Pulmonary thromboembolism (PTE) is a very common disease in China and is a major cause of mortality, although the diagnosis and management of PTE have become more standardized in recent years. Therefore, the pathogenesis and pathophysiology of PTE are currently attracting much attention from researchers. It is evident that, in PTE, the mechanical obstruction of the pulmonary arteries is accompanied by vasoconstriction and an inflammatory response.1-6 As the strongest vasoconstrictor, endothelin-1 (ET-1) was shown to play an important role in PTE. However, the relationship between ET-1 and pulmonary artery pressure (PAP) in PTE, and the role of ET-1 receptor (ET-1R) antagonists in acute PTE (APTE) remain unclear. The aim of this study was to compare the effects of a thrombolytic drug (urokinase (UK)) and a selective ET-1R antagonist (BQ123) in experimental APTE, focusing on their effect on PAP and the production of vasoactive and inflammatory mediators.
Twenty healthy adult hybrid dogs (weighing (12.86±0.35) kg, age 1-3 years) were purchased from the Experimental Animal Center, Shanghai Jiao Tong University School of Medicine. All procedures were approved by the animal care committee at Shanghai Jiao Tong University School of Medicine. The animals were randomly assigned to five groups: sham, model, UK, BQ123 and combination (UK plus BQ123) (4 dogs per group).
Experimental APTE was induced as previously described.7,8 Briefly, the dogs were anesthetized by an intramuscular injection of ketamine (0.1 g) and 3% phenobarbital (1 ml/kg). A 6F Swan-Ganz catheter was placed into the pulmonary artery via the femoral vein, and connected to a pressure transducer and a multi-channel signal analysis system (Shanghai Alcott Biotech, China) to monitor mean pulmonary artery pressure (mPAP). The correctness of the catheter placement was confirmed by detection of a typical pressure wave of the pulmonary artery. 6F central venous catheters were also inserted into the internal jugular vein for the injection of blood clots and fluid infusion.
The dogs in the sham group were given equal volume of normal saline for model preparation and treatment. APTE was induced in the other four groups. First, 20 ml of blood was drawn, mixed with 100 U thrombin and allowed to clot in the syringe at room temperature. The clot was then cut into pieces of (0.5-1.0) cm × 1.0 cm × (1.0-2.0) cm. The autologous blood clots were suspended in normal saline and injected into the pulmonary circulation via the jugular vein within 1-2 minutes.
The dogs in the model, UK, BQ123 and combination groups received normal saline, urokinase (UK, 20 000 IU/kg), BQ123 (2 μg/kg, a generous gift from Dr. YUAN Wen-jun, Second Military Medical University School of Pharmacy, China) or UK + BQ123 in the same volume (50 ml), respectively, 1 hour after the induction of APTE. At the end of experiment, the dog was sacrificed by a lethal dose of phenobarbital. The lungs were harvested to investigate whether thrombi were present in the pulmonary arteries and then fixed in the 4% paraformaldehyde and embedded in paraffin for histological analysis.
Arterial (for blood gas analysis) and venous (for ET-1, thromboxane (TX) B2 and tumor necrosis factor (TNF)-α concentrations) blood samples were collected and mPAP was recorded before (T0) and at 1 (T1), 2 (T2), 3 (T3) or 5 (T4) hours after infusion of the blood clot. Venous blood was centrifuged at 3000 r/min. The supernatant was stored at -70°C. The concentrations of serum ET-1, TXB2 and TNF-α were determined by a radioimmunoassay.
The results were expressed as mean ± standard deviation (SD). SPSS 11.0 software was used in statistical analysis. Differences between the groups were analyzed with one-way analysis of variance (ANOVA) followed by the Least Significance Difference test. Statistical significance was defined as P <0.05.
Paraffin-embedded sections were stained with hematoxylin-eosin and examined microscopically. Emboli in the pulmonary arteries were clearly visualized both grossly and microscopically in each group except the sham group (Figure 1), indicating successful induction of APTE.
Changes in mPAP after APTE induction
The mPAP was significantly higher in the model group than in the sham group at all time points after the induction of APTE, except at baseline (T0) (Figure 2). This confirms that the APTE model used significantly increased the mPAP.
mPAP changes after various treatments
Compared with the model group, the mPAP in the UK, BQ123 and combination groups decreased significantly, almost to the control level in the sham group, at T4 (Figure 2).
The increase of PAP (Δ.PAP) at T1 to T4, which represents the increase of PAP after infusion of the thrombus, was calculated by subtracting the PAP value at T0 from that at T1, T2, T3 or T4. Thus, the ΔPAP value quantitatively demonstrates the change in PAP (Figure 3). At T1, the sham group was significantly different from the other four groups, indicating that the model was appropriate. Both UK and BQ123 significantly reduced ΔPAP. The maximal difference occurred at T2. At T2, the administration of a ET-1R antagonist reduced ΔPAP by similarly as that of a thrombolytic drug ((3.83±1.60) vs (5.56±1.26) mmHg, respectively, at T2), while the combination caused a more significant reduction (-1.70±0.72, P <0.05). At T2, there were significant differences between the combination group and the model group, the UK group, the BQ123 group. Apparently, combination treatment provided better results than the use of UK or BQ123 alone. In addition, at T2, the differences in ΔPAP between the BQ123 group ((3.83±1.60) mmHg) and the model group ((7.70±1.72) mmHg) were significant, indicating that BQ123 inhibited the increase of PAP. At T3 and T4, the comparisons between the model group and the three intervention groups revealed that the ΔPAP values were all significantly different from each other. At T3, ΔPAP was (7.94±2.21) mmHg in the model group, compared with (2.22±1.47), (0.05±1.33) and (-2.60±0.84) mmHg in the UK, BQ123 and combination groups, respectively (P <0.05). At T4, ΔPAP was (6.69±1.84), (2.46±1.95), (-0.48±0.93) and (-2.43±0.78) mmHg, respectively (P <0.05). These findings demonstrate that treatment with thrombolytic drugs and/or ET-1R antagonists can ameliorate the increase of PAP that occurs in response to APTE.
Arterial blood gas analysis
Overall, analysis of arterial blood gases showed no difference in PaO2 pressure among the five groups at any time point.
Changes of serum ET-1, TXB2 and TNF-α levels in response to APTE and effects of UK and BQ123
We calculated the changes in the serum ET-1 concentration after inducing APTE using the equation: ΔET-1 = (ET-1 at T2 + T3 + T4)/3 - (ET-1 at T0). Figure 4 shows that compared with the model group, BQ123 significantly attenuated the increase of ET-1. Interestingly, the level of ET-1 was higher in the UK group than in the model group.
As expected, a significant difference (P <0.05) was observed between the sham ((3.18±2.22) pg/ml) and the model, the UK, combination groups ((57.79±7.57), (70.60±8.31), (46.88±10.46) pg/ml, respectively). The ET-1 concentration in the BQ123 group ((26.24±8.71) pg/ml) was significantly lower than that in the model, UK and combination groups (P <0.05).
In general, there were no significant differences in TXB2 levels among the five groups, except at T4, when the TXB2 level in the combination group ((720.13±15.72) pg/ml) was significantly higher compared with the model group ((574.52±15.68) pg/ml; P <0.05) (data not shown).
As shown in Figure 5 there were significant differences in the TNF-α concentration at T1 and T2 among the five groups. At T1, the TNF-α concentration was significantly lower in the BQ123 group ((467.5±48.2) pg/ml) than in the model group ((745.0±64.0) pg/ml) and the UK group ((712.5±34.0) pg/ml) (P <0.05). At T2, the TNF-α concentration in the BQ123 group ((497.5±37.3) pg/ml) was significantly lower than that in the model, UK and combination groups ((780.0±70.7), (770.0±41.8) and (710.0±30.0) pg/ml, respectively; P=0.026). This indicates that BQ123 inhibited the increase of TNF-α.
In the present study, APTE in dogs was successfully induced by the injection of autologous blood clots into the pulmonary circulation, and confirmed by histological examination of lung tissues. The mPAP in the model group was significantly higher than that in the sham group. Compared with other animal models using glass or polystyrene beads as embolic materials, the experimental model used in the present study more closely mimics clinical APTE.3 Autologous blood clots not only lead to mechanical obstruction of pulmonary arteries, but also induce the release of neurohumoral factors, providing a basis for subsequent experiments.
The development of right ventricular failure after sudden pulmonary hypertension is a major cause of death after PTE.9,10 This sudden pulmonary hypertension occurs not only because of physical obstruction of blood flow, but also because of the release of humoral factors.3 Many experiments showed that, if cardiac output is preserved, PAP does not increase unless 60%-70% of the pulmonary vasculature is obliterated by a non-hematogenous obstruction, but only 25%-30% of the pulmonary vasculature must be obstructed to increase PAP when the obstruction is caused by a thromboembolus.3,4,11 Our results showed that the mPAP was significantly higher in the APTE model group at all time points after induction than in the sham group. For example, mPAP increased from (16.43±0.83) mmHg at T0 to (26.68±1.38) mmHg at T1, confirming the validity of our experimental model.
Many reports have demonstrated that once a thrombus becomes lodged in a pulmonary artery, there is a rapid and complex interaction of cellular and molecular events that cause the release of vasoactive or bronchospastic mediators such as histamine, serotonin, platelet-activating factor, prostaglandins, ET-1, and cytokines, which could explain the pulmonary hypertension and regional ventilation-perfusion mismatch.1-4,10,11
ET-1 is a potent pulmonary vasoconstrictor that is released during pulmonary embolism and is regarded as a promising therapeutic target in cardiovascular diseases such as pulmonary arterial hypertension and heart failure.12 ET-1 activates its receptors on pulmonary vascular smooth muscle cells causing contractile responses and may stimulate the release of TXA2 from pulmonary tissue. It has been reported that there is a positive feedback mechanism between ET-1 and TXA2 production.8,13 ET-1 also activates the cyclooxygenase pathway, which in turn enhances TXA2 formation.14,15 In addition, ET-1 exerts vasoconstrictive effects that are independent of its effects on TXA2 formation. ET-1 has been shown to play a crucial role in the vasoconstriction in response to APTE.4,16 Several animal studies have shown that the plasma and tissue levels of ET-1 were markedly increased in pulmonary arterial hypertension (PAH) followed by pulmonary embolism.8,12,17-19Sofia et al20 also reported that there was an increased release of ET-1 from the lungs of patients following APTE. In humans, plasma ET-1 levels are correlated with markers of disease severity including pulmonary vascular resistance, right atrial pressure and pulmonary artery oxygen saturation.12
Considering the importance of pulmonary artery vasoconstriction in PTE, we found that the use of pulmonary vasodilators or pharmacological antagonism of vasoconstriction may have beneficial effects in patients with PTE, particularly in patients who are not responding to or who have a contraindication to one of the established treatment regimens. Several studies have reported that pulmonary hypertension and elevated levels of TXB2 (internal metabolite of TXA2) after APTE could be attenuated by selective ET-1R antagonists.15,21 However, none has compared the effect of thrombolytic drugs and ET-1R antagonists or evaluated their combined effects during the early phase of APTE. Our study showed that, although both a thrombolytic drug (urokinase) and a selective ET-1R antagonist (BQ123) did not improve alveolar gas exchange, they significantly decreased mPAP. Furthermore, the combination of both drugs provided more beneficial effects. This finding indicates that, during the acute phase of pulmonary embolism, it is reasonable to use an ET-1R antagonist alone or in combination with a thrombolytic drug.
We found that BQ123 reduces not only mPAP, but also the serum ET-1 concentration, indicating that ET-1R antagonists may inhibit positive feedback of ET-1 production to further decrease mPAP. Interestingly, UK alone increased the serum ET-1 level, which may subsequently increase mPAP. Ji et al17 also found that the ET concentration in arterial and venous blood increased transiently when the emboli were resolved. This may explain why the combination of UK and BQ123 may be more beneficial because BQ123 counteracts the harmful effects of UK.
It is known that thrombin induces aggregation of neutrophils and platelets, leading to a local inflammatory response after pulmonary embolism. Moreover, ET-1 itself has pro-inflammatory activity.12,14 Therefore, we compared the TNF-α concentrations in the groups at different time points. We found that there was a significant difference in TNF-α level at T1 and T2 among the five groups, and that the TNF-α level was much lower in the BQ123 group than in the model, UK and combination groups.
Although our study mimics the process of APTE in humans, there are still some distinctions between this model and the actual process. The formation of hominal thromboembolism is a much more sophisticated and multifactorial process than that in the canine model used in our study. Therefore, further studies are needed to provide evidence to support the clinical use of ET-1R antagonists. On the other hand, adding another time point between T0 and T1 would provide valuable information on the acute effects of thromboembolism and the effects of these drugs.
In summary, our study demonstrated that mPAP increases significantly in APTE and that the selective ET-1R antagonist BQ123 significantly decreases mPAP. The mechanism of its effect may be related to the direct antagonistic effect on ET-1R, with decreased production of ET-1 and inhibition of local inflammatory responses. These results obtained in a canine model of APTE indicate that selective ET-1R antagonists may be beneficial to the treatment of APTE, particularly if they are used in combination with a thrombolytic drug.
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