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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1052
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 379-385
Copyright © 2005 by The Endocrine Society

Adenovirally Mediated Expression of Insulin-Like Growth Factors Enhances the Function of First Trimester Placental Fibroblasts

Adrian G. Miller, John D. Aplin and Melissa Westwood

Endocrine Sciences (A.G.M., M.W.) and Academic Unit of Obstetrics and Gynecology (A.G.M., J.D.A.), University of Manchester, Manchester, United Kingdom M13 9PT

Address all correspondence and requests for reprints to: Dr. Melissa Westwood, Endocrine Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester, United Kingdom M13 9PT. E-mail: melissa.westwood{at}manchester.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGFs are critical in fetal growth because of their role in placental development and function. In this study, we used adenovirus (Ad-IGF) to deliver sense or antisense IGF-I or IGF-II cDNA to human primary placental fibroblasts (PPF) in vitro to determine whether this could lead to enhanced placental cell function. PPFs virally transfected with Ad-IGF-I or Ad-IGF-II showed 7-fold (P < 0.01) and 3-fold (P < 0.01) increases in [3H]thymidine incorporation at 48 h post infection compared with nontransfected controls. In a coculture system designed to assess cell migration, nontransfected PPF cells positioned over a monolayer transfected by Ad-IGF-I or Ad-IGF-II showed a more than 10-fold (P < 0.01) and a 7-fold (P < 0.01) increase in migration compared with cells positioned above a nontransfected monolayer. After 96 h in culture, PPFs transfected with sense Ad-IGF-I or Ad-IGF-II showed 2% apoptosis compared with 16% of nontransfected cells, whereas 37% and 25% of cells transfected with antisense Ad-IGF-I or Ad-IGF-II were apoptotic. This work has established that cells of placental origin are amenable to adenoviral transfection and that IGFs exert autocrine and paracrine effects on proliferation, migration, and survival, suggesting that enhancement of IGF levels in the placenta may augment placental function and increase fetal growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRAUTERINE GROWTH RESTRICTION (IUGR) is a manifestation of fetal, maternal, or placental disorders that complicates 5–10% of all pregnancies (1). Fetuses that fail to reach their genetically determined growth potential (2) are at a significantly increased risk of perinatal morbidity, mortality (3), and complications in later life, such as coronary heart disease, type 2 diabetes, and hypertension (4).

Placental dysfunction that presents as a reduction of fetal nutrient supply is a major cause of altered fetal growth (5) and occurs when there are inequalities between maternal supply, placental perfusion, and fetal demand (6). In humans, placental metabolism and substrate transport are altered in IUGR (7, 8), and placentas from such pregnancies are smaller (7, 9); the villi are reduced in number, diameter, and branching complexity (10, 11); and the capillaries within their mesenchymal core have narrower lumens (10). IUGR placentas also show increased lesions (12), vascular damage (13), and apoptosis in cells of the trophoblast lineage (14, 15).

The IGFs stimulate a variety of cellular responses, including proliferation, migration, differentiation (16, 17), and resistance to apoptosis (18), effects predominantly mediated by the type 1 IGF receptor (IGF-1R) (19), using various signaling pathways (20, 21).

Human fetal tissues, including placental villous mesenchymal cells (22), express IGF-I, IGF-II, the IGF-binding proteins (IGFBPs), and IGF receptor mRNA from as early as wk 6 of gestation (23, 24, 25, 26), suggesting the importance of the IGF system in organ growth and differentiation. This is also highlighted by gene-targeting studies. In mice with homozygous deletion of either igf-I or igf-II, fetal growth is impaired, and pups have birth weights approximately 60% those of wild-type animals in both instances (27). The fetal growth-restricted phenotype in mice with homozygous deletion of the igf-II gene is accompanied by small placental size (28) and has led to the hypothesis that the two are causally related (29). In contrast, there is no reduction in placental size in the absence of igf-I, although both in vitro and in vivo studies have shown that IGF-I can modify placental nutrient utilization (30, 31).

In IUGR pregnancies, fetal cord serum levels of IGF-I and IGF-II are reduced, whereas levels of IGFBP-1, which is thought to inhibit IGF activity during pregnancy (32), are considerably increased (33, 34). Reductions in fetal growth associated with changes in IGF and IGFBP levels probably reflect tissue-specific adaptations made to compensate for the altered substrate or oxygen supply (35). Smith et al. (36) suggested that the risk of delivering a low birth weight baby may be determined by placental IGF activity early in pregnancy, and it has been shown that placental production of IGF is aberrant in some IUGR pregnancies (37). Sheikh et al. (30) suggest that this may also be true in the second half of pregnancy, when IGF-I becomes more prominent in regulating fetal growth.

Thus, IGF-I and IGF-II play an integral part in feto-placental growth throughout gestation exerting metabolic, mitogenic, and differentiative effects in an autocrine, paracrine, or endocrine fashion (38) across a wide range of fetal tissues (16, 31). However, their effects have not been investigated in placental stromal fibroblasts. These cells constitute a major population in the human placenta, are more readily cultured and expanded in vitro than trophoblasts, and, given their anatomical location, are likely to be capable of delivering transgene products to other placental cells and into the fetal circulation. In this work we used adenoviruses containing IGF-I or IGF-II cDNA in either the sense or antisense orientation to deliver the genes to primary human placental fibroblasts and characterize the effects of overexpression or inhibition on placental cell proliferation, migration, and survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

All reagents were purchased from Invitrogen Life Technologies (Paisley, UK) or Sigma-Aldrich Corp. (St. Louis, MO) unless otherwise stated.

Cell isolation and cell culture

Placental tissue was obtained with local ethical committee approval and informed consent at elective termination of pregnancy at 8–12 wk. The isolation of primary placental fibroblasts (PPF) was based on a protocol originally designed by Haigh et al. (39). Briefly, approximately 1-mm3 pieces of tissue were transferred to a 10-cm petri dish containing serum-free fibroblast growth medium [DMEM, glucose (4500 mg/liter), 200 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1% (vol/vol) nonessential amino acid solution, and 0.0008% (vol/vol) mercaptoethanol] and subjected to additional selection, based on well defined villous florets, minimal erythrocytes, and an absence of blood clots. Tissue was added to dry wells of a 24-well culture plate and allowed to adhere by incubation at 37 C for 20 min, after which the encapsulating syncytiotrophoblast layer was nicked with a sterile scalpel to facilitate the migration of fibroblasts from the villous mesenchyme. Tissues were subsequently immersed in 1 ml fibroblast growth medium containing 10% (vol/vol) fetal calf serum (FCS) and incubated at 37 C in 5% CO2. The medium was changed every 3–4 d, and the outgrowth of PPF was monitored by light microscopy. After approximately 2 wk in culture, the cells were subcultured, and immunocytochemistry was used to confirm the identity of PPF cells.

Immunocytochemistry of PPF cells

Cell monolayers were grown on glass coverslips, removed from growth medium, fixed in 3.75% (wt/vol) paraformaldehyde (BDH, Poole, UK) and permeabilized in ice-cold methanol for at least 20 min at –20 C. After this time, the coverslips were washed (three times, 5 min each time) in PBS. Nonspecific binding sites were blocked with 1% (wt/vol) BSA in PBS for 1 h at room temperature, then cells were incubated with the following primary antibodies for 2 h at room temperature: mouse antihuman vimentin (1:200; DakoCytomation, Carpinteria, CA), mouse antihuman desmin (1:200; DakoCytomation), mouse antihuman fibroblast surface protein (1:500), or mouse antihuman cytokeratin 7 (1:50; DakoCytomation). After washing five times with PBS/1% BSA, cells were incubated with a sheep antimouse IgG rhodamine-conjugated secondary antibody (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 2 h at room temperature. Cells were counterstained with 4',6-diamidino-2-phenylindole (Vectashield, Vector Laboratories, Inc., Burlingame, CA) for localization of nuclei.

Recombinant adenovirus (Ad-IGF) construction

Reagents for the production of a replication-deficient recombinant adenovirus were purchased from Quantum Biotechnologies (Harefield, UK). IGF-Ia cDNA was obtained by PCR amplification of a sequence contained in pCMV-igf-Ia (a gift from Prof. Stephen Duguay, University of Chicago, Chicago, IL) (40). Similarly, IGF-II cDNA was obtained by PCR of a sequence in pCMV-int-igf-II (gift from Prof. E. Wolf, Ludwig-Maximillian University, Munich, Germany) (41).

The expression cassette pQBI-AdCMV5-igf-(I or II)-IRES-GFP contains a cytomegalovirus (CMV) promoter, IGF transgene cDNA, internal ribosome entry site (IRES), green fluorescent protein (GFP) cDNA, and the bovine GH polyadenylated sequence. After ligation of IGF cDNA with linearized cassette backbone and dideoxynucleotide sequencing to determine insert orientation, cassette and viral DNA were cotransfected into QBI-293A cells, a specialized HEK line, using calcium phosphate precipitation. Transfected cells were overlaid with 1.25% low melting temperature agarose in DMEM/5% FCS and monitored daily for GFP expression. Fluorescent plaques were removed from the agarose, viral particles were propagated in QBI-293A cells and purified by CsCl2 gradient, and titers were determined by the tissue culture infectious dose method.

Multiplicity of infection (MOI)

PPF cells were transfected with Ad-IGF, ranging from 10–100 virion particles/cell, and optimal MOI was determined by monitoring GFP expression and cell morphology to assess cytotoxicity.

Western immunoblotting

Conditioned medium or whole cell lysates were subjected to 12% SDS-PAGE and transferred to 0.2-µm pore size nitrocellulose membrane (Bio-Rad Laboratories, Hemel Hempstead, UK). Membranes were blocked in 5% nonfat milk protein for 1 h and incubated with antibodies specific for IGF-I (I5C9, monoclonal, 1:1000) (42), IGF-II (W3D9, monoclonal, 1:1000; both gifts from Prof. Anne White, University of Manchester) (43), or GFP (polyclonal, 1:1000; Molecular Probes, Leiden, The Netherlands) overnight at 4 C. Membranes were then incubated with secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK; 1:3000) for 1.5 h at room temperature. Proteins were visualized using Supersignal West Femto (Perbio, Tattenhall) or ECL (Amersham Biosciences).

[3H]Thymidine incorporation assay

PPF cells were plated at 1 x 105 cells/well and incubated in fibroblast growth medium (10% FCS) at 37 C in 5% CO2 overnight. The following day, the medium was changed to DMEM/5% FCS and 5 x 106 sense/antisense Ad-IGF plaque-forming units (MOI, 50) were added to the cells. After 6 h, the growth medium was removed and replaced by serum-free DMEM, and cultures were incubated at 37 C in 5% CO2 overnight. Twenty-four or 48 h postinfection, 0.025 µCi [3H]thymidine (Amersham Biosciences) was added, and after an additional 4 h, cells were washed three times in PBS and then incubated with 10% (wt/vol) trichloroacetic acid for 2 h at 4 C. Cells were solubilized in 1 ml 0.1 M NaOH at 4 C and counted on a ß-counter using Optiphase HiSafe liquid scintillant.

In some experiments, virally transfected cells were plated into a Transwell insert, and after the onset of GFP expression, the insert was transferred to a well containing a nontransfected target PPF monolayer for 24 or 48 h before adding 0.025 µCi [3H]thymidine to assess cell proliferation, as described above.

Migration assay

PPF (5 x 104) were seeded into Transwell inserts (8-µm pore size), cultured in DMEM/10% FCS for 6 h, and then exposed to a serum-reduced (1%) medium overnight. The inserts were then positioned over serum-starved PPF cells that had been transfected with 2.5 x 106 plaque-forming units (MOI, 50) of sense Ad-IGF-I or -II 48 h earlier. After 24-h coincubation, the inserts were removed from the wells, washed in PBS, and incubated in ice-cold methanol for at least 20 min at –20 C. The cells were washed twice in PBS and stained with hematoxylin solution for 5 min, and the cells adhering to the top of the insert were removed using a cotton-wool swab. Cells that had migrated to the underside of the insert membrane were counted under white light using a x10 objective; three corresponding areas per membrane were selected for assessment, and where possible, all the cells within the field were counted. However, when the number of cells was too great for accurate quantification, the field was divided into 16 equal sections, and the mean number of cells within five sections was used to calculate the total number of cells within the field.

Apoptosis assay

PPF cells were transfected with sense or antisense adenoviruses (MOI, 50), maintained in DMEM/5% FCS for 48 h to allow expression of the transgene, and then exposed to serum-free medium for 24, 48, 72, or 96 h. After incubation, the medium was removed, and detached cells were pelleted by centrifugation. Attached cells were trypsinized and combined with detached cells. Cells were cytospun onto polysine slides (Merck & Co., Poole, UK) and fixed in 3.7% paraformaldehyde for 10 min. Apoptosis was quantified by examining the nuclear morphology of cells stained with 4 µg/ml Hoechst 33258 (Molecular Probes, Leiden, The Netherlands). Each experiment was repeated at least three times and in each experimental condition, 1000 single cells were scored for apoptosis.

IGF-1R activation assay

PPF cells were transfected with Ad-IGF-I (MOI, 50) and incubated in DMEM/5% FCS for 6 d at 37 C in 5% CO2, after which time the medium was changed to serum-free DMEM and conditioned for 48 h at 37 C in 5% CO2. NWTb3 cells (gift from Prof. D. LeRoith, NIH, Bethesda, MD), which overexpress IGF-1R, were subsequently incubated with conditioned medium, serum-free DMEM with or without 7.5 nM recombinant human IGF-I (rhIGF-I) or medium conditioned by nontransfected PPF cells for 15 min at 37 C in 5% CO2. After this time, cells were rinsed with PBS and lysed with RIPA buffer containing protease and phosphatase inhibitors (Sigma-Aldrich Corp., Poole, UK) for 30 min at 4 C. Lysate was subjected to Western immunoblotting using a rabbit antibody that recognizes only the activated (phosphorylated) isoform of the human IGF-1R ß-subunit (polyclonal, 1:1000; BioSource International, Camarillo, CA).

Data analysis

Statistical analysis was performed on control vs. condition data using the independent samples t test employing SPSS software (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary placental fibroblasts express vimentin and fibroblast surface protein

As we have previously demonstrated, cells isolated from villous explants obtained at termination of pregnancy during the first trimester were positive for vimentin and fibroblast surface protein, which are recognized markers for cells of the fibroblast lineage, but not for desmin or cytokeratin 7, which are markers of smooth muscle actin and trophoblast, respectively (39). The fibroblasts were observed to express coxsackie and adenovirus receptor (CAR) at very low levels, but the alternate adenovirus receptor, integrin {alpha}vß3 was readily detectable (not shown).

PPF cells transfected with Ad-IGF express GFP

Human IGF-I or -II cDNA was ligated into pQBI-AdCMV5-IRES-GFP in the sense or antisense direction and after generation of virus, PCR was used to confirm the presence of the human sequences within the viral DNA (data not shown). Transfection efficiency over a range of MOI (10–100) was assessed by monitoring GFP expression. At an MOI of 50, approximately 60% of PPF cells expressed GFP; higher MOI resulted in unacceptable levels of apoptosis (assessed by cell morphology), whereas at an MOI less than 50, GFP expression was reduced.

Lysates or conditioned medium obtained from PPF cells transfected with virus were analyzed for the presence of GFP and IGF-I or -II, respectively. Figure 1AGo (top) demonstrates that GFP, the secondary transcript within the bicistronic shuttle vector, was expressed and that this was independent of the nature and orientation of the IGF cDNA insert. Expression of the primary transcript (IGF) was demonstrated by Western immunoblot analysis of medium conditioned by virally transfected cells (Fig. 1AGo, bottom) and also by the ability of this medium to activate the tyrosine kinase within the ß-subunit of IGF-1R (Fig. 1BGo). After characterization, the biological consequences of adenovirus-mediated IGF expression were assessed using three assays of PPF cell function.



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  		FIG. 1. A, Characterization of PPF cells transfected with Ad-IGF. Upper panel, Lysates of PPF cells transfected with Ad-IGF-I (lane 3), antisense Ad-IGF-I (lane 4), Ad-IGF-II (lane 5), and antisense Ad-IGF-II (lane 6) were analyzed by Western immunoblot for GFP. A lysate from nontransfected PPF (control PPF) is shown as a negative control and enhanced GFP (~24 kDa), which is approximately 12 kDa smaller than the SuperGlo GFP expressed from the pQBI-AdCMV5-IGF-IRES-GFP vectors, was included as a positive control. Lower panel, Medium conditioned by nontransfected (control PPF) and Ad-IGF-II-transfected PPF cells for 48 h was analyzed for IGF-II (~7 kDa) by Western immunoblot. B, IGF-1R is activated (phosphorylated) by medium conditioned by Ad-IGF-I-transfected PPF cells. Serum-free medium was conditioned by PPF cells transfected with Ad-IGF-I (MOI, 50) for 48 h, then added to serum-starved NWTb3 cells for 15 min; lysates were analyzed by Western blotting using an antibody that only recognizes phosphorylated IGF-1R (lanes 4 and 5). Control samples include lysates of NWTb3 cells incubated with serum-free medium (lane 1), medium conditioned by nontransfected PPF cells (lane 2), and 7.5 nM rhIGF-I (lane 3).

 
Ad-IGF promotes PPF cell proliferation

Twenty-four- and 48-h incubation with recombinant human IGF-I (10 nM) stimulated a 1.8- and 3-fold increase in PPF mitogenesis, respectively (Fig. 2AGo). Cells transfected with Ad-IGF-I (MOI, 50) showed a 3- and 7-fold increase (P < 0.01) in [3H]thymidine incorporation over these time points (Fig. 2AGo).



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  		FIG. 2. A, The effect of rhIGF or sense Ad-IGF on [3H]thymidine incorporation by PPF cells. Recombinant IGF (10 nM) or sense/antisense Ad-IGF (MOI of 50) was incubated with serum-starved PPF cells for 20, 44, 68, or 92 h before the addition of 0.25 µCi/ml [3H]thymidine and incubation for an additional 4 h. [3H]Thymidine incorporation is expressed as proliferation compared with that of nontransfected control cells (percentage) incubated for the same time period and is shown as the mean (±SEM) of three experiments performed in duplicate. B, Proliferation of PPF cells cocultured with Ad-IGF-transfected PPF cells. Nontransfected PPF cells (with or without 10 nM rhIGF) or PPF cells transfected by Ad-IGF (MOI, 50) were positioned above a nontransfected monolayer of PPF cells and incubated for 44 h before the addition of 0.25 µCi/ml [3H]thymidine for an additional 4 h. [3H]Thymidine incorporation by the nontransfected monolayer is expressed as proliferation compared cells cocultured with nontransfected cells and is shown as the mean (±SEM) of three experiments performed in duplicate.

 
Similarly, rhIGF-II (10 nM) and Ad-IGF-II (MOI, 50) significantly enhanced [3H]thymidine incorporation (P < 0.01; Fig. 2AGo). Interestingly, 48 h after transfection with virus containing IGF-I or -II in the antisense orientation, [3H]thymidine incorporation was reduced compared with that in nontransfected cells (Fig. 2AGo). These results suggest that antisense isoforms of Ad-IGF can reduce the levels of endogenous IGF production, presumably at the level of translation, and indicate the importance of endogenous IGF for basal PPF proliferation.

These data demonstrate that both Ad-IGF-I and Ad-IGF-II are capable of eliciting a biological effect in transfected PPF cells. We next investigated whether virally produced IGF could stimulate proliferation in a paracrine manner.

Nontransfected PPF cells were cultured under Transwell inserts containing control PPF cells or cells that had been transfected with sense Ad-IGF-I or Ad-IGF-II. After 48 h, [3H]thymidine incorporation was assessed and was found to be significantly greater (>10-fold; P < 0.01) in cells cocultured with virally transfected PPF cells (Fig. 2BGo).

Virally mediated IGF expression stimulates migration of nontransfected target cells

The IGFs exert chemotactic effects on many cell types, and this function was assessed using Ad-IGF-transfected PPF cells to stimulate the migration of nontransfected PPF cells using a Transwell coculture system. Target cells were seeded into the Transwell inserts and positioned over control cells or cells that had been transfected with sense Ad-IGF-I or Ad-IGF-II.

Migration of PPF cells cultured above a nontransfected PPF monolayer was low (Fig. 3AGo); however, inclusion of 10 nM rhIGF-I or rhIGF-II stimulated a 3-fold increase in migration (P < 0.001; Fig. 3AGo). Similarly, migration of PPF cells positioned above a PPF monolayer transfected by Ad-IGF-I or Ad-IGF-II was enhanced (>10-fold increase; P < 0.001). The migration results are summarized in Fig. 3BGo and suggest that virally mediated IGF expression is capable of stimulating migration in PPF cells.



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  		FIG. 3. Migration of PPF cells in response to recombinant and Ad-IGF. A, Nontransfected PPF cells were seeded into a Transwell insert (8-µm pore size) and then positioned above a nontransfected monolayer of PPF cells (panel 1) or PPF cells transfected with Ad-IGF-I (panel 3) or Ad-IGF-II (panel 5) for 24 h. Cells on the upper surface of the membrane (nonmigratory PPF) were removed, and cells that had migrated to the underside of the Transwell membrane were stained with hematoxylin, then counted using the x10 objective on a light microscope. In some experiments, 10 nM rhIGF-I (panel 2) or IGF-II (panel 4) was included in the culture of nontransfected PPF positioned over a nontransfected PPF monolayer. B, Graphical representation of data presented in A.

 
Virally mediated IGF expression prevents PPF apoptosis

Previous studies have shown that IGF-I and -II protect against apoptosis in several cell types. We therefore examined IGF-mediated survival of PPF cells. PPF cells maintained in DMEM/10% FCS for 96 h showed low levels of apoptosis (<2%); however, serum withdrawal for 48, 72, or 96 h resulted in an 8-fold increase (Fig. 4Go) in the number of cells undergoing apoptosis, as quantified by assessment of nuclear morphology (Fig. 4Go, inset). This could be reversed by transfecting PPF with sense Ad-IGF-I, which suggests that IGF-I is an important survival factor for PPF, and this hypothesis was confirmed by the observation that compared with cells maintained in serum-free DMEM, PPF transduced with antisense Ad-IGF-I demonstrated increased apoptosis after 72 or 96 h of culture in serum-free DMEM. Similar results were obtained in experiments using sense and antisense Ad-IGF-II (Fig. 4Go).



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  		FIG. 4. The effect of sense and antisense AD-IGF on PPF cell survival. The level of apoptosis in nontransfected PPF cells maintained in DMEM/10% FCS or serum-free medium for 48, 72, or 96 h was compared with that observed in serum-starved PPF cells transfected with sense or antisense Ad-IGF-I or -II. Apoptosis was determined by assessing the nuclear morphology (fragmented chromatin, inset) of Hoechst-stained cells (n = 1000), and the results shown are the mean (±SEM) of two experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study show that PPF can be transfected with adenovirus, and aspects of their function are altered when levels of IGF-I or -II are manipulated. The finding that PPF proliferation, migration, and survival are enhanced when transfected with sense isoforms of Ad-IGF indicate that PPF cells are highly responsive to the actions of the IGFs. PPF cells display reduced functionality when transfected with antisense isoforms of Ad-IGF, implying the importance of endogenous IGF levels in normal cells. Su et al. (44) used adenovirus to deliver IGF-I or -II cDNA to cardiomyocytes and also found that the expression of IGFs stimulated migration and angiogenesis while protecting the cells from apoptosis in a hypoxic environment.

The observation that PPF cells could be efficiently transfected at an MOI of 50 was in contrast to the findings of Hikada et al. (45), who investigated the mechanism of adenoviral gene transfer in human skin fibroblasts. They found that fibroblast expression of CAR, the high affinity receptor for adenovirus, was very low on skin fibroblasts and required a 100-fold higher MOI to achieve a level of transfection comparable to that in cells expressing high numbers of CAR. We showed that PPF cells express low levels of CAR, but relatively high levels of integrin {alpha}v, which are probably responsible for the variation in viral titers required to transfect fibroblasts of dermal or placental origin. A possible explanation for this variation in CAR and integrin {alpha}v expression may be that in the native environment, skin fibroblasts would be more likely to come into contact with adenoviruses and may protect themselves by down-regulating both integrin {alpha}v and CAR expression. Conversely, placental fibroblasts are less likely to encounter adenovirus, being protected by the maternal immune system as well as the overlying syncytial barrier.

The feasibility of using gene therapy to enhance placental function was first highlighted by Senut et al. (46), who transplanted virally transfected cells into the rat placenta. The cells contained human GH cDNA, and the group found that within 2 d, the transplanted cells became vascularized and were immunologically tolerated, and the transgene product could be detected in the fetal circulation. We hypothesize that using a similar gene therapy approach to increase placental production of the IGFs may increase fetal growth. The data obtained in this study suggest that secreted transgenic IGF could cause a proliferative effect in the stromal compartment of the placenta. Paracrine effects on other placental cells might also occur as well as export to the fetus. This is relevant to gestational pathology, because a recent study of the placenta-specific igf-II knockout mouse (47) revealed that these animals have a reduced placental volume and, importantly, a decrease in the surface area available for exchange. Furthermore, in humans, small placentas in association with higher rates of apoptosis have been reported in some types of IUGR (48).

It has been shown that systemic administration of IGF-II directly to sheep and monkey fetuses over a 10-d period has no effect on placental weight despite increases in placental amino acid transfer and reduction in catabolic processes such as proteolysis and amino acid oxidation after short-term (4-h) infusion (49). Using gene therapy to supply IGF may sustain IGF bioavailability and have the added advantage of preventing some of the consequences incurred by direct administration of IGF peptide [hypoglycemia, lipohypertrophy (50), and retinal edema, by increasing capillary permeability (51)].

Fetal growth disorders are a major clinical problem, affecting between 5% and 10% of all pregnancies. There is strong evidence showing that fetuses failing to achieve their genetically determined growth potential have an increased risk of developing noninsulin-dependent diabetes, coronary heart disease, and hypertension in later life. These results establish that cells of placental origin are amenable to a viral transfection and that they can respond to the effects of the IGFs in a way that may potentially increase placental function and, subsequently, fetal growth.


   Acknowledgments
 
We are grateful to the Biotechnology and Biologicial Sciences Research Council for funding.


   Footnotes
 
First Published Online October 26, 2004

Abbreviations: Ad, Adenovirus; CAR, coxsackie and adenovirus receptor; CMV, cytomegalovirus; FCS, fetal calf serum; GFP, green fluorescent protein; IGFBP, IGF-binding protein; IGF-1R, type 1 IGF receptor; IRES, internal ribosome entry site; IUGR, intrauterine growth restriction; MOI, multiplicity of infection; PPF, primary placental fibroblast; rhIGF, recombinant human IGF.

Received June 3, 2004.

Accepted October 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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