Uteroplacental adenovirus vascular endothelial growth factor gene therapy increases fetal growth velocity in growth-restricted sheep pregnancies

Abstract

Fetal growth restriction (FGR) occurs in ∼8% of pregnancies and is a major cause of perinatal mortality and morbidity. There is no effective treatment. FGR is characterized by reduced uterine blood flow (UBF). In normal sheep pregnancies, local uterine artery (UtA) adenovirus (Ad)-mediated overexpression of vascular endothelial growth factor (VEGF) increases UBF. Herein we evaluated Ad.VEGF therapy in the overnourished adolescent ewe, an experimental paradigm in which reduced UBF from midgestation correlates with reduced lamb birthweight near term. Singleton pregnancies were established using embryo transfer in adolescent ewes subsequently offered a high intake (n=45) or control intake (n=12) of a complete diet to generate FGR or normal fetoplacental growth, respectively. High-intake ewes were randomized midgestation to receive bilateral UtA injections of 5×10¹¹ particles Ad.VEGF-A165 (n=18), control vector Ad.LacZ (n=14), or control saline (n=13). Fetal growth/well-being were evaluated using serial ultrasound. UBF was monitored using indwelling flowprobes until necropsy at 0.9 gestation. Vasorelaxation, neovascularization within the perivascular adventitia, and placental mRNA expression of angiogenic factors/receptors were examined using organ bath analysis, anti-vWF immunohistochemistry, and qRT-PCR, respectively. Ad.VEGF significantly increased ultrasonographic fetal growth velocity at 3-4 weeks postinjection (p=0.016-0.047). At 0.9 gestation fewer fetuses were markedly growth-restricted (birthweight >2SD below contemporaneous control-intake mean) after Ad.VEGF therapy. There was also evidence of mitigated fetal brain sparing (lower biparietal diameter-to-abdominal circumference and brain-to-liver weight ratios). No effects were observed on UBF or neovascularization; however, Ad.VEGF-transduced vessels demonstrated strikingly enhanced vasorelaxation. Placental efficiency (fetal-to-placental weight ratio) and FLT1/KDR mRNA expression were increased in the maternal but not fetal placental compartments, suggesting downstream effects on placental function. Ad.VEGF gene therapy improves fetal growth in a sheep model of FGR, although the precise mechanism of action remains unclear.

FIG. 1.

Ultrasound assessment of fetoplacental growth and umbilical blood flow. Serial ultrasound measurements of fetal AC (A), biparietal head diameter:AC ratio (B), umbilical artery pulsatility index (C), and placentome index (D) between 83±0.1 and 126±0.3 days of gestation in singleton-bearing adolescent ewes fed a control (C; open squares, n=12) or a high (H) dietary intake to generate normal and compromised fetoplacental growth, respectively. After baseline measurements at 83±0.1 days of gestation, ewes received bilateral uterine artery injections of Ad.VEGF (closed circles, n=18), Ad.LacZ (open triangles, n=14), or saline (open diamonds for H+Saline, n=13, open squares for C+Saline, n=12) at 89±0.2 days of gestation (indicated by arrow). Asterisks denote gestational time points at which there were significant differences in H+Ad.VEGF relative to H+Saline and H+Ad.LacZ groups (p<0.05 for post hoc comparisons). AC, abdominal circumference; Ad, adenovirus; LacZ, β-galactosidase; VEGF, vascular endothelial growth factor.

FIG. 2.

Incidence of marked FGR. Number of fetuses delivered by hysterotomy at 131±0.2 days gestation from singleton-bearing adolescent ewes fed a high (H) dietary intake (to compromise fetoplacental growth) with marked FGR (closed bars) or mild FGR (open bars). Marked FGR was defined as a birthweight >2SD below the mean birthweight of the fetuses of 12 contemporaneous control-intake ewes that demonstrating normal fetoplacental growth (<4222 g for the present study). Fetuses with birthweights >4222 g were of similar weight to normally grown controls and therefore termed “non-FGR.” In midgestation H ewes received bilateral uterine artery injections of Ad.VEGF or control treatment (Ad.LacZ/Saline). Proportions were compared using Fisher's exact test. FGR, fetal growth restriction.

FIG. 3.

In vivo assessment of uterine blood flow. Serial measurements of UBF were recorded between 92±0.2 and 130±0.2 days of gestation using an ultrasonic perivascular flow probe placed around the main trunk of the UtA supplying the gravid horn in singleton-bearing adolescent ewes fed a control (C; open squares, n=12) or a high (H) dietary intake to generate normal and compromised fetal and placental growth trajectories, respectively. UBF determinations were commenced 3 days after bilateral UtA injections of Ad.VEGF (closed circles, n=18), Ad.LacZ (open triangles, n=14), or saline (open diamonds for H+Saline, n=13, and open squares for C+Saline, n=12). Asterisks denote gestational time points at which there were significant differences between the four groups (overall ANOVA p<0.05). UBF, uterine blood flow; UtA, uterine artery.

FIG. 4.

Uterine artery vascular reactivity. Dose–response curves to phenylephrine (A) and bradykinin (B), reflecting vascular contraction and relaxation, respectively, for UtA segments (from gravid and nongravid horns at two levels of branching) from 22 singleton-bearing adolescent ewes fed a control (C; open squares) or a high (H) dietary intake to generate normal and compromised fetoplacental growth, respectively, and necropsied at 131±0.2 days of gestation. At 89±0.2 days of gestation, H ewes received bilateral uterine artery injections of Ad.VEGF (closed circles) or Ad.LacZ/Saline (open circles). Asterisks indicate concentrations of bradykinin at which there were significant differences in H+Ad.VEGF versus H+Ad.LacZ/Saline and C+Saline groups (p<0.05 for overall ANOVA and post hoc comparisons).