Acute inflammation and disruption of vascular integrity are cardinal features of acute lung injury, contributing to the high morbidity and mortality associated with this condition. Using an in vivo
model, we show in this study that a single intravenous injection of OxPAPC significantly attenuates pulmonary inflammation and vascular barrier dysfunction after intratracheal LPS administration. Intravenous OxPAPC significantly reduced tissue and BAL neutrophil counts, BAL levels of IL-6 and IL-1β, and BAL protein concentration after intratracheal administration of aerosolized LPS, with maximal protection at the 1.5- to 3.0-mg/kg dose range. Previous studies showed that administration of a mixture of LPS or a virus-related proinflammatory agent, CpG, and OxPAPC decreased inflammatory cell recruitment and even protected against LPS-mediated lethal shock in various models of acute inflammation (13
). The novel aspect of this study is the use of separate routes of administration for LPS and OxPAPC. To avoid direct competitive inhibition of LPS-mediated proinflammatory signaling by OxPAPC and mimic a more clinical scenario, we used intratracheal administration of aerosolized LPS to induce lung injury and evaluated the ability of concurrent intravenous administration of OxPAPC to attenuate the inflammatory and barrier-disruptive responses.
Oxidative modification of PAPC forms a heterogeneous group of compounds, which can be separated into two major classes: those generated by oxidative fragmentation of sn
-2 fatty acid residues, and those generated by addition of oxygen atoms to the sn
-2 fatty acid. Examples of biologically active “fragmented oxidized phospholipids” are 1-palmitoyl-2-(5-oxovaleroyl)-sn
-glycero-phosphocholine (POVPC), 1-palmitoyl- 2-glutaroyl-sn
-glycero-phosphocholine (PGPC) (17
), and 5-keto- 6-octendioic acid ester of 2-lyso-phosphocholine (KOdiA-PC) (32
). Examples of “oxygenated phospholipids” include 1-palmitoyl- 2-(5,6-epoxyisoprostane E2)-sn
-glycero-3-phosphocholine (5,6-PEIPC) (33
), and esterified epoxycyclopentenones, such as 1-palmitoyl-2-(5,6-epoxycyclopentenone)-sn
-glycero-3-phosphorylcholine (5,6-PECPC) (34
). In addition to these products, rearrangement of endoperoxide intermediates of phospholipid-esterified arachidonic acid during PAPC oxidation results in formation of a third class of esterified γ-ketoaldehydes called isolevuglandins or isoketals (isoketal-PC) (35
), which also exhibit biological activities (36
). The biological activities of oxidized phospholipids belonging to either the fragmented or oxygenated classes sometimes overlap, but are clearly defined by functional groups present in the oxidatively modified fatty acid moiety (see
for review). Examples of the biological effects of oxidized phospholipids include inhibition of LPS-induced E-selectin and vascular cell adhesion molecule expression, blunting the LPS-induced neutrophil adhesion to vascular endothelium, differential regulation of endothelial permeability by oxidized and fragmented PAPC adducts, “platelet activating factor–like” proinflammatory effects, and induction of monocyte adhesion (37
Although OxPAPC was protective in our model over a range of doses, treatment of LPS-challenged animals with high OxPAPC doses revealed a trend to increased levels of protein, cell elements, and cytokines in BAL as compared with LPS-challenged mice treated with lower OxPAPC concentrations. This waning of the protective effect at high OxPAPC doses is consistent with our previous findings (15
), which showed that high OxPAPC concentrations alone caused pulmonary endothelial barrier dysfunction. Although fragmented phospholipids still exhibit potent inhibitory effects on LPS/NF-κB–mediated inflammatory signaling cascade (38
), our previous studies delineated barrier-disruptive effects of specific fragmented phospholipids (POVPC, PGPC, lyso-PC) present in OxPAPC preparation, which appear to dominate over the barrier-protective effects of oxygenated products (PECPC and PEIPC) toward pulmonary vascular endothelium, when OxPAPC was administered at high concentration. Our current study shows that the optimal effects of OxPAPC against LPS-induced lung dysfunction have been achieved in the lower range of concentrations (1.5–3.0 mg/kg).
In our studies, nonoxidized PAPC failed to attenuate an early phase of LPS-induced barrier dysfunction in vitro and LPS-induced increases in BAL protein, cell counts, and IL-6 production in vivo. However, PAPC reduced IL-1β production in vivo and promoted EC barrier recovery at late time points in the in vitro model of LPS-induced endothelial dysfunction. Because PAPC may undergo oxidation in the course of both in vivo and in vitro experiments, we speculate that partial attenuation of LPS-induced effects on some parameters of lung dysfunction is attributed to the partial PAPC oxidation in the course of experiment. Further studies in our lab are underway to monitor oxidation of phospholipids in the biological fluids.
Results from cell culture experiments ( and ) showed that the addition of OxPAPC after LPS treatment abolished LPS-induced barrier disruption in cultured human pulmonary endothelial cells, restoring TER to basal or even supranormal levels. These results complement our published data showing direct barrier enhancement by OxPAPC alone and accelerated barrier restoration by OxPAPC in a model of thrombin-induced endothelial hyperpermeability (15
). That study demonstrated the direct vascular endothelial barrier–enhancing effects of OxPAPC in the absence of inflammatory stimuli, and showed that these effects were mediated by changes in the actin cytoskeleton, focal adhesions, and adherens junctions in a Rac- and Cdc42-dependent fashion (Reference 15
and K. Birukov, unpublished data). Furthermore, the results of our current study strongly suggest a dual nature of OxPAPC protective effects toward lung barrier function: first, via blunting of LPS/TLR4/NF-κB proinflammatory signaling cascade, and second, via direct effects on cytoskeletal remodeling and enhancement of cell–cell interactions (), which are most likely driven by Rac/Cdc42-mediated mechanisms described previously (15
). Taken together, these results suggest that endogenous oxidized phospholipids may act as a negative feedback mechanism, which not only attenuates the acute inflammatory response to endotoxin and gram-negative sepsis associated with activation of TLR4 but also directly reduces the lung vascular leak and neutrophil extravasation. In addition, oxidized phospholipids likely exhibit an even broader range of antiinflammatory effects. Recent studies have demonstrated that OxPAPC may also inhibit inflammatory cytokine response induced by TLR2 and TLR9 ligands (14
The endothelial barrier plays a key role in regulating lung function, both in health and in states of injury, governing vascular permeability and inflammatory cell recruitment to the lung. OxPAPC inhibits LPS-induced E-selectin expression and neutrophil adhesion to endothelial cells in vitro
and induces endothelial cytoskeletal remodeling (11
). However, the proinflammatory effects of oxidized phospholipids in atherogenesis are also well recognized (40
). How do these findings concur with antiinflammatory effects described in this study? OxPAPC alone had no significant effect on neutrophilic inflammation, as measured by tissue and BAL neutrophils, or barrier disruption, as measured by BAL protein, in our in vivo
model. Likewise, OxPAPC alone showed a very modest increase in the IL-6 (14.7 ± 1.93 vs. 8.44 ± 1.62 pg/ml in controls) and IL-1β levels (21.8 ± 5 vs. 4.1 ± 2.1 pg/ml in controls), which was statistically significant by t
test. In comparison, the magnitude of the LPS-induced inflammatory response reflected by cytokine production and neutrophil infiltration was more than two orders of magnitude greater than for OxPAPC alone (, , and ). These results suggest that the minimal acute proinflammatory effects of OxPAPC are negligible when compared with the magnitude of LPS-induced inflammation. Second, OxPAPC-induced chronic vascular inflammation is associated with sustained increases in the local OxPAPC levels associated with accumulation of oxidized lipids in the atherosclerotic plaque. In contrast, intravenous OxPAPC injection in the treatment of LPS-induced lung inflammation is generalized and time-limited, when given concurrent to the inflammatory stimulus. Thus, chronic monocytic inflammation is unlikely to be induced by brief OxPAPC exposure and does not necessarily preclude the potential short-term therapeutic application of oxidized phospholipid compounds in the treatment of acute inflammatory syndromes.
It remains to be elucidated whether OxPAPC is able to traverse the endothelial layer to exert effects directly on alveolar epithelial and inflammatory cells; however, we speculate that intravenously administered OxPAPC exerts its protective effects in our in vivo model by directly enhancing endothelial barrier function, preventing neutrophil recruitment and propagation of the inflammatory cytokine cascades in the lung parenchyma, as well as by directly inhibiting the LPS-induced inflammatory cascades. Although further studies need to be done to identify the specific OxPAPC receptors that mediate its actions on the lung vasculature and potentially the alveolar epithelium, its broad beneficial effects in preventing acute lung injury may have far-reaching clinical implications.
Do oxidized phospholipids have a therapeutic potential? Inflammation appears to be the major problem in a variety of conditions from sepsis and ALI to pancreatitis and burns, even after resolution of the initial insult. In sepsis and ALI, in particular, there has been much interest in the development of therapeutics aimed at attenuating the acute inflammatory response. The recent identification of activated protein C, which exhibits antiinflammatory, antithrombotic, and fibrinolytic activities, represents the first effective antiinflammatory therapy for the treatment of severe sepsis, significantly decreasing morbidity and mortality. However, studies of corticosteroids, well known for their antiinflammatory properties, in the treatment of sepsis and ARDS have shown only modest benefit in human trials despite promising preliminary data from animal studies (41
). Thus, newer treatments aimed specifically at blunting the excessive inflammatory response are being studied. For example, recent studies by Peng and colleagues (44
) showed that, consistent with effects on systemic barrier enhancement, sphingosine 1-phosphate and its pharmacologic analog FTY720 significantly reduced pulmonary/renal vascular leak associated with LPS-induced ALI and inflammation. The efficacy of intravenous OxPAPC in attenuating ALI caused by intratracheal aerosolized LPS suggests a possible therapeutic role for this group of compounds, at least in the acute setting. Moreover, identification of specific oxidized phospholipid species exhibiting potent barrier-protective properties (15
) together with antiinflammatory effects strongly supports clinical significance of oxidized phospholipids as a potential new group of therapeutic agents. Further studies will be needed to determine the specific mechanisms of action of OxPAPC in vivo
, but our current study, coupled with prior work, suggests the possibility of acute administration of exogenous oxidized phospholipids as a novel therapeutic strategy for various conditions leading to ALI and/or sepsis.