Timing of ischemic insult alters fetal growth trajectory, maternal angiogenic balance, and markers of renal oxidative stress in the pregnant rat

Abstract

Increased uterine artery resistance and angiogenic imbalance characterized by increased soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased free vascular endothelial growth factor (VEGF) are often associated with placental insufficiency and preeclampsia but not synonymous with hypertension. We hypothesized chronic reductions in utero-placental perfusion (RUPP) for 5 days (d) during either mid- (d12-d17) or late (d14-d19) gestation would have disparate effects on plasma sFlt-1 and VEGF levels and blood pressure. Five days of chronic RUPP was achieved by placement of silver clips on the abdominal aorta and ovarian arteries on either gestational d12 or d14. Arterial pressure was increased (P < 0.05) in RUPP vs. normal pregnant (NP) in both d17 (10%) and d19 (25%) groups, respectively. Circulating free VEGF was decreased (P < 0.05) and sFlt-1:VEGF ratio increased (P < 0.05) after 5 days of RUPP ending on d19 but not d17 compared with NP controls. Angiogenic imbalance, measured by an endothelial tube formation assay, was present in the d19 RUPP but not the d17 RUPP compared with age-matched NP rats. Five days of RUPP from days 14 to 19 decreased fetal and placental weights 10% (P < 0.01) compared with d19 NP controls. After 5 days of RUPP, from days 12 to 17 of pregnancy, fetal weights were 21% lighter (P < 0.01) compared with d17 NP controls, but placental weight was unchanged. These findings suggest that the timing during which placental insufficiency occurs may play an important role in determining the extent of alterations in angiogenic balance, fetal growth restriction, and the severity of placental ischemia-induced hypertension.

Fig. 1.

Fetal uterine position model. Uterine position was determined by assigning the first position to the implantation closest to the ovary and counting implantations in ascending order to the cervical end of the uterine horn. Both resorbed and viable fetuses were assigned position numbers. This was repeated independently on each uterine horn, and dry weight values were averaged when applicable to yield a mean weight for each position in the uterus. Resorptions at the implantation site were not included in the mean weight analysis.

Fig. 2.

Femoral mean arterial pressure (MAP) measured immediately before and after the clipping procedure in rats on day 12 (•) and day 14 (■) of pregnancy. MAP measured distal to the clip was not different between pregnant rats on day 12 and day 14 of gestation before, immediately, or after 10, 20, and 30 min of flow restriction. Data were analyzed by an unpaired t-test and are expressed as mean ± SE.

Fig. 3.

Blood pressure during late gestation. Arterial pressure was increased in the reduced utero-placental perfusion (RUPP) groups compared with the respective time-controlled normal pregnant (NP) dams [day 17 (d17): 110 ± 3 vs. 100 ± 1 mmHg, P < 0.05; d19: 120 ± 3 vs. 96 ± 3 mmHg, P < 0.001]. Data were analyzed by a two-way between-subjects ANOVA. ^a,b,cSignificant simple effects are noted by different letters (P < 0.05).

Fig. 4.

Conceptus morphometrics. Fetal weights (A) percentage change from the respective normal pregnant averages were decreased in the RUPP d17 more than the d19 (P < 0.01). Percentage change in placental weights from respective normal pregnant controls were decreased greater in the d19 RUPP group compared with the d17 (P < 0.05). Data were analyzed by an unpaired t-test and expressed as means ± SE.

Fig. 5.

Conceptus morphometrics across uterine positions on days 17 and 19. When fetal weight is plotted relative to uterine position (ordered from ovarian to cervical), there are profound treatment (RUPP vs. NP) and timing (d17 vs. d19) differences in fetal size in late gestation. While the slopes of the fetal size relative to uterine position curve were different between d17 and d19 rats (†P < 0.05), only in the d19 rats was a treatment effect observed (*P < 0.05). Data were analyzed by a two-way between-subjects ANOVA and are expressed as means ± SE.

Fig. 6.

Changes in plasma VEGF and sFlt-1/VEGF ratio in response to 5 days of RUPP. A: plasma VEGF levels are lower (P < 0.05) in all groups compared with the NP d19 group. B: angiogenic balance, measured by a ratio of square-root-normalized sFlt-1:VEGF. Only in the d19 RUPP group was the sFlt-1:VEGF higher with respect to the NP control (1.3 ± 0.2 vs. 0.7 ± 0.0, P < 0.05). Data were analyzed by a two-way between-subjects ANOVA and are expressed as means ± SE. ^a,bLetters that differ indicate significant difference (P < 0.05).

Fig. 7.

Changes in endothelial cell tube formation in response to 5 days of RUPP. Tube formation by endothelial cells was decreased in RUPP rats on day 19 (RUPP19) compared with day 19 NP (NP19) controls. No change in tube formation was found between the day 17 RUPP (RUPP17) and NP (NP17) rats. Data were analyzed by a two-way between-subjects ANOVA and are expressed as means ± SE. ^a,bLetters that differ indicate significant difference (P < 0.05).

Fig. 8.

Renal oxidative stress in response to RUPP on d12–d17 and d14–d19. Renal tissue was analyzed for Trolox-equivalent antioxidant capacity (TEAC) (A) and malondialdehyde (MDA; B). No difference was observed between the NP d17 and RUPP d17 TEAC concentration, but RUPP d19 TEAC levels were significantly lower than the NP d19 (0.37 ± 0.04 vs. 0.20 ± 0.02 mM; P < 0.05). MDA was significantly higher in the RUPP d19 compared with the NP d19 group (9.30 ± 1.24 vs. 4.48 ± 1.06; P < 0.05), and no difference was observed between RUPP d17 and NP d17 groups. Data were analyzed by a two-way between-subjects ANOVA and are expressed as means ± SE. ^a,bLetters that differ indicate significant difference (P < 0.05).