Adeno-Associated Virus-2 (AAV-2) Causes Trophoblast Dysfunction, and Placental AAV-2 Infection Is Associated with Preeclampsia
2006; Elsevier BV; Volume: 168; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.050781
ISSN1525-2191
AutoresFabián Arechavaleta-Velasco, Yujie Ma, Jian Zhang, Cindy McGrath, Samuel Parry,
Tópico(s)Prenatal Screening and Diagnostics
ResumoShallow invasion by extravillous trophoblast cells into the uterine wall reduces placental perfusion and causes placental dysfunction, but the one or more causes of shallow placental invasion are unknown. We hypothesized that infection with adeno-associated virus-2 (AAV-2) inhibits trophoblast invasion and is associated with preeclampsia, which is a common obstetric complication resulting from placental dysfunction. We determined that transformed extravillous trophoblast (HTR-8/SVneo) cells were susceptible to AAV-2 infection in the presence or absence of adenovirus, which provides helper function for AAV-2 replication, and that AAV-2 infection reduced invasion of HTR-8/SVneo cells through an extracellular matrix before cytopathic effects were detected. In a case-control study, AAV-2 DNA was found more frequently in trophoblast cells from cases of severe preeclampsia (22/40) than from normal term deliveries (5/27, P = 0.002). These results indicate that AAV-2 infection is a previously unidentified cause of placental dysfunction. Additional studies to determine the susceptibility of extravillous trophoblast to other viruses, and the mechanisms by which viral infection impairs placental function, are warranted. Shallow invasion by extravillous trophoblast cells into the uterine wall reduces placental perfusion and causes placental dysfunction, but the one or more causes of shallow placental invasion are unknown. We hypothesized that infection with adeno-associated virus-2 (AAV-2) inhibits trophoblast invasion and is associated with preeclampsia, which is a common obstetric complication resulting from placental dysfunction. We determined that transformed extravillous trophoblast (HTR-8/SVneo) cells were susceptible to AAV-2 infection in the presence or absence of adenovirus, which provides helper function for AAV-2 replication, and that AAV-2 infection reduced invasion of HTR-8/SVneo cells through an extracellular matrix before cytopathic effects were detected. In a case-control study, AAV-2 DNA was found more frequently in trophoblast cells from cases of severe preeclampsia (22/40) than from normal term deliveries (5/27, P = 0.002). These results indicate that AAV-2 infection is a previously unidentified cause of placental dysfunction. Additional studies to determine the susceptibility of extravillous trophoblast to other viruses, and the mechanisms by which viral infection impairs placental function, are warranted. The development of the human placenta is dependent on the differentiation of trophoblast cells along two pathways. In one pathway, mononucleated cytotrophoblast cells within the placental villi terminally differentiate into the multinucleated syncytiotrophoblast, which forms the only continuous layer separating the maternal intervillous space and the fetal capillary endothelium.1Kaufmann P Mayhew TM Charnock-Jones DS Aspects of human fetoplacental vasculogenesis and angiogenesis. II. Changes during normal pregnancy.Placenta. 2004; 25: 114-126Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar In the other pathway, a subset of undifferentiated cytotrophoblast cells in anchoring villi invades maternal tissues and blood vessels within the uterine decidua and myometrium.2Khong TY Placental vascular development and neonatal outcome.Semin Neonatol. 2004; 9: 255-263Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 3Rama S Rao AJ Regulation of growth and function of the human placenta.Mol Cell Biochem. 2003; 253: 263-268Crossref PubMed Scopus (20) Google Scholar These extravillous, or invasive, trophoblast cells mediate placental attachment to the maternal uterine wall and are responsible for establishing a high flow, low resistance maternal circulation supplying the placenta and fetus. Failed invasion by extravillous trophoblast cells leads to placental dysfunction and adverse obstetric outcomes associated with placental dysfunction, including preeclampsia, fetal growth restriction, and preterm delivery.4Meekins JW Pijnenborg R Hanssens M McFadyen IR van Asshe A A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies.Br J Obstet Gynaecol. 1994; 101: 669-674Crossref PubMed Scopus (779) Google Scholar, 5Salafia CM Minior VK Pezzullo JC Popek EJ Rosenkrantz TS Vintzileos AM Intrauterine growth restriction in infants of less than thirty-two weeks' gestation: associated placental pathologic features.Am J Obstet Gynecol. 1995; 173: 1049-1057Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 6Kim YM Bujold E Chaiworapongsa T Gomez R Yoon BH Thaler HT Rotmensch S Romero R Failure of physiologic transformation of the spiral arteries in patients with preterm labor and intact membranes.Am J Obstet Gynecol. 2003; 189: 1063-1069Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 7Germain AM Carvajal J Sanchez M Valenzuela GJ Tsunekawa H Chuaqui B Preterm labor: placental pathology and clinical correlation.Obstet Gynecol. 1999; 94: 284-289Crossref PubMed Scopus (71) Google Scholar Although some molecular abnormalities associated with failed placental invasion have been reported, the cause of shallow invasion by extravillous trophoblast cells has not been elucidated.3Rama S Rao AJ Regulation of growth and function of the human placenta.Mol Cell Biochem. 2003; 253: 263-268Crossref PubMed Scopus (20) Google Scholar, 8Mayhew TM Charnock-Jones DS Kaufmann P Aspects of human fetoplacental vasculogenesis and angiogenesis. III. Changes in complicated pregnancies.Placenta. 2004; 25: 127-139Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar We hypothesize that viral infections of the placenta induce pathological changes that impair trophoblast invasion into the uterine wall, possibly resulting in adverse obstetric outcomes due to placental dysfunction. Adeno-associated virus (AAV) is a member of the parvovirus family, and several serotypes of AAV have been identified. Of these, types 2 and 3 infect humans, although type 3 is rare and is probably a laboratory contaminant that developed during construction of recombinant AAV vectors.9Berns KI Giraud C Biology of adeno-associated virus.Curr Top Microbiol Immunol. 1996; 218: 1-23Crossref PubMed Scopus (226) Google Scholar, 10Vihinen-Ranta M Suikkanen S Parrish CR Pathways of cell infection by parvoviruses and adeno-associated viruses.J Virol. 2004; 78: 6709-6714Crossref PubMed Scopus (41) Google Scholar AAV-2 is a common human isolate (40 to 80% of adults have been exposed), and infection involves hematogenous seeding.11Erles K Sebokova P Schlehofer JR Update on the prevalence of serum antibodies (IgG and IgM) to adeno-associated virus (AAV).J Med Virol. 1999; 59: 406-411Crossref PubMed Scopus (284) Google Scholar, 12Grossman Z Mendelson E Brok-Simoni F Mileguir F Leitner Y Rechavi G Ramot B Detection of adeno-associated virus type 2 in human peripheral blood cells.J Gen Virol. 1992; 73: 961-966Crossref PubMed Scopus (63) Google Scholar Thus, the placenta may be exposed to AAV-2 during primary or reactivated maternal infection. Although productive AAV-2 infections usually require co-infection with helper viruses (including adenovirus, cytomegalovirus, human papillomavirus, and herpes simplex virus), other investigators demonstrated that AAV-2 can undergo full replicative cycles in keratinocytes, and we demonstrated that trophoblast cells are susceptible to AAV-2 in the presence or absence of helper virus co-infection.10Vihinen-Ranta M Suikkanen S Parrish CR Pathways of cell infection by parvoviruses and adeno-associated viruses.J Virol. 2004; 78: 6709-6714Crossref PubMed Scopus (41) Google Scholar, 13Fisher KJ Gao GP Weitzman MD DeMatteo R Burda JF Wilson JM Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.J Virol. 1996; 70: 520-532Crossref PubMed Google Scholar, 14Leonard CJ Berns KI Adeno-associated virus type 2: a latent life cycle.Prog Nucleic Acid Res Mol Biol. 1994; 48: 29-52Crossref PubMed Scopus (29) Google Scholar, 15Meyers C Mane M Kokorina N Alam S Hermonat PL Ubiquitous human adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model.Virology. 2000; 272: 338-346Crossref PubMed Scopus (63) Google Scholar, 16Parry S Holder J Halterman MW Weitzman MD Davis AR Federoff H Strauss 3rd, JF Transduction of human trophoblastic cells by replication-deficient recombinant viral vectors. Promoting cellular differentiation affects virus entry.Am J Pathol. 1998; 152: 1521-1529PubMed Google Scholar Additionally, there are several preliminary reports linking AAV-2 to adverse reproductive outcomes, including spontaneous miscarriage, gestational trophoblastic disease, and preterm labor.17Burguete T Rabreau M Fontanges-Darriet M Roset E Hager HD Koppel A Bischof P Schlehofer JR Evidence for infection of the human embryo with adeno-associated virus in pregnancy.Hum Reprod. 1999; 14: 2396-2401Crossref PubMed Scopus (55) Google Scholar, 18Tobiasch E Rabreau M Geletneky K Larue-Charlus S Severin F Becker N Schlehofer JR Detection of adeno-associated virus DNA in human genital tissue and in material from spontaneous abortion.J Med Virol. 1994; 44: 215-222Crossref PubMed Scopus (101) Google Scholar, 19Kiehl K Schlehofer JR Schultz R Zugaib M Armbruster-Moraes E Adeno-associated virus DNA in human gestational trophoblastic disease.Placenta. 2002; 23: 410-415Abstract Full Text PDF PubMed Scopus (22) Google Scholar In pregnant mice, infection with AAV induces early abortion, and in humans early miscarriage has been associated with AAV-2 infection of trophoblast cells.18Tobiasch E Rabreau M Geletneky K Larue-Charlus S Severin F Becker N Schlehofer JR Detection of adeno-associated virus DNA in human genital tissue and in material from spontaneous abortion.J Med Virol. 1994; 44: 215-222Crossref PubMed Scopus (101) Google Scholar, 20Botquin V Cid-Arregui A Schlehofer JR Adeno-associated virus type 2 interferes with early development of mouse embryos.J Gen Virol. 1994; 75: 2655-2662Crossref PubMed Scopus (30) Google Scholar Based on our previous findings and the observations of other investigators, we postulated that AAV-2 infection of the placenta may be relatively common and may be associated with an increased risk of obstetric complications associated with placental dysfunction, such as preeclampsia. Thus, we sought to determine 1) if infection of invasive trophoblast cells by AAV-2 reduces cell invasion and/or induces cell death and 2) if placental infection with AAV-2 is associated with severe preeclampsia. The HTR-8/SVneo trophoblast cell line was provided by C.H. Graham (Queen's University, Ontario, Canada). HTR-8/SVneo cells originally were obtained from human first-trimester placenta and immortalized by transfection with a cDNA construct that encodes the simian virus 40 large T antigen.21Graham CH Hawley TS Hawley RG MacDougall JR Kerbel RS Khoo N Lala PK Establishment and characterization of first trimester human trophoblast cells with extended lifespan.Exp Cell Res. 1993; 206: 204-211Crossref PubMed Scopus (827) Google Scholar These cells exhibit a high proliferation index and share phenotypic similarities with nontransfected parent HTR-8 cells, including invasive characteristics.21Graham CH Hawley TS Hawley RG MacDougall JR Kerbel RS Khoo N Lala PK Establishment and characterization of first trimester human trophoblast cells with extended lifespan.Exp Cell Res. 1993; 206: 204-211Crossref PubMed Scopus (827) Google Scholar Cells were cultured in Roswell Park Memorial Institute 1640 medium (Invitrogen, Carlsbad, CA) supplemented with antibiotics and 5% fetal bovine serum as monolayers at 37°C in 5% CO2. Wild-type AAV-2 (wtAAV-2) and wild-type adenovirus-5 (wtAd-5) were purchased from the American Type Culture Collection. Replication-deficient AAV-2 vectors, purchased from the Institute of Human Gene Therapy at the University of Pennsylvania, contained transgenes encoding the following reporter proteins: green fluorescent protein (rAAV-2-GFP) and β-galactosidase (rAAV-2-LacZ). HTR-8/SVneo cells were infected with AAV-2 vectors or wtAAV-2 in the presence or absence of wtAd-5 co-infection. After incubation at 4°C for 30 minutes, the infected cells were plated in 12-well culture plates (5 × 106 cells/well) coated with fibronectin (Invitrogen) for growth. Ninety-six hours after infection with wild-type viruses, the cells were examined for cytopathic effects. Reporter protein expression was detected by either β-galactosidase assay (rAAV-2-LacZ)16Parry S Holder J Halterman MW Weitzman MD Davis AR Federoff H Strauss 3rd, JF Transduction of human trophoblastic cells by replication-deficient recombinant viral vectors. Promoting cellular differentiation affects virus entry.Am J Pathol. 1998; 152: 1521-1529PubMed Google Scholar or fluorescence microscopy to detect green fluorescent protein (rAAV-2-GFP). β-Galactosidase enzyme activity was assessed 24 hours after infection using a solution-based assay system and measuring absorbance at 420 nm (Promega Corp., Madison, WI).16Parry S Holder J Halterman MW Weitzman MD Davis AR Federoff H Strauss 3rd, JF Transduction of human trophoblastic cells by replication-deficient recombinant viral vectors. Promoting cellular differentiation affects virus entry.Am J Pathol. 1998; 152: 1521-1529PubMed Google Scholar To detect GFP, cells were rinsed with phosphate-buffered saline 24 hours after viral infection, and transduction efficiency was determined by observing the cells under fluorescence microscopy. HTR-8/SVneo cells (with or without 1500 particles of wtAAV-2/cell for 2 to 24 hours) were treated with normal goat serum to block nonspecific binding, after which the cells (at least 4 × 104 cells) were incubated with murine monoclonal antibodies against the AAV-2 receptors heparan sulfate (Seikagaku, Falmouth, MA) and αVβ5 integrin (Chemicon International, Temecula, CA).22Kern A Schmidt K Leder C Muller OJ Wobus CE Bettinger K Von der Lieth CW King JA Kleinschmidt JA Identification of a heparin-binding motif on adeno-associated virus type 2 capsids.J Virol. 2003; 77: 11072-11081Crossref PubMed Scopus (298) Google Scholar, 23Summerford C Bartlett JS Samulski RJ AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection.Nat Med. 1999; 5: 78-82Crossref PubMed Scopus (609) Google Scholar, 24Summerford C Samulski RJ Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.J Virol. 1998; 72: 1438-1445Crossref PubMed Google Scholar After treatment with primary antibody, the cells were incubated with fluorescein-conjugated goat antibody to mouse immunoglobulin, and fluorescence was measured using an Epics XL flow cytometer (Coulter Corp., Miami, FL). Adhesion of HTR-8/SVneo cells to fibronectin was measured as previously described.25Burrows TD King A Smith SK Loke YW Human trophoblast adhesion to matrix proteins: inhibition and signal transduction.Hum Reprod. 1995; 10: 2489-2500Crossref PubMed Scopus (53) Google Scholar Briefly, 96-well tissue culture plates were coated with 10 μg/ml fibronectin. Infected and noninfected HTR-8/SVneo cells (4.0 × 104 cells/well) were added to the wells, and the mixtures were incubated at 37°C for 45 minutes. After rinsing the non-bound cells from the wells, the bound cells were quantified by measuring endogenous lactate dehydrogenase (LDH) using the CytoTox 96 nonradioactive cytotoxicity assay (Promega). The invasiveness of HTR-8/SVneo cells through an extracellular matrix was measured using a commercially available cell invasion assay kit (Chemicon). Briefly, HTR-8/SVneo cells (1 × 106 cells/well) were infected with 15 to 1500 particles of wtAAV-2/cell in the presence or absence of 100 particles of wtAd-5/cell and plated on ECMatrix gel-coated layered cell culture inserts. After 24 hours, the noninvading cells and the ECMatrix gel from the interior of the inserts were removed using a cotton-tipped swab. Invasive cells on the lower surface of the membranes were stained by dipping the inserts in 0.2% crystal violet. After 20 minutes, the stained cells were dissolved in 10% acetic acid, and colorimetric absorbance was measured at 560 nm. Human embryonic kidney 293 cells (American Type Culture Collection CRL-1573), which are susceptible to AAV-2 infection and display an invasive phenotype, were used as controls.16Parry S Holder J Halterman MW Weitzman MD Davis AR Federoff H Strauss 3rd, JF Transduction of human trophoblastic cells by replication-deficient recombinant viral vectors. Promoting cellular differentiation affects virus entry.Am J Pathol. 1998; 152: 1521-1529PubMed Google Scholar To determine whether decreased levels of adhesion and/or invasion could be attributed to cell death 24 hours after infection with wtAAV-2, the viability of HTR-8/SVneo cells was determined by measuring LDH leakage into the medium using the CytoTox 96 nonradioactive cytotoxicity assay. After collecting cell culture medium, HTR-8/SVneo cells were lysed with 1% Triton X-100, and equal volumes of medium and lysis buffer (containing LDH released from lysed cells) were transferred to separate wells in 96-well plates. Substrate mix provided with the kit was added, and the enzymatic reaction was measured by absorbance at 490 nm. Results were obtained after subtracting background values. The percentage of cells that remained viable was calculated as follows: 100 × (1 − spontaneous release of LDH/maximum release of LDH). Apoptotic changes in HTR-8/SVneo cells were examined by detecting DNA fragmentation and measuring caspase-3 levels. Infected and noninfected HTR-8/SVneo cells (2.0 × 106 cells/well) were plated on 6-well plates in 2 ml of Roswell Park Memorial Institute 1640 medium. After 96 hours, adherent and floating cells were homogenized in lysis buffer, and DNA was isolated from the lysate by passage through filter tubes (Apoptotic DNA Ladder kit, Roche Diagnostics, Mannheim, Germany). DNA from each sample was electrophoresed in 1.8% agarose gels and visualized by ethidium bromide staining. We conducted a case-control study comparing the rates of AAV-2 infection of trophoblast cells between cases of severe preeclampsia and controls who delivered at term without any medical or obstetric complications. The Institutional Review Board at the University of Pennsylvania approved this study (protocol 700943) in December 1999. We reviewed medical charts at the Hospital of the University of Pennsylvania to identify cases of severe preeclampsia requiring delivery before 37 weeks' gestation in 1998 and 1999. Severe preeclampsia was defined according to criteria outlined by the American College of Obstetricians and Gynecologists: systolic blood pressure >160 mm Hg or diastolic blood pressure >110 mm Hg on two occasions at least 6 hours apart, or end-organ manifestations, including proteinuria (≥3+ by dipstick, ≥5 × g by 24-hour collection), oliguria, cerebral disturbances, pulmonary edema, thrombocytopenia, impaired liver function, or fetal growth restriction, in the setting of preeclampsia.26American College of Obstetricians and Gynecologists Practice Bulletin #33 Diagnosis and Management of Preeclampsia and Eclampsia.Obstet Gynecol. 2002; 99: 159-167Crossref PubMed Google Scholar Because preeclampsia frequently is not a straightforward diagnosis, only severe cases requiring iatrogenic preterm delivery were selected after review of the complete medical record by a certified Maternal-Fetal Medicine specialist (S.P.). Women were excluded if they had other risk factors for developing preeclampsia, including chronic hypertension, renal disease, diabetes, and multiple gestations. We also excluded women whose pregnancies were complicated by fetal malformations. All cases had placental specimens stored in the Pathology Laboratory at the Hospital of the University of Pennsylvania. Controls were selected at random from among uncomplicated term deliveries at the same hospital between July and December 2001, and placentas from these controls were sent to the Pathology Laboratory for storage for our research purposes. The control placentas were fixed and stored as paraffinized tissue blocks according to the same protocol that was used for routine clinical specimens. Histological sections from the basal plate region of placentas were placed on membrane slides for laser microdissection (Molecular Machines and Industries, Knoxville, TN), and trophoblast cells were microdissected from the membranes using the PixCell II Laser Capture Microdissection System (Arcturus Engineering, Mountain View, CA). The laser capture microscope was used to isolate extravillous trophoblast cells from invasive trophoblast columns and villous trophoblast cells from the periphery of chorionic villi (eg, cytotrophoblast and syncytiotrophoblast) in the basal plate region of placentas from cases and controls (Figure 1). DNA was extracted from laser-captured trophoblast cells using DNeasy minikits (Qiagen, Valencia, CA), and 2 ng of total DNA was used to amplify a 379-bp segment of the AAV-2 rep gene by nested PCR. The primers used for the first PCR were forward primer (nucleotide 1421) 5′-CGACTGTGTCGACAAGATGGTGAT-3′ and reverse primer (nucleotide 1885) 5′-TACCTGTCTGCGTAGTTGATCGAAG-3′. The primers used for the nested PCR were forward primer (nucleotide 1473) 5′-AAGGTCGTGGAGTCGGCCAAA-3′ and reverse primer (nucleotide 1851) 5′-GTCGATGGCTGCGCAACTGA-3′. PCR was performed under the following conditions: initial denaturation step at 95°C for 5 minutes, followed by 30 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The PCR products were visualized as a single band by 2% agarose gel electrophoresis, and PCR products were confirmed by sequencing. DNA extracted from wild-type AAV-2 was used as a positive control, whereas water was used as the negative control. To ensure that adequate amounts of DNA were extracted from all samples, we performed nested PCR to detect a 106-bp segment of the β-actin gene. In the viral transduction studies and adhesion, viability, and invasion assays, absorbance values were measured in triplicate samples for each experimental condition, and the experiments were performed three times. Average absorbance values were compared using two-tailed t-tests, and P values < 0.05 were considered statistically significant. We calculated a sample size for a 3:2 case/control design, because placentas from controls had to be collected prospectively and prepared according to the same protocol used for routine clinical specimens (eg, cases). In previous preliminary studies, AAV-2 DNA was detected in 0 to 40% of placentas and amniocytes from early miscarriages, preterm deliveries, and controls.17Burguete T Rabreau M Fontanges-Darriet M Roset E Hager HD Koppel A Bischof P Schlehofer JR Evidence for infection of the human embryo with adeno-associated virus in pregnancy.Hum Reprod. 1999; 14: 2396-2401Crossref PubMed Scopus (55) Google Scholar, 18Tobiasch E Rabreau M Geletneky K Larue-Charlus S Severin F Becker N Schlehofer JR Detection of adeno-associated virus DNA in human genital tissue and in material from spontaneous abortion.J Med Virol. 1994; 44: 215-222Crossref PubMed Scopus (101) Google Scholar, 27Friedman-Einat M Grossman Z Mileguir F Smetana Z Ashkenazi M Barkai G Varsano N Glick E Mendelson E Detection of adeno-associated virus type 2 sequences in the human genital tract.J Clin Microbiol. 1997; 35: 71-78PubMed Google Scholar To demonstrate a 4-fold difference in AAV-2 detection rates (40 vs. 10%) between cases and controls, we determined that we needed to study placentas from 40 cases of preeclampsia and 27 controls (α = 0.05, 1 = β = 0.80). Rates of AAV-2 detection were compared between cases and controls by constructing two-by-two tables and performing χ2 or Fisher exact tests. Flow cytometry studies demonstrated that 99.5 ± 0.5% of HTR-8/SVneo cells expressed the AAV-2 receptor heparan sulfate on cell surfaces, whereas 87.7 ± 2.3% of HTR-8/SVneo cells expressed αVβ5 integrin (Figure 2). Infection with 1500 particles of wtAAV-2/cell for 2 to 24 hours did not affect cell-surface expression of heparan sulfate or αVβ5 integrin. Based on the flow cytometry results, we anticipated that HTR-8/SVneo cells were susceptible to infection by AAV-2, and we observed that recombinant AAV-2 constructs (rAAV-2-LacZ or rAAV-2-GFP, 1 to 100 particles/cell) efficiently transduced HTR-8/SVneo cells in the presence or absence of 100 particles of wtAd-5/cell (Figure 3). The transduction efficiency of rAAV-2-GFP was determined by observing the cells from three separate experiments under fluorescence microscopy at 40× magnification. Fluorescent nuclei were detected in 0.67 ± 1.0% of uninfected cells and 43.7 ± 7.5% of HTR-8/SVneo cells transduced with 100 particles of rAAV-2-GFP/cell (P = 0.01, two-tailed t-test). The pathological effects of AAV-2 infection were assessed by comparing adhesion of infected and noninfected HTR-8/SVneo cells to fibronectin-coated culture plates and invasion of the cells through an extracellular matrix. Viability assays performed 24 hours after infection with wtAAV-2 and/or wtAd-5 demonstrated that >90% of infected and noninfected HTR-8/SVneo cells remained viable (Figure 4A). Nevertheless, HTR-8/SVneo cells infected with 15–1500 particles of wtAAV-2/cell for 24 hours demonstrated significantly decreased adhesion to fibronectin-coated plates compared to controls (Figure 4B). Invasion assays were conducted by plating infected and noninfected HTR-8/SVneo cells and 293 cells in extracellular matrix on semipermeable membranes (Figure 4C). Baseline invasion rates were determined for noninfected HTR-8/SVneo cells and 293 cells. Infection of HTR-8/SVneo cells by wtAAV-2 or wtAd-5 reduced invasion by ∼40% but did not affect invasion through the extracellular matrix by 293 cells. Although the viability of HTR-8/SVneo cells was not reduced 24 hours after infection by wtAAV-2, we sought to determine whether AAV-2 infection induced trophoblast cell death over a longer period of time. The cytopathic effects of AAV-2 infection on extravillous trophoblast cells were evaluated by direct microscopy. At 96 hours after infection with 15–1500 viral particles/cell of wtAAV-2 in the presence or absence of 100 particles/cell of wtAd-5, HTR-8/SVneo cells exhibited marked cytopathic changes, including cell rounding and detachment from the monolayer (Figure 5). To determine whether the cytopathic effects were the result of apoptosis, DNA fragmentation was analyzed by agarose gel electrophoresis (Figure 6). In uninfected cells and cells infected with only wtAd-5, DNA fragments were not detected. However, in cells that were infected for 96 hours with wtAAV-2 with or without wtAd-5, the intensity of bands corresponding to DNA fragmentation was positively correlated with the amount of wtAAV-2 that was used to infect the HTR-8/SVneo cells. The age and gravidity of preeclampsia cases and controls were similar, but a higher percentage of cases (28/40) than controls (6/27) were nulliparous (Table 1). Although the systolic and diastolic blood pressures of cases were greater than controls at their initial prenatal visits, these differences were clinically insignificant, and no subjects had blood pressures greater than 140/90 mm Hg early in pregnancy. As expected, the systolic and diastolic blood pressures of cases were greater than controls at the time of delivery, and the incidence of proteinuria and cesarean delivery was greater in cases than controls. Among cases, the average gestational age at delivery was 32.5 weeks, and 30% had intrauterine growth restriction.Table 1Demographics and Clinical Outcomes for Cases and ControlsCases, N = 40Controls, N = 27P value*Two-tailed t-tests were performed for continuous variables; χ-square (or Fisher's exact) tests were performed for dichotomous variables.Age (year ± SD)25.2 ± 6.926.4 ± 6.10.48Gravidity (± SD)2.4 ± 1.63.0 ± 2.10.22Parity (± SD)0.5 ± 1.01.4 ± 1.4<0.01EGA†EGA = estimated gestational age. 1st visit (week ± SD)12.4 ± 5.415.9 ± 9.40.11Initial SBP‡SBP = systolic blood pressure; DBP = diastolic blood pressure. (mm Hg ± SD)116 ± 10.8107.5 ± 12.0<0.01Initial DBP‡SBP = systolic blood pressure; DBP = diastolic blood pressure. (mm Hg ± SD)70.6 ± 8.558.3 ± 7.5<0.01Initial proteinuria (%)2/40 (5.0%)1/27 (3.7%)0.79SBP‡SBP = systolic blood pressure; DBP = diastolic blood pressure. delivery (mm Hg ± SD)159.7 ± 16.9119.1 ± 10.4<0.01DBP‡SBP = systolic blood pressure; DBP = diastolic blood pressure. delivery (mm Hg ± SD)98.3 ± 10.468.2 ± 8.9<0.01Proteinuria delivery (%)39/40 (97.5%)0<0.01EGA†EGA = estimated gestational age. delivery (week ± SD)32.5 ± 2.339.8 ± 1.3<0.01BW§BW = birth weight. (g ± SD)1588.1 ± 468.73376.9 ± 489.6<0.01IUGR (%)12/40 (30.0%)0<0.01Oligohydramnios∥Oligohydramnios = amniotic fluid index < 5 cm. (%)4/40 (10.0%)00.11Cesarean delivery (%)25/40 (62.5%)6/27 (22.2%)<0.01IUGR, intrauterine growth restriction (birth weight < 10th percentile for gestational age at delivery).* Two-tailed t-tests were performed for continuous variables; χ-square (or Fisher's exact) tests were performed for dichotomous variables.† EGA = estimated gestational age.‡ SBP = systolic blood pressure; DBP = diastolic blood pressure.§ BW = birth weight.∥ Oligohydramnios = amniotic fluid index < 5 cm. Open table in a new tab IUGR, intrauterine growth restriction (birth weight < 10th percentile for gestational age at delivery). Two analyses were performed: 1) comparing rates of AAV-2 detection in extravillous or villous trophoblast cells between cases and controls and 2) comparing rates of AAV-2 detection in extravillous and villous trophoblast cells (possibly indicating greater viral load) between cases and controls. AAV
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