George R. Saade

Using Pravastatin to Improve the Vascular Reactivity in a Mouse Model of Soluble Fms-Like Tyrosine Kinase-1―Induced Preeclampsia

OBJECTIVE: To estimate the effects of pravastatin on the altered vascular function in a mouse model of preeclampsia induced by overexpression of soluble fms-like tyrosine kinase-1 (sFlt-1). METHODS: Pregnant CD1 mice, at day 8 of gestation, were randomly allocated to injection using the tail vein of the adenovirus carrying sFlt-1 (109 plaque-forming units in 100 microliters; sFlt-1 group) or mFc (109 plaque-forming units) as virus control, and then to receive pravastatin (Pra; 5 mg/kg/d) dissolved in drinking water or control. The mice in four groups (sFlt-1, sFlt-1-pravastatin, mFc, and mFc-pravastatin; n=4–6 per group) were killed at day 18 of gestation and 2-mm segments of carotid artery were used for vascular reactivity studies. Serum sFlt-1 levels were also measured by enzyme-linked immunosorbent assay. RESULTS: Mice in the sFlt-1 group had the highest responses to phenylephrine. Treatment with pravastatin decreased the contractile responses to phenylephrine (maximal effect [mean±standard error of the mean] 137.35± 27.70 compared with 42.24±8.76; P=.006) for sFlt-1 compared with sFlt-1-pravastatin, respectively. There were no differences in the contractile responses to thromboxane A2. The vasorelaxant responses to acetylcholine were significantly highest in the mFc-pravastatin group, with a maximal effect of 108.37±5.25 compared with 89.77±3.96 in the mFc group (P=.008), and those with sodium nitroprusside were not different across the four groups. Serum sFlt-1 levels were not different at baseline (day 8) but were significantly lower in sFlt-1-pravastatin compared with sFlt-1 at day 18 (59.42±5.31 compared with 102.59±15.15 ng/mL; P=.01). CONCLUSION: Pravastatin improved the vascular reactivity in this murine model of preeclampsia by decreasing sFlt-1 levels. Statins should be evaluated for the prevention of the vascular abnormalities of preeclampsia.

Amniotic fluid metabolomic analysis in spontaneous preterm birth.

Objective: To identify metabolic changes associated with early spontaneous preterm birth (PTB; <34 weeks) and term births, using high-throughput metabolomics of amniotic fluid (AF) in African American population. Method: In this study, AF samples retrieved from spontaneous PTB (<34 weeks [n = 25]) and normal term birth (n = 25) by transvaginal amniocentesis at the time of labor prior to delivery were subjected to metabolomics analysis. Equal volumes of samples were subjected to a standard solvent extraction method and analyzed using gas chromatography/mass spectrometry (MS) and liquid chromatography/MS/MS. Biochemicals were identified through matching of ion features to a library of biochemical standards. After log transformation and imputation of minimum observed values for each compound, t test, correlation tests, and false discovery rate corrections were used to identify differentially regulated metabolites. Data were controlled for clinical/demographic variables and medication during pregnancy. Results: Of 348 metabolites measured in AF samples, 121 metabolites had a gestational age effect and 116 differed significantly between PTB and term births. A majority of significantly altered metabolites could be classified into 3 categories, namely, (1) liver function, (2) fatty acid and coenzyme A (CoA) metabolism, and (3) histidine metabolism. The signature of altered liver function was apparent in many cytochrome P450-related pathways including bile acids, steroids, xanthines, heme, and phase II detoxification of xenobiotics with the largest fold change seen with pantothenol, a CoA synthesis inhibitor that was 8-fold more abundant in PTB. Conclusion: Global metabolic profiling of AF revealed alteration in hepatic metabolites involving xenobiotic detoxification and CoA metabolism in PTB. Maternal and/or fetal hepatic function differences may be developmentally related and its contribution PTB as a cause or effect of PTB is still unclear.

Long-term maternal cardiovascular function in a mouse model of sFlt-1-induced preeclampsia

Our aim was to evaluate the long-term effects of preeclampsia on vascular function in a mouse model induced by sFlt-1 overexpression. CD-1 mice at day 8 of gestation were injected via the tail vein with adenovirus carrying sFlt1 (AdsFlt1), adenovirus carrying the murine IgG2alpha Fc fragment as the adenovirus control (AdmFc), or saline. Vascular function in the mothers was investigated 6-8 mo after delivery by recording blood pressure (BP) by telemetry (AdsFlt1 n = 8, AdmFc n = 6, saline n = 4) and exploring carotid artery reactivity in a wire myograph (AdsFlt1 n = 6, AdmFc n = 8, saline n = 4). sFlt-1 blood levels at 6-8 mo postpartum had returned to low levels and were comparable between the three groups (P = 0.808). There was no statistically significant difference in BP (P = 0.067) or vascular reactivity between the three groups of postpartum mice (phenylephrine P = 0.079, thromboxane P = 0.979, serotonin P = 0.659, acetylcholine P = 0.795, sodium nitroprusside P = 0.728, isoproterenol P = 0.370). Our results indicate that in a mouse model overexpression of sFlt-1 does not lead to increased in BP and altered vascular function in the absence of the pregnancy and has no long-term effect on BP and vascular function in the postpartum mothers. Our findings favor the hypothesis that increased cardiovascular diseases in women with history of preeclampsia are likely the result of preexisting risk factors common to preeclampsia and cardiovascular diseases.

Long-term alterations in maternal plasma proteome after sFlt1-induced preeclampsia in mice.

OBJECTIVE Preeclampsia is associated with long-term adverse maternal health, such as cardiovascular and metabolic diseases. The objective of this study was to determine whether preeclampsia in a well-characterized animal model that was induced by overexpression of soluble fms-like tyrosine kinase-1 (sFlt1) results in alterations in the maternal circulating proteome that persist long after delivery. STUDY DESIGN CD-1 mice at day 8 of gestation were injected with adenovirus that carried sFlt1 or the murine immunoglobulin G2α Fc fragment as control. Depleted maternal plasma was analyzed 6 months after delivery by label-free liquid chromatography-mass spectrometry assay. The tandem mass spectrometry data were searched against a mouse database, and the resultant intensity data were used to compare abundance of proteins across disease/control plasma pool. Results were analyzed with ingenuity pathways analysis. Right-tailed Fisher exact test was used to calculate a probability value. RESULTS Of 150 proteins that are common for both groups, ingenuity pathways analysis determined 105 proteins that were ready for analysis. Diseases and disorders analysis showed significant enrichment of proteins that are associated with cardiovascular disease. Within this cluster, the most abundant proteins were associated with vascular disease, atherosclerosis, and atherosclerotic lesions. Other top disease clusters were inflammatory response, organismal injury and abnormalities, and hematologic and metabolic disease. CONCLUSION Exposure to sFlt1-induced preeclampsia alters multiple biologic functions in mothers that persist later in life. Our results suggest that some of the long-term adverse outcomes that are associated with preeclampsia actually may be a consequence rather than a mere unmasking of an underlying predisposition. If similar results are found in humans, the development of preventive strategies for preeclampsia should also improve long-term maternal health.

Endothelial growth factor therapy improves preeclampsia-like manifestations in a murine model induced by overexpression of sVEGFR-1

This study examines the effects of VEGF-121 therapy in an animal model of preeclampsia induced by overexpression of soluble VEGF receptor 1 (sVEGFR-1). At day 8 of gestation, CD-1 mice were implanted with subcutaneous osmotic pumps containing either VEGF-121 or vehicle and fitted with telemetric blood pressure (BP) catheters for continuous BP monitoring (days 8-18 of gestation). On day 9, the animals in the VEGF-121 group were randomly allocated for injection with adenovirus carrying sVEGFR-1 or the murine immunoglobulin G2α Fc fragment (mFc) as virus control (Adv-sVEGFR-1; Adv-mFc). Animals in the vehicle group were injected with Adv-sVEGFR-1. On day 18, mice were euthanized, placentas and pups weighted, carotid arteries isolated, and their responses studied in vitro using a wire myograph for isometric tension recording. In mice overexpressing sVEGFR-1, treatment with VEGF-121 significantly reduced BP from days 10 to 18 of gestation compared with that of vehicle. VEGF-sVEGFR-1 animals had significantly higher vasorelaxant response to sodium nitroprusside and significantly lower contractile response to the thromboxane agonist (U-46619) compared with that of the vehicle-sVEGFR-1 mice. Phenylephrine and acetylcholine responses did not significantly vary between the VEGF-sVEGFR-1 and the vehicle-sVEGFR-1 mice. Average pup weight was significantly lower in the vehicle-sVEGFR-1 group compared with the VEGF-sVEGFR-1 and VEGF-mFc groups. In conclusion, VEGF-121 therapy attenuates vascular dysfunction and diminishes intrauterine growth abnormality in an animal model of preeclampsia induced by overexpression of sVEGFR-1. Modulation of VEGF pathway turns into a promising therapeutic approach of preeclampsia.

Maternal pravastatin prevents altered fetal brain development in a preeclamptic CD-1 mouse model

Objective Using an animal model, we have previously shown that preeclampsia results in long-term adverse neuromotor outcomes in the offspring, and this phenotype was prevented by antenatal treatment with pravastatin. This study aims to localize the altered neuromotor programming in this animal model and to evaluate the role of pravastatin in its prevention. Materials and Methods For the preeclampsia model, pregnant CD-1 mice were randomly allocated to injection of adenovirus carrying sFlt-1 or its control virus carrying mFc into the tail vein. Thereafter they received pravastatin (sFlt-1-pra “experimental group”) or water (sFlt-1 “positive control”) until weaning. The mFc group (“negative control”) received water. Offspring at 6 months of age were sacrificed, and whole brains underwent magnetic resonance imaging (MRI). MRIs were performed using an 11.7 Tesla vertical bore MRI scanner. T2 weighted images were acquired to evaluate the volumes of 28 regions of interest, including areas involved in adaptation and motor, spatial and sensory function. Cytochemistry and cell quantification was performed using neuron-specific Nissl stain. One-way ANOVA with multiple comparison testing was used for statistical analysis. Results Compared with control offspring, male sFlt-1 offspring have decreased volumes in the fimbria, periaquaductal gray, stria medullaris, and ventricles and increased volumes in the lateral globus pallidus and neocortex; however, female sFlt-1 offspring showed increased volumes in the ventricles, stria medullaris, and fasciculus retroflexus and decreased volumes in the inferior colliculus, thalamus, and lateral globus pallidus. Neuronal quantification via Nissl staining exhibited decreased cell counts in sFlt-1 offspring neocortex, more pronounced in males. Prenatal pravastatin treatment prevented these changes. Conclusion Preeclampsia alters brain development in sex-specific patterns, and prenatal pravastatin therapy prevents altered neuroanatomic programming in this animal model.

The effect of maternal pravastatin therapy on adverse sensorimotor outcomes of the offspring in a murine model of preeclampsia

Animal and human studies show that in‐utero exposure to preeclampsia alters fetal programming and results in long‐term adverse cardiovascular outcomes in the offspring. Human epidemiologic data also suggest that offspring born to preeclamptic mothers are also at risk of adverse long term neurodevelopmental outcomes. Pravastatin, a hydrophilic lipid‐lowering drug with pleiotropic properties, was found to prevent the altered cardiovascular phenotype of preeclampsia and restore fetal growth in animal models, providing biological plausibility for its use as a preventive agent for preeclampsia. In this study, we used a murine model of preeclampsia based on adenovirus over‐expression of the anti‐angiogenic factor soluble Fms‐like tyrosine kinase 1, and demonstrated that adult offspring born to preeclamptic dams perform poorly on assays testing vestibular function, balance, and coordination, and that prenatal pravastatin treatment prevents impairment of fetal programming.

Prepregnancy obesity and sFlt1-induced preeclampsia in mice: developmental programming model of metabolic syndrome

OBJECTIVE 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). STUDY DESIGN 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. RESULTS Exposure to MO with/without PE resulted in significant increase in progenys 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. CONCLUSION Exposure to maternal prepregnancy obesity and sFlt1-induced preeclampsia alter the offsprings blood pressure, metabolic, inflammatory, and atherosclerotic profiles later in life.

The effect of prepregnancy obesity and sFlt-1–induced preeclampsia-like syndrome on fetal programming of adult vascular function in a mouse model

OBJECTIVE The purpose of this study was to test the hypothesis that prepregnancy obesity and soluble fms-like tyrosine kinase-1 (sFlt-1)-induced preeclampsia lead to altered vascular function in the offspring later in life. STUDY DESIGN CD-1 female mice were placed on a low-fat (LF) or high-fat (HF) diet before mating. On day 8 of pregnancy, the HF mice were injected with adenovirus that carried either sFlt-1 (HF sFlt-1) or murine immunoglobulin G2alpha Fc fragment (HF mFc). LF dams received saline solution. After being weaned, all offspring were placed on a standard diet. At 3 months of age, the carotid artery was isolated for in vitro vascular reactivity studies. RESULTS Among male offspring, the response to phenylephrine was significantly lower in the HF sFlt-1 group. The response to serotonin in males and to thromboxane in females was lower in the HF sFlt-1 and HF mFc groups. In females, the HF sFlt-1 and LF groups displayed less relaxation to acetylcholine. The response to phenylephrine was significantly lower in females than males in the HF mFc and LF groups. The response to thromboxane was significantly lower in the HF sFlt-1 females, compared with males. CONCLUSION Prepregnancy obesity and preeclampsia alter fetal programming of adult vascular function. The mechanism is complex and gender specific.

Effect of age and gender on the progression of adult vascular dysfunction in a mouse model of fetal programming lacking endothelial nitric oxide synthase

The objective of this study was to investigate vascular function at different ages in a transgenic murine model of fetal vascular programming using a model of uteroplacental insufficiency induced by lack of endothelial nitric oxide synthase. Homozygous NOS3 knockout (KO) and wild-type (WT) mice were cross bred to produce WT, KO, and heterozygous that developed in WT (KOP) or KO (KOM) mothers. Male/female offspring from the four groups were killed at 7, 14, and 21 wk of age (n = 5-10/group), and carotid arteries were used for in vitro vascular studies. Responses to phenylephrine (PE), with/without N(G)-nitro-L-arginine methyl ester (L-NAME), angiotensin (ANG), acetylcholine (ACh), sodium nitroprusside, and isoproterenol (ISO) were studied. At 7 wk, only KO offspring showed higher contractile response to PE, whereas, at 14 and 21 wk, both KO and KOM had a higher response. Incubation with L-NAME abolished these differences. ANG contraction was higher in male KO in all age groups and in 21-wk-old females. Relaxation to ACh and ISO was absent in KO, and significantly decreased in KOM offspring in all age groups compared with KOP and WT, independent of gender. Sodium nitroprusside was not different between groups. The effect of the altered intrauterine environment on the development of abnormal vascular function was limited at 7 wk of age and most evident at 14 wk; further deterioration was limited to ANG-mediated vascular contractility in KO offspring. Our findings provide some hope that at least the first seven postnatal weeks may be an appropriate therapeutic window to prevent cardiovascular disease later in life.