EETs Elicit Direct Increases in Pulmonary Arterial Pressure in Mice

Abstract

OBJECTIVE

The biological role of epoxyeicosatrienoic acids (EETs) in the regulation of pulmonary circulation is currently under debate. We hypothesized that EETs initiate increases in right ventricular systolic pressure (RVSP) via perhaps, pulmonary vasoconstriction.

METHODS

Mice were anesthetized with isoflurane. Three catheters, inserted into the left jugular vein, the left carotid artery, and the right jugular vein, were used for infusing EETs, monitoring blood pressure (BP), and RVSP respectively. BP and RVSP were continuously recorded at basal conditions, in response to administration of 4 regioisomeric EETs (5,6-EET; 8,9-EET; 11,12-EET, and 14,15-EET; 1, 2, 5 and 10ng/g body weight (BW) for each EET), and during exposure of mice to hypoxia.

RESULTS

All 4 EETs initiated dose-dependent increases in RVSP, though reduced BP. 11,12-EET elicited the greatest increment in RVSP among all EET isoforms. To clarify the direct elevation of RVSP in a systemic BP-independent manner, equivalent amounts of 14,15-EET were injected over 1 and 2 minutes respectively. One-minute injection of 14,15-EET elicited significantly faster and greater increases in RVSP than the 2-minute injection, whereas their BP changes were comparable. Additionally, direct injection of low doses of 14,15-EET (0.1, 0.2, 0.5, and 1ng/g BW) into the right ventricle caused significant increases in RVSP without effects on BP, confirming that systemic vasodilation-induced increases in venous return are not the main cause for the increased RVSP. Acute exposure of mice to hypoxia significantly elevated RVSP, as well as 14,15-EET-induced increases in RVSP.

CONCLUSIONS

EETs directly elevate RVSP, a response that may play an important role in the development of hypoxia-induced pulmonary hypertension (PH).

Epoxyeicosatrienoic acids (EETs) are metabolized from arachidonic acid by cytochrome P-450 (CYP)/epoxygenases. CYP2C and CYP2J are major families of epoxygenases located in coronary, cerebral, splachnic, as well as pulmonary circulations, and responsible for the synthesis of 4 regioisomeric EETs: 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET.^1–5 EETs possess cardioprotective properties including systemic vasodilation, anti-hypertension, anti-atherosclerosis, anti-inflammation, as well as angiogenesis,^6–8 and are metabolized by soluble epoxide hydrolase (sEH) to their inactive or less active diols called dihydroxyeicosatrienoic acids (DHETs).⁷ To this end, prolonging the biological activity or increasing tissue and circulating levels of EETs, via either genetic disruption of the sEH gene or pharmacological inhibition of sEH activity, is an emerging strategy for the treatment of cardiovascular disease and inflammatory disorders.^7,9,10 However, to date, researchers have not reached a consensus concerning the biological role of EETs in the pulmonary circulation due to results from some studies that show EETs initiate vasoconstriction,^11–14 while in other studies EETs are shown to elicit vasodilation,^15–17 though it was noted that 5,6-EET-elicited pulmonary vasodilation was in most instances, mediated via the cyclooxygenase-dependent pathway. The conflicted findings can be attributed to the different experimental preparations obtained from different species, for example, the use of isolated perfused lungs or isolated pulmonary arteries with different sizes, as well as in response to different isoforms of EETs.¹⁸ Thus, the question arose as to what function EETs have in the regulation of pulmonary circulation; specifically whether EETs possess alternative mechanisms independent of their systemically cardioprotective properties to evoke or participate in some pathological processes, such as pulmonary hypertension (PH).

PH is a progressive disease, characterized by sustained pulmonary vasoconstriction that increases vascular resistance and leads to an elevation in pulmonary artery pressure.¹⁹ In animal models, right ventricular systolic pressure (RVSP) is equal to pulmonary artery systolic pressure under physiological conditions and, therefore, has served as an index for the detection and quantification of pulmonary arterial hypertension.^20,21 In comparison with the systemic circulation, the pulmonary circulation possesses unique features, characterized as low oxygenated blood in pulmonary arteries. Whether this alternate pattern of oxygenated blood flow in the pulmonary circulation is responsible for the pulmonary-specific vascular responses that differ from those observed in systemic circulation needs to be addressed. While the few studies conducted on isolated perfused lungs reported an EET-dependent pulmonary vasoconstriction to increase RVSP,¹¹ in vivo investigations of pulmonary hemodynamic changes in response to each specific regioisomeric EET have never been carried out. Consequently, the controversial conclusions drawn from conflicting literature, along with the lack of in vivo evidence for pulmonary hemodynamic responses to EETs, formed the basis of the present study, which aimed to clarify a direct effect of each EET regioisomer on RVSP and systemic blood pressure (BP) under normal and hypoxic conditions.

MATERIALS AND METHODS

Animals

Male C57Bl/6J mice (12–15 weeks old) used in the present study were purchased from the Jackson laboratory (Bar Harbor, ME). All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the guidelines of the National Institutes of Health and the American Physiological Society for the use and care of laboratory animals

Surgery and experimental preparations

Mice were placed in an isoflurane induction chamber with the vaporizer and the flow of oxygen set at 5% and 500ml/min, respectively. The anesthetized mice were then transferred to an operation table (37 °C) and masked with an isoflurane nose cone. The isoflurane vaporizer and oxygen flow were then adjusted to 2% and 200ml/min respectively, and the mice were breathing room air mixed with oxygen (100% O₂) enriched isoflurane vapor. After shaving the neck area, a middle incision was made to expose the right and left external jugular veins and left common carotid artery through blunt dissection. A fluid-filled catheter with a flame-smoothed glass tip (inside and outside diameter of 300 and 400 µm respectively) was placed in the left carotid artery for monitoring heart rate (HR) and arterial BP. Another polyethylene line (PE-10) was placed in the left jugular vein for infusion of EETs. The PE-10 catheter was measured to contain a volume of 20 µl. A third fluid-filled catheter was inserted into the right jugular vein and advanced into the right ventricle for monitoring right RVSP. This catheter was made of Supramid Extra Tubing with inside and outside diameters of 580 and 780 µm, respectively. The proximal end of the tube was pulled, slit at an angle, and heated to make the edge smooth with inside and outside diameters of 300 and 370 µm, respectively (Figure 1a). Successful entry of the catheter into the right ventricle was evidenced by typical recordings of RVSP waves (Figure 1b,d). The pressure catheters were filled with heparinized saline and connected with pressure transducers (TRN050; Kent Scientific, Torrington, CT) and transducer amplifiers (Kent Scientific). Hemodynamic parameters including the systemic BP, HR, and RVSP were recorded on PowerLab (ADInstruments, Colorado Springs, CO), and analyzed using Chart V8 software (ADInstruments).

Figure 1.

Right ventricular pressure catheter and representative recordings. (a) Schematic illustration of the fluid-filled catheter used in the experiments for the measurement of right ventricular systolic pressure (RVSP). (b) Original tracing for changes in RVSP and systemic arterial blood pressure (BP) in control and in response to 14,15-EET (2ng/g of body weight). (c and d) Original tracings of RVSP recorded from a mouse using a commercial available solid-state pressure transducer and own-designed fluid-filled catheter respectively.

Experimental Protocols

The first protocol was to assess changes in BP and RVSP in response to in vivo administration of EETs. After catheterization, the isoflurane vaporizer was adjusted to a level that maintained the HR constant around 500 bpm. After stable baselines of hemodynamic parameters were recorded, the 4 regioisomeric EETs (14,15-EET, 11,12-EET, 8,9-EET, or 5,6-EET) were separately administered to 4 groups of mice, and subsequent changes in BP and RVSP were recorded. EETs were dissolved in ethanol and further diluted in saline in a final volume of 20 µl containing 1, 2, 5, or 10ng per gram of body weight (ng/g BW) and injected into the catheter placed in the left jugular vein using a Hamilton syringe. The catheter was then connected to a syringe pump that was managing a constant infusion rate of 10 µl/minute. The final concentration of ethanol in the EET solution was less than 0.1%. The saline containing 0.1% ethanol as the vehicle was used in each animal before injection of EETs. Changes in RVSP and BP were continuously monitored.

The second protocol was conducted to clarify that the EET-dependent increase in RVSP is due to their direct impact on pulmonary circulation, via perhaps pulmonary vasoconstriction, rather than their ability to increase systemic vasodilation, which promotes venous return to increase right ventricular pressure. Thus, specific time-course experiments were performed. A final dose of 2ng/g BW of 14,15-EET in a total volume of 20 µl was injected into the left jugular vein catheter that was connected to a syringe pump and was infused at the rate of 10 and 20 μl/min respectively. In this experiment, equivalent amounts of EETs were injected over 1 minute (at 20 μl/minute) and over 2 minutes (at 10 μl/minute) in order to create different local (pulmonary circulation) concentrations of EETs. There was a 15-minute interval between the 2 injections, and changes in RVSP, mean arterial BP (MABP), and HR were continually recorded before, during, and after each injection (Figure 3). Hemodynamic data were analyzed and averaged at every 10-second interval. Additionally, in separate experiments, low doses of 14,15-EET (0.1, 0.2, 0.5, and 1ng/g BW respectively, in a final volume of 20 μl) were directly injected into the right ventricle via the right jugular vein catheter through a “T” adaptor located between the Supramid Extra Tubing and pressure transducer. After the EET was injected into the catheter, the “T” adaptor was connected with a piece of Teflon tubing to the syringe pump that had been adjusted to an infusion rate of 10 μl/minute. Due to adequate stiffness of the Teflon tubing, its buffering effect on RVSP, if any, could be neglected. Changes in RVSP, MABP, and HR in response to intraventricular injection of different doses of EET were continuously recorded.

Figure 3.

Time-dependent changes in RVSP (a), MABP (b), and heart rate (HR; c) in response to a total volume of 20 µl 14,15-EET in the final dose of 2ng/g BW, injected within 1 and 2 minutes respectively. *Significant difference from 2-minute delivery group at the same time point (n = 7).

In the third protocol, hypoxia-induced changes in RVSP and BP were assessed. Under control conditions, mice were breathing room air mixed with oxygen enriched (100% O₂) isoflurane vapor through a cone mask, and then hypoxia was generated by replacing the oxygen tank with a tank containing 10% oxygen balanced with nitrogen, while gas flow and isoflurane vaporizer settings remained unchanged. Baselines of RVSP and BP and their changes in response to 14,15-EET (2ng/g BW) were recorded in both control and hypoxic conditions.

Materials and statistical analysis

The 4 EETs ((±)14(15)-EpETrE, (±)11(12)-EpETrE, (±)8(9)-EpETrE, and (±)5(6)-EpETrE,) were purchased from Cayman chemical company (Ann Arbor, MI; catalog numbers: 50651, 50511, 50351, and 50211 respectively). Gas tanks were purchased from Airgas USA (Bronx, NY). Data are represented as mean ± SEM and n refers to the number of mice. Statistical analyses were performed using GraphPad Prism 5 software. Student’s t-test was used to compare the difference between 2 groups. Two-way analysis of variance followed by the Tukey–Kramer post hoc test was used to compare differences among multiple groups. Statistical significance was accepted at a level of P < 0.05.

RESULTS

Figure 1a is a schema of the fluid-filled catheter used for measuring RVSP. The dimension of the proximal segment of catheter is ~0.37×5mm, which is small enough to allow the catheter to be advanced through the tricuspid valve into the right ventricle without interfering with blood flow. The internal diameter of the tip is ~0.3mm and was able to successfully sense and record changes in RVSP. We have measured RVSP using our fluid-filled catheter (Figure 1d) and a 1.2F solid-state pressure transducer (Scisence Inc, London, Ontario, Canada; Figure 1c) in the same animal. Comparable results were obtained by the 2 measurements, thereby confirming the feasibility, as well as the accuracy of our experiments. Figure 1b shows the representative tracing of RVSP and BP in response to 14,15-EET. This single dose of 2ng/g BW 14,15-EET elicited an increase in RVSP from 27.8mm Hg (basal RVSP) to 34.8mm Hg, associated with a simultaneous reduction of MABP from 93.2 to 87.4mm Hg. We observed that basal RVSP ranged from 27 to 30mm Hg with an average of 29mm Hg, and intravenous administration of 14,15-EET elicited an increase in RVSP but a decrease in systemic BP. Neither RVSP nor MABP was significantly affected by the injection of saline vehicle containing 0.1% ethanol, as indicated by the data of RVSP (29.6±0.8 vs. 29.4±0.8mm Hg) and MABP (89.1±1.3 vs. 89.7±1.4mm Hg) before and after administration of the vehicle. Figure 2 summarized changes in RVSP (Figure 2a) and MABP (Figure 2b), in response to the 4 isoforms of EETs. All 4 EETs initiated dose-dependent increases in RVSP. In particular, 11,12-EET elicited the greatest increase in RVSP at each dose point, compared to the other 3 EET isoforms. Furthermore, administration of the lowest dose of 11,12-EET (1ng/g BW) initiated a significant increase in RVSP, while this statistical significance was not observed in response to the remaining EET isoforms at the same low dose. In the systemic circulation, EETs dose-dependently reduced BP. All EETs reduced MABP significantly at the dose of 10ng/g BW. Significant reduction of MABP was also observed in response to 5ng/g BW of 14,15-EET, 11,12-EET and 5,6-EET, as well as to 2ng/g BW of 14,15-EET. Ultimately, dose–response curves were comparable among the 4 EET regioisomers. Collectively, these data show that EETs are able to work as pulmonary vasoconstrictors and increase RVSP, but act as systemic vasodilators that can reduce peripheral vascular resistance and lower BP. Since 2ng/g BW of 14,15-EET initiated significant changes in both RVSP and MABP, this dose was specifically selected for use in the studies represented in Figures 3 and 5 respectively.

Figure 2.

Dose-dependent increases in right ventricular systolic pressure (RVSP; a) and decreases in mean arterial blood pressure (MABP; b) in response to 4 EET regiosomers (n = 6–9 in each EET group).

Figure 5.

Changes in RVSP (a and b) and MABP (c and d) in normal (air + 100% O₂/isoflurane vapor) and hypoxic conditions (air + 10% O₂/isoflurane vapor). (b) Delta changes of 14,15-EET-induced increases in RVSP in normal and hypoxic conditions. (d) Delta changes of 14,15-EET-induced reduction of MABP in normal and hypoxic conditions. *Significant difference from controls (100% O₂). #Significant difference between 100% O₂ and 10% O₂. @Significant difference from the control at 10% O₂.

In order to exclude the possibility that EET-induced increases in RVSP (Figure 2a) resulted from an increase in systemic venous return, as a function of EET-induced systemic vasodilation, we performed specific time-course experiments by administering a total volume of 20 µl of 14,15-EET in a final dose of 2ng/g BW over 1 and 2 minutes respectively. Thus, Figure 3 provides evidence confirming that the EET-induced increase in RVSP is not attributed to systemic vasodilation-induced increase in venous return. As described in experimental protocol 2, an equal amount of 14,15-EET (2ng/g BW) injected over 1-minute was expected to generate a higher pulmonary concentration of the EET than that injected over a 2-minute duration. Indeed, the 1-minute injection initiated a significantly faster and greater elevation of RVSP than the 2-minute injection (Figure 3a). However, this difference did not coincide with a reduction in MABP, as evidenced by comparable MABP curves (Figure 3b), which are associated with constant HR (Figure 3c). Moreover, 14,15-EET-induced increases in RVSP took precedence over its reduction of MABP, excluding the possibility that the increase in RVSP is caused by an increase in venous return. Additionally, Figure 4 shows that low doses of 14,15-EET injected directly into the right ventricle elicited dose-dependent increases in RVSP (Figure 4a) without effects on MABP (Figure 4b). There were no significant changes in RVSP in response to intraventricular injection of vehicle (29.8±0.8 vs. 29.9±0.8mm Hg before and after vehicle injection respectively).

Figure 4.

Dose-dependently increased RVSP (a) and unchanged MABP (b) in response to a direct administration of 14,15-EET into the right ventricle. *Significant difference from the basal condition (n = 5).

Finally, the effects of EETs on RVSP were evaluated in control and hypoxic conditions. As shown in Figure 5a, RVSP was increased significantly in hypoxia, suggesting that hypoxia per se causes pulmonary vasoconstriction. In response to 14,15-EET, an augmented RVSP was observed in both oxygen tensions, but was more predominant in hypoxic conditions. As a result, an absolute increment of RVSP in response to 14,15 EET was significantly greater in hypoxia than in controls (Figure 5b). In systemic circulation, however, hypoxia initiated a significant reduction of MABP (Figure 5c). There were no differences in 14,15-EET-induced attenuation of MABP in both oxygen conditions (Figure 5d). Thus, Figure 5 indicates that hypoxia potentiates 14,15-EET-induced PH.

DISCUSSION

The major findings of this study are: (i) All of the 4 EET regioisomers administered in in vivo conditions elicit a direct increase in RVSP (Figure 2a). (ii) EETs lower BP via systemic vasodilation (Figure 2b), however the response is not responsible for the EET-induced increase in RVSP (Figures 3 and 4). (iii) Hypoxia increases RVSP and potentiates EET-induced PH, but does not significantly affect EET-induced peripheral vasodilator responses (Figure 5). Additionally, we have successfully developed a sensitive and economical approach, using a fluid-filled catheter to continuously monitor pulmonary hemodynamic changes in mice. The present study provides solid evidence confirming the properties of EETs in the elevation of RVSP, possibly involving EET-induced pulmonary vasoconstriction and clarifies specific roles of EETs in the potentiation of hypoxia-induced PH.

EET-dependent increases in RVSP

Due to the controversy regarding the bioactivity of EETs in the pulmonary circulation,^{12,13,15–18} we examined the direct effect of EETs on pulmonary circulation and indicated that all of the 4 EET regioisomers are able to increase RVSP (Figure 2). Additionally, all EETs elicited a reduction in BP via systemic vasodilation.²² In this context, it is necessary to exclude the possibility that the increase in RVSP by EETs resulted from systemic vasodilation that increases venous return and consequently RVSP, by way of the Frank–Starling mechanism. We therefore designed specific experiments to clarify the issue. As shown in Figure 3, the local pulmonary concentration of 14,15-EET is proportional to the elevation of RVSP and inversely proportional to its delivery rate (Figure 3a). Typically, this time-specific responsive phenotype of changes in RVSP was not observed in changes of MABP (Figure 3b) due to a fast dilution of EET in systemic circulation. In regard to the unchanged HR (Figure 3c), we interpret the result to mean that anesthesia may have desensitized the baro-reflex in response to such small reductions of BP. Moreover, the EET-induced increases in RVSP preceded the changes in MABP, further demonstrating that under a constant HR, an increase in venous return, if any, as a function of systemic vasodilation is not a primary determinant in the EET-induced elevation of RVSP presented in this study. The same conclusion could also be drawn from the results shown in Figure 4, in which increased RVSP resulted from the direct infusion of low doses of 14,15-EET into the right ventricle; the doses used (0.1–1ng/g BW) are approximately 100–1,000 fold lower than the total amount of EETs measured from mouse lungs (85±20ng/g tissue, in the form of incorporated with cellular phospholipids; unpublished data) and show no impact on systemic BP. Additionally, in comparison to endogenous EETs that are released in response to any stimuli that activates phospholipases²³ and work in concert to impose their bioactivities, exogenous administration of sub-physiological levels of EETs also elicited increases in RVSP, indicating EETs as potent activators in promoting pulmonary arterial pressure. Although the present study does not provide direct evidence for the increase in RVSP being a result of EET-induced pulmonary vasoconstriction, some in vitro studies indeed demonstrate an EET-induced vasoconstriction in isolated pulmonary arteries and increased pulmonary artery pressure in isolated mouse lungs.^11–13 In this context, we speculate that EETs directly elevate RVSP via perhaps, the mechanism(s) involving pulmonary vasoconstriction.

Synergistic effects of EET and hypoxia in pulmonary hypertension

The pathophysiological relevance of EET-dependent increases in RVSP was revealed in our studies. Changes in sEH expression and EET bioavailability were intimately linked to pathophysiology of hypoxia-induced PH, as evidenced by results that acute hypoxic vasoconstriction was potentiated by inhibition of sEH and attenuated by an EET antagonist.^11,24 Moreover, overexpression of an EET synthase (CYP2C29) in mice significantly elevated mean pulmonary artery pressure and total pulmonary resistance, responses that were also initiated during exposure of mice to hypoxia for 2 hours.²⁵ Notably, previous studies have reported the actions of endogenous EETs in the potentiation of PH via a hypoxia-induced upregulation of CYP/epoxygenase, and therefore, we evaluated actions of exogenously administered EET in the hypoxia-induced PH. We found that hypoxia and 14,15-EET synergistically enhanced RVSP (Figure 5a) but reduced MABP (Figure 5c). Importantly, 14,15-EET has an enhanced function in hypoxic pulmonary circulation, manifested by a more than 2-fold increase in RVSP in hypoxia than controls (Figure 5b). In the systemic circulation, however, 14,15-EET caused a comparable reduction of MABP in both oxygen tension conditions (Figure 5d). The difference in magnitude of EET-dependent responsiveness between the pulmonary and systemic vasculatures, as a function of hypoxia, points to a strong influence of EETs in promoting a pathological state in the pulmonary circulation. The different actions of EETs between the 2 circulations may serve as a promising approach when attempting to clarify the mechanism(s) responsible for the EET-dependent or hypoxia-induced, along with their interactions during the development of PH. Since the expression of epoxygenase is sensitive to oxygen tension,²⁶ hypoxia-induced PH has been reported to result from increases in endogenous EET synthesis or reduction in EET degradation.^11,24,25 The consequence of increases in EETs is then correlated to the activation of Rho-kinase signaling that is considered as a primary determent of PAH in human and animal models with different etiologies, including hypoxia-induced PH.^27,28 Indeed, both hypoxia and exogenous application of 11,12-EET-elicited activation of Rho kinase in pulmonary smooth muscle cells,¹¹ which indicates Rho kinase as the intrinsic link between EETs and hypoxia-induced PH. Such a link provides a mechanistically based explanation for our results, showing that in hypoxia, 14,15-EET-induced increases in RVSP was significantly potentiated (Figure 5b). Moreover, during the process of hypoxia-induced upregulation of EET synthesis, an increased release of superoxide, as a function of activated CYP2C29 signaling²⁹ may also contribute significantly to the development of PH.

In conclusion, the present study provides a general profile illustrating the vascular activities of EETs in the intact pulmonary and systemic circulations. EETs initiate a direct elevation of pulmonary arterial pressure in a hypoxia-facilitated manner, while systemically act as vasodilators to lower BP. The conclusion drawn from the study may have far-reaching pathophysiological significance in revealing a crucial role of EETs in the pathogenesis of PH.

DISCLOSURE

The authors declared no conflict of interest.