Angiotensin-II-dependent Hypertension Requires Cyclooxygenase 1-derived Prostaglandin E₂ and EP₁ Receptor Signaling in the Subfornical Organ of the Brain

Abstract

Cyclooxygenase (COX)-derived prostanoids have long been implicated in blood pressure (BP) regulation. Recently prostaglandin E₂ (PGE₂) and its receptor EP₁R have emerged as key players in angiotensin II (Ang-II)-dependent hypertension (HTN) and related end-organ damage. However, the enzymatic source of PGE₂, ie COX-1 or COX-2, and its site(s) of action are not known. The subfornical organ (SFO) is a key forebrain region that mediates systemic Ang-II-dependent HTN via reactive oxygen species (ROS). We tested the hypothesis that cross-talk between PGE₂/EP₁R and ROS signaling in the SFO is required for Ang-II HTN. Radiotelemetric assessment of BP revealed that HTN induced by infusion of systemic “slow-pressor” doses of Ang-II was abolished in mice with null mutations in EP₁R or COX-1 but not COX-2. Slow-pressor Ang-II-evoked HTN and ROS formation in the SFO were prevented when the EP₁R antagonist SC-51089 was infused directly into brains of wild-type mice, and Ang-II-induced ROS production was blunted in cells dissociated from SFO of EP₁R^−/− and COX-1^−/− but not COX-2^−/− mice. In addition, slow-pressor Ang-II infusion caused a ~3-fold increase in PGE₂ levels in the SFO but not in other brain regions. Finally, genetic reconstitution of EP₁R selectively in the SFO of EP₁R-null mice was sufficient to rescue slow-pressor AngII-elicited HTN and ROS formation in the SFO of this model. Thus, COX-1-derived PGE₂ signaling through EP₁R in the SFO is required for the ROS-mediated HTN induced by systemic infusion of Ang-II, and suggests that EP₁R in the SFO may provide a novel target for antihypertensive therapy.

Introduction

Hypertension is a global health problem, afflicting nearly a third of the population and predisposing to serious diseases affecting the brain, heart and kidneys¹. Cyclooxygenase (COX)-derived prostanoids, endogenous fatty acid metabolites known to play critical roles in a wide variety of biological processes, have long been implicated in blood pressure (BP) regulation². Clinical use of prostanoid synthesis-inhibiting nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with hypertension³, suggesting that endogenous prostanoids generally reduce blood BP. However, recently a more complex picture has emerged in which specific components of the prostanoid system have divergent effects and can be pro-hypertensive. For example, the major prostanoid prostaglandin E₂ (PGE₂) and its receptor subtype 1 (EP₁R), one of four G-protein-coupled receptors (EP_1–4R) mediating the effects of PGE₂⁴, are now considered key players in hypertension and related end-organ damage^5–7. In particular, recent studies utilizing mice with global targeted disruption of EP₁R revealed a critical role for this receptor subtype in systemic Ang-II-dependent hypertension⁸. However, the underlying mechanisms involved in this, including the enzymatic source of PGE₂, i.e. COX-1 or COX-2 and its site(s) of action at EP₁R, remain poorly defined.

There is abundant evidence that neurohumoral dysfunction is a key contributor to Ang-II-dependent hypertension⁹. In particular, regions devoid of a blood-brain-barrier can be activated by elevated levels of blood-borne Ang-II, triggering alterations in downstream signaling pathways and hypertension¹⁰. One of these regions, the subfornical organ (SFO), is strongly implicated in sympathoexcitation and hypertension caused by elevated levels of circulating Ang-II, particularly the model involving chronic infusion of subpressor doses of Ang-II, ie “slow-pressor”, which is thought to recapitulate key features of human essential hypertension^11–13. Reactive oxygen species (ROS) signaling in the SFO is clearly involved in this model^{11, 14, 15}; however, the factors regulating ROS production evoked by Ang-II in this brain region and contributing to neural dysregulation and hypertension remain poorly understood.

Here we sought to determine if COX-derived PGE₂ and EP₁R signaling in the SFO provide an essential link between Ang-II, ROS and the central neural changes that give rise to slow-pressor Ang-II hypertension. Utilizing genetic and pharmacologic tools to selectively target distinct components of the PG system in mice, we provide evidence that COX-1-derived PGE₂ signaling through EP₁R in the SFO is required for the ROS-mediated hypertension induced by systemic infusion of Ang-II.

Methods

An expanded Materials and Methods section is available in the Online Supplement at http://hyper.ahajournal.org/.

Animals

Adult EP₁R-null, COX-1-null and COX-2-null mice (8–10 weeks old) were obtained from in-house colonies. Mice were congenic with the C57Bl/6 strain and age-matched C57Bl/6 mice were used as wild-type (WT) controls. All procedures were approved by the Animal Care and Use Committee at Cornell University. Care of the mice met or exceeded standards set forth by the NIH Guide for the Care and Use of Laboratory Animals, USDA regulations, and the AVMA Panel on Euthanasia.

Pharmacological agents

Inhibitors of EP₁R (SC-51089), COX-1 (SC-560), COX-2 (NS398) and Ang-II type 1 receptors (AT₁R, losartan) were utilized.

Blood pressure studies

Mice were anesthetized and instrumented with radiotelemetry devices as described¹¹. After 7 days recovery, baseline BP measurements were taken over 3–4 days, after which mice were implanted subcutaneously with osmotic minipumps loaded with the slow-pressor dose of Ang-II (600ng/kg/min, 14 days) as described¹¹. BP was recorded daily for 3 weeks to monitor the effects of Ang-II during the entire infusion period as well as several days post-infusion. In studies using intracerebroventricular (i.c.v) infusion of SC-51089, mice were instrumented with i.c.v. cannulae¹¹ during the same surgical session as radiotelemeter implantation. For these studies, at the time of Ang-II pump installation, a second 14-day osmotic minipump containing SC-51089 (144µg/day) was implanted and connected to the i.c.v. cannulae and BP monitoring was carried out as described above.

Meaurement of dipsogenic responses

WT mice were instrumented with i.c.v. cannulae and allowed 7 days recovery. Mice were administered either vehicle, SC-51089 (10µg/kg), SC-560 (10mg/kg) or NS-398 (10mg/kg) by i.p. injection (200 nl) 30 minutes prior to i.c.v. bolus administration of Ang-II (200ng, 200nl). Water drinking responses were measured over 1 hour as described previously^{14, 16}.

Quantitative real-time PCR detection of prostanoid-related transcripts

WT mice were decapitated and brains flash frozen. SFO tissue was collected by micropunch as described¹⁴. Total RNA was harvested and cDNA was generated using random hexamers. Templates (25ng) were subjected in triplicate to real-time RT-PCR using Power SYBR Green and specific primers for COX-1, COX-2, EP_1–4R and PGE synthases as described¹⁴. β-actin was used for relative quantification by ΔΔCt method¹⁴.

PGE₂ assay

WT mice were implanted with osmotic minipumps loaded with the 2 week slow-pressor dose of Ang-II (see above) or saline. Mice were euthanized at 3, 7 or 14 days after start of infusions and brains flash frozen. Micropunches of SFO, paraventricular nuclei (PVN), somatosensory cortex (CTX) and cerebellum (CBM) were collected from 2 mice per biological sample and weighed. Samples were processed and PGE₂ concentration was determined using an enzyme immunoassay kit as described¹⁷.

ROS detection

ROS production was assessed in dissociated SFO cells and in SFO-containing tissue sections using dihydroethidium (DHE) as an indicator. For in vitro ROS detection, WT, COX-1-null, COX-2-null or EP₁R-null mice were sacrificed, brains removed and coronal slices containing the SFO obtained. SFO cells were dissociated, incubated with DHE and time-resolved fluorescence was measured every 30s before (vehicle) and after addition of Ang-II (100nM) as described¹⁸. Additional in vitro studies were performed the same way except pre-treatment with SC-51089 (10µM) or losartan (3 µM) were used, and PGE₂ (100nM) was also applied. For in situ ROS detection, brains were removed on day 16 of Ang-II or vehicle infusions, frozen sections were incubated with DHE and fluorescence was visualized and quantified as described¹¹. Data are expressed as DHE fluorescence intensity relative to control samples.

Adenoviral-mediated reconstitution of EP₁R in EP₁R-null mice

A recombinant adenoviral vector encoding murine EP₁R tagged with HA on the N-terminus (AdEP₁R; Fig S4A) was engineered and then generated by the Iowa Gene Transfer Vector Core (IGTVC). AdEP₁R potency and stability was validated both in vitro and in vivo (Fig S4). An Ad vector encoding GFP (AdGFP) obtained from IGTVC was used as the control vector. EP₁R-null mice underwent SFO-targeted injection of AdEP₁R (5×10¹⁰ pfu/mL, 500nl) or titer-matched AdGFP as described^{11, 14}. During the same surgical session, radiotelemeters were implanted as described above. Nine days later, osmotic minipumps loaded with the 2 week slow-pressor Ang-II dose were implanted. BP recording and ROS measurements were performed as described above.

Data analysis

Data are expressed as mean ± SEM. Comparisons between two groups were evaluated using the Student’s t test. Multiple comparisons were evaluated by ANOVA followed by Dunnett’s or Tukey’s test. Differences were considered statistically significant at p<0.05.

Results

Genetic disruption of EP₁R prevents hypertension during slow-pressor Ang-II infusion

EP₁R are implicated in the BP elevation induced by high doses of Ang-II⁸. Here, using EP₁R-null and WT mice, we determined whether EP₁R are involved in hypertension caused by chronic slow-pressor doses of Ang-II, a model thought to mimic human hypertension and in which there is a strong CNS component⁹. Baseline mean arterial pressure (MAP) was not different between the groups (EP₁R^−/− 98±2, WT 97±3mmHg; p>0.05). In accordance with previous studies^{11, 19}, Ang-II induced a gradual rise in MAP in WT mice that peaked at ~30mmHg above baseline following 2 weeks Ang-II infusion (Fig 1A). In contrast, this Ang-II-induced rise in BP was absent in EP₁R-null mice (Fig 1A).

Figure 1. Slow-pressor Ang-II hypertension is prevented in mice with null mutations in EP₁R or COX-1 but not COX-2.

A) Summary of MAP before, during and after 2 wk slow-pressor Ang-II infusions in WT (n=5) and EP₁R^−/− (n=7) mice. B) MAP throughout slow-pressor Ang-II in WT (n=7), COX-2^−/− (n=4) and COX-1−/− (n=5) mice. *p<0.05 vs. baseline (WT in panels A and B; COX-2 in panel B); †p<0.05 vs. WT or COX-2−/−. Arrow indicates start of Ang-II infusions.

Ang-II slow-pressor hypertension is ameliorated in COX-1-null but not COX-2-null mice

Data in Figure 1A suggest that PGE₂ signaling is needed for slow-pressor Ang-II hypertension. Since PGE₂ is a major reaction product of both COX isozymes²⁰, we next sought to determine the enzymatic source of PGE₂ involved in the slow-pressor effects of Ang-II using mice with null mutations in either COX-1 or COX-2. Baseline MAP did not differ between the groups (COX-1^−/− 93±2, COX-2^−/− 96±7, WT 98±3 mmHg; p>0.05). Similar to WT mice in Figure 1A, Ang-II induced the classic slow rise in MAP which peaked during the second week of infusion (Fig 1B). This response was abolished in COX-1-null mice, whereas it remained intact in COX-2-null mice (Fig 1B).

EP₁R and COX-1 in the CNS are implicated in Ang-II-induced cardiovascular and dipsogenic effects

Data in Figure 1 suggest that COX-1-derived PGE₂ and EP₁R are involved in the rise in BP during slow-pressor Ang-II infusion, but the use of global knockouts prevents us from pinpointing the site(s) of these effects. To test the hypothesis that slow-pressor Ang-II hypertension is caused by a PGE₂/EP₁R mechanism operating in the CNS, we utilized three separate approaches. First, given abundant evidence that slow-pressor Ang-II hypertension is mediated via CNS-driven increases in sympathetic activity⁹, power spectral analysis was used to assess slow-pressor Ang-II-induced sympathetic responses in EP₁R-null vs WT mice. Increased low frequency (LF)/high frequency (HF) oscillations of arterial pressure reflect increased sympathetic activity²¹. Consistent with previous findings, Ang-II infusions caused a doubling of the LF/HF ratio in WT mice by the end of the 2 week infusion period (pumps empty completely on day 16) (Fig 2A) when the hypertensive response is maximal (see Fig 1 and 2B). In contrast, the LF/HF ratio was unchanged in EP₁R−/− mice over the course of Ang-II infusions (Fig 2A). Second, using chronic infusion of the EP₁R antagonist SC51089 into brains (i.c.v.) of WT mice, data shown in Figure 2B demonstrate that the slow-pressor Ang-II-induced rise in MAP observed in i.c.v. vehicle-treated controls was prevented by blockade of EP₁R in the CNS. It should be noted that i.c.v. infusions at this volume do not escape into the peripheral circulation¹¹. Third, given the well established role of the CNS in mediating Ang-II effects on dipsogenesis^{16, 22}, we employed the classic assay of bolus injection of Ang-II in the brain (i.c.v.) coupled with measurement of drinking responses in WT mice pre-treated (i.p.) with either SC51089 or the selective inhibitors of COX-1 (SC-560) or COX-2 (NS-398). As seen in Figure 2C, i.c.v. Ang-II elicited the well-established dipsogenic response in vehicle-treated mice. This response was intact in mice treated with the COX-2 inhibitor, whereas it was markedly attenuated in mice treated with either the COX-1 inhibitor or the EP₁R antagonist.

Figure 2. EP₁R and COX-1 in the CNS are involved in Ang-II-induced cardiovascular and dipsogenic effects.

A) Power spectral analysis of arterial pressure variability at several time-points before, during and after slow-pressor Ang-II infusion in WT (n=5) and EP₁R^−/− mice (n=7). Data are presented as the LF/HF ratio relative to day 0. *p<0.05 vs. day 0; †p<0.05 vs. WT at day 16. B) Summary of MAP in WT mice with chronic i.c.v. infusion of the EP₁R antagonist SC-51089 (n=7) or saline (n=6) at the same time as systemic slow-pressor Ang-II infusions. *p<0.05 vs. baseline; †p<0.05 vs. saline. Arrow indicates start of i.c.v. and Ang-II infusions. C) Drinking responses elicited by bolus i.c.v. injection of Ang-II in WT mice treated 30 min earlier with i.p. injections of vehicle (Veh, n=11), EP₁R antagonist SC51089 (n=11), COX-1 inhibitor SC560 (n=4) or COX-2 inhibitor NS398 (n=4). Data are expressed as the total time drinking (seconds) for 30 min after i.c.v. injection of Ang-II. †p<0.05 vs. vehicle; n.s., not significant.

PGE₂ synthetic enzymes and receptors are expressed in the SFO under basal conditions, and PGE₂ production is augmented in this brain region during slow-pressor Ang-II infusion

Data in Figures 1 and 2 suggest that COX-1-derived PGE₂ acting at EP₁R in the CNS is important in slow-pressor Ang-II hypertension. Since the SFO is a key region of the CNS mediating this form of hypertension^{11, 12}, next we examined the capacity of the SFO for PGE₂ formation and signaling. Real-time qPCR was performed to determine basal mRNA levels of COX isozymes, PGE synthases and EP receptor subtypes in SFO tissue harvested from adult WT mice. First, COX-1 was expressed at ~7-fold higher levels in SFO than COX-2 (Fig 3A). Second, of the PGE synthases that convert PGH₂ to PGE₂, ie PGES-1, PGES-2 or cytosolic PGES (c-PGES), the latter was expressed at much higher levels in the SFO than either of the other two isoforms (Fig 3B). Third, of the four receptor subtypes mediating PGE₂ effects, mRNA levels of EP₁R were more than 10-fold higher than EP₂R, EP₃R or EP₄R in SFO tissue (Fig 3C). Importantly, all four of the EPR subtypes were expressed at very low levels in organum vaculosum of the lamina terminalis (Fig S1). Finally, we sought to directly evaluate the effects of slow-pressor Ang-II infusions on PGE₂ formation in the SFO. As seen in Figure 3D, Ang-II caused a significant increase in PGE₂ levels in the SFO as early as day 3 of the infusion compared to saline controls. This was sustained through 7 days of the infusion, but by day 14, PGE₂ levels were not different from controls. PGE₂ levels were also measured in PVN, CTX and CBM of WT mice infused with slow-pressor doses of Ang-II or vehicle, but no significant Ang-II-induced changes in PGE₂ levels were observed in any of these regions compared to controls (Fig S2).

Figure 3. PGE₂ synthetic enzyme and receptors are expressed at high levels in the SFO under basal conditions, and PGE₂ levels are increased in the SFO early after slow-pressor Ang-II infusion.

A–C) Basal mRNA levels of EPR_1–4, COX isozymes and PGE synthases in adult WT SFO tissue (n=3) as analyzed by quantitative real-time PCR. †p<0.05 vs. EP_2–4R (A), vs. COX-2 (B), vs. PGES1 and PGES2. D) PGE₂ levels measured by ELISA in micropunches of SFO from WT mice at day 3, 7 and 14 of slow-pressor Ang-II (n=3) or saline infusion (n=3). *p<0.05 vs. saline. In all assays, two brains were pooled for per biological sample.

PGE₂/EP₁R signaling is coupled to Ang-II-induced ROS accumulation in the SFO

Slow-pressor Ang-II infusion causes increased ROS formation in the SFO^{11, 15}. Thus, several experiments were performed to test the hypothesis that PGE₂/EP₁R are required for ROS formation in the SFO in response to Ang-II. First, ROS production was assessed in vitro in single cells dissociated from the SFO of adult WT and null mice. Consistent with earlier reports^{14, 16}, Ang-II caused a significant increase in DHE signal in WT SFO cells compared to control (Fig 4A). This response was absent in SFO cells dissociated from either EP₁R^−/− or COX-1^−/− mice, whereas it was intact in COX-2^−/− SFO cells (Fig 4A). Pharmacological studies using the EP₁R antagonist SC-51089 in SFO cells dissociated from WT mice confirmed the results observed in the EP₁R^−/− mice (Fig S3), and further verified earlier data¹⁶ that Ang-II-induced increases in ROS formation are sensitive to losartan (Fig S3). Interestingly, PGE₂ elicited increases in DHE intensity to a similar extent as Ang-II, and while this response was inhibited by SC-51089, it was unaffected by losartan (Fig S3). Next, in situ DHE microfluorography was used to assess Ang-II-induced ROS production in the SFO of WT mice receiving systemic slow-pressor Ang-II infusions concomitant with i.c.v. infusions of either SC-51089 or vehicle. Consistent with earlier reports¹¹, DHE fluorescence intensity was ~2.5-fold higher in the SFO of mice receiving Ang-II (day 16) compared to untreated mice (Fig 4B). This response was abolished in mice receiving i.c.v. infusions of SC-51089 (Fig 4B).

Figure 4. PGE₂/EP₁R signaling is required for Ang-II-induced ROS formation in the SFO.

A) Summary of the effects of Ang-II vs. vehicle (Veh) on ROS production as measured by DHE fluorescence intensity in cells dissociated from SFO of adult WT (n=24), EP₁R^−/− (n=9), COX1^−/− (n=9) and COX2^−/− (n=9) mice. *p<0.05 vs. Veh; n.s., not significant. B) Left: Representative confocal images showing DHE fluorescence in SFO tissue at day 16 of slow-pressor Ang-II infusion in mice treated concomitantly with either i.c.v. vehicle or SC-51089. Mice left untreated served as controls. Right: Summary of DHE fluorescence intensity in SFO tissue of mice with no treatment (n=7) or at day 16 of slow-pressor Ang-II infusions treated concomitantly with either i.c.v. vehicle (Veh, n=6) or SC-51089 (n=7). †p<0.05 vs.untreated; #p<0.05 vs. Ang-II + i.c.v. Veh. Scale bar: 50 µm.

Virally-mediated reconstitution of EP₁R selectively in the SFO rescues slow-pressor Ang-II hypertension and ROS formation in EP₁R-null mice

Our data thus far show that global knockout of EP₁R prevents slow-pressor AngII-induced hypertension, and the CNS, particularly the SFO, may be involved. To directly test the hypothesis that SFO-selective expression of EP₁R is sufficient to induce gradual hypertension elicited by slow-pressor doses of Ang-II, we utilized a genetic rescue approach to reconstitute EP₁R selectively in the SFO with AdEP₁R in EP₁R-null mice. First, the potency and stability of AdEP₁R were evaluated. AdEP₁R increased exogenous EP₁R mRNA levels as measured by qPCR in Neuro2A cells in a concentration-dependent manner (Fig S4B). Furthermore, in vivo targeting of the virus to the SFO induced highly robust HA-EP₁R levels in the SFO as revealed by immunohistochemistry (Fig S4C). This was confirmed at the mRNA level, with qPCR showing highly abundant expression of the HA-EP₁R transgene in SFO and barely detectable levels in PVN, rostral ventrolateral medulla and CTX (Fig S4D). Transgene expression was also stable over time as indicated by similar high levels of HA-EP₁R mRNA in SFO at 9 and 28 days post-transduction (Fig S4D). Having verified the potency and stability of the virus, EP₁R^−/− mice underwent SFO-targeted injections of AdEP₁R or control vector AdGFP. Prior to initiating slow-pressor Ang-II infusions 9 days after viral transduction, baseline MAP was not different in the two groups (AdGFP 97±5 mmHg, AdEP₁R 105±2; p>0.05). In control AdGFP-treated EP₁R^−/− mice, there was no change in MAP at any time throughout the Ang-II infusion (Fig 5A), verifying data in Figure 1 showing that slow-pressor Ang-II hypertension is abolished in EP₁R^−/− mice. In contrast, the classic gradual rise in MAP was restored in AdEP₁R-treated EP₁R^−/− mice (Fig 5A). This was accompanied by LF/HF ratios that were similar to those observed in WT mice at the end of the 2 week infusion period (1.72±0.6, p>0.05). To determine whether this was accompanied by restoration of Ang-II-induced ROS accumulation in the SFO, a separate cohort of EP₁R^−/− mice with SFO-targeted AdEP₁R or AdGFP were subjected to DHE studies as described above. In AdGFP-treated EP₁R^−/− mice, DHE fluorescence intensity in the SFO did not change from baseline, confirming in this null strain findings in Figure 4B obtained using the EP₁R antagonist. In contrast, ROS levels in the SFO of AdEP₁R-treated EP₁R^−/− mice were re-established to that of Ang-II-treated WT mice (see Fig 4B), with a ~3-fold increase in DHE intensity compared to controls (Fig 5B).

Figure 5. Adenoviral-mediated reconstitution of EP₁R selectively in the SFO rescues slow-pressor Ang-II hypertension and ROS formation in EP₁R^−/− mice.

A) Summary of MAP before, during and after slow-pressor Ang-II infusion in EP₁R^−/− mice with SFO-targeted AdGFP (n=6) or AdEP₁R (n=6). *p<0.05 vs. baseline; †p<0.05 vs. AdEP₁R. B) Left: Representative confocal images showing DHE fluorescence in the SFO at the end of the slow-pressor Ang-II infusion period in EP₁R^−/− mice with SFO-targeted AdGFP or AdEP₁R. Right: Summary of DHE fluorescence intensity in the SFO of EP₁R^−/− mice with SFO-targeted AdGRP (n=3) or AdEP₁R (n=4) at the end of the slow-pressor Ang-II infusion period. †p<0.05 vs. AdGFP. Scale bar: 50µm.

Discussion

Brain Ang-II, prostanoids and ROS have each been proposed as important mediators of BP regulation and hypertension^{2, 9, 23}. Here we provide evidence that these factors are mechanistically linked in the pathogenesis of slow-pressor Ang-II-elicited hypertension. We show that elevations in BP during slow-pressor Ang-II infusions are abolished in mice with global null mutations of EP₁R or COX-1 but not COX-2. Pharmacologic inhibition of EP₁R selectively in the CNS prevents slow-pressor Ang-II hypertension, and central Ang-II-driven sympathetic and dipsogenic responses are also mediated by brain EP₁R. Markedly elevated levels of COX-1, cPGES and EP₁R are observed in the SFO relative to other PGE₂ synthetic enzymes and receptors, making this forebrain structure an ideal platform for COX1-derived PGE₂ signaling through EP₁R. Indeed, slow-pressor Ang-II infusions induce early robust PGE₂ production in the SFO. Both in vitro and in vivo inhibition of EP₁R prevents Ang-II-induced ROS accumulation in the SFO, a response that is known to have a causative role in slow-pressor Ang-II hypertension^{11, 15}. Finally, virally-mediated reconstitution of EP₁R selectively in the SFO of EP₁R-null mice restores hypertension and SFO ROS formation in response to slow-pressor Ang-II infusions. This provides the first evidence that COX-1-derived PGE₂/EP₁R signaling in the SFO is required for the ROS-mediated hypertension elicited in the slow-pressor Ang-II model.

The significance of these findings lies in the complex picture that has recently emerged concerning specific components of the prostanoid system and their divergent effects on BP. COX-inhibiting NSAIDs are among the most widely prescribed classes of therapeutic agents, many of the effects of which are mediated by their actions in the CNS². Their general association with hypertension in humans has suggested that endogenous prostanoids lower BP^{2, 3}. However, the ubiquitous tissue distribution and biological complexity of the prostanoid system², coupled with recent evidence that certain components are pro-hypertensive^{6–8, 24} underscores the importance of understanding how prostanoids influence BP regulation, particularly as newer agents with a higher selectivity of action within the prostanoid system are being developed. Our data bolster the emerging concept that endogenous PGE₂-mediated EP₁R activation contributes to Ang-II-dependent hypertension and related end-organ damage^{5, 8}. Although the study by Guan et al. established that the pressor effects of high-dose systemic Ang-II are blunted in EP₁R^−/− mice⁸, the enzymatic source of PGE₂ and the tissue site(s) of action remained poorly defined. Here, utilizing tissue-specific reconstitution of EP₁R in EP₁R-null mice, our data now point to a key role for PGE₂/EP₁R signaling in the CNS, particularly the SFO, in mediating systemic Ang-II-dependent hypertension. Whereas these studies do not rule out the possibility that other EPR subtypes and/or other tissues sites are involved in this model of Ang-II hypertension, the complete restoration of the slow-pressor response, coupled with the markedly higher levels of EP₁R expression in SFO compared to the other subtypes strongly supports this concept. This is important information in considering EP₁R as a novel target for treatment of hypertension.

Another major finding of the present study is that we identified COX-1 in the SFO as the sole source of the PGE₂ required for Ang-II slow-pressor hypertension. Although it is well established that Ang-II stimulates PGE₂ synthesis²⁵, the enzymatic source of PGE₂ in Ang-II-evoked hypertension has remained poorly defined. Our data showing an absence of slow-pressor Ang-II-evoked hypertension in COX-1-null mice but an intact response in COX-2^−/− mice implicates COX-1 as the source of PGE₂ and mediator of hypertension in this model. The relative roles of COX-1 versus COX-2-derived products in Ang-II-dependent responses have been controversial, but our findings are consistent with previous studies showing that pharmacological blockade or genetic deletion of COX-1, but not COX-2, reduced the acute pressor effects of Ang-II in mice²⁴. In addition, Capone et al. recently showed that selective pharmacological inhibition of COX-1, but not COX-2, prevented the cerebrovascular effects of Ang-II, and that COX-1 is the major source of PGE₂ in the somatosensory cortex⁵. Our evidence that COX-1 is expressed at much higher levels than COX-2 in the SFO, and that the COX-1-coupled PGE synthase cPGES²⁶ is the predominant isoform in the SFO suggests COX-1 as the source of increased PGE₂ levels in this brain region during slow-pressor Ang-II infusion. The importance of COX-1 is further suggested by our in vitro data demonstrating that Ang-II-evoked ROS formation in dissociated SFO cells is prevented by inhibition of COX-1 but not COX-2. Thus, our data suggest that at least in the SFO, COX-1 predominates under basal conditions. Further studies will be required to define the cell-type localization of these enzymes in the SFO before and after Ang-II infusion to better understand the cellular mechanisms involved.

It is notable that systemic Ang-II induced increases in PGE₂ production in the SFO but not in other brain regions including the PVN, cortex and cerebellum, despite these regions being enriched in PGE₂ synthesis enzymes and receptors^{27, 28}. This suggests that the links between Ang-II and PGE₂ are specific rather than due to generalized CNS activation in this model, and is also consistent with the fact that the SFO lacks a blood-brain-barrier and can be accessed by circulating Ang-II via AT₁ binding⁹. Indeed Ang-II has been shown to elicit PGE₂ synthesis in cultured CNS cells via stimulation of AT₁ receptors^{29, 30}, and interestingly, autoradiographic studies have demonstrated intense PGE₂ binding in the anteroventral region of the third ventricle (AV3V), a region that encompasses the SFO³¹. It is also important to note that the Ang-II-induced increase in PGE₂ production in the SFO occurred early in the infusion period (3 and 7 days), prior to a significant rise in sympathetic outflow and BP. Furthermore, PGE₂ levels had returned to baseline by 14 days, a time when hypertension is at its peak. This suggests that PGE₂ in the SFO per se is not directly causing neural changes leading to hypertension in this model, but rather serves as a critical signaling intermediate that then triggers downstream pathways involved in central Ang-II-mediated hypertension. Indeed, essential hypertension has a slow and insidious onset, and its underlying pathophysiological mechanisms precede the elevation in BP³². Increasingly, adaptive changes in CNS neurons are considered highly relevant to hypertension^{9, 33}. Determining whether early induction of PGE₂ in the SFO by slow-pressor Ang-II is involved in such changes in CNS circuitries involved in the delayed sympathetic activation and hypertension in this model will require further investigation.

We and others have demonstrated that Ang-II induces ROS formation in the SFO via the activation of NADPH oxidase, and this is a key signaling event in the hypertensive and dipsogenic actions of Ang-II in the brain^{11, 14–16, 34}. A key finding of the present study demonstrates that COX-1-derived PGE₂ and EP₁R are required for this Ang-II-evoked ROS formation in the SFO in vitro and in vivo. This is consistent with recent studies demonstrating a similar mechanism in Ang-II-mediated ROS formation in cerebral blood vessels, which likely involves NADPH oxidase⁵. Given that Ang-II-induced NADPH oxidase activation in neurons is Ca²⁺-dependent¹⁸, along with evidence that activation of EP₁R results in IP3-mediated release of intracellular Ca²⁺³⁵ and reduced Ca²⁺ efflux through the Na/Ca²⁺ exchanger¹⁷, it is reasonable to speculate that NADPH oxidase mediates PGE₂/EP₁R-mediated ROS formation in the SFO through Ca²⁺ signaling. Further studies will be required to elucidate the detailed cellular mechanisms linking Ang-II, prostanoids, ROS and neuronal signaling in the SFO. For example, it will be important to define the relationship between early pre-hypertensive induction of PGE₂ in the SFO by slow-pressor Ang-II and ROS accumulation in this region during the later hypertensive phase in the model.

Perspectives

COX-derived prostanoid signaling has long been implicated in the pathogenesis of Ang-II-dependent hypertension, and this study provides evidence for the first time that a mechanism involving increased COX-1-dependent PGE₂ formation and EP₁R signaling in the SFO region of the forebrain is a key underlying mechanism. Determining how these various players are spatially and functionally linked in the SFO to provide the substrate for adaptive neural changes that lead to gradually developing Ang-II hypertension is a critical next step. However, in the meantime, this is important information as new therapeutic agents targeting the prostanoid system are being developed.