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AICAR (5-aminoimidazole-4-carboxamide-3-ribonucleoside) administration ameliorates1

hypertension and angiogenic imbalance in a model of preeclampsia in the rat2

3

Christopher T Banek1,2

, Ashley J Bauer2, Karen M. Needham

1, Hans C. Dreyer

1, Jeffrey S4

Gilbert1,2

*5

6

1Department of Human Physiology, University of Oregon;

2Department of Biomedical Sciences,7

University of Minnesota Medical School8

9

Short title:AICAR and angiogenic balance in pregnancy10

11

Keywords:Preeclampsia, AICAR, hypertension, angiogenic balance12

13

Figures 5; Tables 114

15

*Corresponding author address:16

Jeffrey S Gilbert, PhD17

Department of Human Physiology18

University of Oregon19

1240 University of Oregon20

Eugene, OR 97402-124021

[email protected]

23

24

Articles in PresS. Am J Physiol Heart Circ Physiol (February 15, 2013). doi:10.1152/ajpheart.00903.2012


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ABSTRACT28

Previous studies suggest restoration of angiogenic balance can lower blood pressure and improve29

vascular endothelium function in models of preeclampsia. Our laboratory has recently reported30

exercise training mitigates hypertension in an animal model of preeclampsia, but the mechanisms31

are unknown. AMP-activated protein kinase (AMPK) is stimulated during exercise and has been32

shown to increase expression of vascular endothelial growth factor (VEGF). Therefore, the33

purpose of this study was to determine if AICAR (5-aminoimidazole-4-carboxamide-3-34

ribonucleoside), a potent AMPK stimulator, would increase circulating VEGF, improve35

angiogenic potential, decrease oxidative stress and abrogate placental ischemia-induced36

hypertension. In rats, reduced uteroplacental perfusion pressure (RUPP) was induced on day 1437

of gestation by introducing silver clips on the inferior abdominal aorta and ovarian arteries.38

AICAR was administered i.p. (50mg/kg b.i.d.) days 14-18, and blood pressure and tissues were39

collected on day 19. RUPP-induced hypertension was ameliorated (P


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INTRODUCTION50

Preeclampsia, a pregnancy-specific syndrome, is the leading cause of fetal and maternal51

morbidity and mortality across the world, and unfortunately the incidence has been increasing in52

recent decades (32; 35). Currently, there are limited treatments available, and early delivery is53

often indicated to prevent further progression of the syndrome. Consequently, preeclampsia is54

the leading cause of pre-mature delivery, which is well-known to have numerous deleterious55

effects on neonatal and life-long health (32; 35).56

Recent clinical and experimental studies suggest dysregulation of circulating angiogenic57

factors results in an angiogenic imbalance that is most notably characterized by an altered ratio58

of pro-angiogenic (e.g. vascular endothelial growth factor; VEGF) and anti-angiogenic factors59

(e.g. soluble VEGF receptor-1; sFlt-1). An imbalance favoring increased anti-angiogenic factors60

has been strongly linked to the etiology of hypertension during preeclampsia (3; 18; 27; 29).61

Recent studies also indicate restoration of angiogenic balance can mitigate an increase in blood62

pressure observed in several rodent models of preeclampsia (17; 18; 27; 28; 37). Moreover,63

recent experimental and clinical observations of hypertension in pregnancy and preeclampsia64

have shown exercise can mitigate hypertension, restore angiogenic balance, and endothelial65

function (9; 17). Despite these recent findings, the exact mechanisms by which physical activity66

improves angiogenic balance and decreases blood pressure in pregnancy remain unclear (15; 17).67

Recent studies have revealed some of the metabolic changes observed due to exercise68


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4-carboxamide-3-ribonucleoside (i.e. AICAR) (22; 30), an adenosine mimetic (8; 23), and has73

been shown to decrease mean arterial pressure in hypertensive rats (10). Further, activation of the74

AMPK pathway has been shown to regulate increases in VEGF expression (39), but whether or75

not this occurs with AICAR administration remains unknown. Therefore, we hypothesized76

AICAR, a potent AMPK stimulator, would stimulate the bioavailability of VEGF in circulation,77

improved endothelial function, and, most importantly, abrogate the placental ischemia-induced78

hypertension.79

METHODS AND APPARATUS80

Animals81

Studies were performed in timed-pregnant Sprague-Dawley rats purchased from Charles River82

(Wilmington, MA) or Harlan (Indianapolis, IN). Animals were housed in a temperature-83

controlled room (23C) with a 12:12 light:dark cycle. All experimental procedures executed84

were completed in accordance with National Institutes of Health guidelines for use and care of85

animals and were approved by the Institutional Animal Care and Use Committee at both86

University of Minnesota and the University of Oregon. Dams were assigned to normal pregnancy87

(NP) (n=8) and RUPP (n=8) groups with and without AICAR (Santa Cruz Biotechnology Inc.,88

Santa Cruz, CA) treatment (NP+A (n=6); RUPP+A (n=8)). AICAR treatment was introduced by89

intraperitoneal (i.p.) injection at 50mg/kg twice a day (4; 10; 30) mixed in 0.9% sterile saline90

solution, and controls were treated with a 0.9% sterile saline solution vehicle. Half of the in vivo91


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Reduced Uterine Perfusion Pressure (RUPP) procedure94

The RUPP procedure is a robust model for studying the link between placental ischemia and95

hypertension in the pregnant rat and has been described in detail previously (3; 17). In brief,96

silver clips were placed on the lower abdominal aorta (0.203-mm inner diameter (ID)) above the97

iliac bifurcation and also on branches (0.100 mm ID) of both the right and left ovarian arteries98

supplying the uterus on day 14 of gestation (term = 21 days). Four normal pregnant dams99

underwent a sham surgery, which included the midline incision and suture. After observing no100

differences in the angiogenic factors and blood pressures these animals were grouped with the101

normal pregnant rats.102

Measurement of MAP in chronically instrumented conscious rats103

Animals were instrumented on day 17 of gestation with an indwelling catheter and arterial104

pressure was determined in both groups of rats on day 19 of gestation as described previously105

(17). Briefly, on day 17, V-3 tubing (SCI) catheters we introduced to the carotid artery while the106

animal was under isoflurane anesthesia. Catheters were exteriorized through the back of the neck107

following subcutaneous tunneling. On day 19, animals were placed in restraining cages, and108

direct pressures were monitored using a blood pressure transducer (ADInstruments) for one hour109

following a 30 minute stabilization period (16). Mean arterial pressure (MAP) was averaged over110

the hour time period. Additionally, heart rate was analyzed over the hour period from the arterial111

pressure measurements using LabChart 7.0.112


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reported previously (3; 17). Blood was collected for subsequent assays into Corvac sterile116

serum separator tubes (Sherwood Davis, St. Louis, MO). Blood and amniotic fluid glucose117

concentrations were measured as reported previously (21). Fetal weight, placental weight, and118

number of resorptions were recorded in the manner described previously (3; 17). All collected119

tissues for subsequent analysis were flash frozen in liquid nitrogen and stored at -80C.120

Enzyme-linked immunosorbant assays121

Plasma concentrations of free VEGF and sFlt-1 were measured using commercial enzyme linked122

immunosorbant assay kits (R&D Systems, Quantikine; Minneapolis, MN) according to the123

manufacturers directions as described previously (3; 17).Oxidative stress assays124

Renal and placental oxidative stress were assessed by measuring total antioxidant capacity and125

catalase activity, carried out as we have previously reported (21). Total antioxidant capacity was126

assessed in renal and placental tissue by measuring Trolox-equivalent antioxidant capacity127

(TEAC) assay kit (Cayman Chemical Company, Ann Arbor, MI) according to the128

manufacturers directions. In addition, catalase activity was measured in renal and placental129

tissue using a commercial assay kit (Cayman Chemical Company, Ann Arbor, MI) according to130

the manufacturers directions. All values were normalized to total protein concentration in tissue131

lysate.132

Protein extraction and quantitation133

As described previously (15; 17), total soluble protein was extracted from whole placentas and134


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cellular protein concentration was determined using the bicinchoninic acid (BCA) method138

(Pierce Biotechnology, Rockford, IL). Renal tissue position (right or left) was chosen at random,139

and placental samples were carefully selected for middle position on either side of the uterine140

horns (15; 17).141

Western Blot142

Western blotting was carried out as previously reported (2; 15). Protein (50 g) was separated by143

electrophoresis on 4-20% sodium dodecyl sulfate (SDS) polyacrylamide separating gels (Life144

Technologies, Grand Island, NY) then transferred to nitrocellulose membranes (BioRad,145

Hercules, CA) and Ponceau stained to assess the transfer across each gel. The images of the146

Ponceau stained membranes were digitized with a flatbed scanner.147

The membranes were then incubated one hour in casein blocking solution (BioRad). Membranes148

were incubated in blocking solution containing commercially available antibodies overnight at149

4C (Anti-phospho(Thr172) -AMPK (40H9), 0.1g/ml; Anti-AMPK (23A3), 0.1g/ml; Cell150

Signaling Technology). Membranes were washed and incubated 1 hour with the appropriate151

horseradish peroxidase conjugated secondary antibodies (0.01g/ml) (Cell Signaling152

Technology, Danvers, MA) and incubated in chemiluminescent substrate (West-Femto, Pierce,153

Grand Island, NY). The immunoreactive bands were digitized using an Alpha-Innotech digital154

imaging system. All digitized images were quantified using Un-Scan-It gel 6.1 software (Silk155

Scientific, Orem UT). Specificity of primary antibodies (negative controls) was evaluated by156


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Angiogenic balance was further assessed in the serum of pregnant rats in vitro as previously159

reported (17) in two separate experiments and each was performed in duplicate. Firstly, the sera160

collected from the treatment groups (NP, RUPP, NP+A, RUPP+A) were introduced to human161

umbilical vascular endothelial cell (HUVEC) solution (plated at 5x105cells/mL) and monitored162

for eight hours for tubule formation. In addition, the direct effect of AICAR was also assessed by163

treating cells with serum from NP and RUPP and adding 20M AICAR directly to the media.164

Total number of tubule formations per frame was assessed at 40X optical zoom with a digital165

inverted compound microscope and ImageJ analysis software (National Institutes of Health,166

Bethesda, MD). Total tube count was assessed by at least two individual investigators that were167

blinded to the identity of the experimental groups. Values from each observer were averaged to168

obtain final counts.169

Statistical analysis and calculations170

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


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rate observed between or within treatment groups (NP 39418; RUPP 43722; NP+A 45110;181

RUPP+A 44119 beats per minute).182

Effects of AICAR on RUPP-induced angiogenic imbalance183

Free VEGF levels in maternal plasma were decreased (P


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vs. NP, and AICAR remediated the RUPP resorption. Lastly, blood glucose and amniotic fluid203

glucose were not changed with AICAR treatment in either normal pregnant or RUPP. The204

previous data is summarized in Table 1.205

Tissue AMPK activation206

The ratio of phosphorylated AMPK (pAMPK) to total AMPK, was increased with AICAR207

treatment in RUPP placenta (P


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AICAR treatment may have several beneficial effects on the hypertensive phenotype of a model225

of placental ischemia-induced hypertension.226

Administration of an adenosine mimetics similar to AICAR, such as Metformin, have227

long been recognized to improve cardiovascular function and lower blood pressure in non-228

pregnant hypertensives (20; 36), but the mechanisms remain uncharacterized (1; 19; 36; 38).229

Further, recent studies have shown AICAR can directly improve endothelial function and lower230

blood pressure in rodent models of hypertension and improved conduit vessel function (10; 11).231

Therefore, our observation that AICAR treatment lowers blood pressure is in agreement with232

previous reports in other models of hypertension (4; 6; 10), and we are the first to report these233

effects in any model of hypertension during pregnancy. It has been noted that the blood pressure234

effects observed in this model could potentially be directly from AICAR interaction on the235

vascular endothelial cells, as endothelial-cell-mediated vasorelaxation to AICAR has been shown236

to be dependent on both NO and EDHF production (11). However, the vasodilatory properties237

observed by Ford and colleagues ex vivo are transient (11), and the mechanisms mediating238

chronic effects of AICAR remain unclear (6). In the present study blood pressure was recorded239

more than 12 hours after the final intraperitoneal injection of AICAR, suggesting the maternal240

cardiovascular effects of AICAR in the present study were due to chronic signaling changes such241

as restoration of angiogenic balance rather than previously reported acute mechanisms (11). In242

particular, our present data suggests the effects of AICAR administration may be due to chronic243

changes in factors such as circulating free VEGF and sFlt-1 concentrations that in turn can244


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while acute treatment with AICAR in the media has no effect. This also suggests that cells other248

than ECs may be responsible for the changes that promote tube formation. Further studies are249

underway to identify which cells respond to AICAR in a manner consistent with pro-angiogenic250

function.251

It has been well established AICAR can mimic many of the metabolic effects observed in252

moderate physical activity, such as mitochondrial biogenesis (12; 13; 24), decreased lipogenesis253

(30), increased glucose tolerance (4; 6; 24; 30; 33; 34), and AMPK stimulation (11; 26; 30; 33).254

Moreover, our lab has recently reported exercise training prior to and during gestation has255

several beneficial effects on maternal blood pressure and angiogenic balance (17). While the256

present study reports that AICAR administration appears to have similar cardiovascular and257

angiogenic balance effects as exercise in RUPP animals (15; 17), an interesting difference is258

AICAR did not directly stimulate the production of VEGF in the normal pregnant rats (17). In259

comparison to a recent study of adenosine administration and hypoxia-dependent mechanisms in260

placental explants (14), the current study also suggests these effects on the angiogenic factors261

may be ischemia- or hypoxia-dependent. In addition, this study also suggests gestational AICAR262

treatment may hold promise as a therapeutic independent of its use as a mimetic or model of263

exercise. While these findings are intriguing, further studies are required to elucidate the264

underlying mechanisms by which AICAR lowers blood pressure and restores angiogenic265

balance.266

Th ff f AICAR i i b l i i AICAR


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on the angiogenic balance and blood pressure were only observed in the RUPP, and not the271

normal pregnant (NP). This further suggests a possible mechanism that is mediated by the272

placental ischemia and/or hypoxia. Interestingly, AICAR has been used to improve ischemia-273

reperfusion injury in cardiac tissue during coronary bypass surgery (7), which, unsurprisingly,274

may have similar effects in other tissues such as the placenta. Moreover, these specific placental275

ischemia-dependent mechanisms have been proposed before in in vitromodels (14), but, we are276

the first to report this effect of adenosine-mimetic administration in a model of placental277

ischemia-induced hypertension. These observations are further supported by our placental tissue278

analysis of AMPK phosphorylation, in which AICAR-induced increases in phosphorylation of279

AMPK was dependent on the placental ischemia induced by the RUPP procedure and not280

observed in the NP placenta. Taken together with the MAP and angiogenic balance data, AICAR281

has no measured effect on the normal pregnant dams in this study. We also observed that chronic282

AICAR did not stimulate AMPK phosphorylation in skeletal muscle in either NP or RUPP283

groups. Although these findings are interesting, further studies are required to determine if this is284

a tissue or pregnancy specific observation or if the molecular changes are dependent on hypoxia285

or chronic ischemia.286

In addition to the effects on the angiogenic balance, RUPP-associated renal and placental287

markers of oxidative stress were reduced with AICAR treatment. Previous work has shown that288

oxidative stress plays an important role in preeclampsia and RUPP hypertension (25; 31) and our289

present observations suggest that the effects of AICAR may be mediated by a combination of the290


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placental-ischemia. While the attenuation of these oxidative stress markers in the RUPP+AICAR294

coincide with the stimulation of bioavailable VEGF and a concomitant production or295

maintenance of downstream antioxidative molecules, whether the observed effects are dependent296

on the VEGF remains unclear. Further studies in direct and indirect the antioxidant properties of297

AICAR are required to identify the exact pathways underlying the present observations.298

While we feel the present results are exciting, it is important to recognize they are not299

without limitations. While our current hypothesis focused on the previously reported links300

between purinergic signaling and VEGF, other angiogenic factors important in preeclampsia301

such as sEng (5; 37) may also be affected by AICAR and further studies are planned to evaluate302

these possibilities. Moreover, this study does not specifically address the exact role of AMPK303

signaling and additional studies are planned to evaluate the exact role of that pathway in304

angiogenic balance. Lastly, the source of the changes in VEGF and sFlt-1 in the present study305

remain unknown, so further studies are required to isolate the source. Nevertheless, studies are306

planned to further investigate the role of AMPK on AICARs effects in vivo. Further studies are307

currently aimed at the effects of AICAR specific to pregnancy, the cell-type-specific (e.g.308

placental, vascular endothelial cell, skeletal muscle) effects, and the source of and contribution to309

VEGF and sFlt-1 control.310

In conclusion, the effects of AICAR reveal interesting data on the role of AMPK311

activation on angiogenic balance and normotension restoration. Also presented are encouraging312


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required to elucidate the exact mechanisms by which AICAR lowers blood pressure and restores316

angiogenic balance.317

Source of Funding318

This work was supported in part by grants from the American Heart Association 10SDG2600040319

to JSG and the National Institutes of Health HL114096 to JSG and HD057332 to HCD.320

Disclosures321

CTB, AJB, KM: None322

HCD: Funding from NICHD and NHLBI323

JSG: Funding from AHA and NHLBI324

Acknowledgements325

The authors would like to thank Susan Capoccia and Haley Gillham for their technical assistance326

in this study.327

328

329


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460

461


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TABLE LEGENDS462

Table 1.Maternal and conceptus morphometric and metabolic data at necropsy.463

*P


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between NP, NP+A, and RUPP+A. (Panel B) Direct AICAR (DA) treatment onto HUVECs483

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


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Table. 1

TreatmentsMaternal

Weight (g)

Fetal

Weight (g)

Placental

Weight (g)

% Viability

Maternal

Blood

Glucose

(mg/dL)

Amniotic

Fluid

Glucose

(mg/dL)

NP 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

RUPP+A *305.04.7 *2.080.06 0.460.02 69.811.1 79.09.2 99.731.7


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MAP(mmHg)

NP RUPP NP+A RUPP+A80

100

120

140

*

Figure 1.


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NP RUPP NP+A RUPP+A

0

250

500

750

1000

1250

BioavailableVEGF(pg/ml)

*

NP RUPP NP+A RUPP+A

0

100

200

300

400

500

PlasmasF

lt-1(pg/ml)

Figure 2.

*

B

A


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Figure 3.

Tubules/Frame

NP RUPP NP+A RUPP+A

0

20

40

60

80

100

*

*

Tubulesperfram

e

NP RUPP NP+DA RUPP+DA

0

10

20

30

BA

*

i


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RenalCatalase

Activity

(uM

formald

ehyde)

NP RUPP NP+A RUPP+A

0

50

100

150

200

Figure 4.

D

C

RenalAntioxidantCapacity

(TroloxEqu

ivalence(mM))

NP RUPP NP+A RUPP+A

0.0

0.2

0.4

0.6

0.8

1.0

PlacentalAnt

ioxidantActivity

(TroloxEqu

ivalence(mM))

NP RUPP NP+A RUPP+A

0.0

0.2

0.4

0.6

PlacentalCatala

seActivity

(MF

ormaldehyde)

NP RUPP NP+A RUPP+A

0

10

20

30

40

B

A

*

*

*

Fi 5


31/31

pAMPK-Total AMPK-

PlacentapAMPKa/AM

PK

NP RUPP NP+A RUPP+A

0.0

0.1

0.2

0.3

0.4

0.5

Figure 5.

Documents

ajpheart.00903.2012.full.pdf