We sought to establish a model of fetal programming of metabolic syndrome by exposure to soluble fms-like tyrosine kinase-1 (sFlt1)-induced preeclampsia (PE) and preexisting maternal obesity (MO).
CD-1 female mice were placed on either standard or high-fat diet for 3 months. On day 8 of pregnancy, mice were injected with either adenovirus-carrying sFlt1 or adenovirus-carrying murine immunoglobulin G2α Fc fragment. Offspring were studied at 6 months of age.
Exposure to MO with/without PE resulted in significant increase in progeny’s weight and adiposity. Blood pressure in males was significantly increased due to MO with PE. Metabolic blood analytes were affected in males and females exposed to only PE or MO with/without PE; inflammatory—in females exposed to MO with/without PE and males born to MO with PE; atherosclerotic—in females exposed to MO.
Exposure to maternal prepregnancy obesity and sFlt1-induced preeclampsia alter the offspring’s blood pressure, metabolic, inflammatory, and atherosclerotic profiles later in life.
The metabolic syndrome (the presence of ≥2 of the following components: obesity, hypertension, glucose intolerance, and dyslipidemia) reached epidemic proportions in many developed nations, therefore, the understanding of the pathogenic potential of obesity and its adverse consequences is of great importance. Pregnant, obese women are at risk for a wide array of potential medical and obstetric problems, which also have adverse effects on the fetus during prenatal as well as postnatal development. Epidemiological and animal studies have shown that offspring born of pregnancies complicated by obesity are at increased risk of obesity and other features of the metabolic syndrome, and consequently are at higher risk to develop cardiovascular diseases. Similar to obesity, associations between maternal preeclampsia and hypertension in offspring have been described. However, when adjusted for offspring’s body mass index (BMI), the differences in blood pressure between the offspring of preeclamptic and normotensive pregnancies were attenuated. In the study, published in 2009, Øglaend et al found that higher blood pressure in offspring of preeclamptic mothers decreased when adjusted for maternal BMI and maternal blood pressure. Findings suggest that higher blood pressure in offspring could be attributed to maternal body mass or blood pressure–2 high-risk factors to develop preeclampsia in pregnancy. In light of increasing rates in obesity and the fact that maternal prepregnancy weight is a strong independent risk factor for preeclampsia, investigations into how both conditions together affect offspring’s health later in life are necessary.
The proportion of women who are obese in the United States has doubled since the 1960s. In 2003, the prevalence of being overweight or obese among women (BMI >25 kg/m 2 and >30 kg/m 2 , respectively) was 61.6%. Obesity and being overweight affect multiple organ systems and are associated with an increased risk of cardiovascular diseases, diabetes, gallstones, etc. During gestation obese women are at risk for a wide array of potential medical and obstetric problems, which may have adverse effects on the fetus. One of those problems is preeclampsia. Recent studies have shown that the maternal prepregnancy weight is a strong independent risk factor for preeclampsia. The incidence of preeclampsia almost doubles compared to that of nonobese pregnancies. After adjusting for maternal race and prepregnancy smoking status, the risk of preeclampsia doubled at a BMI of 26 and almost tripled at a BMI of 30 when compared with women with a BMI of 21. A dose-dependent relation between prepregnancy BMI and severity of preeclampsia has also been demonstrated. The risk of severe preeclampsia was 2-fold for BMI of 25, 3-fold for BMI of 30, and 5-fold for BMI of 35 when compared with women with BMI of 20. Similar effects of BMI were observed with regard to mild preeclampsia and transient hypertension in pregnancy.
One more rationale to look at the combined model of obesity and preeclampsia is based on a common physiological pathway for both conditions: inflammation.
Obesity is associated with systemic inflammation, because adipose tissue is a source of proinflammatory cytokines and metabolic mediators, such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, leptin, and plasminogen activator-1. The levels of leptin, TNF-alpha, IL-6, and IL-8 have also been shown to be significantly increased in preeclamptic subjects when compared with healthy control pregnant and nonpregnant women. BMI is strongly correlated with circulating leptin, TNF-alpha, and IL-6, each of which has proinflammatory action. The C-reactive protein (CRP) is increased in obesity. The CRP also was noted to be significantly higher during the first trimester in women who subsequently developed preeclampsia, compared to those who remained normotensive. Ramsay et al found that obesity in pregnancy is associated with endothelial dysfunction and activation of the inflammatory system (increased CRP and IL-6 levels). In summary, obesity triggers systemic inflammation that has been implicated in the development of preeclampsia. In turn, these events could lead to the cardiovascular dysfunction in the offspring exposed to prepregnancy obesity and preeclampsia.
Preeclampsia is a human-specific disease. Except for patas monkeys, none of the animal species exhibit preeclampsia during their pregnancies. Therefore, surgical (ligation of abdominal aorta, internal iliac arteries, or uterine arteries), genetic (knocking out the endothelial nitric oxide synthase gene), or pharmacological (use of a nitric oxide synthase inhibitor N -nitro-L-arginine methyl ester, activation of the renin-angiotensin system or systemic inflammatory responses) models have been used to mimic preeclampsia symptoms in animals. In this study, we used a mouse model developed in our laboratory, where dams are injected during pregnancy with either adenovirus-carrying soluble fms-like tyrosine kinase–1 (sFlt1) or adenovirus carrying the murine immunoglobulin G2α Fc fragment (mFc) as the adenovirus control. Data suggest that an excess of circulating sFlt1 may have a pathogenic role in the development of preeclampsia. The sFlt1 model closely resembles the clinical situation of hypertension in pregnancy and minimizes manipulation. Of note, lower sFlt1 levels had been demonstrated in obese pregnant women than controls. We advanced this model to include maternal obesity by placing a group of females on a high-fat diet (HF) before pregnancy. We previously demonstrated that prepregnancy obesity and sFlt1 overexpression alter fetal programming of adult vascular reactivity. For this study, we chose to further evaluate the influence of maternal prepregnancy obesity and preeclampsia-like syndrome on the health of offspring later in life by examining offspring’s blood pressure and other metabolic parameters.
Materials and Methods
The study protocol and all related procedures were approved by the Animal Care and Use Committee at the University of Texas Medical Branch, Galveston, TX. The mice were maintained in the animal care facility at the University of Texas Medical Branch. They were housed separately in temperature- and humidity-controlled quarters with constant light:dark cycles of 12:12 hours. Animals were provided with food and water ad libitum. Mice were fed either a standardized diet (SF) (5.6 gm%; Teklad 7012: Harlan Teklad LM-485 Mouse/Rat Sterilizable Diet; Harlan Teklad, Madison, WI) or a HF (34.9 gm%, D12492 ; Research Diets Inc, New Brunswick, NJ). The source of fat in the HF is lard, while the standard fat chow does not contain animal product, and the source of fat is soybean oil.
Female CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA) at approximately 4-5 weeks of age ( Figure 1 ). Mice were randomly assigned either to the SF group (control mice) or to the HF group (diet-induced obesity mice). After 3 months on the assigned diet, mice were mated with a CD-1 male, maintained on SF. We expected female mice after 12-14 weeks on HF to weigh significantly more than those in SF group, and, therefore, we categorized them as obese mice.
The day when a vaginal plug was noted was considered day 1 of pregnancy. On day 7 of gestation (E7), blood was collected from the tail vein to establish a baseline for sFlt1 levels, and then on E8, mice in each diet group were injected via tail vein with adenovirus vector (10 9 platelet-forming units in 100 μL) carrying sFlt1 or mFc. A standardized procedure to prepare adenovirus-carrying sFlt1 and mFc vector was followed as described previously by our laboratory. Blood from pregnant mice were collected on E14 (midterm) and E18 (term) of pregnancy.
Pregnant mice delivered 4 groups of offspring: HF sFlt1, exposed to maternal obesity and sFlt1 overexpression; HF mFc, exposed to maternal obesity only; SF sFlt1, exposed to SF and sFlt1 overexpression; and SF mFc, exposed to SF (normal intrauterine environment).
After delivery, pups were weighed at 1 day of age. During lactation, mothers were fed the originally assigned diet. At the age of 21 days, offspring in all 4 groups were weaned from the mothers onto a SF, containing 5.6 gm% fat. At 6 months of age, both female and male offspring were studied for the manifestations of metabolic syndrome.
In vivo telemetric blood pressure measurement experiments
To implant the internal blood pressure transducers, the mice were anesthetized with a mixture of ketamine (80-100 mg/kg, Ketalar; Parke-Davis, Morris Plains, NJ) and xylazine (5-10 mg/kg, Gemini; Rugby, Rockville Center, NY). A vertical midline skin incision along the neck was made and the submaxillary glands were gently separated. The left common carotid artery located next to the trachea was carefully isolated. The catheter (diameter 0.4 mm) then was introduced into the carotid artery through a small incision in the vessel wall, while the body of the transducer (PA-C10 model; Data Systems International, Overland Park, KS) was secured in a subcutaneous pouch along the animal’s right flank through the same ventral neck incision. The neck incision was closed using 6-0 silk. Mice were kept warm on a heating pad and monitored closely until full recovery from anesthesia.
Recording of blood pressure in males and females from all 4 groups (HF sFlt1, HF mFc, SF sFlt1, and SF mFc) began 48-72 hours after surgical implantation of the pressure transducer and was continuously monitored for 5 consecutive days using RLA 1020 telemetry receivers (Data Systems International). The information was fed to data acquisition and recording system, Dataquest software (A.R.T.3.1; Gartner Dataquest, Stamford, CT). Then, the mice were sacrificed, and blood and tissues were collected for later analysis.
Blood was collected on E7, E14, and E18 from pregnant mice and at 6 months of age from offspring.
Glucose blood levels were measured using OneTouch Ultra, a blood glucose monitoring system (LifeScan, Milpitas, CA), after mice had fasted overnight (16-18 hours). Commercially available kits were used according to the manufacturer’s instructions to determine serum levels of sFlt1 (R&D Systems, Minneapolis, MN), cholesterol (Cayman, Ann Arbor, MI), triglycerides (BioAssay Systems, Hayward, CA), CRP (ICL Inc, Newberg, OR), soluble intercellular adhesion molecular-1 (sICAM-1) (Endogen Pierce, Woburn, MA), and insulin, leptin, adiponectin, and IL-6 (Millipore, Billerica, MA).
Blood pressure data obtained from the telemetry system were plotted as mean values over each 24-hour period and were expressed as mean ± SEM using software (GraphPad Prism 4, version 4.00 for Windows; GraphPad Software, La Jolla, CA). For statistical analysis, 1-way analysis of variance with Newman-Keuls multiple comparison test was applied. Serological results were expressed as the mean ± SEM and compared between various groups by 1-way analysis of variance followed by Holm-Sidak, Tukey, Newman-Keuls, or Dunnett multiple comparison tests as appropriate unless otherwise stated (GraphPad Prism 4, version 4.00 for Windows). A probability ( P ) value of < .05 was considered statistically significant.
Female mice on a HF gained significantly more weight during the 3-month prepregnancy period–the increase from original weight at week 0 was 272.5 ± 15.4%, while weight in mice on SF changed 165.4 ± 6.5% ( P < .0001). HF mothers weighed significantly more than SF mothers on E1 (SF 31.8 ± 1.1 g vs HF 46.2 ± 2.2 g, P < .0001) and had significantly higher total cholesterol levels than dams on SF (0.6 ± 0.1 mg/dL vs HF 1.1 ± 0.1 mg/dL, P = .01). Maternal serum levels of sFlt1 during pregnancy are shown in Figure 2 . In SF sFlt1 (n = 6) group sFlt1 levels were significantly higher than in SF mFc (n = 7) on day 18 of pregnancy, while mice in HF sFlt1 (n = 6) group had significantly higher sFLt1 levels on E14 and E18 when compared to HF mFc (n = 4) ( Figure 2 ).
There were no differences in average pup weight at birth (SF mFc 1.6 ± 0.05 g, SF sFlt1 1.6 ± 0.03 g, HF mFc 1.5 ± 0.04 g, HF sFlt1 1.6 ± 0.06 g, P = .69) nor in the number of pups born to each group of dams (SF mFc 12.4 ± 0.1, SF sFlt1 11.2 ± 1.6, HF mFc 11.8 ± 1.4, HF sFlt1 9.8 ± 1.5, P = .58). At 3 weeks of age the resulting offspring was weaned onto a standard chow containing 5.6 gm fat. At weaning, males born to both HF groups were significantly heavier than the ones born to mothers on SF (SF mFc 13.3 ± 1.7 g, SF sFlt1 13.0 ± 0.94 g, HF mFc 23.3 ± 5.64 g, HF sFlt1 21.7 ± 1.52 g, P = .03). Females born to HF sFlt1 mothers were significantly heavier than other groups, followed by HF mFc group, and then both SF groups (SF mFc 13.6 ± 1.13 g, SF sFlt1 12.9 ± 0.58 g, HF mFc 14.8 ± 1.84 g, HF sFlt1 19.3 ± 1.57 g, P = .01).
Six-month-old offspring characteristics
Average body and visceral adipose tissue weights, heart weight/body weight, and adipose tissue weight/body weight ratios (adiposity) are shown in Table 1 (males) and Table 2 (females). In both sexes, offspring born to both groups of obese mothers were significantly heavier, had considerably more adipose tissue, and adipose tissue/body mass ratio was significantly higher. There were no differences between the groups in the heart/weight ratio.
|Characteristic||HF sFlt1||HF mFc||SF sFlt1||SF mFc|
|Weight, g||49.6 ± 1.6 a||50.8 ± 1.5 a||42.8 ± 1.4 b||41.4 ± 2.2 b|
|Heart/body weight ratio||0.4 ± 0.01||0.4 ± 0.02||0.4 ± 0.01||0.4 ± 0.01|
|Weight of visceral adipose tissue, g||2.6 ± 0.15 a||2.6 ± 0.16 a||1.7 ± 0.28 b||1.1 ± 0.25 b|
|Adipose tissue per body weight, %||5.2 ± 0.19 a||5.1 ± 0.22 a||3.7 ± 0.57 b||2.8 ± 0.52 b|
|Characteristic||HF sFlt1||HF mFc||SF sFlt1||SF mFc|
|Weight, g||40.4 ± 1.5 a||42.6 ± 2.0 a||32.0 ± 1.9 b||32.6 ± 1.3 b|
|Heart/body weight ratio||0.4 ± 0.01||0.4 ± 0.03||0.5 ± 0.02||0.4 ± 0.02|
|Weight of visceral adipose tissue, g||3.1 ± 0.44 a||3.3 ± 0.88 a||1.2 ± 0.24 b||1.0 ± 0.19 b|
|Adipose tissue per body weight, %||7.6 ± 0.84 a||7.4 ± 1.74 a||2.9 ± 0.48 b||3.2 ± 0.61 b|