2012
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Figures 5; Tables 1
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University of Oregon
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Eugene, OR 97402-1240
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jgilbe@uoregon.edu
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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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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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INTRODUCTION
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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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the leading cause of pre-mature delivery, which is well-known to have numerous deleterious
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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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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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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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Animals
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Studies were performed in timed-pregnant Sprague-Dawley rats purchased from Charles River
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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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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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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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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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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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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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(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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As described previously (15; 17), total soluble protein was extracted from whole placentas and
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gastrocnemius
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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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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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Hercules, CA) and Ponceau stained to assess the transfer across each gel. The images of the
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The membranes were then incubated one hour in casein blocking solution (BioRad). Membranes
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Signaling Technology). Membranes were washed and incubated 1 hour with the appropriate
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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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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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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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RESULTS
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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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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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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 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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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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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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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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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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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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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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ischemia-induced hypertension. These observations are further supported by our placental tissue
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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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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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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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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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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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TABLE LEGENDS
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*P<0.05 indicates different from NP. P<0.05 indicates different from RUPP.
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FIGURE LEGENDS
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The RUPP-induced increase in blood pressure was ameliorated with AICAR (50mg/kg b.i.d.)
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treatment. *P<0.05 indicates different from NP. P<0.05 indicates different from RUPP. Data
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presented as meanSEM; n = 8 RUPP, 8 normal pregnant (NP), 6 NP+ AICAR (A), and 8
472
RUPP+A..
473
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
480
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
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
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
498
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.
A
1250
1000
750
500
250
0
NP
RUPP
NP+A
RUPP+A
500
400
300
200
100
0
NP
RUPP
NP+A
RUPP+A
Figure 3.
80
60
40
20
0
NP
RUPP
30
Tubules/Frame
100
NP+A
RUPP+A
20
10
NP
RUPP
NP+DA RUPP+DA
0.0
0.4
0.2
NP
30
NP
RUPP
RUPP
NP+A
40
20
10
NP+A
RUPP+A
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