTTGCCGATT-3' and 5'-CCGAGTGAAGATCCCCTTTTA-3'. The primers were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA). The TaqMan fluorogenic probe consisted of the following sequences: Ang2, 5'-FAM-CCTAGAAAAGAAGGTGCTAGCTATGGAA-6-carboxy tetramethyl rhodamine-3' (TAMRA); GUS, 5'-FAM-TGAACAGTCACCGACGAGAGTGCTGG-TAMRA. All probes were purchased from IDT. Under these PCR cycling conditions the real-time TaqMan RT-PCR reaction for Ang2 is unable to detect the presence of transcripts from the mouse gene, only those from the human gene (Elson et al., 2001).
Ang2 protein quantification
Human Ang2 protein in injected placentas was detected and quantified using a dual epitope two-site ELISA (Thurston et al., 2000).
Immunostaining
Fixed tissue sections were immunostained with a polyclonal rabbit anti-human Ang2 (Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA), goat anti-mouse Ang2 (Research Diagnostics Inc, Flanders, NJ, USA) and polyclonal rabbit anti-GFP (Clontech Laboratories, Palo Alto, CA, USA) antibodies. Bound antibody was visualized using an ABC kit (Vector Laboratories Inc., Burlingame, CA, USA) and DAB (Zymed Laboratories Inc., South San Francisco, CA, USA), as described previously (Geva et al., 2002).
Placental ultrastructure
Transmission electron microscopy was performed using a JEM 1200 EX microscope (JEOL Ltd, Tokyo, Japan).
Blood vessel luminal area quantification
Blood vessel luminal area (the area of the blood vessel within the placental labyrinth) was quantified using a Leica Microsystem microscope (Buffalo, NY, USA) and OpenLab Software (Improvision Inc., Lexington, MA, USA).
Statistical analysis
The temporal changes in the Ang2 during pregnancy were evaluated using log-linear regression, with the log of the amount regressed against time. Group comparisons were calculated using a two-sided sample t-test. Calculations were carried out in Data Desk (Vellman, 1997).
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Results |
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Similar Ang2 transcription profiles and localization in murine and human placentas
The relationship between the placental Ang2 expression and the localization throughout human and murine pregnancy was compared to validate a mouse model to study the vascular phenotype. In humans, the placental Ang2 transcripts are high in the first trimester and then decrease 10-fold in a log-linear pattern during the third trimester (human Ang2=616 x 10(–0.035 week), r2=51%, P<0.001; n=22; Figure 1A) (Geva et al., 2002). A similar pattern of the Ang2 expression was observed in murine placentas (murine Ang2=134 x 10(–0.111 day), r2=88%, P<0.001; n=18; Figure 1B). The human Ang2 protein is localized in chorionic villous syncytiotrophoblasts and endothelial cells throughout gestation (Figure 1C and E) (Geva et al., 2002). The mouse Ang2 protein localizes to trophoblasts and endothelial cells within the placental labyrinth (Figure 1D and F). These data demonstrate similar Ang2 cellular localization patterns and similar temporal declines in humans and mice, suggesting that the time-dependent regulation, localization and function of Ang2 are species-independent. Based on these studies, Ad-Ang2 was delivered in utero into E14 placentas, a time when endogenous Ang2 expression is minimal, permitting study of the possible role of the Ang2 overexpression in placental angiogenesis.
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Ang2 expression after gene delivery in vitro and in vivo
To validate the Ang2 expression within the murine-transfected placenta, we quantified the Ang2 transcripts and protein using real-time quantitative PCR and ELISA, and localized Ang2 ligand within the mouse placenta using immunostaining. Human Ang2 was delivered directly into mouse placentas using an Ad-Ang2 (n=37). Control mice were injected with the Ad-GFP (n=31) or saline (n=16) or were untreated (n =37). The Ad-GFP serves as a control for potential effects of adenovirus and expression of a non-targeted reporter gene GFP, while the saline injections serve as controls for the potential effects of surgery. Ang2 mRNA levels were 154.95±79.96% relative to ß-GUS. No Ang2 transcripts were present in control placental tissues (n=6). Ang2 protein levels were 60-fold higher (2.99±1.26 ng/ml; n=18) in the Ad-Ang2 transfected group than in adenovirus expressing GFP (Ad-GFP; 0.05 ± 0.02 ng/ml; n=18) and other controls (0.06 ± 0.03 ng/ml; n=20; P<0.02) (Figure 2). Both Ang2 and GFP are localized in trophoblast cells within the placental labyrinth (Figure 3).
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Intrauterine Ang2 gene delivery increases placental weight
Placentas from Ad-Ang2-injected mice were 1.5-fold heavier than controls (Ad-Ang2, 0.119±0.006 g; n=31; Ad-GFP, 0.083±0.004 g; n=24, and saline 0.074±0.006 g; n=15, respectively, P<0.0001) (Figure 4A).
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Fetal survival 72 h following injection (95%) was independent of treatment. The injected fetuses were grossly normal, and fetal weight was similar among treatment groups (Ad-Ang2, 0.793±0.018 g; n=31; Ad-GFP, 0.722±0.022 g; n=24, and saline 0.809 ± 0.029 g; n=15) (Figure 4B). Histological analyses of fetal tissues (liver, spleen, heart, lung, kidney, brain) confirmed that delivery of Ad-Ang2, Ad-GFP or saline did not adversely affect fetal development (data not shown). These data demonstrate that the intraplacental Ang2 gene delivery during gestation selectively induces a unique placental phenotype.
Ang2 remodels mature placental blood vessels and increases blood vessel luminal area
Our model system allows us to address directly the effect of local vascular endothelial-specific ligand overexpression on placental vasculature in vivo. Three days following Ad-Ang2 delivery (n=24), the placental labyrinth displays remarkable histologic and cellular changes: it appears honeycombed with abnormally dilated maternal and fetal vessels, which contain increased numbers of red blood cells (Figure 5A). Ang2 overexpression selectively and rapidly remodels the labyrinth perivascular ECM, subsequently promoting interstitial oedema (Figure 5A, D and G) that is particularly noticeable at the local injection site. These data are concordant with the increase in placental weight. In placentas expressing higher levels of Ang2, the endothelial cells are detached from the underlying trophoblast cell layer (Figure 5A, inset). The ultrastructural distribution of perivascular and interstitial oedema in the Ad-Ang2 group (n=9) was documented using transmission electron microscopy (Figure 5D). The trophoblast cells in Ang2 overexpressing placentas display increased sarcoplasmic reticulum and secondary liposomes, consistent with high protein (Ang2) synthesis and metabolism. Perivascular ECM remodelling with tissue oedema, separation of the endothelial cells from the surrounding pericytes and smooth muscle cells within the maternal blood vessels are also present (Figure 5G). These changes do not occur in the Ad-GFP (n=21; Figure 5B, E and H) or control (n=26; Figure 5C, F and I) groups. Quantitative image microscopy analysis revealed that the blood vessel luminal area is significantly (2-fold) elevated in the Ang2-transfected placentas compared to the Ad-GFP and other control (saline and untreated) groups (34.9±2.7%, n=14; 12.9±1.0%, n=10 and 18.3±2.7%, n=15, respectively, P<0.0001; Figure 6). These data clearly demonstrate that the Ang2 overexpression in the placenta dramatically remodels both fetal and maternal vasculature.
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Fetal–maternal gene transfer and fetal and maternal phenotype
The placental circulation is the conduit for gas and nutrient exchange between fetal and maternal compartments. If fetal–maternal gene transfer occurs, gene delivery into the placenta may have adverse fetal and/or maternal consequences that might preclude any conclusions regarding the effects of placental blood vessel modulation on fetal and maternal phenotype. Therefore, human Ang2 expression in fetal and maternal tissues was evaluated using quantitative real-time PCR. All dams (n=3) expressed human Ang2 transcripts (Table I). Human Ang2 expression varies between mice and among organs; however, Ang2 is consistently expressed at minimal levels in the maternal liver. The umbilical vein directly transports blood from the placenta to the fetal liver. Thus, if maternal–fetal transfusion of adenovector occurs, the fetal liver will express the highest level of gene product. Ang2 transcripts were quantified in fetal livers (n=9) from each transfected dam. Two fetal livers from different transfected dams expressed Ang2 (3.39 and 0.98%, relative to GUS). Trace levels of Ang2 transcripts were detected in two other fetal livers (0.01 and 0.02%). Ang2 was not expressed in five of nine (55%) fetal livers from the Ad-Ang2 group. Although fetal–maternal transfer of human Ang2 occurs, Ang2 levels in maternal and fetal tissues are low (0.00–0.36% and 0.00–2.18%, respectively, of those in the transfected placentas).
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Discussion |
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The birth of a healthy infant at term is dependent upon normal placental development and maternal–fetal oxygen and nutrient exchange. Abnormal placental function is responsible for a variety of pregnancy complications ranging from miscarriage and second trimester fetal death to pre-eclampsia and IUGR (Ahmed and Perkins, 2000
; Ong et al., 2000). Elucidation of transcript profiles involved in placental angiogenesis throughout normal and abnormal pregnancies has provided insight into the factors that regulate development of placental blood vessels, development and differentiation of the placental villous tree and fetal growth (Dunk et al., 2000; Geva et al., 2002; Wulff et al., 2002; Maynard et al., 2003; Levine et al., 2004). These data also provide a plausible explanation for the mechanism by which the extent of oxygen saturation in intervillous space (maternal) blood regulates chorionic villous (fetal) vascular morphology (Kingdom and Kaufmann, 1997; Ahmed and Perkins, 2000; Ong et al., 2000; Geva et al., 2002). Furthermore, the Ang2 plays a central role in determining the angiogenic state of maternal blood vessels and endovascular component of uterine invasion (Goldman-Wohl et al., 2000; Zhou et al., 2003).
Angiogenesis refers to the formation of capillaries from pre-existing vessels and vascular remodelling and is an essential component of embryogenesis, normal physiologic development, and tumour growth and spread (Yancopoulos et al., 2000; Ferrara et al., 2004). Ang2 is a complex regulator of blood vessel plasticity that plays a pivotal role in both vessel sprouting (in the presence of VEGF-A) and vessel regression (in the absence of VEGF-A). However, unlike Ang1, Ang2 appears to phosphorylate the Tie2 receptor on some cells, while acting as a natural antagonist on others, suggesting that the pro-angiogenic action of Ang2 depends on the differentiation state of endothelial cells (Maisonpierre et al., 1997; Yancopoulos et al., 2000; Gale et al., 2002; Lobov et al., 2002).
Molecular and structural analysis of placentas from patients who underwent pregnancy complicated by pre-eclampsia and IUGR revealed increases in VEGF-A (3-fold) and Ang2 (1.5-fold) expression with increases in fetal blood vessel diameter (Geva et al., 2002). These observations suggest an important role for Ang2 (in concert with VEGF-A), in a placental angiogenic (branching and non-branching) switch and in compensatory angiogenesis that may serve as a mechanism to compensate for the hypoxia associated with development of IUGR. We hypothesize that this increase may be related to low oxygen tension, as hypoxia up-regulates both molecules via activation of hypoxia-inducible factor-1
(Geva et al., 2002). The current study provides support for the concept that a major function of Ang2 in the placenta is to destabilize blood vessels and promote vascular plasticity. Ang2, in the absence of VEGF-A, initially induces a rapid change in the perivascular ECM. Subsequently, the blood vessels are remodelled, resulting in an increase in vessel diameter. These events closely reproduce the steps of angiogenesis and suggest a direct pro-angiogenic role for Ang2, maintaining the vessels receptive to angiogenic modulators (e.g. VEGF-A) (Lobov et al., 2002).
In the present study, we have developed a novel murine model system in which in utero gene delivery via a non-replicating adenoviral expression vector has the potential to affect the activities of placental vascular modulators in vivo and to manipulate blood vessel phenotype during pregnancy. To that end, we made use of a non-replicating adenoviral Ang2 gene expression vector (Thurston, 2000; Nagy, 2002; Cao, 2004). The endogenous murine and human Ang2 ligand were detected by immunostaining in trophoblast cells of maternal vasculature and in endothelial cells within the fetal vessels (Figure 1), while no expression was detected in endothelial cells following Ad-Ang2 transfections (Figure 3), since adenoviral vectors do not transduce endothelial cells in vivo, unless some special manipulations are used. These studies will enhance our knowledge of the molecular and morphologic alterations that characterize placental vasculature and the pathogenesis of conditions associated with abnormal angiogenesis.
We suggest two plausible molecular mechanisms for Ang2-mediated placental blood vessel modulation and vascular leakage: (1) In a Tie2-dependent mechanism, Ang2 antagonizes Ang1-mediated phosphorylation of the Tie2 receptor (Sato et al., 1995; Asahara et al., 1998; Witzenbichler et al., 1998). Ang1 is responsible for blood vessel stabilization and maturation by recruitment of pericytes and smooth muscle cells (Sato et al., 1995; Asahara et al., 1998; Witzenbichler et al., 1998; Yancopoulos et al., 2000). The vasculopathy phenotype in Tie2 (Sato et al., 1995) and Ang1 (Suri et al., 1996) deficient mice is accentuated in Ang2 embryonic lethal transgenic mice (Maisonpierre et al., 1997). This vasculopathy is characterized by endothelial cell detachment and disrupted blood vessel formation. Inducing Ang2 overexpression by direct placental injection induces a similar phenotype. (2) A Tie2-independent mechanism has been suggested recently based upon Ang2 modulation of endothelial cell adhesion (Carlson et al., 2001). These in vitro data demonstrated that the Ang2 binds to Vß5 and vitronectin integrins, which mediate migration and spreading of both endothelial and non-endothelial cells. Our findings that the Ang2 gene delivery in vivo modulates fetal and maternal vascular integrity in the placental labyrinth and uterine arteries and causes endothelial cell detachment, coupled with expression of Vß5 and vitronectin integrins on placental trophoblast cells (Bowen and Hunt, 1999; Douglas et al., 1999; Thirkill and Douglas, 1999; Wixler et al., 1999) are also consistent with a Tie2-independent mechanism.
Although there is species-dependent cytologic variation between murine chorioallantoic and human haemochorial placentas, the vascular structures are similar (Cross, 2000; Rossant and Cross, 2001). Human and mouse Ang2 are highly homologous (85%) (Maisonpierre et al., 1997) and likely perform similar functions during placental development (Figure 1). These observations, together with the present data, indicate that in utero molecular manipulation of murine placentas in a temporal fashion throughout gestation might facilitate functional genetic studies of placental biology and fetal phenotype.
In conclusion, we now provide direct evidence that the Ang2 gene overexpression promotes placental vascular plasticity by remodelling the perivascular ECM and increasing vascular luminal area, subsequently promoting interstitial oedema which increases placental weight. Further, we suggest that in utero gene delivery has the potential to modulate the placental vasculature during an ongoing pregnancy, and suggests a new in vivo model for studying the pathobiology of placental vascularization and its effects on fetal phenotype.
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Acknowledgements |
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We thank J.S. Rudge, E. Ioffe and G.D. Yancopoulos (Regeneron Pharmaceuticals Inc.) for helpful scientific discussions and gifts of reagents; D. Hylton (Regeneron) for performing the ELISAs; J.A. Weimann and G. Keshet (Department of Molecular Pharmacology, Stanford University School of Medicine); Maria Pallavicini and Robert N Taylor (UCSF) for their invaluable suggestions and assistance; and M. Fung, C.M. Espiritu and S.K. Apgar (Comprehensive Cancer Center, UCSF) for performing quantitative real-time PCR, immunostaining and graphics production.
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Notes |
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This study was supported, in part, by a Serono Foundation Fellowship in Reproductive Endocrinology (to E. Geva). This work is in partial fulfillment of the requirements of the PhD degree for E. Geva, MD, Weizmann Institute of Science, Rehovot, Israel. Presented in part at the 49th Annual Meeting of the Society for Gynecologic Investigation, 19–22 March 2002, Los Angeles, CA, USA.
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References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ahmed A and Perkins J (2000) Angiogenesis and intrauterine growth. Baillieres Best Pract Res Clin Obstet Gynaecol 14, 981–998.[CrossRef][Medline]
Asahara T, Chen D, Takahashi T, Fujikawa K, Kearney M, Magner M, Yancopoulos GD and Isner JM (1998) Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res 83, 233–240.
Bowen JA and Hunt JS (1999) Expression of cell adhesion molecules in murine placentas and a placental cell line. Biol Reprod 60, 428–434.
Cao L, Jiao X, Zuzga DS, Liu Y, Fong DM, Young D and During MJ (2004) VEGF links hippocampal activity with neurogenesis, learning and memory. Nat Genet 36, 827–835.[CrossRef][Web of Science][Medline]
Carlson TR, Feng Y, Maisonpierre PC, Mrksich M and Morla AO (2001) Direct cell adhesion to the angiopoietins. J Biol Chem 276, 26516–26525.
Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C et al. (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 4, 435–439.
Cross JC (2000) Genetic insights into trophoblast differentiation and placental morphogenesis. Semin Cell Dev Biol 11, 105–113.[CrossRef][Web of Science][Medline]
Douglas GC, Thirkill TL and Blankenship T (1999) Vitronectin receptors are expressed by macaque trophoblast cells and play a role in migration and adhesion to endothelium. Biochim Biophys Acta 1452, 36–45.[Medline]
Dunk C, Shams M, Nijjar S, Rhaman M, Qiu Y, Bussolati B and Ahmed A (2000) Angiopoietin-1 and angiopoietin-2 activate trophoblast Tie-2 to promote growth and migration during placental development. Am J Pathol 156, 2185–2199.
Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald DM, Johnson RS and Arbeit JM (2001) Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor-1alpha. Genes Dev 15, 2520–2532.
Ferrara N (2004) Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25, 581–611.
Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ and Moore MW (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439–442.[CrossRef][Medline]
Gale NW, Thurston G, Hackett SF, Renard R, Wang Q, McClain J, Martin C, Witte C, Witte MH, Jackson D et al. (2002) Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev Cell 3, 411–423.[CrossRef][Web of Science][Medline]
Geva E, Ginzinger DG, Zaloudek CJ, Moore DH, Byrne A and Jaffe RB (2002) Human placental vascular development: vasculogenic and angiogenic (branching and non-branching) transformation is regulated by vascular endothelial growth factor (VEGF)-A, angiopoietin-1 and angiopoietin-2. J Clin Endocrinol Metab 87, 4213–4224.
Goldman-Wohl DS, Ariel I, Greenfield C, Lavy Y and Yagel S (2000) Tie-2 and angiopoietin-2 expression at the fetal-maternal interface: A receptor ligand model for vascular remodelling. Mol Hum Reprod 6, 81–87.
Kingdom JC and Kaufmann P (1997) Oxygen and placental villous development: origins of fetal hypoxia. Placenta 18, 613–621.[CrossRef][Web of Science][Medline]
Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH et al. (2004) Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 350, 672–683.
Lobov IB, Brooks PC and Lang RA (2002) Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci USA 99, 11205–11210.
Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N et al. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55–60.
Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE et al. (2003) Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 111, 649–658.[CrossRef][Web of Science][Medline]
Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Metmar MJ, Lawitts JA, Benjamin L, Tan X, Manseau EJ et al. (2002) Vascular permeability factor/vascular endothelial growth factor induces lymphagiogenesis as well as angiogenesis. J Exp Med 196, 1497–1506.
Ong S, Lash G and Baker PN (2001) Angiogenesis and placental growth in normal and compromised pregnancies. Baillieres Best Pract Res Clin Obstet Gynaecol 14, 969–980.
Rossant J and Cross JC (2001) Placental development: lessons from mouse mutants. Nat Rev Genet 2, 538–548.[Web of Science][Medline]
Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maguire M, Gridley T, Wolburg H, Risau W and Qin Y (1995) Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376, 70–74.[CrossRef][Medline]
Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN and Yancopoulos G (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171–1180.[CrossRef][Web of Science][Medline]
Thirkill TL and Douglas GC (1999) The vitronectin receptor plays a role in the adhesion of human cytotrophoblast cells to endothelial cells. Endothelium 6, 277–290.[Web of Science][Medline]
Thurston G, Rudge JS, loffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM and Yancopoulos GD (2000) Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med 6, 460–463.[CrossRef][Web of Science][Medline]
Vellman PF (1997) Data Desk Statistics Guide version 6.0. Data Description Inc., Ithaca, New York, pp 20/A–20/10.
Wixler V, Laplantine E, Geerts D, Sonnenberg A, Petersohn D, Eckes B, Paulsson M and Aumailley M (1999) Identification of novel interaction partners for the conserved membrance proximal region of alpha-integrin cytoplasmic domains. FEBS Letters 445, 51–55.
Witzenbichler B, Maisonpierre PC, Jones P, Yancopoulos GD and Isner JM (1998) Chemotactic properties and angiopoietin-1 and -2, ligands for the endothelial-specific receptor tyrosine kinase Tie2. J Biol Chem 273, 18514–18521.
Wulff C, Wilson H, Dickson SE, Wiegand SJ and Fraser HM (2002) Hemochorial placentation in the primate: Expression of vascular endothelial growth factor, angiopoietins, and their receptors throughout pregnancy. Biol Reprod 66, 802–812.
Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ and Holash J (2000) Vascular-specific growth factors and blood vessel formation. Nature 407, 242–248.[CrossRef][Medline]
Zhou Y, Bellingard V, Feng KT, McMaster M and Fisher SJ (2003) Human cytotrophoblasts promote endothelial survival and vascular remodelling through secretion of Ang2, PIGF, and VEGF-C. Dev Biol 263, 114–125.[CrossRef][Web of Science][Medline]
Submitted on October 14, 2004; accepted on January 21, 2005.
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