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Articles in PresS. Am J Physiol Heart Circ Physiol (February 15, 2013). doi:10.1152/ajpheart.00903.

2012

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AICAR (5-aminoimidazole-4-carboxamide-3-ribonucleoside) administration ameliorates


hypertension and angiogenic imbalance in a model of preeclampsia in the rat

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Christopher T Banek1,2, Ashley J Bauer2, Karen M. Needham1, Hans C. Dreyer1, Jeffrey S


Gilbert1,2*

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Department of Human Physiology, University of Oregon; 2Department of Biomedical Sciences,


University of Minnesota Medical School

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Short title: AICAR and angiogenic balance in pregnancy

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Keywords: Preeclampsia, AICAR, hypertension, angiogenic balance

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Figures 5; Tables 1

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*Corresponding author address:

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Jeffrey S Gilbert, PhD

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Department of Human Physiology

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University of Oregon

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1240 University of Oregon

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Eugene, OR 97402-1240

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jgilbe@uoregon.edu

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Copyright 2013 by the American Physiological Society.

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ABSTRACT

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Previous studies suggest restoration of angiogenic balance can lower blood pressure and improve

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vascular endothelium function in models of preeclampsia. Our laboratory has recently reported

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exercise training mitigates hypertension in an animal model of preeclampsia, but the mechanisms

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are unknown. AMP-activated protein kinase (AMPK) is stimulated during exercise and has been

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shown to increase expression of vascular endothelial growth factor (VEGF). Therefore, the

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purpose of this study was to determine if AICAR (5-aminoimidazole-4-carboxamide-3-

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ribonucleoside), a potent AMPK stimulator, would increase circulating VEGF, improve

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angiogenic potential, decrease oxidative stress and abrogate placental ischemia-induced

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hypertension. In rats, reduced uteroplacental perfusion pressure (RUPP) was induced on day 14

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of gestation by introducing silver clips on the inferior abdominal aorta and ovarian arteries.

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AICAR was administered i.p. (50mg/kg b.i.d.) days 14-18, and blood pressure and tissues were

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collected on day 19. RUPP-induced hypertension was ameliorated (P<0.05) with AICAR vs.

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RUPP. AICAR increased (P<0.05) plasma VEGF and decreased (P<0.05) plasma sFlt-1 in the

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RUPP+AICAR vs. RUPP. Antioxidant capacity was restored (P<0.05) by AICAR in RUPP

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placenta. Renal and placental catalase activity was decreased (P<0.05) in RUPP+AICAR vs.

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RUPP. Angiogenic potential was increased (P<0.05) in RUPP+AICAR vs. RUPP. Fetal and

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placental weights were unaffected by AICAR. Placental AMPK phosphorylation was increased

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(P<0.05) in RUPP+AICAR vs. normal pregnant and RUPP. These findings suggest AICAR may

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be useful to mitigate angiogenic imbalance, renal and placental oxidative stress, and increase in

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blood pressure associated with RUPP hypertension. Further, placental AMPK phosphorylation

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was observed only in the setting of ischemia.

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INTRODUCTION

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Preeclampsia, a pregnancy-specific syndrome, is the leading cause of fetal and maternal

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morbidity and mortality across the world, and unfortunately the incidence has been increasing in

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recent decades (32; 35). Currently, there are limited treatments available, and early delivery is

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often indicated to prevent further progression of the syndrome. Consequently, preeclampsia is

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the leading cause of pre-mature delivery, which is well-known to have numerous deleterious

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effects on neonatal and life-long health (32; 35).

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Recent clinical and experimental studies suggest dysregulation of circulating angiogenic

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factors results in an angiogenic imbalance that is most notably characterized by an altered ratio

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of pro-angiogenic (e.g. vascular endothelial growth factor; VEGF) and anti-angiogenic factors

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(e.g. soluble VEGF receptor-1; sFlt-1). An imbalance favoring increased anti-angiogenic factors

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has been strongly linked to the etiology of hypertension during preeclampsia (3; 18; 27; 29).

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Recent studies also indicate restoration of angiogenic balance can mitigate an increase in blood

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pressure observed in several rodent models of preeclampsia (17; 18; 27; 28; 37). Moreover,

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recent experimental and clinical observations of hypertension in pregnancy and preeclampsia

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have shown exercise can mitigate hypertension, restore angiogenic balance, and endothelial

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function (9; 17). Despite these recent findings, the exact mechanisms by which physical activity

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improves angiogenic balance and decreases blood pressure in pregnancy remain unclear (15; 17).

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Recent studies have revealed some of the metabolic changes observed due to exercise

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training can be stimulated pharmacologically (22; 30). Of the several pharmacological models of

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exercise in recent literature, AMP-activated protein kinase (AMPK) has been reported as a major

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regulator of or contributor to several essential metabolic adaptations observed in exercise (4; 6;

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12; 13; 24; 33). Moreover, the pharmacological activation of AMPK through 5-aminoimidazole-

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4-carboxamide-3-ribonucleoside (i.e. AICAR) (22; 30), an adenosine mimetic (8; 23), and has

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been shown to decrease mean arterial pressure in hypertensive rats (10). Further, activation of the

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AMPK pathway has been shown to regulate increases in VEGF expression (39), but whether or

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not this occurs with AICAR administration remains unknown. Therefore, we hypothesized

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AICAR, a potent AMPK stimulator, would stimulate the bioavailability of VEGF in circulation,

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improved endothelial function, and, most importantly, abrogate the placental ischemia-induced

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hypertension.

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METHODS AND APPARATUS

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Animals

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Studies were performed in timed-pregnant Sprague-Dawley rats purchased from Charles River

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(Wilmington, MA) or Harlan (Indianapolis, IN). Animals were housed in a temperature-

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controlled room (23C) with a 12:12 light:dark cycle. All experimental procedures executed

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were completed in accordance with National Institutes of Health guidelines for use and care of

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animals and were approved by the Institutional Animal Care and Use Committee at both

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University of Minnesota and the University of Oregon. Dams were assigned to normal pregnancy

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(NP) (n=8) and RUPP (n=8) groups with and without AICAR (Santa Cruz Biotechnology Inc.,

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Santa Cruz, CA) treatment (NP+A (n=6); RUPP+A (n=8)). AICAR treatment was introduced by

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intraperitoneal (i.p.) injection at 50mg/kg twice a day (4; 10; 30) mixed in 0.9% sterile saline

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solution, and controls were treated with a 0.9% sterile saline solution vehicle. Half of the in vivo

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studies for each group were completed at each institution.

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Reduced Uterine Perfusion Pressure (RUPP) procedure

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The RUPP procedure is a robust model for studying the link between placental ischemia and

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hypertension in the pregnant rat and has been described in detail previously (3; 17). In brief,

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silver clips were placed on the lower abdominal aorta (0.203-mm inner diameter (ID)) above the

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iliac bifurcation and also on branches (0.100 mm ID) of both the right and left ovarian arteries

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supplying the uterus on day 14 of gestation (term = 21 days). Four normal pregnant dams

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underwent a sham surgery, which included the midline incision and suture. After observing no

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differences in the angiogenic factors and blood pressures these animals were grouped with the

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normal pregnant rats.

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Measurement of MAP in chronically instrumented conscious rats

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Animals were instrumented on day 17 of gestation with an indwelling catheter and arterial

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pressure was determined in both groups of rats on day 19 of gestation as described previously

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(17). Briefly, on day 17, V-3 tubing (SCI) catheters we introduced to the carotid artery while the

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animal was under isoflurane anesthesia. Catheters were exteriorized through the back of the neck

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following subcutaneous tunneling. On day 19, animals were placed in restraining cages, and

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direct pressures were monitored using a blood pressure transducer (ADInstruments) for one hour

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following a 30 minute stabilization period (16). Mean arterial pressure (MAP) was averaged over

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the hour time period. Additionally, heart rate was analyzed over the hour period from the arterial

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pressure measurements using LabChart 7.0.

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Conceptus measurements & serum collection

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After the measurement of MAP, the dams were placed under isoflurane anesthesia and a midline

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ventral incision was made to isolate the abdominal aorta for plasma and serum collection as

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reported previously (3; 17). Blood was collected for subsequent assays into Corvac sterile

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serum separator tubes (Sherwood Davis, St. Louis, MO). Blood and amniotic fluid glucose

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concentrations were measured as reported previously (21). Fetal weight, placental weight, and

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number of resorptions were recorded in the manner described previously (3; 17). All collected

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tissues for subsequent analysis were flash frozen in liquid nitrogen and stored at -80C.

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Enzyme-linked immunosorbant assays

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Plasma concentrations of free VEGF and sFlt-1 were measured using commercial enzyme linked

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immunosorbant assay kits (R&D Systems, Quantikine; Minneapolis, MN) according to the

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manufacturers directions as described previously (3; 17).Oxidative stress assays

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Renal and placental oxidative stress were assessed by measuring total antioxidant capacity and

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catalase activity, carried out as we have previously reported (21). Total antioxidant capacity was

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assessed in renal and placental tissue by measuring Trolox-equivalent antioxidant capacity

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(TEAC) assay kit (Cayman Chemical Company, Ann Arbor, MI) according to the

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manufacturers directions. In addition, catalase activity was measured in renal and placental

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tissue using a commercial assay kit (Cayman Chemical Company, Ann Arbor, MI) according to

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the manufacturers directions. All values were normalized to total protein concentration in tissue

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lysate.

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Protein extraction and quantitation

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As described previously (15; 17), total soluble protein was extracted from whole placentas and

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gastrocnemius

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phenylmethanesulphonylfluoride (PMSF) in dimethyl sulfoxide (DMSO), sodium orthovanadate

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and a protease inhibitor cocktail (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Total soluble

in

radioimmunoprecipitation

assay

(RIPA)

lysis

buffer

containing

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cellular protein concentration was determined using the bicinchoninic acid (BCA) method

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(Pierce Biotechnology, Rockford, IL). Renal tissue position (right or left) was chosen at random,

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and placental samples were carefully selected for middle position on either side of the uterine

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horns (15; 17).

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Western Blot

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Western blotting was carried out as previously reported (2; 15). Protein (50 g) was separated by

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electrophoresis on 4-20% sodium dodecyl sulfate (SDS) polyacrylamide separating gels (Life

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Technologies, Grand Island, NY) then transferred to nitrocellulose membranes (BioRad,

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Hercules, CA) and Ponceau stained to assess the transfer across each gel. The images of the

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Ponceau stained membranes were digitized with a flatbed scanner.

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The membranes were then incubated one hour in casein blocking solution (BioRad). Membranes

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were incubated in blocking solution containing commercially available antibodies overnight at

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4C (Anti-phospho(Thr172) -AMPK (40H9), 0.1g/ml; Anti-AMPK (23A3), 0.1g/ml; Cell

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Signaling Technology). Membranes were washed and incubated 1 hour with the appropriate

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horseradish peroxidase conjugated secondary antibodies (0.01g/ml) (Cell Signaling

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Technology, Danvers, MA) and incubated in chemiluminescent substrate (West-Femto, Pierce,

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Grand Island, NY). The immunoreactive bands were digitized using an Alpha-Innotech digital

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imaging system. All digitized images were quantified using Un-Scan-It gel 6.1 software (Silk

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Scientific, Orem UT). Specificity of primary antibodies (negative controls) was evaluated by

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imaging membranes with the primary antibody omitted.

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Endothelial tube formation assay

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Angiogenic balance was further assessed in the serum of pregnant rats in vitro as previously

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reported (17) in two separate experiments and each was performed in duplicate. Firstly, the sera

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collected from the treatment groups (NP, RUPP, NP+A, RUPP+A) were introduced to human

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umbilical vascular endothelial cell (HUVEC) solution (plated at 5x105 cells/mL) and monitored

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for eight hours for tubule formation. In addition, the direct effect of AICAR was also assessed by

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treating cells with serum from NP and RUPP and adding 20M AICAR directly to the media.

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Total number of tubule formations per frame was assessed at 40X optical zoom with a digital

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inverted compound microscope and ImageJ analysis software (National Institutes of Health,

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Bethesda, MD). Total tube count was assessed by at least two individual investigators that were

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blinded to the identity of the experimental groups. Values from each observer were averaged to

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obtain final counts.

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Statistical analysis and calculations

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All data are presented as mean SEM and statistical significance was accepted when P<0.05.

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Data that were not normally distributed (e.g. sFlt-1) were square root transformed prior to

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statistical analysis. Comparisons between the groups were made with a two-way between-

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subjects analysis of variance and Newman-Keuls post-hoc tests were employed with groups

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consisting of NP or RUPP +/- AICAR. Statistical calculations were made with GraphPad Prism

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version 5.00 for Windows (GraphPad Software, San Diego, CA USA).

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RESULTS

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Effect of AICAR on blood pressure and heart rate

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RUPP-induced hypertension was ameliorated with AICAR treatment (P<0.05) (Figure 1) and

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AICAR had no effect on blood pressure in NP rats. There were no differences in resting heart

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rate observed between or within treatment groups (NP 39418; RUPP 43722; NP+A 45110;

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RUPP+A 44119 beats per minute).

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Effects of AICAR on RUPP-induced angiogenic imbalance

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Free VEGF levels in maternal plasma were decreased (P<0.05) in RUPP vs. NP, and AICAR

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increased (P<0.05) VEGF in the RUPP when compared to RUPP untreated. Plasma sFlt-1 levels

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were increased (P<0.05) in RUPP vs. NP, and RUPP+AICAR circulating sFlt-1 was decreased

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(P<0.05) compared to RUPP. (Figure 2A-B). Moreover, angiogenic potential was increased

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(P<0.05) with AICAR treatment in the RUPP compared to the untreated RUPP (Figure 3A).

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Also, there was no difference observed between the NP and NP+A. Additionally, AICAR did not

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have a direct effect on HUVEC tubule formation in either NP or RUPP sera treated cells (Figure

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3B).

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Measurement of oxidative stress

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Placental trolox-equivalent antioxidant capacity (TEAC) levels were decreased in the RUPP

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compared to the NP (P<0.05), and AICAR treatment obviated the decrease in the RUPP+A

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(Figure 4A). No effect on TEAC in the kidney was observed (Figure 4B). Additionally,

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placental (Figure 4C) and renal (Figure 4D) catalase activity (measured by formaldehyde

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production) was increased by RUPP, and decreased (P<0.05) in RUPP+A with respect to the

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RUPP group.

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Maternal and fetal morphometric and metabolic data at necropsy

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Maternal weights and fetal weights were decreased (P<0.05) in the RUPP compared to NP, and

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AICAR had no significant effect. Placental weight was decreased (P<0.05) in RUPP vs. NP, and

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AICAR mitigated this decrease. Total litter viability per cent was decreased (P<0.05) in RUPP

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vs. NP, and AICAR remediated the RUPP resorption. Lastly, blood glucose and amniotic fluid

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glucose were not changed with AICAR treatment in either normal pregnant or RUPP. The

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previous data is summarized in Table 1.

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Tissue AMPK activation

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The ratio of phosphorylated AMPK (pAMPK) to total AMPK, was increased with AICAR

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treatment in RUPP placenta (P<0.05) compared to untreated RUPP, NP, and NP+AICAR

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animals (Figure 5). Additionally, the ratio of phosphorylated AMPK to total AMPK in

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skeletal muscle was not increased in either NP or RUPP treated with AICAR, respective to their

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untreated controls (NP 0.200.01; RUPP 0.190.01; NP+A 0.200.01; RUPP+A 0.21 0.01).

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DISCUSSION

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The present study is the first to investigate the treatment of placental ischemia induced

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hypertension with 5-aminoimidazole-4-carboxamide-3-ribonucleoside (AICAR), which has

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revealed novel evidence of restoration of angiogenic balance and abrogation of hypertension in

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an experimental model of hypertension in preeclampsia. Foremost, the hypertension observed in

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our reduced uteroplacental perfusion pressure (RUPP) model was abrogated with intraperitoneal

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50mg/kg b.i.d. AICAR treatment. Secondly, maternal angiogenic balance was restored in the

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RUPP following AICAR treatment, by both increasing the circulating free VEGF and decreasing

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circulating sFlt-1. These observations were further supported by the increased microtubule

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formation in HUVECs treated with serum from the RUPP+AICAR vs. RUPP, which indicates an

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increased angiogenic potential and suggests an increased endothelial function with AICAR in the

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RUPP. Next, the RUPP-associated increase in oxidative stress markers were attenuated in both

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placenta and kidney when treated with AICAR. When viewed in concert, these findings suggest

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AICAR treatment may have several beneficial effects on the hypertensive phenotype of a model

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of placental ischemia-induced hypertension.

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Administration of an adenosine mimetics similar to AICAR, such as Metformin, have

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long been recognized to improve cardiovascular function and lower blood pressure in non-

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pregnant hypertensives (20; 36), but the mechanisms remain uncharacterized (1; 19; 36; 38).

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Further, recent studies have shown AICAR can directly improve endothelial function and lower

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blood pressure in rodent models of hypertension and improved conduit vessel function (10; 11).

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Therefore, our observation that AICAR treatment lowers blood pressure is in agreement with

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previous reports in other models of hypertension (4; 6; 10), and we are the first to report these

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effects in any model of hypertension during pregnancy. It has been noted that the blood pressure

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effects observed in this model could potentially be directly from AICAR interaction on the

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vascular endothelial cells, as endothelial-cell-mediated vasorelaxation to AICAR has been shown

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to be dependent on both NO and EDHF production (11). However, the vasodilatory properties

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observed by Ford and colleagues ex vivo are transient (11), and the mechanisms mediating

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chronic effects of AICAR remain unclear (6). In the present study blood pressure was recorded

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more than 12 hours after the final intraperitoneal injection of AICAR, suggesting the maternal

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cardiovascular effects of AICAR in the present study were due to chronic signaling changes such

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as restoration of angiogenic balance rather than previously reported acute mechanisms (11). In

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particular, our present data suggests the effects of AICAR administration may be due to chronic

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changes in factors such as circulating free VEGF and sFlt-1 concentrations that in turn can

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influence cardiovascular and renal function, rather than direct, acute vasodilatory effects of

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AICAR administration. These observations are further supported by our tube formation data

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which shows chronic in vivo treatment alters circulating factors and increases tubule formation

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while acute treatment with AICAR in the media has no effect. This also suggests that cells other

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than ECs may be responsible for the changes that promote tube formation. Further studies are

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underway to identify which cells respond to AICAR in a manner consistent with pro-angiogenic

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function.

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It has been well established AICAR can mimic many of the metabolic effects observed in

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moderate physical activity, such as mitochondrial biogenesis (12; 13; 24), decreased lipogenesis

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(30), increased glucose tolerance (4; 6; 24; 30; 33; 34), and AMPK stimulation (11; 26; 30; 33).

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Moreover, our lab has recently reported exercise training prior to and during gestation has

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several beneficial effects on maternal blood pressure and angiogenic balance (17). While the

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present study reports that AICAR administration appears to have similar cardiovascular and

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angiogenic balance effects as exercise in RUPP animals (15; 17), an interesting difference is

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AICAR did not directly stimulate the production of VEGF in the normal pregnant rats (17). In

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comparison to a recent study of adenosine administration and hypoxia-dependent mechanisms in

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placental explants (14), the current study also suggests these effects on the angiogenic factors

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may be ischemia- or hypoxia-dependent. In addition, this study also suggests gestational AICAR

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treatment may hold promise as a therapeutic independent of its use as a mimetic or model of

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exercise. While these findings are intriguing, further studies are required to elucidate the

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underlying mechanisms by which AICAR lowers blood pressure and restores angiogenic

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balance.

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The effects of AICAR on angiogenic balance present an interesting story as AICAR

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administration appears to regulate both VEGF and sFlt-1. Indeed, our initial hypothesis only

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considered effects on VEGF via AMPK activation, as the regulation of sFlt-1 via AMPK is

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unclear at this point. Also, it is important to note the changes following AICAR administration

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on the angiogenic balance and blood pressure were only observed in the RUPP, and not the

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normal pregnant (NP). This further suggests a possible mechanism that is mediated by the

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placental ischemia and/or hypoxia. Interestingly, AICAR has been used to improve ischemia-

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reperfusion injury in cardiac tissue during coronary bypass surgery (7), which, unsurprisingly,

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may have similar effects in other tissues such as the placenta. Moreover, these specific placental

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ischemia-dependent mechanisms have been proposed before in in vitro models (14), but, we are

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the first to report this effect of adenosine-mimetic administration in a model of placental

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ischemia-induced hypertension. These observations are further supported by our placental tissue

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analysis of AMPK phosphorylation, in which AICAR-induced increases in phosphorylation of

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AMPK was dependent on the placental ischemia induced by the RUPP procedure and not

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observed in the NP placenta. Taken together with the MAP and angiogenic balance data, AICAR

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has no measured effect on the normal pregnant dams in this study. We also observed that chronic

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AICAR did not stimulate AMPK phosphorylation in skeletal muscle in either NP or RUPP

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groups. Although these findings are interesting, further studies are required to determine if this is

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a tissue or pregnancy specific observation or if the molecular changes are dependent on hypoxia

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or chronic ischemia.

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In addition to the effects on the angiogenic balance, RUPP-associated renal and placental

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markers of oxidative stress were reduced with AICAR treatment. Previous work has shown that

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oxidative stress plays an important role in preeclampsia and RUPP hypertension (25; 31) and our

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present observations suggest that the effects of AICAR may be mediated by a combination of the

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inhibition of the formation of reactive oxygen species and the stimulation of antioxidant

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molecules. Similar to our observations with angiogenic balance this effect was only observed in

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the RUPP groups following AICAR treatment, suggesting a dependence on the presence of

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placental-ischemia. While the attenuation of these oxidative stress markers in the RUPP+AICAR

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coincide with the stimulation of bioavailable VEGF and a concomitant production or

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maintenance of downstream antioxidative molecules, whether the observed effects are dependent

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on the VEGF remains unclear. Further studies in direct and indirect the antioxidant properties of

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AICAR are required to identify the exact pathways underlying the present observations.

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While we feel the present results are exciting, it is important to recognize they are not

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without limitations. While our current hypothesis focused on the previously reported links

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between purinergic signaling and VEGF, other angiogenic factors important in preeclampsia

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such as sEng (5; 37) may also be affected by AICAR and further studies are planned to evaluate

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these possibilities. Moreover, this study does not specifically address the exact role of AMPK

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signaling and additional studies are planned to evaluate the exact role of that pathway in

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angiogenic balance. Lastly, the source of the changes in VEGF and sFlt-1 in the present study

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remain unknown, so further studies are required to isolate the source. Nevertheless, studies are

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planned to further investigate the role of AMPK on AICARs effects in vivo. Further studies are

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currently aimed at the effects of AICAR specific to pregnancy, the cell-type-specific (e.g.

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placental, vascular endothelial cell, skeletal muscle) effects, and the source of and contribution to

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VEGF and sFlt-1 control.

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In conclusion, the effects of AICAR reveal interesting data on the role of AMPK

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activation on angiogenic balance and normotension restoration. Also presented are encouraging

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therapeutic potential for placental ischemia-induced hypertension, by restoring angiogenic

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balance, abrogating RUPP-induced hypertension, and having no reportable deleterious effects on

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the fetal-placental unit. While these results are promising, further pharmacological studies are

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required to elucidate the exact mechanisms by which AICAR lowers blood pressure and restores

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angiogenic balance.

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Source of Funding

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This work was supported in part by grants from the American Heart Association 10SDG2600040

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to JSG and the National Institutes of Health HL114096 to JSG and HD057332 to HCD.

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Disclosures

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CTB, AJB, KM: None

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HCD: Funding from NICHD and NHLBI

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JSG: Funding from AHA and NHLBI

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Acknowledgements

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The authors would like to thank Susan Capoccia and Haley Gillham for their technical assistance

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in this study.

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34. Salt IP, Connell JM and Gould GW. 5-aminoimidazole-4-carboxamide ribonucleoside

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pregnancy. Metabolism 43: 647-654, 1994.

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angiogenic response to exercise. The Journal of Physiology 586: 6021-6035, 2008.

460
461

462

TABLE LEGENDS

463

Table 1. Maternal and conceptus morphometric and metabolic data at necropsy.

464

*P<0.05 indicates different from NP. P<0.05 indicates different from RUPP.

465

Data presented as meanSEM.

466

FIGURE LEGENDS

467

Figure 1. AICAR mitigates increase in blood pressure in reduced uterine perfusion

468

pressure (RUPP) rats

469

The RUPP-induced increase in blood pressure was ameliorated with AICAR (50mg/kg b.i.d.)

470

treatment. *P<0.05 indicates different from NP. P<0.05 indicates different from RUPP. Data

471

presented as meanSEM; n = 8 RUPP, 8 normal pregnant (NP), 6 NP+ AICAR (A), and 8

472

RUPP+A..

473

Figure 2. Chronic AICAR administration restores angiogenic balance in RUPP rats

474

(Panel A) Free VEGF levels in maternal plasma were restored to NP levels in the RUPP treated

475

with AICAR (50mg/kg b.i.d.), and significant increased (P<0.05) compared to RUPP controls.

476

(Panel B) Plasma sFlt-1 levels were restored back to NP levels in the RUPP treated with AICAR,

477

and significantly decreased (P<0.05) compared to RUPP controls. *P<0.05 indicates different

478

from NP. P<0.05 indicates different from RUPP. Data presented as meanSEM; n = 8 RUPP, 8

479

normal pregnant (NP), 6 NP+ AICAR (A), and 8 RUPP+A.

480

Figure 3. Angiogenic potential restored by chronic AICAR administration in RUPP rats

481

(Panel A) Angiogenic potential was increased (P<0.05) with chronic AICAR (50mg/kg b.i.d.)

482

treatment in the RUPP compared to the untreated RUPP. No statistical difference was observed

483

between NP, NP+A, and RUPP+A. (Panel B) Direct AICAR (DA) treatment onto HUVECs

484

treated with NP or RUPP serum had no effect on angiogenic potential. *P<0.05 indicates

485

different from NP. P<0.05 indicates different from RUPP. Data presented as meanSEM; n = 8

486

RUPP, 8 normal pregnant (NP), 6 NP+ AICAR (A), and 8 RUPP+A.

487

Figure 4. Markers of oxidative stress in the RUPP rat were reduced by chronic AICAR

488

administration

489

(Panel A) Trolox-equivalent antioxidant capacity (TEAC) levels were decreased in the RUPP

490

placenta compared to the NP (P<0.05), and AICAR (50mg/kg b.i.d.) treatment obviated the

491

decrease in the RUPP+A. (Panel B) Renal TEAC levels were unaffected by AICAR treatment.

492

(Panel C) Placental catalase activity (measured by formaldehyde production) was increased

493

(P<0.05) in the RUPP vs. NP, and decreased (P<0.05) in RUPP+A with respect to RUPP. (Panel

494

D) Renal catalase activity was increased (P<0.05) in the RUPP vs. NP, and decreased (P<0.05)

495

in RUPP+A with respect to RUPP. *P<0.05 indicates different from NP. P<0.05 indicates

496

different from RUPP. Data presented as meanSEM; n = 8 RUPP, 8 normal pregnant (NP), 6

497

NP+ AICAR (A), and 8 RUPP+A.

498

Figure 5. AICAR administration increased placental tissue AMPK phosphorylation in

499

RUPP

500

The ratio of phosphorylated AMPK (pAMPK) to total AMPK, was increased with AICAR

501

(50mg/kg b.i.d.) treatment in RUPP placenta (P<0.05) compared to untreated RUPP animals.

502

*P<0.05 indicates different from NP. P<0.05 indicates different from RUPP. Data presented as

503

meanSEM; n = 8 RUPP, 8 normal pregnant (NP), 6 NP+ AICAR (A), and 8 RUPP+A.

Table. 1

Treatments

NP

Maternal
Weight (g)

Fetal
Weight (g)

Placental
Weight (g)

% Viability

Maternal
Blood
Glucose
(mg/dL)

Amniotic
Fluid
Glucose
(mg/dL)

337.84.5 2.270.04

0.480.01

95.61.9

82.67.4

137.415.1

RUPP

*294.510.8 *2.050.06

*0.420.02

*47.98.5

101.77.7

129.813.6

NP+A

342.37.7 2.340.05

0.500.03

98.81.2

81.013.8

166.733.5

*305.04.7 *2.080.06

0.460.02

69.811.1

79.09.2

99.731.7

RUPP+A

Figure 1.

MAP (mmHg)

140

120

100

80

NP

RUPP

NP+A

RUPP+A

Figure 2.

Bioavailable VEGF (pg/ml)

A
1250

1000
750

500
250
0

NP

RUPP

NP+A

RUPP+A

Plasma sFlt-1 (pg/ml)

500
400
300
200

100
0

NP

RUPP

NP+A

RUPP+A

Figure 3.

80
60
40

20
0

NP

RUPP

30

Tubules per frame

Tubules/Frame

100

NP+A

RUPP+A

20

10

NP

RUPP

NP+DA RUPP+DA

Placental Catalase Activity


(PM Formaldehyde)
0.6

0.0

0.4

0.2

NP

30

NP

RUPP

RUPP

NP+A

40

20

10

NP+A
RUPP+A

Renal Antioxidant Capacity


(Trolox Equivalence (mM))

Renal Catalase Activity


(uM formaldehyde)

Placental Antioxidant Activity


(Trolox Equivalence (mM))

Figure 4.
B

1.0

0.8

0.6

0.4
0.2

RUPP+A

0.0

NP

200

NP

RUPP

RUPP

NP+A

50

NP+A

RUPP+A

150

100

RUPP+A

Placenta pAMPKa/AMPK

Figure 5.

pAMPK-
Total AMPK-

0.5

0.4
0.3
0.2
0.1
0.0

NP

RUPP

NP+A

RUPP+A

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