Impact of epoxyeicosatrienoic acids in lung ischemia-reperfusion injury

Abstract

Objective: Epoxyeicosatrienoic acids (EETs) are protective in both myocardial and brain ischemia, variously attributed to activation of K(ATP) channels or blockade of adhesion molecule upregulation. In this study, we tested whether EETs would be protective in lung ischemia-reperfusion injury.

Methods: The filtration coefficient (K(f)), a measure of endothelial permeability, and expression of the adhesion molecules vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) were measured after 45 minutes ischemia and 30 minutes reperfusion in isolated rat lungs.

Results: K(f) increased significantly after ischemia-reperfusion alone vs time controls, an effect dependent upon extracellular Ca(2+) although not on the EET-regulated channel TRPV4. Inhibition of endogenous EET degradation or administration of exogenous 11,12- or 14,-15-EET at reperfusion significantly limited the permeability response to ischemia-reperfusion. The beneficial effect of 11,12-EET was not prevented by blockade of K(ATP) channels nor by blockade of TRPV4. Finally, 11,12-EET-dependent alteration in adhesion molecules expression is unlikely to explain its beneficial effect, since the expression of the adhesion molecules VCAM and ICAM in lung after ischemia-reperfusion was similar to that in controls.

Conclusion: EETs are beneficial in the setting of lung ischemia-reperfusion, when administered at reperfusion. However, further study will be needed to elucidate the mechanism of action.

Figure 1

Dependence of ischemia-reperfusion injury on extracellular Ca²⁺. In rat lung, 45 min ischemia (45′ I) followed by 30 min reperfusion (30′ R) increased the filtration coefficient (K_f) nearly 8-fold (n=15) compared to that in time controls (n=7). Although low Ca ²⁺ perfusate (LoCa) had no impact in time control lungs (n=5), this perfusate did blunt the permeability response to 45 (n=4) or 60 min (n=4) ischemia followed by reperfusion. In contrast, the TRPV antagonist ruthenium red (RR) had no effect. *P<0.05 vs control (one-way ANOVA). Thus, while the permeability response to ischemia-reperfusion in rat lung requires Ca²⁺ entry, the Ca²⁺ channel TRPV4 does not appear to play a role.

Figure 2

Endogenous and exogenous EETs protect against ischemia-reperfusion in rat lung. Results in controls and lungs challenged with 45′ ischemia-30′ reperfusion (IR, from Figure 1) are repeated here for ease of comparison. Inhibition of soluble epoxide hydrolase (AEPU) limited the permeability response to ischemia-reperfusion, supporting a protective role for endogenous EETs. Although 11,12-EET did not have marked impact when given prior to ischemia (Pre I), it was effective given at reperfusion (Pre R). Neither glibenclamide (Glib) nor ruthenium red (RR) prevented the protective effect of 11,12-EET in this model. *P<0.05 vs control (one-way ANOVA).

Figure 3

VCAM and ICAM expression in rat lung. In control lungs, VCAM was expressed predominantly in bronchiolar epithelium and type II alveolar epithelium, with some positive staining across sections of the alveolar septal wall (A). In lungs subjected to ischemia-reperfusion, the pattern of VCAM expression in airways and the alveolar septal wall did not change, though positive staining was occasionally observed in mononuclear cells situated around the septal wall, in perivascular/peribronchiolar interstitial cuffs, and in endothelium of small extra-alveolar vessels (B). In contrast to the pattern of VCAM staining, ICAM expression in controls appeared to be limited to alveolar type I epithelium (D), a pattern which did not change after ischemia-reperfusion (E). Sections of lung exposed to only secondary antibodies showed no positive staining at either the 1:200 (C) or 1:500 dilutions (F). Br, bronchiole; Pa, pulmonary arteriole; *areas enlarged in insets. Additional images for these two groups, as well as representative images from lungs exposed only to ischemia or in time control lungs, are available in Supplemental material (Figure 5 and Figure 6).

Figure 4

VCAM and ICAM volume fractions in the alveolar septal wall. A morphometric approach was used to quantitatively assess volume fractions for adhesion molecule expression in the alveolar septal wall (see text for details). Neither the VCAM nor ICAM volume fraction differed among the four experimental groups: control lungs fixed after isolation, lungs exposed to ischemia alone or ischemia-reperfusion, and time controls (one-way ANOVA).