Adeno-associated virus-2 (AAV-2) causes trophoblast dysfunction, and placental AAV-2 infection is associated with preeclampsia

Abstract

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.

Figure 1

Laser capture microdissection of trophoblast cells. a: Photomicrograph of placental villi from the placental basal plate region (magnification: 20×). b: Photomicrograph of extravillous trophoblast column from the placental basal plate region (magnification: 100×). Extravillous trophoblast cells were immunostained with anti-cytokeratin-18 antibodies for identification. Arrows indicate regions where cells were captured.

Figure 2

Indirect immunofluorescence assays indicating HTR-8/SVneo cell-surface expression of AAV-2 receptors. Fluorescence intensity was measured in cells that were incubated with monoclonal antibodies against heparan sulfate (a) or αVβ5 integrin (b) and fluorescein-conjugated secondary antibody (blue-shaded curves) and in cells that were incubated with fluorescein-conjugated secondary antibody alone (green-shaded curves). The y axis indicates cell number, and the x axis indicates logarithm of fluorescence intensity.

Figure 3

Transduction of HTR-8/SVneo cells by recombinant AAV-2 constructs (rAAV-2-LacZ and rAAV-2-GFP). A: Successful transduction of the Lac-Z transgene was measured by β-galactosidase activity along the y axis. HTR-8/Svneo cells were infected for 24 hours with 1–100 viral particles/cell of rAAV-2-LacZ in the presence or absence of 100 viral particles/cell of wtAd-5 (Ad), and significantly greater transduction rates compared to uninfected controls are indicated by asterisks. B: Successful infection and production of the GFP transgene was detected by green fluorescent staining. HTR-8/SVneo cells were infected for 24 hours with 100 viral particles/cell of rAAV-2-GFP in the absence of wtAd-5. Results are: from a light microscope (a and c), from a fluorescence microscopy (b and d), uninfected cells (a and b), and cells infected with rAAV-2-GFP (c and d).

Figure 4

Adhesion and invasion of HTR-8/SVneo cells. A: Viability of HTR-8/SVneo cells was determined by measuring LDH leakage into cell culture medium 24 hours after infection with 15 to 1500 viral particles/cell of wtAAV-2 and/or 100 viral particles/cell of wtAd-5. B: Adhesion of HTR-8/SVneo cells to fibronectin-coated plates 24 hours after infection with 15 to 1500 viral particles/cell of wtAAV-2 and/or 100 viral particles/cell of wtAd-5 was compared to noninfected controls. Significant differences (P < 0.05) are indicated by an asterisk. C: Invasion assays were performed 24 hours after viral infection. Invasion was measured spectrophotometrically according to the optical density for crystal violet at 550 nm (y axis). Baseline invasion rates were determined for noninfected HTR-8/SVneo cells and 293 cells, and infection of HTR-8/SVneo cells by wtAAV-2 (150 viral particles/cell) and/or wtAd-5 (100 viral particles/cell) reduced invasion by ∼40%. Significantly different invasion rates between infected cells and controls (P < 0.05) were indicated by an asterisk.

Figure 5

Cytopathic effect of wtAAV-2. HTR-8/SVneo cells were infected with 15 to 1500 viral particles/cell of wtAAV-2 in the presence or absence of 100 viral particles/cell of wtAd-5. Cells were photographed at 10× magnification 96 hours after infection. Control (a); wtAd-5 (b); wtAAV-2 (1500 particles/cell) plus wtAd-5 (c); wtAAV-2 (1500 particles/cell) (d); wtAAV-2 (150 particles/cell) (e); and wtAAV-2 (15 particles/cell) (f).

Figure 6

DNA ladder assay demonstrating nucleosomal fragments in apoptotic cells. HTR-8/SVneo cells were infected for 96 hours with 15 to 1500 viral particles/cell of wtAAV-2 and/or 100 viral particles/cell of wtAd-5. The intensity of bands corresponding to DNA fragmentation positively correlated with the amount of AAV-2 that was used to infect the HTR-8 cells.