Preeclampsia alters fetal programming and results in long-term metabolic consequences in the offspring. Pravastatin has been shown to prevent preeclampsia in animal models. Our aim was to characterize the effects of preeclampsia on fetal programming of adult growth and metabolic function, and evaluate the role of preventive pravastatin therapy, using a well characterized murine model.
CD-1 mice were injected through the tail vein with adenovirus carrying soluble fms-like tyrosine kinase 1 (sFlt-1) and randomly allocated to pravastatin (5 mg/kg/day; sFlt-1/prav, n = 7) or water (sFlt-1, n = 6) until weaning. A control group was injected with adenovirus carrying the murine immunoglobulin G2α Fc fragment (mFc, n = 8). Male and female offspring (6-8/group) were weighed every month until 6 months of age. Intraperitoneal glucose tolerance testing was performed after 16 hours of fasting at 3 and 6 months of age; glucose and insulin responses were measured.
sFlt-1 offspring weight was lower than mFc control ( P < .001) until 2 months of age for females and 5 months of age for males ( P < .001). There were no differences in postnatal growth between mFc and sFlt-1/prav offspring. At 3 and 6 months, female sFlt-1 offspring had higher glucose response compared with mFc and sFlt-1/prav. Three-month-old male sFlt-1 had lower insulin response compared with mFc offspring.
Preeclampsia alters postnatal growth and metabolic function in the adult offspring in this animal model. Maternal therapy with prav prevents some of these alterations in the offspring.
As the incidence of metabolic abnormalities in the population rises, investigation into their cause and prevention becomes increasingly important. Although genetic and postnatal environmental and lifestyle events contribute to an individual’s risk of metabolic disorders, the uterine environment is also essential in determining the susceptibility to adult diseases. Barker and others hypothesized that an abnormal intrauterine environment leads to long-term effects in adult life. This theory of developmental origin of adult diseases implies that chronic conditions in adulthood such as diabetes, hypertension, or cardiovascular disease have their origin in utero because of insults during critical periods of development that cause changes in fetal structure, physiology, and metabolism.
In preeclampsia, the hostile intrauterine environment because of a hypoxic placenta and angiogenic imbalance is thought to lead to long-term adverse effects on the offspring. The association between maternal preeclampsia and offspring cardiometabolic abnormalities is supported by multiple epidemiologic studies that show that infants born to preeclamptic mothers are at increased risk of hypertension, obesity, metabolic syndrome, hyperlipidemia, and insulin resistance later in their life. The risk is increased further if the infant was also to be born small for gestational age; a condition strongly associated with preeclampsia. Further evidence of metabolic abnormalities in offspring of preeclamptic mothers has been demonstrated in multiple animal models of preeclampsia.
Using a murine model of preeclampsia, we and others have previously shown that pregnant rodents that overexpress the soluble fms-like tyrosine kinase 1 (sFlt-1) demonstrate a preeclampsia like phenotype that includes angiogenic imbalance, hypertension, abnormal vascular profile, kidney injury, and other manifestations of preeclampsia. In addition, we have shown that offspring of these dams are at risk for metabolic abnormalities later in life.
Attempts for primary and secondary prevention of preeclampsia have had limited success. As preeclampsia and cardiovascular disease share a similar pathophysiology, the use of statins has been investigated in preeclampsia prevention. We, and others, have demonstrated that prenatal treatment with pravastatin, a hydrophilic HMG-CoA reductase inhibitor, of dams destined to develop preeclampsia led to improved maternal blood pressure, vascular reactivity, and pup growth. Further, it increased placental growth factor and endothelial nitric oxide synthase expression although decreasing sFlt-1 and soluble endoglin. These findings have occurred with no increase in pup resorption, deformation, or changes in maternal cholesterol levels.
Therefore, our objective in the current study was to determine whether antenatal treatment of dams, destined to develop preeclampsia, with pravastatin prevents the long-term offspring adverse metabolic outcomes associated with preeclampsia, using this well characterized mouse model of sFlt-1 induced preeclampsia.
The study protocol and procedures were approved by the institutional Animal Care and Use Committee of the University of Texas Medical Branch. Pregnant CD1 mice were obtained from Charles River (Wilmington, MA), housed separately in temperature and humidity controlled quarters with constant light/dark cycles of 12 hours/12 hours, and provided with food and water ad libitum. Maintenance and care were provided by certified personnel and veterinary staff according to the guidelines of the Animal Care and Use Committee.
sFlt-1 model and experimental protocol
The preparation of adenovirus carrying sFlt-1 and murine immunoglobulin G2α Fc (mFc) fragments has been described in detail elsewhere. Briefly, we used a replication-deficient recombinant adenovirus vector that leads, after a single injection in the tail vain, to hepatocyte transduction that then allows sustained in vivo secretion of antiangiogenic sFlt-1.
On day 8 of gestation, pregnant CD-1 mice were randomly allocated to injection via the tail vein with either adenovirus carrying sFlt-1 (10 9 plaque-forming units in 100 μL) or adenovirus carrying the mFc fragment (10 9 plaque-forming units in 100 μL; mFc virus control group, n = 8). From the next day (day 9), mice from the sFlt-1 group were randomly assigned to receive either water (sFlt-1 group, n = 8) or pravastatin (Sigma-Aldrich, St. Louis, MO) dissolved in their drinking water at a concentration that gave a final dose of 5 mg/kg/d based on the daily water consumption of pregnant mice as previously described (sFlt-1/pravastatin group, n = 7). The mFc control group received water. One hundred microliters of blood were collected from the mice tail vein at day 8 (baseline) and at day 18 (of 20-21 days of pregnancy for mice). Collected blood was centrifuged at 10,000 rpm for 15 minutes, and serum was collected and stored at minus 80°C until time of testing. Serum concentrations of sFlt-1 were measured using mouse soluble vascular endothelial growth factor R1 enzyme-linked immunosorbent assay immunoassays per the manufacturer’s guidelines (R and D systems, Minneapolis, MN). All assays were run in duplicate.
Dams were allowed to delivery vaginally, and treatment allocation was continued through weaning at day 21. Pups were then culled into sibling groups and allowed to grow to adulthood and receive regular water and feed. Pups were weighed monthly to monitor postnatal growth pattern.
Intraperitoneal glucose tolerance testing
At 3 and 6 months of age, 1-2 males and females from each dam underwent intraperitoneal glucose tolerance testing (IPGTT) after 16 hours of fasting. Pups were injected with a 1 mg/kg of 40% glucose solution. Blood was collected via tail vein at time 0 (before the injection) and then serially at 30, 60, and 120 minutes postinjection. Glucose was measured via glucometer (Accucheck One Touch Ultra; Life Scan Johnson and Johnson, Milpitas, CA) and serum was obtained and stored at each time point as previously described. Insulin concentrations were measured in serum using mouse insulin immunoassays per the manufacturer’s guidelines (Crystal Chem Inc, Downers Grove, IL). Investigators were blinded to study group at time of testing.
Analysis was performed using Prism 6 (GraphPad Software Inc, La Jolla, CA) and SigmaPlot (Systat 11.0, Chicago, IL). Normality was assayed using Shapiro-Wilk test. Area under the dose response curve (AUC) was used to assess the overall glucose and insulin responses in the IPGTT. Comparisons between the 3 groups of the study were done using 1-way analysis of variance with posthoc Tukey testing or Kruskal-Wallis tests as appropriate. Data are reported as mean plus or minus standard error of the mean. A 2 tailed P < .05 was considered statistically significant.
Litters were obtained from 6 to 8 dams for each study group. There was no difference in maternal weights at baseline or day 18, average litter size, or total litter weight at birth between the 3 groups ( Table 1 ). Day 8 maternal sFlt-1 concentrations were not different between groups, but day 18 concentrations were significantly higher in the sFlt-1 group as compared with the mFc group (119.00 ± 8.00 vs 55.55 ± 7.63 ng/mL, P < .0001). Furthermore, and as we have previously shown, pravastatin treatment in the sFlt-1 group reduced sFlt-1 concentrations at day 18 in the sFlt-1/pravastatin group to levels similar to the mFc group (sFlt-1 vs sFlt-1/pravastatin; 119.0 ± 8.00 vs 63.24 ± 7.52 ng/mL, P < .0001; Figure 1 ). Two sFlt-1 and 1 mFc male offspring was killed before 3 months of age because of injuries received from cage mates. In addition, 4 sFlt-1 and 3 mFc male offspring died during procedures performed for other research purposes.
|Maternal weight, day 8, g||31.96 ± 0.86||32.13 ± 1.22||33.66 ± 0.65||.25|
|Maternal weight, day 18, g||56.14 ± 1.69||58.03 ± 1.21||58.66 ± 2.06||.56|
|Litter weight, g||21.993 ± 1.15||26.16 ± 2.42||24.17 ± 1.75||.25|
|Litter size||13 (11.75–14.25)||14 (13.25–14)||13 (12.5–14.5)||.70|
Figure 2 displays female and male growth curves from postweaning to 6 months of age. Female sFlt-1 offspring were significantly smaller at 1 month of age compared with mFc control and sFlt-1/pravastatin groups. After 2 months of age, there were no differences between the weights of the female offspring from the 3 groups of the study ( Figure 2 , A). Male sFlt-1 offspring remained smaller through 5 months of age compared with mFc controls, and antenatal pravastatin treatment restored their weight to levels similar to mFc control ones. At 6 months, the weights were not significantly different between the 3 groups of the study ( Figure 2 , B).
Results of the IPGTT are displayed in Figure 3 . At 3 months of age, the AUC of the glucose response was higher for female sFlt-1 (n = 8) compared with mFc control (n = 7), and treatment with pravastatin restored the glucose response to levels similar to control (n = 8) ( P = .01, Figure 3 , A). In addition, female sFlt-1 offspring had higher fasting and peak glucose values compared with both mFc control and sFlt-1/pravastatin offspring ( P = .008 and .006, respectively). In contrast, there were no differences in the glucose response, whether assessed by the AUC, fasting, or peak glucose for the 3-month-old male offspring from the 3 groups of the study (sFlt-1, n = 7; mFc, n = 7; sFlt-1/pravastatin, n = 9) ( Figure 3 , B).
At 6 months of age, the female sFlt-1 offspring continued to have higher glucose response as measured by the AUC compared with mFc controls and this was again restored to control levels by maternal treatment with pravastatin (sFlt-1, n = 7; mFc, n = 10; sFlt-1/pravastatin, n = 8) ( Figure 3 , C). There were no differences in the male glucose response between the 3 groups (sFlt-1, n = 8; mFc, n = 11; sFlt-1/pravastatin, n = 8) ( Figure 3 , D).
Table 2 displays serum fasting insulin concentrations and the area under the insulin response curve after intraperitoneal glucose administration. At 3 months of age, sFlt-1 female offspring had a trend toward a lower insulin AUC compared with both mFc and sFlt-1/pravastatin offspring, but the difference did not reach significance ( P = .06). Whereas the 3-month-old male sFlt-1 offspring had significantly lower fasting insulin compared with mfc control group ( P = .02), and treatment with pravastatin did not correct that in sFlt-1/pravastatin offspring. Similar findings were also seen in 6-month-old male sFlt-1 offspring but the results did not reach statistical significance ( P = .07).