Trauma and hemorrhagic shock activate molecular association of 5-lipoxygenase and 5-lipoxygenase-Activating protein in lung tissue

Abstract

Background: Post-traumatic lung injury following trauma and hemorrhagic shock (T/HS) is associated with significant morbidity. Leukotriene-induced inflammation has been implicated in the development of post-traumatic lung injury through a mechanism that is only partially understood. Postshock mesenteric lymph returning to the systemic circulation is rich in arachidonic acid, the substrate of 5-lipoxygenase (ALOX5). ALOX5 is the rate-limiting enzyme in leukotriene synthesis and, following T/HS, contributes to the development of lung dysfunction. ALOX5 colocalizes with its cofactor, 5-lipoxygenase-activating protein (ALOX5AP), which is thought to potentiate ALOX5 synthetic activity. We hypothesized that T/HS results in the molecular association and nuclear colocalization of ALOX5 and ALOX5AP, which ultimately increases leukotriene production and potentiates lung injury.

Materials and methods: To examine these molecular interactions, a rat T/HS model was used. Post-T/HS tissue was evaluated for lung injury through both histologic analysis of lung sections and biochemical analysis of bronchoalveolar lavage fluid. Lung tissue was immunostained for ALOX5 and ALOX5AP with association and colocalization evaluated by fluorescence resonance energy transfer. In addition, rats undergoing T/HS were treated with MK-886, a known ALOX5AP inhibitor.

Results: ALOX5 levels increase and ALOX5/ALOX5AP association occurred after T/HS, as evidenced by increases in total tissue fluorescence and fluorescence resonance energy transfer signal intensity, respectively. These findings coincided with increased leukotriene production and with the histological changes characteristic of lung injury. ALOX5/ALOX5AP complex formation, leukotriene production, and lung injury were decreased after inhibition of ALOX5AP with MK-886.

Conclusions: These results suggest that the association of ALOX5/ALOX5AP contributes to leukotriene-induced inflammation and predisposes the T/HS animal to lung injury.

Keywords: ALOX5; Hemorrhagic shock; Leukotrienes; Lipoxygenase; Post-traumatic lung injury; Trauma.

Figure 1. Experimental design

A standardized rodent model of trauma/sham shock (T/SS) and trauma/hemorrhagic shock (T/HS) was utilized. Control animals were immediately sacrificed. After anesthesia, tracheostomy and femoral arterial and venous cannulation was performed. Next, a laparotomy was created to simulate trauma. Hemorrhagic shock was induced to achieve a mean arterial pressure of 30–32 mmHg for 45 minutes. Animals were then resuscitated with crystalloid followed by a period of observation. Vehicle and the ALOX5AP inhibitor MK-886 were administered prior to initiation of shock.

Figure 2. Subcellular localization of FRET signal intensity

A) Pulmonary localization of ALOX5 and ALOX5AP in Trauma and Hemorrhagic Shock-induced lung injury. Quantification and the intracellular location of ALOX5 and ALOX5AP are shown. The cellular structures were partitioned into (A) cytosolic (B) perinuclear and (C) nuclear domains that were mutually exclusive (illustrated in these representative images in green). Nuclei are blue and the cytosolic domains are gray (B and C). B) The molecular association between ALOX5 and ALOX5AP in the cytoplasm increases after T/HS when compared to Control and T/SS (*, p<0.05). FRET intensity significantly decreased after administration of MK-886 when compared T/HS+Vehicle (**, p<0.01). C) The molecular association between ALOX5 and ALOX5AP in the perinuclear domain increases after T/HS when compared to Control and T/SS (*, p<0.01). FRET intensity significantly decreased after administration of MK-886 when compared T/HS+Vehicle (**, p<0.01). D) The molecular association between ALOX5 and ALOX5AP in the nuclear domain increases after T/HS when compared to Control (*, p<0.01) but not T/SS (p>0.05). FRET intensity significantly decreased after administration of MK-886 when compared T/HS+Vehicle (**, p<0.01). ALUFI, arbitrary linear units of fluorescence intensity.

Figure 3. Trauma and Hemorrhagic Shock induce lung injury

A) Representative images of lung sections stained with H&E from control, trauma/sham shock (Trauma), trauma/hemorrhagic shock (T/HS), T/HS + DMSO (T/HS+Vehicle) and T/HS + MK-886 groups are shown. Control and Trauma animals showed little derangement in cellular architecture. In contrast, the T/HS and T/HS+Vehicle groups demonstrate neutrophil infiltration, alveolar thickening, and intra-alveolar cellular debris. The administration of MK-886 decreases neutrophil infiltration, tissue edema, and alveolar debris. B) Alveolar wall thickness is increased by T/HS compared to Control or T/SS alone (* p<0.0001). MK-886 decreased alveolar wall thickness compared to Vehicle (** p<0.0001) C) PMNs are increased in lung tissue (PMNs/high power field) by T/HS compared to Control or T/SS alone (* p<0.0001). MK-886 decreased alveolar wall thickness compared to Vehicle (** p<0.0001)

Figure 4. T/HS increases cysteinyl leukotriene production

Cysteinyl leukotrienes increase sequentially after T/HS when compared to Control and T/SS (*, p < 0.05). MK-886 administration prior to induction of hemorrhage decreases the amount of leukotriene produced when compared to T/HS+Vehicle (**, p < 0.05).

Figure 5. Trauma/hemorrhagic shock (T/HS) increases expression of ALOX5; and administration of MK-886 reduces the increase in ALOX5

Total fluorescence of tagged ALOX5 and ALOX5AP was quantified in all images. Total ALOX5 did not increase after T/SS; however, T/HS resulted in a significant increase in ALOX5 fluorescence over both Control and T/SS (*, p<0.05). Total ALOX5 fluorescence was attenuated after administration of ALOX5AP inhibitor, MK-886 compared to T/HS+Vehicle (**, p < 0.05). The total amount of ALOX5AP increased after T/HS compared to control (*, p <0.01), but not when compared to T/SS (p>0.05). With the administration of MK-886, there was no significant decrease in ALOX5AP fluorescence when compared to T/HS+Vehicle. ALUFI, arbitrary linear units of fluorescence intensity.

Figure 6. Hemorrhagic shock results in ALOX5 and ALOX5AP association and MK-886 attenuates these interactions

Representative images are shown above for Control, trauma/sham shock (Trauma), and trauma with hemorrhagic shock (T/HS) tissues. In all images, nuclei are blue and tissue is gray. In the first column, ALOX5 distribution is labeled red. In the middle column, ALOX5AP is labeled green. The third column demonstrates fluorescence resonance energy transfer (FRET) signal intensity, normalized to the donor channel (FRETn/d), via a pseudo-color intensity scale with increasing intensity of the FRET signal transitioning from green (low) to yellow to red (high). In the T/HS groups (with and without DMSO), FRET signal intensity increases over both control and T/SS. MK-886 administration prior to hemorrhagic shock attenuates this effect and decreases the FRET signal intensity.