Increased Angiotensin II Contraction of the Uterine Artery at Early Gestation in a Transgenic Model of Hypertensive Pregnancy Is Reduced by Inhibition of Endocannabinoid Hydrolysis
Increased vascular sensitivity to angiotensin II (Ang II) is a marker of a hypertensive human pregnancy. Recent evidence of interactions between the renin–angiotensin system and the endocannabinoid system suggests that anandamide and 2-arachidonoylglycerol may modulate Ang II contraction. We hypothesized that these interactions may contribute to the enhanced vascular responses in hypertensive pregnancy. We studied Ang II contraction in isolated uterine artery (UA) at early gestation in a rat model that mimics many features of preeclampsia, the transgenic human angiotensinogen×human renin (TgA), and control Sprague–Dawley rats. We determined the role of the cannabinoid receptor 1 by blockade with SR171416A, and the contribution of anandamide and 2-arachidonoylglycerol degradation to Ang II contraction by inhibiting their hydrolyzing enzyme fatty acid amide hydrolase (with URB597) or monoacylglycerol lipase (with JZL184), respectively. TgA UA showed increased maximal contraction and sensitivity to Ang II that was inhibited by indomethacin. Fatty acid amide hydrolase blockade decreased Ang IIMAX in Sprague–Dawley UA, and decreased both Ang IIMAX and sensitivity in TgA UA. Monoacylglycerol lipase blockade had no effect on Sprague–Dawley UA and decreased Ang IIMAX and sensitivity in TgA UA. Blockade of the cannabinoid receptor 1 in TgA UA had no effect. Immunolocalization of fatty acid amide hydrolase and monoacylglycerol lipase showed a similar pattern between groups; fatty acid amide hydrolase predominantly localized in endothelium and monoacylglycerol lipase in smooth muscle cells. We demonstrated an increased Ang II contraction in TgA UA before initiation of the hypertensive phenotype. Anandamide and 2-arachidonoylglycerol reduced Ang II contraction in a cannabinoid receptor 1–independent manner. These renin–angiotensin system-endocannabinoid system interactions may contribute to the enhanced vascular reactivity in early stages of hypertensive pregnancy.
Preeclampsia is a common disorder of pregnancy that manifests with hypertension and proteinuria. The renin–angiotensin system (RAS) plays an important role in the normal and pathological regulation of the female reproductive system1 and an increased response to activation of the RAS is characteristic of pregnancies at risk of developing preeclampsia. Thus, since the pioneering work of Gant et al,2,3 an increased sensitivity to angiotensin II (Ang II), one of the main agonists of the RAS, was early recognized as a marker for the development of a hypertensive pregnancy.4
The transgenic female rat containing the human angiotensinogen gene mated with the male transgenic containing human renin (hREN), the hAGT×hREN rat (TgA) mimics many features of human preeclampsia. This model shows increased blood pressure, proteinuria, and placenta alterations of edema and necrosis in the last half of gestation.5 Mean blood pressure increases abruptly ≈10 days before delivery reaching values of 160±10 mm Hg.5 Among the vascular effects observed in this model, a prostanoid-mediated endothelial dysfunction of the uterine artery (UA) has been described at late gestation.6,7
The endocannabinoid system is composed of mediators such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), cannabinoid receptors (CB1, CB2 and non-CB1/CB2), and enzymes in charge of synthesis or hydrolysis of these mediators, that is, fatty acid amide hydrolase (FAAH) for hydrolysis of AEA and monoacylglycerol lipase (MAGL) for 2-AG.8 Thus, AEA and 2-AG act as endogenous ligands for cannabinoid receptors. Endocannabinoids possess vasoactive, mitogenic, and differentiating properties and are implicated in placentation9 and in several pregnancy disorders including preeclampsia.10 Endocannabinoids participate in the regulation of angiogenesis during implantation and decidualization,11 control myometrial contractility,12 and regulate uterine and umbilical blood flow.13 All these events are determinants of a successful pregnancy and may have devastating consequences when altered.14
Once generated, the actions of AEA and 2-AG are terminated by specific reuptake15 and subsequent degradation by either FAAH16 or MAGL,17 respectively. The ability to manipulate in vivo endocannabinoid levels by blocking their degradation makes these enzymes attractive therapeutic targets for several pathologies where AEA and 2-AG are involved,18–20 thus emphasizing the relevance of studying FAAH and MAGL blockers.21 Notably, the localization and expression pattern of FAAH in the blastocyst suggests a role for this enzyme in limiting AEA levels as a protection for a successful implantation.22 Data also suggest that these metabolizing enzymes regulate endocannabinoid levels in maternal tissues during pregnancy.23
Recent evidence of interactions between the RAS and endocannabinoid system suggests a regulatory role for endocannabinoids in modulating Ang II vascular responses. Thus, the contraction to Ang II in mice gracilis arteries is increased by blockade of the CB1 receptor suggesting a vasodilatory role of endocannabinoids modulating contraction to Ang II in arteries from the systemic circulation.24
In this study, we compared the Ang II–mediated contraction of isolated UA from TgA and Sprague–Dawley (SD) rats at early gestation and the effects of blocking AEA and 2-AG hydrolysis, by inhibiting FAAH or MAGL. We hypothesize that prostanoids have a greater impact in modulating Ang II contractions in preeclamptic rats and that blocking endocannabinoid degradation antagonizes Ang II contraction.
Material and Methods
Female human angiotensinogen transgenic rats, when mated with a hREN transgenic male (human angiotensinogen×hREN), develop hypertension and proteinuria in the second half of pregnancy.5 Seven-day pregnant preeclamptic human angiotensinogen×hREN (TgA, n=9) and 7-day pregnant SD (n=6) rats were used. All experiments were performed in accordance with the guidelines of the Wake Forest School of Medicine Institutional Animal Care and Use Committee (see online-only Data Supplement for details).
Vascular Reactivity Experiments
Segments of the main UA, a maximum of 2 mm in length, were mounted between an isometric force transducer (Kistler Morce DSC 6, Seattle, WA) and a displacement device on a myograph (Multi Myograph, Model 620M Danish Myo Technologies, Aarhus, Denmark), using 2 stainless steel wires (diameter 40 μm), as described previously.25 In a subgroup of arteries from the control and TgA rats, the endothelium was destroyed by passing a human hair through the lumen (see online-only Data Supplement for details).26
Response to Potassium Chloride
After equilibration, to test the viability of the arterial preparations and determine the response to nonreceptor-mediated contraction, UA segments were exposed to 75 mmol/L potassium chloride in Krebs-Henseleit Buffer for 5 minutes, and after washing the incubation was repeated twice. Contraction measured at the third incubation was recorded as maximal contraction to potassium chloride (KMAX).
Response to Ang II
After washing and resting for 30 minutes, UA segments were exposed to a cumulative concentration–response curve of Ang II by exposing arteries to eleven (10–11–10−8 mol/L) increasing concentrations in fourth-log steps, with each subsequent dose being introduced only after a steady response had been reached (2 minutes). Because at higher concentrations of ligand the Ang II contraction decreased as a result of desensitization, only doses as high as 10 nmol/L were used. In parallel experiments, different arterial segments were denuded or preincubated for 15 minutes with the cyclooxygenase inhibitor indomethacin (10–5 mol/L) or the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (10–4 mol/L). Additional arterial segments were preincubated for 15 minutes with the FAAH blocker URB597 ((3’-(aminocarbonyl)[1,1’-biphenyl]-3-yl)-cyclohexylcarbamate) at 1×10−6 mol/L or the MAGL blocker JZL184 (4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate) at 1×10−6 mol/L. Some TgA UAs were preincubated with the CB1 receptor blocker SR141716A at 1×10−6 mol/L or SR141716A at 1×10−6 mol/L plus URB597 at 1×10−6 mol/L.
Expression of FAAH and MAGL in UA was detected by immunostaining using commercial antibodies. For details see the online-only Data Suplement. Images were acquired at ×400 magnifications using a Leica DM 4000B upright microscope (Leica Microsystems, Bannockburn, IL). Illumination settings were held constant for image capture sessions (Retiga 1300R CCD Digital camera, QImaging, Surrey, BC, Canada; SimplePCI v6 software Cranberry Twp, PA). Regions of interest were defined using the open source Fiji software (ImageJ, National Institutes of Health; http://fiji.sc/Fiji) covering smooth muscle and endothelial layers in each arterial segment. Intensity of the staining in 5 regions of interest per segment was quantified following the reciprocal intensity method.27
All data analysis was performed using the GraphPad Prism v5 statistical analysis package (GraphPad Software Inc, La Jolla, CA). See the online-only Data Suplement for details. Data are expressed as mean±SEM. One-way ANOVA with Bonferroni multiple comparisons was used to determine significant differences. A P value of <0.05 was accepted as an indication of statistical significance.
Blood Pressure in TgA Transgenic Rats at Early Gestation
Mean blood pressure values were not different between SD (n=6) and TgA (n=7) animals at 7 days of gestational age (98.8±2 versus 97±3 mm Hg; P>0.05).
Contractile Response to Ang II in UA at Early Gestation in Control Rats
Optimal diameters of the isolated UA segments used in these studies were not different between control and TgA (339±13 versus 317±4 μm; P>0.05). Maximal response to potassium chloride was increased in TgA compared with control UA (4.6±0.5 versus 3.3±0.3 mN/mm; P<0.05). In control arteries from SD rats, Ang II (10−11–10−8 mol/L) elicited a dose-dependent contraction that reached a plateau around 90% of KMAX. Arterial denudation increased maximal Ang II contraction and sensitivity (Figure 1A; Table 1; P<0.05), whereas preincubation of intact arteries with indomethacin increased Ang II sensitivity (Figure 1A; Table 1; P<0.05). Blockade of nitric oxide production with Nω-nitro-L-arginine methyl ester in intact arteries increased maximal contraction and sensitivity to Ang II (Figure 1A; Table 1; P<0.05).
|Ang IIMAX, % KMAX||pD2|
Contractile Response to Ang II in TgA UA at Early Gestation
In UAs from TgA rats, the contraction to Ang II was increased compared with SD controls. Maximal response and sensitivity values were higher in TgA UA (Figure 1B; Table 1; P<0.05). In denuded arteries, contraction was similar to intact arteries, whereas preincubation of intact arteries with indomethacin diminished maximal response and sensitivity compared with TgA intact. Compared with intact arteries, sensitivity to Ang II in indomethacin-treated arteries was lower in TgA (Figure 1E; Table 1; P<0.05). Blockade of nitric oxide production with Nω-nitro-L-arginine methyl ester in intact arteries did not alter Ang II contraction in TgA (Figure 1F; Table 1; P<0.05). Ang II contraction in SD and TgA UA was completely abolished by preincubation with losartan 10−6 mol/L (data not shown).
Effects of Blocking Endogenous Production of AEA and 2-AG on Ang II Contraction
Preincubation with the FAAH blocker URB597 10−6 mol/L reduced maximal response to Ang II in SD UA, whereas preincubation with the MAGL blocker JZL184 10−6 mol/L had no effect (Figure 2A and 2B; Table 2). In contrast, both FAAH and MAGL blockade inhibited maximal response and sensitivity to Ang II (Figure 3A and 3B; Table 3) in TgA UA. The inhibitory effect of blocking FAAH on Ang II in TgA UA was not modified by concomitant blockade of the CB1 receptor with SR141716A 10−6 mol/L. We observed no effect of blocking CB1 receptor alone on Ang II contraction (Figure 4; Table 3).
|Ang IIMAX, % KMAX||91±5||61±12*||85±20|
|Ang IIMAX, % KMAX||109±4||86±8*||78±10*||98±4||76±7*|
Expression of MAGL and FAAH in Control and TgA UA at Early Gestation
Immunolocalization of FAAH and MAGL revealed the presence of both enzymes in UA. FAAH was localized predominantly in the endothelium (Figure 5B and 5E), whereas MAGL was detected predominantly in smooth muscle cells (Figure 5C and 5F). A similar intensity of FAAH and MAGL signals was observed in both control and TgA arteries with stronger staining for MAGL than FAAH in both groups. (Figure S1 in the online-only Data Supplement).
For the first time we report that the transgenic hAGTxhREN rat, a rodent model that mimics many features of preeclampsia, displayed increased contraction to Ang II in the UA at early gestation, before the onset of blood pressure rise. Herein, we also present additional novel findings of functional interactions between RAS and endocannabinoid system in the uterine vasculature, with an increased role for the endocannabinoids AEA and 2-AG in reducing Ang II contraction in this preeclamptic model.
Increased vascular sensitivity to Ang II was reported in human pregnancies with an increased risk of developing hypertension.2–4 We observed this effect at early gestation in the TgA rat before the hypertensive phenotype is established. Because increased sensitivity to Ang II constitutes a hallmark for the development of a hypertensive pregnancy, our results contribute to the characterization of the hAGTxhREN rat as a suitable model of preeclampsia. The presence of agonistic autoantibodies to the angiotensin 1 (AT1) receptor (AT1-AA) is another characteristic of human preeclampsia that this model replicates.28 AT1-AA have been shown to modulate vascular responses in systemic and placental vessels29,30 and increase Ang II sensitivity in pregnant rats.31 The effects of AT1-AA on blood pressure and Ang II sensitivity in isolated vessels of late pregnant rats required the presence of both AT1-AA and Ang II,32 and these vascular responses are blocked by the epitope peptide AFHYESQ.32 Because AT1-AA seem to be secondary to a hypoxic/ischemic vascular disorder,33 it is less probable that AT1-AA are playing a role in the increased Ang II sensitivity in early gestation TgA UA before the pathogenic syndrome is established.
The effects of endothelium denudation or inhibition of nitric oxide production on Ang II contraction were lower in TgA than in controls, suggesting an endothelium-derived dysfunction in TgA UA, consistent with a previous report in this model at late gestation.6 Ang II contraction was dependent on prostaglandin generation. Indomethacin preincubation shifted Ang II contraction to the left in arteries from control animals and to the right in arteries from TgA animals. This suggests a change in the type of prostanoids being generated in UA: from vasodilatory prostanoids in control UA to vasoconstrictor prostanoids in TgA arteries. This observation is consistent with the reported imbalance in the production of prostanoids described in preeclampsia: endothelial production of prostacyclin is decreased, whereas thromboxane A2 levels are increased,34,35 an effect proposed to be mediated by increased reactive oxygen species generation in preeclamptic pregnancies. Interestingly, prostacyclin levels decreased months before the clinical onset of preeclampsia.36 TgA rats seem to reproduce this imbalance before the installation of the hypertensive phenotype. Recent reports also support the contribution of CYP subfamily 2J polypeptide 2 epoxygenase to the effects on blood pressure, albuminuria, and vascular function observed in the TgA rat at late gestation.7
The actions of endocannabinoids in the vasculature are complex and not explained by a single mechanism or target tissue.37 An endothelium-dependent vasodilatory action involving a CB1 receptor-dependent pathway, as well as nitric oxide generation and K+ channels activation has been demonstrated for endocannabinoids.38 Although the development of highly specific blockers of FAAH and MAGL has allowed the study of the mechanisms mediated by AEA and 2-AG,39,40 one limitation of our study is that our approach to examine endocannabinoid -mediated responses relies exclusively on pharmacological blockade. Using this approach, however, an efficient catabolism of AEA and 2-AG by their corresponding hydrolyzing enzymes was demonstrated in vascular tissues.38,41 Thus, in the mesenteric artery AEA and 2-AG are able to relax preconstricted arteries, an effect that is potentiated by blockade of the hydrolases FAAH and MAGL.38 We used the enzyme blockers, URB597 and JZL184, at concentrations previously shown effective in vascular studies; that is, URB597 10−6 mol/L abolished AEA vascular actions in rat gracilis arteries.42 Our observations of a reduced Ang II contraction in TgA UA on blockade of FAAH or MAGL suggest the induction of a vasodilatory mechanism or the attenuation of vasoconstrictor mediators. Studies of coronary arteries showed that the vasodilatory effects of endocannabinoids, particularly AEA, are mediated by their catabolism to arachidonic acid and subsequent conversion to vasodilatory eicosanoids such as prostacyclin or epoxyeicosatrienoic acids.43 Interestingly, these effects in coronary arteries are not mediated by the CB1 receptor,43 in agreement with the absence of effects of CB1 receptor antagonism on the reduction of Ang II contraction induced by FAAH blockade in TgA UA. Vasodilatory effects of AEA in rat aorta,44 as well as AEA-mediated nitric oxide production in endothelial cells,45 have both been described as being independent of CB1 or CB2 receptors, making it possible to explain the involvement of non-CB1/CB2-mediated vasodilatation in the reduction of Ang II contraction that we observed after blockade of endocannabinoid degradation.
It has also been described that vasodilatory prostanoids released by endocannabinoid hydrolysis would cause vasorelaxation in part via opening K+ channels.46 Given the important effect of blockade of prostaglandin generation we observed in TgA, it is conceivable that similar mechanisms may be operating in UA. Thus, increased endocannabinoid levels, via inhibition of their degradation, would increase vasodilatory prostanoids in UA that in turn would limit Ang II contraction.
Our immunohistochemistry data indicate differences in tissue localization for FAAH and MAGL in UA. FAAH was mainly localized to the cells lining the arterial lumen consistent with endothelial location and sparsely located in smooth muscle cells in SD and TgA arteries. MAGL was observed along the whole arterial wall spanning both endothelial and smooth muscle cells. Previous evidence indicated the presence of FAAH in bovine coronary arteries, kidney endothelial cells, and human umbilical vein endothelial cells.43,47–49 Recently, FAAH expression has also been reported in arterial smooth muscle cells.42 The localization we observed for FAAH agrees with the endothelium-dependent effects described for the FAAH blocker URB597.42 As a key mediator of 2-AG degradation,50 MAGL is ubiquitously expressed.51 In the vasculature, 2-AG relaxation of mesenteric artery has been shown to be endothelium-independent,52 in agreement with the localization in smooth muscle cells we observed in UA. In both SD and TgA UA it seems that the staining for MAGL was higher than what we observed for FAAH, and this may be related to the reported greater levels of 2-AG compared with AEA observed in the rodent uterus.53 Thus, a higher expression of MAGL would contribute to modulate 2-AG levels. Similar intensity of immunohistochemistry signals between arterial segments from control and TgA animals suggests that the observed differences in vascular effects are not related to different levels of expression of FAAH or MAGL in arteries from preeclamptic animals.
In terms of the possible prostanoid compounds involved in the vascular responses to endocannabinoid hydrolysis, AEA and 2-AG are also substrates of the enzyme cyclo-oxygenase-2.54 Thus, if endocannabinoids are not able to be metabolized by FAAH or MAGL because of their blockade, more substrate would be available for cyclo-oxygenase-2. AEA and 2-AG are oxidized by cyclo-oxygenase-2 to prostaglandins-ethanolamides (prostamides) and prostaglandins-glyceryl esters, respectively.54 These compounds do not interact with cannabinoid or prostaglandin receptors, suggesting additional pathways involved in their cellular function.55 Interestingly, the effects of URB597 on myogenic tone of mouse gracilis arteries are inhibited by indomethacin,42 suggesting the involvement of prostaglandins-ethanolamides and prostaglandins-glyceryl esters on endocannabinoid-derived vascular responses. In the mesenteric artery the vasodilatory responses to endocannabinoids are endothelium-dependent;38 however, we did not test the influence of endothelium on the responses to FAAH and MAGL blockers. Thus, the role of the prostanoids families of compounds in the regulation of the vascular actions of Ang II by endocannabinoids warrants further investigation.
We observed a functional interaction between RAS and endocannabinoid system in the uterine circulation. As in the clinical manifestation of preeclampsia, an increased role of prostanoids in the vasculature may help explain the enhanced effects of endocannabinoids we observed on Ang II contraction. The vascular response to Ang II is a clinical target for antihypertensive therapies, and because FAAH inhibitors have no hemodynamic effects under normotensive conditions,56 our results also point to the use of endocannabinoid degradation blockers as effective pharmacotherapies for hypertension.
We gratefully acknowledge grant support in part provided by Unifi, Inc Greensboro, NC and Farley-Hudson Foundation, Jacksonville, NC.
Sources of Funding
This work was funded by the
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Novelty and Significance
What Is New?
Increased contraction to angiotensin II in the uterine artery at early gestation in a rat model of preeclampsia.
The endogenous cannabinoids anandamide and 2-arachidonoylglycerol reduced contraction of the uterine artery to angiotensin II.
What Is Relevant?
By increasing levels of endogenous cannabinoids, the inhibition of the enzymes fatty acid amide hydrolase and monoacylglycerol lipase would counteract enhanced contractile responses to angiotensin II.
These effects are enhanced in a model of preeclampsia at early gestation, before the hypertensive phenotype initiates.
A functional interaction between the renin–angiotensin system and the endocannabinoid system was observed in the uterine circulation.