Journal of Applied Physiology
Vol. 82, No. 2, pp. 592-598, February 1997
PULMONARY CIRCULATION AND LUNG FLUID BALANCE

Thromboxane A₂ mediates increased pulmonary microvascular permeability after intestinal reperfusion

Richard H. Turnage¹, John L. Lanoue¹, Kevin M. Kadesky¹, Yan Meng¹, and Stuart I. Myers²

¹ Department of Surgery, University of Texas Southwestern Medical School, Dallas 76235-9031; ¹ Dallas Veterans Administration Medical Center, Dallas, Texas 75216; and ² Department of Surgery, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Turnage, Richard H., John L. LaNoue, Kevin M. Kadesky, Yan Meng, and Stuart I. Myers. Thromboxane A₂ mediates increased pulmonary microvascular permeability after intestinal reperfusion. J. Appl. Physiol. 82(2): 592-598, 1997.This study examines the hypothesis that intestinal reperfusion (IR)-induced pulmonary thromboxane A₂ (TxA₂) release increases local microvascular permeability and induces pulmonary vasoconstriction. Sprague-Dawley rats underwent 120 min of intestinal ischemia and 60 min of IR. Sham-operated animals (Sham) served as controls. After IR or Sham, the pulmonary vessels were cannulated, and the lungs were perfused in vitro with Krebs buffer. Microvascular permeability was quantitated by determining the filtration coefficient (K_f), and pulmonary arterial (Ppa), venous (Ppv), and capillary (Ppc) pressures were measured to calculate vascular resistance (Rt). After baseline measurements, imidazole (TxA₂ synthase inhibitor) or SQ-29,548 (TxA₂-receptor antagonist) was added to the perfusate; then K_f, Ppa, Ppv, and Ppc were again measured. The K_f of lungs from IR animals was four times greater than that of Sham (P = 0.001), and Rt was 63% greater in the injured group (P = 0.01). Pc of IR lungs was twice that of controls (5.4 ± 1.0 vs. 2.83 ± 0.3 mmHg, IR vs. Sham, respectively; P < 0.05). Imidazole or SQ-29,548 returned K_f to baseline measurements (P < 0.05) and reduced Rt by 23 and 17%, respectively (P < 0.05). IR-induced increases in Pc were only slightly reduced by 500 µg/ml imidazole (14%; P = 0.05) but unaffected by lower doses of imidazole (5 or 50 µg/ml) or SQ-29,548. These data suggest that IR-induced pulmonary edema is caused by both increased microvascular permeability and increased hydrostatic pressure and that these changes are due, at least in part, to the ongoing release of TxA₂.

capillary filtration coefficient; hydrostatic pressure; pulmonary vascular resistance; pulmonary edema

INTRODUCTION

INTESTINAL REPERFUSION (IR) injury induces pulmonary microvascular dysfunction manifested by fluid and protein extravasation, leukosequestration, and histological and ultrastructural evidence of pulmonary capillary endothelial cell injury (7, 15, 26). More recent studies have related IR-induced pulmonary edema to increased pulmonary artery pressure (Ppa; 24) and enhanced microvascular permeability to fluid (3). A variety of circulating inflammatory mediators have been incriminated in the pathogenesis of this injury, particularly neutrophils and complement (3, 9, 11, 15, 25, 26). Paracrine inflammatory and vasoactive substances released by the lung also appear to be important mediators of pulmonary injury in this model (10, 23, 24).

Thromboxane A₂ (TxA₂) is one such paracrine substance that likely contributes to IR-induced lung injury. Evidence for this includes the observations that pulmonary TxA₂ release is upregulated in this and similar injuries (10, 12, 24); inhibition of TxA₂ synthesis or TxA₂-receptor blockade prevents pulmonary injury in these models (12, 24, 29); inflammatory mediators active in IR [e.g., complement (14) and oxygen-derived free radicals (17)] upregulate pulmonary TxA₂ release; and TxA₂ induces many of the pathophysiological phenomena characteristic of IR-induced lung injury, including increased microvascular permeability, vasoconstriction, and neutrophil infiltration (12, 13).

This study examines the pathophysiological mechanisms by which IR-induced TxA₂ release alters local pulmonary microvascular function in this model. Specifically, this study examines the hypothesis that IR-induced TxA₂ release enhances pulmonary microvascular permeability, as measured by the capillary filtration coefficient (K_f), and induces pulmonary vasoconstriction.

MATERIALS AND METHODS

Animal Model

Quantitation of Pulmonary Microvascular Dysfunction In Vitro

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Ppv and the Calculation of Vascular Resistance

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As described by Allison et al. (2), the pulmonary circulation can be represented as a simple linear model in the isogravimetric state. In this model, Ppa is separated from Ppc by a precapillary resistance (Ra) and Ppc is separated from Ppv by a postcapillary resistance (Rv). Therefore, where Rt is determined in the isogravimetric state, Ra and Rv can be calculated as follows

(3)

(4)

(5)

All resistance calculations (Rt, Ra, and Rv) were normalized for body weight and expressed as mmHg ^· ml^{1 ·} min^{1 ·} 100 g body wt¹.

In these determinations of K_f and vascular resistance, Ppc was measured by using the double occlusion method as described by Allison et al. (2) and Townsley et al. (19). This methodology has been demonstrated to correlate closely with measurements of isogravimetric capillary pressure (Ppc_i; 6, 18) in both normal (19) and acutely injured lungs (2).

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Measurement of Pulmonary TxA₂ Release

Pulmonary TxA₂ release was also quantitated by measuring TxB₂ in perfusate obtained from both the arterial (afferent) and venous (efferent) sides of the isolated perfused lung. This strategy allowed the detection of a TxB₂ gradient across the lung. The concentration of TxB₂ in BAL fluid and in perfusate was expressed as picograms per milliliter.

Statistical Analysis

All experiments were approved by the Committee on the Care and Use of Animals at the University of Texas Southwestern Medical School and Dallas Veterans Administration Medical Center.

RESULTS

In Vitro Measurement of IR-Induced Pulmonary Microvascular Dysfunction

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Table 1. Hemodynamic parameters in lungs of animals sustaining intestinal reperfusion before and 20 min after addition of thromboxane synthetase inhibitor imidazole or thromboxane receptor antagonist SQ-29,548 Condition Pulmonary Capillary Pressure, mmHg Total Vascular Resistance, mmHg · ml1 · min1 · 100 g body wt Precapillary Resistance, mmHg · ml1 · min1 · 100 g body wt Postcapillary Resistance, mmHg · ml1 · min1 · 100 g body wt Baseline Posttreatment Baseline Posttreatment Baseline Posttreatment Baseline Posttreatment Sham 2.83 ± 0.3  0.49 ± 0.06  0.41 ± 0.05  0.07 ± 0.02  IR 5.4 ± 1.0* 0.80 ± 0.07* 0.51 ± 0.06* 0.30 ± 0.04* IR + imidazole, 5 µg/ml 9.6 ± 1  9.1 ± 1.3  0.77 ± 0.08  0.65 ± 0.07  0.46 ± 0.07  0.36 ± 0.06  0.36 ± 0.02  0.34 ± 0.03  IR + imidazole, 50 µg/ml 5.4 ± 1.0  4.2 ± 1.13  0.80 ± 0.07  0.72 ± 0.09  0.5 ± 0.06  0.47 ± 0.07  0.27 ± 0.04  0.19 ± 0.06  IR + imidazole, 500 µg/ml 8.5 ± 1.3  7.3 ± 0.9 0.82 ± 0.1  0.63 ± 0.06 0.47 ± 0.1  0.38 ± 0.06  0.38 ± 0.04  0.25 ± 0.03  IR + SQ-29,548 7.71 ± 0.92  8.42 ± 0.65  0.69 ± 0.06  0.57 ± 0.04 0.46 ± 0.06  0.32 ± 0.03 0.22 ± 0.03  0.24 ± 0.02 Values are means ± SE; n  5 per group. Data in first 2 rows are measurements from lungs of animals sustaining intestinal reperfusion (IR) or sham operation (Sham). Following rows, measurements from lungs of animals sustaining IR before (Baseline) and 20 min after (posttreatment) addition of imidazole or SQ-29,548. * P  0.05 vs. sham, analyzed with 2-tailed, unpaired Student's t-test; P  0.05 vs. baseline measurements, analyzed with 2-tailed, paired Student's t-test.

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Measurement of Pulmonary TxB₂ Release

The concentration of TxB₂ in the perfusate by lungs from IR animals was nearly 50% greater than that of sham-operated controls (87.8 ± 7.2 vs. 59.74 ± 9.0 pg/ml, respectively; P = 0.05). There was no difference in the concentration of TxB₂ in perfusate sampled from the arterial and venous sides of the lungs from the Sham group (46.5 ± 7.3 vs. 59.7 ± 9.0 pg/ml, arterial vs. venous ports, respectively; P = 0.1), whereas the concentration of TxB₂ in perfusate distal to the IR lungs was 33% greater than that obtained from the arterial port (71.5 ± 14 vs. 94.4 ± 14 pg/ml, arterial vs. venous ports, respectively; P = 0.006).

Despite the relatively brief duration of treatment (20 min), imidazole resulted in a significant reduction in TxB₂ concentration within the BAL of animals sustaining IR. The addition of 50 µg/ml of imidazole to the perfusate of lungs isolated from IR animals resulted in a 63% reduction in TxB₂ concentration within the BAL fluid (4,151 ± 925 vs. 1,536.9 ± 221.4 pg/ml, untreated and 50 µg/ml imidazole, respectively; P < 0.05). The addition of 500 µg/ml imidazole resulted in a nearly 70% reduction in TxB₂ concentration within the BAL fluid (4,151 ± 925 vs. 1,331 ± 186.9 pg/ml in untreated and 500 µg/ml imidazole, respectively; P = 0.007). Treatment with SQ-29,548 or 5 µg/ml imidazole had no effect on TxB₂ release into the BAL of IR animals (data not shown).

DISCUSSION

Reperfusion of ischemic intestine induces pulmonary microvascular dysfunction characterized by interstitial edema and ultrastructural evidence of endothelial cell injury (3, 7, 15, 26). The most commonly utilized parameter of IR-induced pulmonary microvascular dysfunction has been the transvascular leakage of protein quantitated by assays utilizing radiolabeled albumin (15) or vital dyes such as Evans blue (23). In the present study, the isolated perfused lung preparation was employed to examine the physiological determinants of pulmonary edema, i.e., altered microvascular permeability and hydrostatic pressure. Determination of the capillary K_f quantitates microvascular permeability to fluid (2-4, 6, 18, 19, 30), whereas measurement of pulmonary Rv and Ppc quantitates changes in intravascular hydrostatic pressure (1, 2, 6, 18, 19).

The K_f of lungs of animals sustaining IR was four times greater than that of sham-operated controls (Fig. 2), a finding consistent with that reported by Carden et al. (3) in a similar model of IR injury. These data suggest that a change in pulmonary microvascular permeability to fluid is one mechanism in the pathogenesis of pulmonary edema in this model. This finding is consistent with the experience of other investigators utilizing analogous models in the rat and other animals (2, 28, 30).

IR injury had a profound effect on pulmonary Rv. Rt was 63% greater in the lungs of animals sustaining IR when compared with controls (P = 0.01; Table 1). This was primarily due to a fourfold greater Rv in the lungs of animals sustaining IR when compared with controls (P = 0.001). In contrast, there was no difference in the Ra between these two groups. The effect of this increase in Rv was a doubling of hydrostatic pressure in the capillaries of injured animals compared with controls, a change that may have exacerbated the movement of fluid from the intravascular space into the pulmonary interstitium (5). These findings are consistent with previously reported experience with this model (24) and other models of acute lung injury (12, 17, 29, 30).

The measurement of Ppc by using the double occlusion technique correlates very well with classical isogravimetric techniques in both normal (19) and injured (2) lungs. Baseline Ppc measurements in the experiments reported in this manuscript ranged from 2.83 ± 0.3 in sham-operated controls to 9.6 ± 1.0 in one group of animals sustaining IR. The measurements in the sham-operated animals appeared particularly low, and more recent experiments in which Ppc was measured in Sham and IR animals yielded values of 4.6 ± 0.5 and 10.8 ± 1.5 for these two groups, respectively (n = 7 per group; P = 0.002). Review of the literature demonstrates significant variability in the measurement of Ppc in normal (or control) animals with values ranging from 4.6 to 7.5 (1, 2, 19, 30). The paired nature of the observations reported in these experiments (i.e., Ppc was measured before and after treatment with either imidazole or SQ-29,548) reduces the impact of this variability on the interpretation of these data.

The inflammatory mediators responsible for this alteration in pulmonary microvascular function have not been definitively defined. Previous in vivo (10) and in vitro (24) studies have demonstrated that pulmonary TxA₂ release is increased during IR. The present study demonstrated a >100% increase in TxB₂ in the BAL of animals sustaining IR (P < 0.01), 50% greater TxB₂ concentration in the perfusate of IR lungs when compared with Sham (P = 0.05), and in paired experiments a 50% increase in TxB₂ in perfusate collected from the efferent side of isolated, perfused IR lungs when compared with that from the afferent side (P = 0.006). The generation of TxA₂ by the lung has been described previously after a variety of inflammatory injuries including pulmonary ischemia-reperfusion (29), aspiration (8), and skeletal muscle reperfusion (12). Thus the endogenous release of TxA₂ by the lung appears to be a common mechanism by which this organ responds to a heterogeneous group of local and remote injuries.

Recent studies in our laboratory have demonstrated that one source of pulmonary TxA₂ generation may be alveolar macrophages. Alveolar macrophages isolated from the BAL fluid from animals sustaining IR generated greater than twice as much TxB₂ as did those from sham-operated controls (preliminary data). Furthermore, this increase in TxA₂ release is due, at least in part, to an increase in the content of the synthetic enzyme thromboxane synthase within the lungs of animals sustaining IR (10).

The role of TxA₂ release in the pathogenesis of IR-induced pulmonary microvascular dysfunction is suggested by experiments in which the TxA₂ synthetase inhibitor imidazole or the TxA₂-receptor antagonist SQ-29,548 was added to the perfusate of the isolated perfused lung. In paired experiments, the addition of 500 µg/ml imidazole reduced the K_f of lungs of injured animals by 40% when compared with baseline measurements (P = 0.03). Treatment with SQ-29,548 had similar effects on the microvascular permeability of lungs from animals sustaining IR. In these experiments, the addition of SQ-29,548 to the perfusate of an IR lung resulted in a 50% reduction in K_f from pretreatment values (P < 0.05). Despite reducing BAL TxB₂ concentration to nearly the same degree as the 500 µg/ml dose (i.e., 63% reduction), the addition of 50 µg/ml of imidazole to the perfusate of the isolated perfused IR lung reduced K_f by 25% when compared with baseline values, a reduction that approached statistical significance (P = 0.08). This possibly represents a type II error because the sample size was only five animals. The implications of these experiments are that TxA₂, at least in part, mediates the increase in microvascular permeability associated with IR and that this increase in permeability is due to an ongoing process that is at least partially reversible.

Potential mechanisms by which TxA₂ alters pulmonary microvascular permeability have been postulated by a variety of investigators. In other injury models, TxA₂ has been shown to directly increase microvascular permeability by altering the endothelial cell cytoskeleton with microfilament disassembly and widening of interendothelial tight junctions (27, 29). In an in vitro perfused lung model of pulmonary ischemia-reperfusion, endogenous pulmonary TxA₂ release was associated with ultrastructural evidence of abnormal endothelial cell tight junctions, changes ameliorated by TxA₂- receptor blockade (29).

The influence of local TxA₂ release on pulmonary Rv and Ppc appears to be less than its influence on microvascular permeability. The addition of imidazole (500 µg/ml) or SQ-29,548 to the perfusate of the isolated perfused lung resulted in a modest reduction in total pulmonary Rv in the lungs of animals sustaining IR (23 and 17% for imidazole and SQ-29,548, respectively; P < 0.05). Treatment with these agents reduced IR-induced increases in Ra by 19% (P = 0.15) and 30% (P = 0.03), respectively, but had no apparent effect on Rv. It is interesting that neither of these agents significantly altered Rv, since it is at this site that TxA₂ exerts its greatest effect of vascular tone. These data suggest that other paracrine mediators are likely to be involved in the regulation of pulmonary vascular tone in this model. A reduction in the constitutive release of nitric oxide or prostacyclin by the pulmonary endothelium would be expected to significantly increase pulmonary Rv. Furthermore, the generation of potent vasoconstrictors, such as endothelin, in response to the systemic inflammatory state would also contribute to the vasoconstriction associated with this and similar injury models.

The complexity of injury-induced pulmonary microvascular dysfunction is suggested by the present study as well as that of other investigators utilizing a variety of injury models. Stephenson et al. (16) found that inhibition of thromboxane synthetase with OKY-046 or TxA₂-receptor blockade with ONO-3708 attenuated but did not prevent phorbol myristate acetate (PMA)-induced pulmonary hypertension in vivo, results that were similar to that reported by Allison et al. (1) in a blood-perfused isolated lung model. In this latter study, PMA-induced changes in pulmonary microvascular permeability were dependent on the presence of cellular components of blood (perhaps platelets or leukocytes), whereas PMA-induced pulmonary vasoconstriction occurred in a cell-free perfusate (1). In this model, inhibition of TxA₂ release prevented PMA-induced increases in K_f and resulted in a 78% reduction in pulmonary Rv (1). Other investigators have related endogenous pulmonary TxA₂ release to increased Rv after pulmonary ischemia-reperfusion injury (29), skeletal muscle reperfusion injury (12), and oxidant exposure (17).

In conclusion, this study suggests that, during IR, pulmonary microvascular permeability and hydrostatic pressure are both significantly increased when compared with controls. Inhibition of TxA₂ synthesis or blockade of TxA₂ receptors reduces IR-induced changes in microvascular permeability with relatively minimal changes in pulmonary Rv or Ppc. The observation that in vitro inhibition of TxA₂ synthesis or TxA₂-receptor blockade reduces K_f suggests that the increase in pulmonary microvascular permeability associated with IR is due to the ongoing release of TxA₂ and not due to an irreversible microvascular injury.

ACKNOWLEDGEMENTS

Portions of this work were presented at the 18th Annual Conference on Shock, Asheville, NC, June 1995 and the Clinical Congress of the American College of Surgeons, New Orleans, LA, October 1995.

FOOTNOTES

Address for reprint requests: R. H. Turnage, Dept. of Surgery, Univ. of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, TX 75235-9031.

Received 9 July 1996; accepted in final form 30 September 1996.

REFERENCES