Endothelial Cell Prostaglandin I₂ and Platelet-Activating Factor Production Are Markedly Attenuated in the Calcium-Independent Phospholipase A₂β Knockout Mouse

Abstract

Damage and activation of lung endothelium can lead to interstitial edema, infiltration of inflammatory cells into the interstitium and airways, and production of inflammatory metabolites, all of which propagate airway inflammation in a variety of diseases. We have previously determined that stimulation of human microvascular endothelial cells from lung (HMVEC-L) results in activation of a calcium-independent phospholipase A 2 (iPLA 2 ), and this leads to arachidonic acid release and production of prostaglandin I 2 (PGI 2 ) and platelet-activating factor (PAF). We stimulated lung endothelial cells isolated from iPLA 2 β-knockout (KO) and wild type (WT) mice with thrombin and tryptase to determine the role of iPLA 2 β in endothelial cell membrane phospholipid hydrolysis. Thrombin or tryptase stimulation of WT lung endothelial cells resulted in increased arachidonic acid release and production of PGI 2 and PAF. Arachidonic acid release and PGI 2 production by stimulated iPLA 2 β-KO endothelial cells were significantly reduced compared to WT. Measured PLA 2 activity and PGI 2 production by iPLA 2 β-KO cells were suppressed by pretreatment with (R)-bromoenol lactone (R-BEL), which is a selective inhibitor of iPLA2γ. In contrast to the increase in PAF production induced by stimulation of WT endothelial cells, none was observed for KO cells, and this suggests that endothelial PAF production is entirely dependent on iPLA 2 β activity. Because inflammatory cell recruitment involves the interaction of endothelial cell PAF with PAF receptors on circulating cells, these data suggest that iPLA 2 β may be a suitable therapeutic target for the treatment of inflammatory lung diseases.Airway inflammation is involved in the pathogenesis of several acute and chronic lung diseases that include asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, emphysema, cystic fibrosis, pneumonia, and interstitial fibrosis. Exposure to injurious stimuli activates a variety of cells, including eosinophils, macrophages, mast cells, fibroblasts, smooth muscle cells, and endothelial cells, and this results in the release of vasoactive mediators, toxic metabolites, and cytokines that are involved in acute and chronic bronchoconstriction (1,2). Lung endothelial injury can result in interstitial edema which contributes to increased morbidity and mortality in pulmonary diseases (3). In addition, neutrophil infiltration facilitated by endothelial cell barrier dysfunction contributes to tissue damage in the acute phase of lung injury (4-6). † This work was supported by United States Public Health Service Grants R37-DK34388, P41-RR00954, P60-DK20579, and P30- Serine proteases such as thrombin and tryptase are released in inflammatory lung diseases. Increased numbers of mast cells are frequently observed in terminal airways, bronchoalveolar lavage fluid, and sputum of asthmatic patients (7). Allergen inhalation activates resident mast cells that release a variety of mediators, including arachidonic acid, PAF,1 histamin...

Molecular Biology of Atherosclerosis

“…Decreased VSMC proliferation after placement of a carotid cast was seen with cPLA 2 deficiency, but the relationship with atherosclerosis is unclear (816). Recent data suggest that a calcium-insensitive PLA 2 (iPLA2␤) may actually be the predominant isoform responsible for PGI 2 synthesis in endothelial cells (1603), but its relation to atherosclerosis is also unclear.…”

Molecular Mechanisms Regulating the Vascular Prostacyclin Pathways and Their Adaptation during Pregnancy and in the Newborn

“…PLA 2 enzymes carry out the first step in eicosanoid synthesis. Depending on their primary structure, cellular localization, and Ca 2ϩ requirement, PLA 2 enzymes are classified into secretory sPLA 2 , cytosolic cPLA 2 , and Ca 2ϩ -independent iPLA 2 (Buczynski et al, 2009;Sharma et al, 2010). The PLA 2 enzymes are further divided into groups (I, II, III, IV, …) and subgroups (A, B, C, …) on the basis of their amino acid sequence.…”

“…Group IV-A cPLA 2 (cPLA 2␣ ) is a key player in eicosanoid production and is constitutively expressed in most cells especially ECs. In addition to cPLA 2 , eicosanoid production also involves sPLA 2 (Wheeler-Jones, 2008;Sharma et al, 2010). Ca 2ϩ -independent iPLA 2 is widely distributed in many tissues and cells and may contribute to overall PLA 2 activity and PGI 2 production in lung ECs (Sharma et al, 2010).…”

“…In addition to cPLA 2 , eicosanoid production also involves sPLA 2 (Wheeler-Jones, 2008;Sharma et al, 2010). Ca 2ϩ -independent iPLA 2 is widely distributed in many tissues and cells and may contribute to overall PLA 2 activity and PGI 2 production in lung ECs (Sharma et al, 2010). Studies in knockout and transgenic mice support a role of PLA 2 in physiological processes, such as host defense and pathological processes such as inflammation and atherosclerosis (Murakami et al, 2010).…”

Analysis of Two Major Intracellular Phospholipases A2 (PLA2) in Mast Cells Reveals Crucial Contribution of Cytosolic PLA2α, Not Ca2+-independent PLA2β, to Lipid Mobilization in Proximal Mast Cells and Distal Fibroblasts

“…A high glucose-induced iPLA 2 ␤ activates RhoA/Rho kinase via 12/15-lipoxygenase metabolites, which contributes to vascular smooth muscle hypercontractility in diabetic animals (43). In lung endothelial cells, thrombin-or tryptase-stimulated synthesis of PGI 2 and platelet-activating factor is attenuated by iPLA 2 ␤ deficiency (83). Even in nonvascular cells, scavenger receptor A-dependent adhesion, a process that requires 12/15-lipoxygenase but not COX products, is absent in Pla2g6 Ϫ/Ϫ macrophages (84).…”