Acute pulmonary embolism (APE) is a major clinical problem resulting from the migration of emboli to the lungs and obstruction of pulmonary blood vessels. Although this critical condition is a leading cause of death, (1) little progress has been made in the pharmacological management of the acute right heart failure and circulatory shock of this high-mortality condition (2).
APE-induced pulmonary hypertension is the cumulative result of at least three main factors: the mechanical obstruction of pulmonary vessels, general pulmonary arteriolar constriction attributable to a neurogenic reflex, and the release of vasoconstrictors by activated platelets, leukocytes, and endothelial and lung cells (3,4). Although the current treatment of APE is focused on removing the mechanical obstruction of pulmonary vessels (1), pharmacological blockade of the pulmonary vasoconstriction has been suggested as a coadjuvant therapy (4–8).
In addition to the pharmacological antagonism of pulmonary vasoconstrictors released during APE, selective pulmonary vasodilators may attenuate the pulmonary hypertension and produce beneficial effects after APE. For example, we and others have demonstrated that inhaled nitric oxide (NO) consistently attenuates pulmonary hypertension in different animal models of APE (9–12). In this regard, many of the biological effects of NO, including the regulation of vascular tone and platelet function, are mediated by cyclic guanosine 3′,5′-monophosphate (cGMP), which is produced after NO activates soluble guanylate cyclase. However, cGMP is short-lived because of the rapid degradation by phosphodiesterases (PDE) (13). Therefore an alternative to achieve increased endogenous cGMP levels in pulmonary vessels may be to inhibit cGMP specific phosphodiesterase type 5 (PDE5), which is the predominant PDE in the pulmonary vasculature.
In this study, we hypothesized that PDE5 inhibition with sildenafil would attenuate the APE-induced hemodynamic derangements. This hypothesis is supported by several studies showing that sildenafil, or a sildenafil analog, caused preferential pulmonary vasodilation in a variety of clinical and experimental settings, including primary pulmonary hypertension (14,15), chronic thromboembolic pulmonary hypertension (16), meconium aspiration syndrome (17), hypoxia (18), and during an IV infusion of the thromboxane analog U46619 (19,20). Moreover, given that both 8-Br-cGMP (the cGMP analog) (21,22) and sildenafil (23,24) reduce lipid peroxidation, we hypothesized that sildenafil would attenuate the increase in oxidative stress after APE (25).
This study was approved by our institutional animal investigation committee. In the first series of experiments we used a whole animal model of APE to study the effect of sildenafil on APE-induced hemodynamic changes. Twenty-five mongrel dogs (12.8 ± 1.1 kg) of either sex were anesthetized with ketamine (10–15 mg/kg, IM), xylazine (1,5 mg/kg, IM), and pancuronium (0.1 mg/kg, IV), tracheally intubated, and their lungs mechanically ventilated with room air using a volume-cycled respirator (C. F. Palmer, London, UK). The tidal volume was 15 mL/kg and the respiratory rate was adjusted to maintain a baseline physiologic arterial carbon dioxide tension. Anesthesia was maintained with an IM injection of ketamine (3 mg/kg) and xylazine (0.3 mg/kg) every 30 min. Fluid-filled catheters were placed into the left femoral artery and right femoral vein for mean arterial blood pressure (MAP) monitoring via a pressure transducer and fluid administration, respectively. A 7.5F balloon-tipped Swan-Ganz thermodilution catheter was placed into the pulmonary artery via the left femoral vein; its correct location was confirmed by detection of the typical pressure wave of this artery. The catheter was connected to pressure transducers to allow the monitoring of mean pulmonary artery pressure (MPAP), central venous pressure, and pulmonary capillary wedge pressure (PCWP). The transducers were zeroed at the level of the right heart and recalibrated before each set of measurements. Thermodilution cardiac output was determined in triplicate by injecting 3 mL of saline and the results recorded on a computerized system (Monitor DC Baxter, Edwards Critical Care Vigilance, Irvine, CA). Heart rate (HR) was measured using a surface electrocardiogram (lead I). Blood samples were drawn from the femoral artery at predetermined times for blood gas analysis. Arterial oxygen tension (Pao2), arterial oxygen saturation, carbon dioxide tension (Paco2), pH, and hematocrit (Hct) were determined using a blood gas analyser (Stat Profile 5 Analyser; Biomedical, Waltham, MA).
After at least 20 min for stabilization, a baseline hemodynamic evaluation was performed. Thereafter, APE was induced by repeated injections (every 30 s) of 300 μm microspheres (Sephadex G50; Pharmacia Fine Chemicals; Uppsala, Sweden) into the inferior vena cava over 3–5 min. The amount of microspheres infused in each dog was adjusted to induce an increase of 20 mm Hg in MPAP. This model of APE is very similar to that previously reported (10). Hemodynamic evaluation was performed 30 min (30E time point) after APE was induced. The animals were randomly separated into the four following conditions: 1) Sildenafil 0.25 group (n = 8), sildenafil 0.25 mg/kg (Pfizer, São Paulo, Brazil; infused IV over 15 min); 2) Sildenafil 1 + 0.3 group (n = 8), a bolus injection of 1 mg/kg of sildenafil (infused IV over 15 min) followed by a 30 min infusion of sildenafil (0.3 mg · kg−1 · h−1) (26); 3) Control group (n = 9), the same volume of saline; or 4) shams (n = 3), saline infusions only. Hemodynamic evaluations were performed 45 and 90 min (45S and 90S time points, respectively) after the sildenafil (or saline) infusion started. Cardiac index (CI), systemic vascular resistance index (SVRI), and pulmonary vascular resistance index (PVRI) were calculated by standard formulae. Arterial blood samples were drawn at baseline, 30E, 45S, and 90S time points for blood gas analysis and determination of plasma thiobarbituric acid reactive species (TBA-RS) levels as described below.
To further validate our findings, we performed another series of experiments in the rat isolated perfused lung preparation. Male Wistar rats (270–330 g) were anesthetized with urethane (1 g/kg intraperitoneally). Their tracheas were cannulated with polyethylene tubing and connected to an animal ventilator (60 breaths/min). The rats were heparinized (500 U of heparin) and a midsternal thoracotomy was performed as previously described (27). Blood (7 mL) was withdrawn from the right ventricle and mixed with 7 mL of normal saline containing 1.5% serum albumin. Although this mixture has a smaller Hct and protein concentration than blood, plasma osmolarity does not change significantly (27). A cannula was inserted into the pulmonary artery via the right ventricle and a tight ligature was placed around the main trunk of the pulmonary artery. The lungs were continuously perfused with a peristaltic pump (Incibras, Sao Paulo, Brazil) at a constant flow rate (9 mL/min) and pulmonary venous outflow was diverted into a reservoir via a cannula that was inserted in the left atrium through the left ventricle and fixed with a ligature at the apex of the heart. Another ligature was placed above the atrioventricular junction to prevent the perfusate mixture from flowing into the ventricles. The perfusate mixture was maintained at 37°C by a heat exchanger. MPAP was measured from a side arm of the inflow cannula with pressure transducers (COBE, Arvada, CO) zeroed at the level of pulmonary artery cannula. The preparation was allowed to equilibrate for 20 min before the experiments were begun. In this series of experiments (n = 5–7/group), sildenafil (1 mg/kg body weight) or saline was added to the perfusate mixture 5 min before lung embolization (or saline injection) with 6.6 mg/kg of a suspension of Sephadex microspheres (or saline) injected into the pulmonary artery via a side arm of the inflow cannula after stabilization. Perfusate samples were collected in tubes containing ethylenediamine tetraacetic acid at baseline and at the end of the experiments. After centrifugation at 800g for 5 min, aliquots were removed and stored at −70°C until used for the determination of TBA-RS levels.
TBA-RS levels were determined in plasma samples from the first series of experiments and in lung perfusate samples by modifications of the method of Lapenna et al. (28). In short, 0.2 mL of sample was added to reaction mixture containing 0.5 mL of 1% phosphoric acid (pH 2.0) and 0.25 mL aqueous solution of 0.6% TBA (final volume of 1.0 mL), followed by 60 min heating at 95°C. After cooling, samples and standards of malondialdehyde were read at 532 nm against the blank of the standard curve.
All the results are expressed as mean ± sem and the between groups comparisons were assessed by two-way analysis of variance (SigmaStat for Windows; Jandel Scientific, Richmond, CA). One-way analysis of variance for repeated measures was used to determine the changes in the hemodynamic and respiratory variables in each group. When the one-way analysis of variance for repeated measures was significant, the differences were tested by the Dunnett multiple comparisons test. A probability value < 0.05 was considered the minimum level of statistical significance.
In the first protocol, baseline hemodynamic variables were similar in the four experimental groups (Table 1). The hemodynamic and respiratory data from the sham-operated animals showed no significant changes during the 120 min of monitoring. Thus, to simplify the data interpretation, the results of sham-operated animals are not presented.
None of the animals died as a consequence of the APE. Stepwise injections of 300 μm microspheres into the inferior vena cava over a period of <5 min induced sustained pulmonary hypertension (MPAP increased from 13 ± 1 mm Hg to 34 ± 2 mm Hg; P < 0.001) 30 min after the end of microspheres administration in the 3 embolized experimental groups. In addition, lung embolization increased PVRI by approximately 330% (from 203 ± 24 dynes · s · cm−5 · m−2 to 877 ± 154 dynes · s · cm−5 · m−2; P < 0.01). No other significant hemodynamic changes were seen 30 min after APE (Table 1). Although the animals in the control group showed no further changes in MPAP and PVRI after the APE-induced pulmonary hypertension, significant decreases in MPAP and PVRI were observed 45 min and 90 min after IV administration of both doses of sildenafil (Fig. 1; both P < 0.05). Interestingly, both doses of sildenafil produced no significant changes in MAP, PCWP, SVRI, CI, and HR (Table 1).
APE produced marked decreases in Pao2 (from 104 ± 8 mm Hg to 55 ± 8 mm Hg; P < 0.01) in the 3 embolized experimental groups. Treatment with sildenafil did not significantly change blood oxygenation.
Lung embolization was associated with significant increases in plasma TBA-RS levels in the control group (Fig. 2). Conversely, treatment with sildenafil significantly attenuated APE-induced increase in oxidative stress (P < 0.05; Fig. 2).
As shown in Figures 3 and 4, the results we observed in the second protocol (rat isolated perfused lung preparation) were similar to those found in the first protocol. Although lung embolization increased MPAP by almost 20 mm Hg in the control group, sildenafil significantly attenuated APE-induced pulmonary hypertension (Fig. 3) and increase in oxidative stress (Fig. 4) (both P < 0.05).
The main finding of this study was that IV administration of sildenafil significantly reduced APE-induced pulmonary hypertension and oxidative stress without evident effects on systemic circulation. Consistent with this finding, sildenafil produced similar effects in rat isolated perfused lung preparation.
Although APE can increase PVRI by different mechanisms, current treatment of APE is focused on the mechanical obstruction of pulmonary vessels. However, the relevance of active participation of pulmonary vasoconstriction in the hemodynamic responses to APE has been valued as a new target for pharmacological interventions (4–6). Pulmonary vasodilators that have been tested in animal models of APE include NO, prostacyclin, ketanserin, hydralazine, amrinone, isoproterenol, nitroglycerine, nitroprusside, and captopril (4). Although it is generally accepted that vasodilators may be effective in hemodynamically stable patients with APE, only inhaled NO produced selective pulmonary vasodilation during experimentally induced APE (4,10). The results of the present study show that the two doses of IV sildenafil decrease MPAP and PVRI without significantly affecting MAP and SVRI. These observations suggest that sildenafil is a selective pulmonary vasodilator that can produce beneficial effects during APE. In addition, the use of an infusion of sildenafil in the present study establishes that the effect can be sustained.
The beneficial hemodynamic effects of sildenafil we observed are analogous to those previously described after the inhalation of NO during microsphere embolism in piglets (10)—namely, a significant decrease in MPAP and PVRI without systemic vasodilation. Similarly, previous studies have shown that sildenafil improves the hemodynamics in other clinical settings and animal models of pulmonary hypertension (14–20). However, an important feature of the model we have used in the present study is the presence of a mechanical obstructive component causing pulmonary hypertension. Many of these conditions share pathophysiological mechanisms causing pulmonary hypertension, especially the release of vasoconstrictors or the loss of pulmonary vasodilation. Both mechanisms can alter the vasoregulatory function of pulmonary vascular endothelium (29,30). For example, endothelin-1, a potent pulmonary vasoconstrictor involved in the pulmonary hypertension after APE, significantly attenuated the increase in cGMP levels after vasorelaxant agonist stimulation (31). Therefore, in the present study, the inhibition of PDE5 by sildenafil may have caused pulmonary vasodilation by enhancing and sustaining the levels of cGMP in pulmonary vessels.
The importance of increased oxidative stress in pulmonary microembolism-induced lung vascular injury is well established (25,32). In the present study, sildenafil attenuated APE-induced increase in lipid peroxidation, thus suggesting a protective effect for sildenafil involving antioxidant mechanisms (23,24). Therefore, it is possible that sildenafil may have protected against an impaired vascular NO bioavailability related to its oxidative inactivation by reactive oxygen species (ROS) released during APE (33). Consistent with this suggestion, increased ROS generation leads to endothelial NO synthase uncoupling through increased oxidative stress-induced oxidation and depletion of BH4, a cofactor required for NO synthesis. Although previous studies have found protective effects of increased cGMP levels or sildenafil in conditions associated with increased oxidative stress (21–24), the mechanisms contributing to such an effect are not clear.
In conclusion, we found that IV sildenafil can selectively attenuate the increases in MPAP and PVRI after APE in dogs. These beneficial effects may result from antioxidant effects of sildenafil and provide new insight into the treatment of APE-induced pulmonary hypertension. Because particulate emboli differ from thromboemboli, in that the latter contains cellular and molecular constituents that interact differently with pulmonary endothelial cells than relatively inert particulate matter, the conclusions drawn from this study may be limited to this particular model. We suggest that clinical studies should be performed to assess the effectiveness of sildenafil in the treatment of patients with APE or other diseases associated with increased oxidative stress.
We thank Laboratorios Pfizer Ltda. for providing sildenafil and Fernanda Viaro and Paulo Evora for technical support.
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