Placental gene transfer: transgene screening in mice for trophic effects on the placenta

doi:10.1016/j.ajog.2009.06.029

American Journal of Obstetrics and Gynecology

Volume 201, Issue 5, November 2009, Pages 499.e1-499.e8

https://doi.org/10.1016/j.ajog.2009.06.029 Get rights and content

Objective

We hypothesized that gene transfer of select growth factors to the placenta may enhance placental and fetal growth. Thus, we examined the effect of 8 growth factor transgenes on murine placenta.

Study Design

Adenoviral-mediated site-specific intraplacental gene transfer of 8 different growth factor transgenes at embryonic day (e) 14 was performed. Transgenes included angiopoietin-1, angiopoietin-2 (Ang-2), basic fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-1 (IGF-1), placenta growth hormone, platelet-derived growth factor-B (PDGF-B), and vascular endothelial growth factor₁₂₁. Fetuses and placentas were harvested at e17 and assessed for survival, gene transfer efficiency, placenta area, and fetal and placental weights.

Results

Efficient gene transfer to the placenta was detected with minimal dissemination to the fetus. Overexpression of IGF-1, PDGF-B, and Ang-2 resulted in an increase in placenta cross-sectional area. Only Ang-2 gene transfer resulted in increased fetal weight, and only Ang-2 and basic fibroblast growth factor resulted in a change in placental weight.

Conclusion

Site-specific placental gene transfer results in efficient gene transfer with minimal dissemination to the fetus. Adenoviral-mediated IGF-1, adenoviral-mediated PDGF-B, and adenoviral-mediated Ang-2 significantly increase placenta growth.

Section snippets

Adenoviral constructs

As previously described,²² all constructs used in this study were first-generation recombinant replication defective, serotype 5 adenovirus vectors. All adenoviral genomes had either E1 or both E1 and E3 regions deleted. All transgenes were driven by the cytomegalovirus (CMV) promoter. The parental adenovirus was either dl7001 (E1 and E3 deleted), AdEasy-1 (E1 and E3 deleted), in340 (E1 and E3 deleted), or PJM17 (E1 deleted). Viruses included in the study are listed in Table 1.

Viruses were

Survival

All dams for all groups survived to the time of harvest (e17) except in the Ad-VEGF₁₂₁-treated group, which had a 40% (2 of 5) survival rate. There was 100% survival rate for groups treated with PBS, Ad-IGF-1, and Ad-PDGF-B. There was an 88% survival rate for Ad-bFGF-treated animals, a 67% survival rate for Ad-Ang-2- and Ad-HGF-treated animals, and a 47% survival rate for Ad-Ang-1- and Ad-PlGF-treated animals.

Fetal and placenta weight analysis

Only Ad-Ang-2 resulted in a significant increase in fetal wet weight compared with

Comment

This is the first demonstration of direct intraplacental adenoviral-mediated gene transfer used as a tool to overexpress a growth factor transgene to stimulate placental growth and development in the mouse. Our findings suggest a significant increase in the placental cross-sectional area observed in the Ad-IGF-1–, Ad-PDGF-B–, and Ad-Ang-2–treated groups compared with controls. It is remarkable that any differences in placental cross-sectional area were observed in those normal mice, because

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Additionally, using the same adenoviral vector expressing VEGF but injected directly into the proximal part of the uterine artery, they demonstrated increased uterine blood flow and vasodilation in a sheep model counteracting the abnormalities in FGR [100]. Direct placental injection of adenoviral vectors containing eight different growth factor transgenes have also been performed to examine the effects of overexpressing genes including IGF-1, placental growth factor (PGF) and angiopoietin 2 (ANG-2) on placental and fetal growth [101]. Following on from this study, we performed intraplacental delivery, through direct injection of the placenta, of adenoviral vectors containing IGF-1 have been performed in various animal [76,102] and in vitro human models [77,78] with positive effects on fetal growth and placental nutrient transport.
Pregnancy complications adversely impact both mother and/or fetus throughout the lifespan. Fetal growth restriction (FGR) occurs when a fetus fails to reach their intrauterine potential for growth, it is the second highest leading cause of infant mortality, and leads to increased risk of developing non-communicable diseases in later life due ‘fetal programming’. Abnormal placental development, growth and/or function underlies approximately 75% of FGR cases and there is currently no treatment save delivery, often prematurely. We previously demonstrated in a murine model of FGR that nanoparticle mediated, intra-placental human IGF-1 gene therapy maintains normal fetal growth. Multiple models of FGR currently exist reflecting the etiologies of human FGR and have been used by us and others to investigate the development of in utero therapeutics as discussed here. In addition to the in vivo models discussed herein, utilizing human models including in vitro (Choriocarcinoma cell lines and primary trophoblasts) and ex vivo (term villous fragments and placenta cotyledon perfusion) we have demonstrated robust nanoparticle uptake, transgene expression, nutrient transporter regulation without transfer to the fetus. For translational gene therapy application in the human placenta, there are multiple avenues that require investigation including syncytial uptake from the maternal circulation, transgene expression, functionality and longevity of treatment, impact of treatment on the mother and developing fetus. The potential impact of treating the placenta during gestation is high, wide-ranging across pregnancy complications, and may offer reduced risk of developing associated cardio-metabolic diseases in later life impacting at both an individual and societal level.
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Regulation of amino acid transporters by adenoviral-mediated human insulin-like growth factor-1 in a mouse model of placental insufficiency in vivo and the human trophoblast line BeWo in vitro
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Chen et al. [19] demonstrated successful transfection of Ad-Timp3 into BeWo cells but the time period of infection was short and their focus was on targeted death in the BeWo cell line induced by TIMP3 expression. Adenoviral-associated virus has also successfully been used to transfect trophoblast previously [20], however we chose to use Ad-hIGF-1 as it demonstrated trophic effects in mouse placenta in a previous study [18]. It is unknown if the responses in human placenta would reflect those in the mouse model or use alternate mechanisms, therefore we chose to include Ad-hIGF-1 treatments in both a mouse model of placental insufficiency and in vitro in a widely-accepted and published model of human trophoblast, the BeWo Choriocarcinoma cell line in the current study.
Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor-11 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined amino acid transporter expression and localization in both a mouse model of placental Insufficiency (PI) and a model of human trophoblast, the BeWo Choriocarcinoma cell line.
For in vitro human studies, BeWo Choriocarcinoma cells were maintained in F12 complete medium + 10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 h. MOIs of 10:1 and 100:1 were utilized. In BeWo, transfection efficiency was 100% at an MOI of 100:1 and Ad-IGF-1 significantly increased IGF-1 secretion, proliferation and invasion but reduced apoptosis compared to controls. In vitro, amino acid uptake was increased following Ad-IGF-1 treatment and associated with significantly increased RNA expression of SNAT1, 2, LAT1 and 4F2hc. Only SNAT2 protein expression was increased but LAT1 showed relocalization from a perinuclear location to the cytoplasm and cell membrane.
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Enhanced amino acid isoform transporter expression and relocalization to the membrane may be an important mechanism contributing to Ad-hIGF-1 mediated correction of placental insufficiency.
Lentivirus-Mediated Transduction of Optical Reporter Genes in Blastocysts for Placental Studies
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Genetic manipulation methods in rodents have greatly enhanced understanding of pathophysiological processes. However, although placental dysfunction underlies many pregnancy disorders, understanding molecular functions in the placenta has been limited by difficulties in gene manipulation in this tissue. Recently developed placenta-specific gene manipulation methods have addressed many of these limitations, enabling analysis of gene functions specifically in the placenta, without confounding effects from embryonic gene functions. Here, we describe methods recently used to alter gene function in rodent placentas, with particular emphasis on lentivirus-based gene transduction. This method enables consistent, high levels of transgene expression in all trophoblast lineages throughout pregnancy and serial, quantitative monitoring of optical reporter gene expression in live pregnant animals by bioluminescence imaging. The addition of inducible placenta-specific transgene expression using an advanced doxycycline-inducible transgene system makes it possible to alter gene expression at multiple, discrete stages of pregnancy with on-off control and serial live imaging of optical reporters.
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Adenoviruses are capable of infecting both dividing and non-dividing cells at high efficiency but cannot integrate their genetic material into that of the host cells; the carried genes will therefore not be transmitted to the daughter cells. Direct in utero inoculation with recombinant adenoviruses has been conclusively demonstrated to successfully alter gene expression specifically in the placenta (Katz et al., 2009; Xing et al., 2000). Intra-placental injection can, however, cause injuries that may reduce fetal viability if performed earlier than E13.5.
The placenta is a transient organ that forms during pregnancy to support the growth and development of the fetus. During human placental development, trophoblast cells differentiate through two major pathways. In the villous pathway, cytotrophoblast cells fuse to form multinucleated syncytiotrophoblast. In the extravillous pathway, cytotrophoblast cells acquire an invasive phenotype and differentiate into either (1) interstitial extravillous trophoblasts, which invade the decidua and a portion of the myometrium, or (2) endovascular extravillous trophoblasts, which remodel the maternal vasculature. These differentiation events are tightly controlled by the interplay of oxygen tension, transcription factors, hormones, growth factors, and other signaling molecules. More recently, microRNAs have been implicated in this regulatory process. Abnormal placental development, particularly the limited invasion of trophoblast cells into the uterus and the subsequent failure of the remodeling of maternal spiral arteries, is believed to cause preeclampsia, a severe pregnancy related disorder characterized by hypertension and proteinuria. Oxidative stress, the abnormal production and/or function of signaling molecules, as well as aberrant microRNAs expression have been suggested to participate in the pathogenesis of preeclampsia. Several potential biomarkers for preeclampsia have been identified, creating new opportunities for the development of strategies to diagnose, prevent, and treat this disorder.
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The findings of the present study will also be of interest to scientists engaged in fundamental research into placentation, placental pathology, or fetal development. Ad vector-mediated gene transfer to the placenta has been tried previously in pregnant mice, rats, and rabbits by intraplacental or maternal intravenous injection [43–46]. Intraplacental injection of Ad showed highly efficient transduction into the placenta, and the transgene was detected at low levels in the maternal liver.
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: This study was supported in part by Grant R01-DK59242 from the National Institute of Diabetes and Digestive and Kidney Diseases (to T.M.C.).

: The first 2 authors contributed equally to the study and article.

: Cite this article as: Katz AB, Keswani SG, Habli M, et al. Placental gene transfer: transgene screening in mice for trophic effects on the placenta. Am J Obstet Gynecol 2009;201:499.e1-8.

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ResearchBasic science: ObstetricsPlacental gene transfer: transgene screening in mice for trophic effects on the placenta

Objective

Study Design

Results

Conclusion

Section snippets

Adenoviral constructs

Survival

Fetal and placenta weight analysis

Comment

Am J Pathol

Placenta

J Surg Res

Placenta

Mol Cell Endocrinol

Dev Biol

J Invest Dermatol

Cell

Obstet Gynecol

Blood

J Invest Dermatol

Fetal growth and growth restriction

The contribution of low birth weight to infant mortality and childhood morbidity

N Engl J Med

Fetal origins of coronary heart disease

Br Med J

Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth

Diabetologia

Fetal responses to placental insufficiency: an update

BJOG

Gene therapy: therapeutic mechanisms and strategies

Human placental vascular development: vasculogenic and angiogenic (branching and nonbranching) transformation is regulated by vascular endothelial growth factor-A, angiopoietin-1, and angiopoietin-2

J Clin Endocrinol Metab

Soluble flt-1 and the angiopoietins in the development and regulation of placental vasculature

J Anat

Influence of maternal diabetes on placental fibroblast growth factor-2 expression, proliferation, and apoptosis

J Soc Gynecol Investig

The differential expression of hepatocyte growth factor and met in human placenta

J Clin Endocrinol Metab

A role for hepatocyte growth factor during early postimplantation growth of the placental lineage in mice

Biol Reprod

Research
Basic science: Obstetrics
Placental gene transfer: transgene screening in mice for trophic effects on the placenta