In various pathological conditions, especially during inflammation and oxidative stress, circulating and cell membrane phospholipids undergo oxidation to form a diverse group of oxidized phospholipids (OxPLs). These bioactive OxPLs possess profound biological activities and exert both beneficial and harmful effects on human body governed by their biochemical and biophysical properties. It has been long recognized that OxPLs accumulate in atherosclerotic lesions (1–3), and later studies have shown OxPLs elevation in other cardiopulmonary disorders driven by increased inflammatory responses [Reviewed in (4)]. However, emerging evidence suggests that OxPLs not only induce and propagate inflammatory response but may also contribute to the host response, resolution of inflammation and protection of vascular endothelial barrier properties (5). Endothelial cell (EC) lining of the vascular lumen forms a barrier that controls the passage of fluids, macromolecules, and immune cells between the blood and underlying tissue. The disruption of this selective and semi-permeable barrier results in uncontrolled passage of harmful substances leading to the development of pulmonary edema and acute lung injury (ALI) observed in many disorders: sepsis, severe infection, trauma, toxin inhalation, etc., and culminating in acute respiratory distress syndrome (ARDS) (6, 7). OxPLs play a dual role in regulating endothelial function: some species of OxPLs have been involved in enhancing endothelial barrier integrity while others increased endothelial permeability. The wide structural heterogeneity of OxPLs has been suggested to be responsible for their contrasting biological activities (8, 9). For instance, full length oxidation products of a major membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC, focus of this review) possess potent endothelial barrier protective and anti-inflammatory properties (10). On the other hand, truncated or fragmented products of PAPC oxidation induce acute endothelial barrier disruption and inflammation (11). Multiple signaling pathways including receptor-mediated and cytoskeletal reorganization have been implicated in OxPAPC-induced upregulation of endothelial function which will be discussed in detail in the following sections.

A diverse spectrum of OxPLs are generated from the oxidation of phospholipids that contain polyunsaturated fatty acids (PUFA) at their sn-2 position. PUFAs are highly prone to oxidative modifications by a group of specific enzymes such as cyclooxygenases (COX) and lipoxygenases (LOX) or reactive oxygen species (ROS)-mediated non-enzymatic lipid oxidation (9, 12, 13). Briefly, fatty acids such as arachidonic acid (AA) and linoleic acid (LA) are released from membrane phospholipids by phospholipase A2 (PLA2). In turn, COX-mediated oxidation of AA produces prostaglandins (PGs) and thromboxanes whereas LOX-catalyzed metabolic pathways yield leukotrienes, lipoxins, resolvins, protectins and eoxins. Multiple studies have shown the modulation of endothelial function by PGs but it is out of the scope of this manuscript. Briefly, PGI2, PGE2 and PGA2 exhibited potent, although transient barrier protective activities in pulmonary endothelium in comparison to OxPAPC effects (14, 15). These PGs enhanced endothelial barrier by stimulating cAMP production leading to PKA-dependent activation of Rac and PKA-independent activation of Epac/Rap1/Rac1 signaling cascade (14). PGE2, PGI2, and PGA2 also protect against thrombin-induced hyperpermeability in vitro and LPS-induced lung injury in vivo (15, 16). Similarly, PGD2 has been shown to enhance endothelial barrier function and protect against ALI (17–19). Furthermore, stable analogs of PGs such as beraprost and iloprost possess protective and anti-inflammatory activities in pulmonary endothelium (20–23). Both COX and LOX are involved in the generation of hydroxyeicosatetraenoic acids (HETEs) from AA oxidation and hydroxyoctadecadienoic acids (HODEs) from LA oxidation. Free radicals-mediated non-enzymatic oxidation of phospholipids produces a heterogenous mixture of bioactive OxPLs species through the classical pathway of lipid peroxidation characterized by initiation, propagation, and termination steps (24).

PAPC, a major membrane phospholipid, undergoes oxidation resulting in generation of wide varieties of full length as well as fragmented oxidized products. The full-length OxPAPC products contain same number of carbon atoms in oxidized arachidonic fatty acid chain as in their precursor. Examples of such products include 1-palmitoyl-2-(5, 6-epoxyisoprstane E2)-sn-glycero-3-phosphatidyl choline (PEIPC) and 1-palmitoyl-2-(5,6 epoxycyclopentenone) sn -glycero-3-phsphocholine (5,6-PECPC), among others. Conversely, fragmented OxPAPC products are oxidatively truncated at the sn-2 position. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-phosphatidylchonine (POVPC), 1-palmitoyl-2glutaroyl sn-glycero-phosphocholine (PGPC), 1-(palmitoyl)-2-(5-keto-6-octene-dioyl)phosphatidylcholine (KOdiA-PC) represent examples of such products (25). Besides phosphatidylcholine, other phospholipids containing different polar groups such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylethanolamine (PAPE) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylserine (PAPS) are also subjected to oxidative modifications and exhibit similar effects on endothelial barrier regulation and inflammation (15). Furthermore, lipid peroxidation also results in the production of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) (24).

A continuous monolayer of EC covers vascular lumen and provides a highly selective semi-permeable barrier between the circulation and underlying tissues. EC barrier controls the passage of fluids, solutes, and cells across the vascular endothelium. Various injurious stimuli disrupt the endothelial barrier integrity leading to an increased endothelial permeability for macromolecules and immune cells that is a hallmark of numerous disorders such as pulmonary edema, ARDS, sepsis, and other pathologies (26–29). Bioactive lipid mediators may have both, positive and negative impact on endothelial barrier function ( Figure 1 ). The principal reason behind the contrasting effects of various species of OxPLs is best explained by structure-function analysis. It appears that different molecular species present in OxPAPC govern its function on endothelial barrier. Precisely, full-length OxPLs species such as PEIPC and PECPC mediate barrier protective and barrier-enhancing effects of OxPAPC whereas sn-2-fragmented OxPLs such as PGPC and POVPC induce endothelial permeability (11, 30). Likewise, polar head groups present in OxPAPC also modulate its barrier function. OxPLs with negatively or positively charged polar head groups such as oxidized phosphocholine and phosphoserine exerted potent and sustained barrier-protective effects (31). But oxidized glycerophosphate lacking polar head group had only transient EC barrier protective effects (31).

A role of OxPLs in various cardiopulmonary disorders including ALI (32), ARDS (33), pulmonary hypertension (34), asthma (35), and cystic fibrosis (36) has been suggested by many studies showing the presence of elevated levels of lipid peroxidation products. Furthermore, OxPLs have a direct impact on EC function, as evidenced by OxPLs-driven effects in chronic vascular inflammation associated with atherosclerosis and manifested by enhanced adhesion of monocytes to EC, augmented expression of several inflammatory genes and secretion of inflammatory cytokines and chemokines such as interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) (2, 8, 37). OxPLs are also shown to cause pro-thrombotic phenotype of EC (38, 39) and act as potent oxidative stress inducers in EC (40, 41).

In contrast to these pathological roles of OxPLs, multiple studies have demonstrated potent anti-inflammatory and endothelial barrier protective functions of OxPLs in host defense against bacterial pathogens. Initial studies demonstrated a protective role of OxPAPC against endothelial dysfunction caused by bacterial wall lipopolysaccharide (LPS) in cultured EC as well as in murine models of ALI (30, 38, 39). Later studies have reported the involvement of OxPAPC in rescue and repair of endothelial function disrupted by various injurious factors: live and killed bacteria, components of bacterial wall, edemagenic agonists (thrombin), inflammatory cytokines (TNFα, IL-6), and pathologic mechanical forces: high amplitude cyclic stretch, disturbed flow [Reviewed in (42)]. Several signaling pathways mediating beneficial effects of OxPLs have been described in endothelium (14). Anti-inflammatory roles of OxPAPC have been summarized in our recent reviews (10, 42). This review will solely focus on OxPAPC-mediated endothelial barrier function.

Endothelial barrier is a dynamic structure that constantly undergoes remodeling in response to mechanical forces and various agonists that positively or negatively regulate barrier function. Altered expression of cell junction proteins, the assembly-disassembly dynamics of adherens junction (AJ - VE-cadherin, α, β, γ, and p120-catenins, nectin) and tight junction (TJ - claudin-5, ZO-1, 2, 3, afadin) protein complexes and reorganization of endothelial cytoskeleton in response to chemical and mechanical stimulation are the major determinants of endothelial barrier integrity. While barrier-disruptive insults (edemagenic agonists or high magnitude cyclic stretch) stimulate stress fiber formation, actomyosin contraction, disassembly of cell junction complexes leading to cell retraction and formation of inter-cellular gaps, barrier-enhancing agonists (OxPAPC, hepatocyte growth factor, sphingosine 1-phosphate) or physiologic mechanical forces (laminar flow, low magnitude cyclic stretch) stimulate enhancement of cortical cytoskeleton, assembly and cooperation of AJ and TJ protein complexes (43, 44). Remodeling of cell junctions and actomyosin cytoskeleton is precisely controlled by signaling protein kinases and small GTPases.

As discussed above, truncated products of phospholipids oxidation possess endothelial barrier disruptive properties. Moreover, even OxPAPC at higher concentration (≥ 50 µg/ml) causes barrier disruption as opposed to its barrier enhancing function at lower concentrations (5-20 µg/ml) (11, 68). These contrasting biological effects of OxPLs on lung endothelium are largely due to the different signaling pathways activated by them. This notion was best exemplified by one of our studies where the same protein vinculin associated with two distinct proteins- with FA protein talin during thrombin-induced barrier disruption and with VE-cadherin during OxPAPC-induced barrier enhancement (63). The selective activation of various signaling pathways depending on the dose and structural variations of OxPLs appears to be the primary determinants of their biological function.

It is now widely accepted that OxPLs play dual roles on regulating the function of vascular endothelium and thus they can be either potentially developed into therapeutics or utilized as targets of therapeutic interventions for various cardiopulmonary diseases that are developed secondary to endothelial dysfunction. In particular, with its proven endothelial barrier-protective and anti-inflammatory activities against a broad spectrum of injurious insults such as bacterial pathogens (76), thrombin (30), LPS (45, 77, 78), and mechanical forces represented by VILI or cyclic stretch (67), OxPAPC holds a strong therapeutic potential against these agonists-induced ALI, ARDS and sepsis. The central role of Rac/Rap1-mediated cytoskeletal reorganization in OxPAPC-induced upregulation of endothelial barrier function makes this molecule attractive therapeutic candidate since the activation of these GTPases act as a common platform for conveying barrier enhancing signals originating from numerous barrier protective agents. The involvement of a number of receptors and kinases in mediating the beneficial actions of OxPAPC on the lung endothelium further provides the opportunity to consider these multiple molecules and associated pathways for the therapeutics.

Some OxPLs products are detected at the site of tissue injury, inflammation and their deleterious effects on vascular endothelium has been well documented (2, 4). More recent studies suggest that these groups of OxPLs can also act as secondary injurious insults and exacerbate endothelial dysfunction. For instance, EC or mice lungs exposed to particulate matter from polluted air resulted in the generation of truncated OxPLs species such as POVPC, PGPC, and lyso-PC that caused acute endothelial barrier disruption (75). Furthermore, when combined with suboptimal dose of particulate matter that does not cause endothelial barrier dysfunction on its own, OxPLs augmented endothelial permeability, indicating their additive role in exacerbation of endothelial function in pre-existing disease conditions. This phenomenon was further evident with the presence of higher basal level of truncated OxPLs in aged mice and corresponding enhanced increase and delayed clearance of these phospholipids in LPS-treated aged mice compared to their younger counterparts (79). Aged mice were more susceptible to TNF-α-induced lung injury and non-toxic dose of POVPC when combined with TNF-α caused similar levels of lung inflammation. These observations suggest that generation of truncated OxPLs and their additive harmful effects with inflammatory agents further exacerbate lung injury/inflammation in aged population who may already have impaired anti-oxidant system. The definite role of truncated OxPLs as secondary injurious agonist was established by the selective removal of these lipids with platelet-activating factor acetyl hydrolase 2 (PAFAH2) which specifically hydrolyzes truncated OxPLs. The overexpression of PAFAH2 in EC rescued particulate matter- or inflammatory cytokines-induced endothelial barrier disruption, while pharmacological inhibition of PAFAH2 worsened lung injury in TNF-α-challenged mice (75, 79). A few other studies have also reported the presence of higher levels of truncated OxPLs in aged mice (80, 81), and increased production of fragmented phosphatidylcholine in human blood plasma as well as higher levels of ester-linked but lower level of ether-linked phosphatidylcholine in aged human individuals (82, 83). The changes in the levels of lipid profile of ester- vs ether-linked with aging has been attributed as a potential risk factor for the development of various diseases in elderly population. Specifically, ether-linked phospholipids are known to possess anti-oxidant activity and decreased serum levels of these PLs is linked to type 2 diabetes and hypertension (84, 85). Likewise, the oxidation of mitochondrial phospholipid cardiolipin has been shown to contribute to EC necrotic death and increased permeability (86), and hyperoxic lung injury (87). The profile of cardiolipin also undergoes changes with ageing as evidenced by the decrease of total cardiolipin content in mitochondria accompanied by an increase in oxidized forms in heart and brain from aged rats (88, 89). All of these findings suggest that altered lipid profile caused by excessive accumulation of bioactive truncated OxPLs due to exaggerated oxidative stress during aging may play a critical role in propagating associated adverse pathologies. On a positive note, targeting such lipid program switch to selectively inhibit the production or remove pre-formed harmful OxPLs products may provide a potential therapeutic avenue.

Although OxPLs oxidized in vitro are increasingly recognized for their anti-inflammatory and lung barrier-protecting activities in vitro and in vivo, they have a number of serious limitations precluding their therapeutic use. PAPC oxidation in vitro yields a complex, structurally diverse mixture of OxPLs. Such natural OxPLs are ineffective for in vivo therapy due to their various shortcomings. First, natural OxPLs with either barrier-protective or barrier-disruptive properties are rapidly degraded by phospholipases A that are very abundant in blood plasma, tissues, and cells. Second, natural OxPL mixtures contain a large proportion of oxidatively fragmented molecular species which demonstrate low anti-inflammatory activity in combination with increased toxicity and unwanted effects such as disruption of lung endothelial barrier. Therefore, engineering and synthesis of novel class of phospholipase-resistant phospholipids that have enhanced stability and better biological activities could lead to the discovery of effective drugs that preserve and protect endothelial function and ultimately can be used in the prevention and treatment of endothelial dysfunction-driven diseases. To overcome these limitations, we designed, synthesized, and experimentally tested biological properties of a phospholipase resistant phospholipid incorporating a prostaglandin I₂ stable analog iloprost (ILO) as a prototype synthetic phospholipid compound with barrier-protective and anti-inflammatory properties (90). Such incorporation of ILO into synthetic phospholipid backbone led to more prolonged EC barrier enhancement and more pronounced anti-inflammatory effect, credited to its ability to cause a prolonged activation of Rap1 and Rac. In our continued efforts to synthesize such optimal phospholipids derivatives, recently an alkyl-amide OxPLs was synthesized that retained endothelial barrier enhancement and anti-inflammatory effects (91). As demonstrated by these examples, chemically modified phospholipase-resistant phospholipids may exhibit simultaneously two types of activities. First, these compounds inhibit signaling induced by TLRs (92). Second, these synthetic compounds enhance vascular endothelial barrier, reverse action of edemagenic mediators and prevent formation of lung edema in vivo (90, 91). Such poly-pharmacological mode of action can make these compounds especially effective for treatment of severe infections leading to the development of lung edema.

The generation of chemically diverse species of OxPLs and corresponding contrasting biological effects requires precise structure-function analysis of these lipid mediators to define their accurate pathophysiological roles. The advancements in liquid chromatography-mass spectrometry (LC-MS) techniques along with the use of sophisticated bioinformatics analysis has now made possible to detect minor modifications in OxPLs structure and its possible impact on biological activities. These advanced analysis and detection techniques combined with omics analysis approaches including lipidomics, metabolomic, transcriptomic, and proteomics will reveal the signaling cascades associated with OxPLs that will assist in identifying potential therapeutic targets for OxPLs-derived diseases. Furthermore, oxidative phospholipidomics analysis may serve as a valuable tool for identification of biomarkers as suggested by a recent study characterizing oxygenated cardiolipins and phosphatidylethanolamines as predictive biomarkers of apoptotic and ferroptotic cell death, respectively (93). Future projects also should consider exploring the role of OxPLs in the pathogenesis of other diseases besides its established role in cardiopulmonary disorders since recent findings suggest their involvement in other diseases including aging (79) and traumatic brain injury (94). The translation of potent beneficial effects of OxPAPC into clinics is the most exciting challenge ahead and other additional molecular targets such as receptors and Rho GTPases that mediate OxPAPC actions on lung endothelium could also be considered for therapeutic targets. Moreover, enzymes such as PAFAH2 represent another category of potential therapeutic candidates to prevent excessive accumulation of deleterious OxPL products.

It is now widely appreciated that the complexity of contrasting biological effects of OxPLs largely relies on their structural heterogeneity with full-length oxidized products exerting beneficial effects and truncated species acting as pathogenic factor in various cardiopulmonary disorders. The studies so far have established that OxPAPC itself or signaling intermediates that mediate its functions on endothelium, barrier-disruptive OxPLs targeting enzymes such as PAFAH2, and structure-based synthetic analogues of barrier-protective OxPLs could be developed into therapeutics against acute endothelial dysfunction associated with injury, infection, or sepsis. The utilization of LC-MS advanced detection techniques in combination with next generation omics analytical tools has enabled to precisely monitor the lipid modifications and possibly use these modifications as biomarkers for various pathologies. Thus, the dual biological nature of OxPLs presents the opportunity to consider both “good” and “bad” oxidized products of circulating or membrane phospholipids for therapeutic interventions.

PK and KB outlined the review. PK drafted the manuscript and KB finalized. All authors contributed to the article and approved the submitted version.

The studies in authors laboratory have been supported by RO1HL076259, R01HL146829, and RO1HL087823 grants from National Heart, Lung, and Blood Institute and RO1GM122940 from National Institute of General Medical Sciences to KB.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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