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Macrophage-derived LTB4 Contributes to Pulmonary Hypertension

Mark Nicolls


X Jiang

Rasa Tamosiuniene


Yon Sung


G Dhillon

L Gera

Norbert Voelkel


W Tian

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Conference: 2012 International PHA Conference and Scientific Sessions

Release Date: 06.22.2012

Presentation Type: Abstracts

BACKGROUND: A prominent pathological feature of PAH is periarteriolar accumulations of macrophages. Because PAH is associated with chronic inflammation and because eicosanoids are markers and mediators of lung damage, several studies previously examined the role of 5-lipoxygenase (5-LO) and its lipid products in the pathogenesis of PAH. Enzymatic activity of 5-LO results in the generation of leukotriene B4 (LTB4) and the cysteinyl leukotrienes (LTC4, LTD4, and LTE4). While the cysteinyl leukotrienes have been well studied in PAH, LTB4 has not received attention, perhaps, because it is primarily considered for its role as a leukocyte attractant. Similarly, prior studies have not elucidated if and how leukotrienes might specifically injure blood vessels in PAH.

METHODS: Both human and rodent PAH histology were analyzed. Athymic (T-cell deficient) rats were treated with the vascular endothelial growth factor receptor (VEGFR) inhibitor, SU5416 and analyzed at multiple time points. Ex vivo co-culturing experiments utilized macrophages from PAH lungs as well as transfected macrophages that specifically mimic 5-LO phosphorylation states. Results from the ex vivo studies directed subsequent intervention studies.

RESULTS: We showed both in clinical PAH tissue and in a rodent model of severe PAH, intense expression of 5-LO in perivascular macrophages mostly in a serine 271 phosphorylated form (p5-LO) resulting from p38 activation. Increased p5-LO, led to macrophage secretion of LTB4 via LTA4 hydrolase (LTA4H). High levels of LTB4 were detected in the bronchoalveolar lavage fluid, whereas LTC4 was unchanged. In transwell macrophage/endothelial cell co-culture experiments, we showed that LTB4, rather than LTC4, induced pulmonary artery endothelial cell apoptosis through inhibition of the endothelial sphingosine kinase 1 (Sphk1)-endothelial nitric oxide synthase (eNOS) pathway. Exogenous S1P added to transwell cultures rescued cells from LTB4- mediated apoptosis. We next demonstrated that inhibition of LTA4 hydrolase reversed fulminant PAH in the animal model by reducing LTB4, preventing endothelial cell apoptosis and restoring the Sphk1- eNOS pathway. After success with systemic therapy, an inhaled formulation of the LTB4 antagonist was created and was highly effective when initiated after disease was already advanced (when given three times per week). Histological analyses were consistent with occluded arterioles being recanalized after the initiation of therapy. Animals not receiving this therapy, died within two weeks whereas treated animals survived with normalized hemodynamics.

CONCLUSIONS: This is the first demonstration that lung macrophages observed in PAH directly induce pulmonary artery cell apoptosis and endothelial death is attributable to macrophage LTB4. LTB4-mediated pulmonary artery endothelial cell apoptosis was induced through inhibition of protective endothelial Sphk1-eNOS signaling. These results led to the rational design of a therapy which uniquely targeted LTB4 concentrated in pulmonary macrophages. Blocking LTB4-mediated inflammation is a promising new approach for treating PAH associated with immune dysregulation.