Advancements in understanding the role of lysophospholipids and their receptors in lung disorders including bronchopulmonary dysplasia

Abstract

Bronchopulmonary dysplasia (BPD) is a devastating chronic neonatal lung disease leading to serious adverse consequences. Nearly 15 million babies are born preterm accounting for more than 1 in 10 births globally. The aetiology of BPD is multifactorial and the survivors suffer lifelong respiratory morbidity. Lysophospholipids (LPL), which include sphingosine-1-phosphate (S1P), and lysophosphatidic acid (LPA) are both naturally occurring bioactive lipids involved in a variety of physiological and pathological processes such as cell survival, death, proliferation, migration, immune responses and vascular development. Altered LPL levels have been observed in a number of lung diseases including BPD, which underscores the importance of these signalling lipids under normal and pathophysiological situations. Due to the paucity of information related to LPLs in BPD, most of the ideas related to BPD and LPL are speculative. This article is intended to promote discussion and generate hypotheses, in addition to the limited review of information related to BPD already established in the literature.

1. Introduction

Annually worldwide, about 15 million infants are born preterm, out of which about 2.4 million babies are born as extreme preterm before 32 weeks of post menstrual age (PMA) [1]. BPD is a condition affecting the lung development in premature neonates treated for respiratory distress syndrome with an incidence of 5–68%, which increases with declining gestational age [2]. Currently, BPD is defined as the requirement of supplemental oxygen at 36 weeks PMA or treatment with supplemental oxygen for more than 28 days after delivery [3]. Mechanical ventilation and supplemental oxygen contribute significantly to the aetiology of BPD along with other factors such as maternal chorioamnionitis, postnatal infections, persistent ductus arteriosus and malnutrition [3]. BPD is characterised by structural anomaly of the lung comprising of alveolar simplification, in addition to sequelae such as wheezing, increased frequency of lung infections and pulmonary hypertension resulting in lifelong morbidity [4][2][5]. With the advancements of technology in neonatal care such as less invasive ventilation, use of prenatal steroids, surfactants and better nutrition, even the extremely premature infants have improved survival, though with sequelae [2]. Currently available treatment modalities are not efficacious in reducing the prevalence of BPD and such children are at increased risk of long term morbidity, including brain development and learning disabilities [2][3]. Hence, it is imperative that research strategies at basic, translational and clinical levels be accelerated to address this major therapeutic challenge. Identification and understanding of signalling pathways that are modulated in BPD pathology are necessary to establish new therapeutic approaches and improve the existing strategies. This review focuses on understanding the role of S1P and LPA as well as its cognate G-protein receptors in the development of BPD.

The functional importance of lipids as signalling molecules, in addition to their role as structural membrane components or a source of stored energy, is better understood these days. The membrane lipids consist primarily of phospholipids, sphingolipids and cholesterol, which, along with membrane proteins, constitute the functioning lipid bilayer membrane [6][7].

Phospholipids, predominantly found in the plasma membrane, are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. These constitute more than half the mass of lipids in most membrane structures. LPL and inositol phospholipids, though present in smaller quantities, are functionally very important and play a crucial role in cell signalling [5][8]. LPLs share modest chemical structures of having a 3-carbon glycerol or a C18 sphingoid backbone, to which is attached a single acyl chain of varied length and saturation. Important among the group are LPA, S1P, lysophosphatidylcholine (LPC), and sphingosylphosphorylcholine (SPC) [9]. LPLs were initially identified both as precursors and metabolites in the de novo biosynthesis of phospholipids. Recently, LPLs have been identified to play a significant role in processes such as organization of membrane-bound signalling complexes, formation of lipid signalling molecules serving as second messengers, modulation of cell growth, proliferation and motility as well as pro-apoptotic and anti-proliferative actions [10][11][12].

In general, for a particular LPL ligand, the LPL receptors share high amino acid similarities. More than one LPL receptor is expressed by many cell types and, upon binding of the ligand, each receptor can couple with multiple types of G proteins to activate a range of downstream effectors that mediate a variety of cellular responses [13]. Studies on LPL receptors are giving a better picture on their role in lipid signalling, thus serving as potential targets for drug discovery. LPL receptors are known to play a major role in the pathobiology of various diseases affecting the nervous system, angiogenesis, cardiovascular development, wound healing, immunity, ovarian cancer and reproduction, and in a several of lung diseases [14][15][16]. S1P and LPA are shown to act through cognate G protein-coupled receptors (GPCRs) in an autocrine or paracrine fashion [5][17]. This review focuses on the biological importance of S1P as well as LPA and their receptors on lung diseases with special emphasis to BPD.

2. Pathophysiology of BPD

To develop novel treatment strategies for BPD, animal models that mimic the clinical pathogenesis of BPD are essential [18][19][20]. The stage of lung development in the neonatal mice at birth is in the late canalicular stage, corresponding to that of a preterm neonate born at 24 to 26weeks of gestation [21]. Severe experimental BPD can be induced in new born rat or mouse pups by exposure to supraphysiological levels of oxygen (>80%) for 8 days [22][23][24]. The impact of lung injury induced by hyperoxia is determined by the cumulative neonatal oxygen exposure [37]. Thus, these animal models that primarily replicate the human disease morphologically have helped us immensely to better understand the clinical pathophysiology of BPD.

The disease is characterised by reduced secondary septation of alveoli resulting in alveolar simplification with reduced area for gas exchange, reduced alveolar numbers, and increased alveolar size accompanied by increased pulmonary resistance [25]. Thickened interstitium, parenchymal fibrosis, oedema, and airway inflammation are also characteristic abnormalities of BPD [26][27]. Abnormal pulmonary vasculature/impaired angiogenesis characterised by loss of small pulmonary arteries and reduced capillary density is seen, and babies show lower lung volumes and decreased forced expiratory flows during the first 2 years of life, with lung function abnormalities often persisting into adolescence and adulthood [2][28][29]. A high incidence of air trapping, parenchymal abnormalities, and emphysema-like structural defects has been reported in majority of BPD survivors [27][30]. The infants with BPD have higher re-hospitalization rates because of asthma, infection, pulmonary hypertension, and other respiratory tract ailments. It is interesting to note that BPD survivors also develop severe asthma, pulmonary hypertension, and emphysema at later stages of life, in addition to neurodevelopmental and cardiovascular disorders [31][28]. The pathophysiological mechanisms of BPD are summarised in Figure 1. In addition to the primary complications of BPD, the lung pathology in survivors may be complicated by an aggravated response towards a second hit. This was proven in animal models where aggravated second hit response led to shorter life span. Neonatal hyperoxia enhanced the inflammatory response in adult mice infected with influenza A virus, as seen with significantly increased number of macrophages, neutrophils, and lymphocytes in the airways of infected mice which had been exposed to hyperoxia compared to controls [32], and also contributed additively to cigarette smoke-induced chronic obstructive pulmonary disease changes in adult mice [33].

Figure 1. A schematic overview of the pathophysiology of bronchopulmonary dysplasia (BPD).

Multiple risk factors lead to lung injury, resulting in apoptosis of distal lung cells, inflammation, extracellular matrix remodeling and altered growth factor signalling. This cripples lung growth and function for life.

3. Sphingolipid metabolism and signalling in BPD

The lung is one of the most widely studied organs contributing to the understanding of the role of sphingolipids in cellular functions. Sphingolipids are important structure-bearing constituents of the cell membrane that also function as regulatory molecules in cell proliferation and cell death, endothelial barrier function, angiogenesis, and immune response [34][35]. Sphingolipids are comprised of a hydrophobic sphingoid long chain base linked by an amide bond to a hydrophilic fatty acid that varies in chain length, degree of hydroxylation, and saturation, thereby creating a large sphingoid family [36]. The important sphingolipids are sphingomyelin (SM), ceramides, sphingosine, and S1P [37][38]. Ceramides act as a precursor for all other sphingolipids, and S1P is generated from ceramide via sphingosine. The dysregulation of the sphingolipids contribute to various pathological processes underlying injury, inflammation, oedema, infections, and cancer [38][39][40]. Ceramides and S1P play an important role in apoptosis, with ceramides stimulating apoptosis and cell cycle arrest, and S1P modulating cell survival and proliferation, especially in sepsis [41][42]. S1P levels are elevated in tracheal aspirates of preterm babies [43] and in the lung tissues of neonatal mice exposed to hyperoxia [5]. Interestingly, S1P plays a detrimental role in conditions such as BPD [5], pulmonary fibrosis [44], pulmonary hypertension [45], and asthma related airway remodelling [46]. Thus, it is the inciting stimulus that determines the role played by S1P in the given disease condition. Only one study has reported the role of sphingomyelin in BPD patients that looked at the PC/sphingomyelin ratio from tracheal aspirates and the compliance of the respiratory system [47]. In another study, S1P was noted to be elevated in the tracheal aspirates of neonatal infants going on to develop BPD [43].

3.1. Sphingolipid metabolism

The major sphingolipid, SM, is synthesized by the condensation of serine and palmitoyl coenzyme A, catalysed by the rate limiting enzyme serine palmitoyl transferase, resulting in 3–keto-dihydro sphingosine. This is reduced to dihydro sphingosine and acylated to dihydroceramide by ceramide synthase and, subsequently desaturated to ceramide. Ceramide is then converted to SM, or hydrolysed to sphingosine through ceramidases, or channelled to glycosphingolipids [48]. Figure. 2 details the metabolic pathways of LPL. While sphingosine kinases, (SPHKs) 1 and 2, phosphorylate sphingosine to S1P, specific S1P phosphatases 1 and 2, and non-specific lipid phosphate phosphatases dephosphorylate S1P back to sphingosine [49]. S1P is also terminally hydrolysed by S1P lyase [50], a pyridoxal phosphate dependent enzyme localised mainly in endoplasmic reticulum, to trans-2-hexadecenal and ethanolamine phosphate in mammalian cells [50]. Ethanolamine phosphate is used for the biosynthesis of ethanolamine phospholipids, and trans–2-hexadecenal is oxidized to trans–2- hexadecenoic acid, which is recycled into glycerolipid or sphingolipid metabolic pathways. The signalling mechanism of S1P is shown in Figure. 3. S1P is primarily stored in platelets and erythrocytes which lack S1P lyase [51]. The plasma concentration of S1P is at 0.2 – 0.8μM range and the serum concentrations are 3–4 times higher [37]. S1P metabolism and signalling get altered in various pathological conditions such as sepsis, BPD, asthma, pulmonary hypertension, and radiation-induced lung injury, suggesting the importance of this pathway in lung diseases.

Figure 2. Pathways of sphingolipid metabolism.

Ceramide is first synthesised from which all other sphingolipids such as Sph, S1P and SM would be synthesised. Sph: sphingosine; S1P: sphingosine-1-phosphate; SM: sphingomyelin.

Figure 3. Sphingosine-1-phosphate (S1P) signalling.

The S1P produced by sphingosine kinase 1 or 2 (SPHK1 /SPHK2) act intracellularly in the cytoplasm or get exported out of the cell by ABC transporters/Spns2 and act extracellularly on S1P receptors to produce various cellular responses. In BPD there is increased SPHK1 and S1P affecting cellular responses leading to altered lung function. S1P: sphingosine-1-phosphate

3.1.1. Sphingosine-1-phosphate receptors

S1P has a diverse range of functions, which are mediated in a receptor dependent fashion through GPCR (S1PR1–5) or receptor independent manner through intracellular targets such as HDACs, TRAF2, and telomerase [52][53]. S1P generated in the cell is transported to the extracellular milieu by ABC transporters [54], and Spns2 [37][55] where it acts in an autocrine or paracrine manner through GPCR on the cell surface.

S1P acts extracellularly through receptor- dependent and intracellularly via receptor independent pathways [56][54], initiating cellular responses like calcium homeostasis, cell growth and survival, motility, cytoskeletal organisation, adherens junction, and tight junction assembly, autophagy, immune regulation, and morphogenesis [54]. Most of the effects of extracellular S1P are mediated via a family of S1P1–5 receptors [57]. All the five S1PRs are expressed in plasma membrane as well as the cytoplasm and nucleus of benign and malignant tissues from multiple human organs [58]. S1P and its receptors have drawn major attention these days as they play important roles in regulating diverse disease pathological conditions such as inflammatory stimuli [59], mitochondrial functions [60], endothelial cell barrier function [61][62][63][64], angiogenesis[65][66], retinal neovascularisation [67], neuroprotection [68], tumour growth [69], and cardiovascular functions [70]. In lung, the role of S1P and its receptors in the pathophysiology of various conditions such as acute lung injury, inflammation, fibrosis, emphysema, airway inflammation, and acute respiratory distress syndrome has been studied [71][72][73][37]. Significantly lower levels of S1P in plasma and lung tissues were reported in a murine model of lipopolysaccharide (LPS) induced lung injury which was ameliorated upon infusion with S1P [74][75]. The decrease in S1P could be due to increased expression of S1P lyase [76] or S1P phosphatases. Taken together, these results suggest a protective role for S1P in LPS-mediated lung injury. Hyperoxia is also known to cause lung injury, however, with a different underlying pathological process. Hyperoxia enhanced S1P levels in lung tissues of neonatal mice as compared to controls exposed to normoxia [5], and Sphk1 knockout mice showed protection against hyperoxic injury in both neonates and adults [55][5], suggesting altered S1P metabolism due to increased SPHK1 activity and/or expression and decreased S1P lyase or S1P phosphatase activities.

3.1.2. Sphingosine 1 phosphate/Sphingosine-1-phosphate receptor (S1P1–5) signalling in BPD

Not much is known on the role played by S1P signalling via its receptors in the pathogenesis of BPD. We showed that neonatal Sphk1^−/− mice compared to Sphk1^+/+ had improved alveolarisation under hyperoxia resisting BPD like morphology [5]. In vitro studies suggested a role for S1P receptor 1 and 2 in p47^phox component of NADPH activation leading to reactive oxygen species (ROS) formation [55]. Differential gene expression study using neonatal Sphk1^−/− mice showed the importance of S1P/SPHK1 signalling in promoting hyperoxia-induced DNA damage, inflammation, apoptosis, and ECM remodelling in neonatal lungs, while suppressing pro-survival cellular responses involved in normal lung development. The study also proposed SPHK1 as a therapeutic target for drug development to combat BPD [77]. The importance of S1P signalling in BPD has been confirmed by showing the deleterious effect of enhanced S1P generation in the hyperoxia-induced mice model. It was noted that partial deletion of S1P lyase (Sgpl1^+/−) in mice accentuated hyperoxia-induced lung injury. Hyperoxia- induced increase in NOX2 and NOX4, leading to enhanced ROS generation, was mediated through S1P [5]. S1P accumulation in human lung microvascular endothelial cells (HLMVECs), upon hyperoxia stimulation and downregulation of S1P transporter spinster homolog 2 (Spns2) or S1P receptors S1P1 and 2, but not S1P3, using specific siRNA, attenuated hyperoxia-induced ROS generation. A novel role for Spns2 and S1P1and 2 in the activation of p47^phox leading to the production of ROS in hyperoxia-mediated lung injury is also of interest in defining the mechanism by which S1P mediates lung inflammation and injury [55]. Sphk1 deficiency decreased hyperoxia-induced accumulation of interkeukin-6 (IL-6) and ROS levels in bronchoalveolar lavage (BAL) fluids and NOX2 and NOX4 protein expression in lung tissues, indicating involvement of S1P in ROS generation through p47^phox. Exogenous S1P stimulated intracellular ROS generation in HLMVEC, whereas Sphk1 siRNA or inhibition of SPHK1 activity attenuated hyperoxia-induced S1P generation. Also, siRNA-knockdown of NOX2 and NOX4 reduced both basal and S1P-induced ROS formation, suggesting an important role for SPHK1-mediated S1P signalling and ROS in the development of hyperoxia-induced neonatal murine lung injury [5]. Thus, an in-depth understanding of the mechanisms of S1P signalling would clarify the therapeutic targets, further facilitating drug development.

3.1.3. Sphingosine-1-phosphate receptors as drug targets

S1PRs are differentially expressed in cells and binding of ligand causes differential regulation of downstream effector molecules resulting in various cellular responses [78]. Although this differential expression and effects of S1PRs underscore the complexity of S1P signalling, they present a valuable approach for targeting S1PRs for drug development [79]. The effects of these agents in different lung diseases have been widely studied [80][54]and are summarised here (Table 1).

Table 1: Effect of various small molecule inhibitors on different lung diseases.

The specificity of each drug to its receptors, and its mechanism of action on in vitro and in vivo models are briefed.

Drug	Specificity	Disease	Model	Mechanism of action
FTY720	S1P1 antagonist (S1P analogue)	Asthma	Antigen-sensitized murine asthma model	Inhibition of Th1- and Th2- mediated airway inflammation, inhibition of T cell, and eosinophil infiltration into bronchial tissue, bronchial hyper responsiveness [73] [122]
		Lung cancer	House dust mite model of allergic lung inflammation in C57BL/6J mice	Attenuates 0RMDL3 expression [123]
			Urethane-induced lung cancer in BALB/c mice	Decreased PCNA, increased caspase expression, and impaired tumor development [124]
FTY720-P	S1P1, and S1P3 agonist	Lung	HLMVEC	Enhanced endothelial barrier function and reversed endothelial barrier dysfunction [59]
SEW2781	S1P receptor agonist	ALI	LPS induced vascular leakage	Reduced vascular leakage and strengthening of endothelial barrier [124]
RP-002, CYM-5442, AAL-R	S1P1 agonist	Influenza	C57BL/6 mice infected with influenza virus	Reduction in cytokine and chemokine production, inhibition of macrophage, and natural killer cell accumulation in lungs [124]
BML-241	S1P1–3 antagonist	Asthma	Antigen challenged mice	Substantiated the role of S1P2 involvement in bronchial smooth muscle contractility [116]
JTE-013	S1P2 antagonist	Asthma	Human bronchial smooth muscle (BSM) cells	Suppression of S1P-induced inhibition of RANTES production [116]
		Pulmonary hypertension (PH)	Mouse model of allergic airway inflammation	Inhibition of S1P-mediated bronchial smooth muscle contraction [125]
		Cystic fibrosis (CF)	Hypoxia-mediated PH model	Prevention of development of hypoxia-mediated PH [126]
			Lung dendritic cells	Reduced expression of MHCII and CD40 [127]
SB-649146	S1P1 antagonist	ALI	LPS lung injury	Reduced barrier function [75]
VPC23019	S1P1 and 3 antagonist	CF	Lung dendritic cells	Reduced expression of MHCII and CD40 [127]

In BPD, the role of S1P signalling via its receptors is not well understood. Experiments using HLMVECs showed that exogenous S1P stimulated intracellular ROS generation, and use of SPHK1 inhibitors, SKI-II and PF543 attenuated hyperoxia-induced S1P generation [5]. S1P signalling is critical in lung diseases, and so is the role of the receptors. The non-specificity of the available drugs to receptors was earlier a problem. A better understanding of the role of S1P receptors in the pathogenesis of BPD, combined with the development of receptor-specific drugs, could serve as a milestone in the path towards ameliorating BPD.

3.1.4. Ceramides in BPD

Ceramides play an important role in apoptosis, emphysema, and lung inflammation and mediate acute lung injury by increasing alveolar permeability and pro-inflammatory cytokine production [81][82][83]. Long chain ceramides (Cer 16:0, Cer 18:0 and Cer 20:0) have anti-proliferative and pro-apoptotic effects, whereas very long chain ceramides (Cer 22:0, Cer 24:0 and Cer 24:1) promote cell proliferation [84]. In new born mice exposed to 80% oxygen for 4 weeks, lung function measurements and morphometry were drastically affected, in addition to transient increase in ceramide levels, which improved upon treatment with D-sphingosine during the recovery phase [85]. A subsequent study investigated whether ceramides are detectable in tracheal aspirates (TAs) of preterm infants and, if so, whether there was a difference between infants with and without BPD. Infants born at ≤ 32 weeks of gestational age in need of mechanical ventilation in the first week of life were included. A pattern with early increase and subsequent decrease in ceramides seemed to predispose for BPD development suggesting the ceramide profiles in TAs to be a new early marker for BPD and also that the concentrations of ceramides might not be influenced by local inflammation in the lungs [86]. Fenretinide, a synthetic retinoic acid derivative, induced ceramide upregulation accompanied by a decrease in vascular endothelial growth factor (VEGF) along with HDAC-2 and Nrf-2 through suppression of hypoxia-inducible factor-1 α causing alveolar septal cell apoptosis leading to pulmonary emphysema in rats [87]. VEGF plays an important role in pulmonary vascular development, and even the deletion of a single VEGF allele during embryogenesis is lethal [88] and decreased postnatal VEGF expression reduces alveolarization [89]. Decreased levels of VEGF were measured in a rodent animal model of BPD [90] and inhibition of VEGF-receptor 1 led to lung cell apoptosis and emphysema [91][92]. These studies suggested that sphingolipids may regulate lung vascular development through VEGF. Ceramides also decreased surfactant protein B (SP-B) formation by reducing DNA binding and transcriptional activity of NKX2–1, an important transcription factor for SP-B gene expression, thereby contributing to decreased SP-B production in a lung already deficient in surfactant [93]. Intra-amniotic exposure to LPS in a sheep model, which induced antenatal chorioamnionitis, significantly increased ceramide concentrations in the foetal lungs of sheep, suggesting the importance of ceramides in the development of BPD and their potential as a target for new therapeutic interventions [94]. Interestingly, ceramides can be increased by increased sphingomyelin metabolism via sphingomyelinases, or from sphingosine by ceramide synthase.

4. Lysophosphatidic acid signalling in BPD

LPA (1-acyl-2-hydroxy-sn-glycero-3-phosphate) is a simple naturally occurring bioactive LPL, present in almost all tissues and biological fluids including plasma (0.14–1.64μM) [95]. An earlier study showed a much higher level of LPL in women compared to men [96]. It is involved in both de novo biosynthesis of membrane phospholipids and in modulation of signalling pathways by serving as a second-messenger. LPA is involved in several cellular effects including growth, proliferation, migration, cytokine synthesis, cell contraction/rounding, retraction, and stress fibre formation [97][98]. The biological effects produced are mediated through the binding of LPA ligand to G-protein coupled LPA1–6 receptors expressed on the surface of a wide variety of cells [99][100]. The effects could be through a range of downstream signalling cascades such as mitogen-activated protein kinase activation, adenylyl cyclase inhibition/activation, phospholipase C activation/Ca2+ mobilization, arachidonic acid release, Akt/ PKB activation, and the activation of small GTPases, Rho, Rac, and Ras [101]. LPA is generated from LPC by autotaxin (ATX) or lysophospholipase D. The identification and characterisation of cloned receptors, and genetic engineering strategies to develop receptor-deficient mice as well as development of LPA receptor agonists/ antagonists helped in identifying the physiological and pathological roles of LPA. Experimental and clinical evidence strongly suggests that LPA, through LPAR signalling, is involved in lung pathology and diseases, including airway repair and remodelling, inflammation [97], and fibrosis [102][103].

4.1. Lysophosphatidic acid metabolism in BPD

A major pathway of LPA synthesis involves conversion of precursor phospholipids to their corresponding LPL. This conversion is catalysed by phosphatidylserine-specific phospholipase A1 or secretory phospholipase A2, in platelets, or by lecithin cholesterol acyltransferase (LCAT) and PLA1 in plasma [104]. The LPLs are then converted to LPA via ATX activity. In another pathway, phosphatidic acid (PA) is first produced from either phospholipids or diacylglycerol through phospholipase D or diacylglycerol kinase. Thereafter, by the actions of either PLA1 or PLA2, PA is converted to LPA [104]. Also, LPA can be generated through acylation of glycerol-3-phosphate by glycerophosphate acyltransferase or the phosphorylation of monoacylglycerol by acylglycerol kinase [105]. LPA generation from membrane phospholipids occurs both intracellularly and extracellularly. It is the intracellular LPA that serves as an important intermediate for the de novo biosynthesis of complex glycerolipids as well as phospholipids [106]. Extracellular LPA mediates its bioactive effects through LPA receptors [107]. LPA degradation is mediated by lipid phosphate phosphatases (LPPs) [108], LPA-acyltransferase (LPAAT), and various phospholipases (e.g., PLA1 or PLA2) [109]. LPA may be converted back to phosphatidic acid by LPAAT, hydrolysed by LPP, or converted by lipases into glycerol-3-phosphate. LPA is also released from activated platelets, which can further induce platelet activation through positive feedback. In general, LPA production and signalling can induce mitogenic and migratory effects on numerous cell types involved in angiogenesis and tissue repair. Increased LPA levels can rapidly and reversibly increase blood brain barrier permeability, suggesting a concentration-dependent mechanism. They have opposing actions, which may be context-dependent [110].

4.1.1. Biological significance of Autotaxin

ATX is one of the well-studied enzymes associated with LPA signalling, which contribute to extracellular production of LPA. ATX-mediated autocrine signalling induces cell motility through LPA mediated signalling. Partial genetic deletion of ATX in mice (Enpp2^−/+ mutants) has contributed significantly to the understanding of this enzyme’s physiological role in vascular and neural development [111][112]. Total ATX deficiency is embryonically lethal and ATX knock out caused death in mice at embryonic day 9.5 with profound vascular defects indicating its role in stabilization of blood vessels [113]. Furthermore, secretory PLA2 activity was increased in BAL fluid after inhaled antigen challenge in asthmatics, indicating its role in the generation of arachidonic acid, which serves as a substrate for the synthesis of leukotrienes, responsible for bronchoconstriction and airway oedema in clinical asthma [114]. Late-phase allergic reactions were also characterised by increased phospholipids and LPL in BAL fluid [115]. Increased concentrations of ATX were detected in both murine and human fibrotic lungs. The genetic deletion of ATX gene from bronchial epithelial cells or macrophages attenuated disease severity, establishing its role in the pathogenesis of idiopathic pulmonary fibrosis [102]. Segmental allergen challenge in humans enhanced ATX expression and LPA levels, and blocking ATX in mice reduced allergen-induced asthmatic responses [116].

Studies related to ATX expression and its role in BPD is limited. Exposure of rat pups to hyperoxia enhanced ATX expression and activity 72 h and 120 h post-hyperoxia exposure [117]; however, the relationship of increased ATX expression/activity to BPD development has not been investigated. Therefore, ATX is a consequential area for therapeutic interventions and its role in BPD is yet to be explored in detail.

4.2. Lysophosphatidic acid receptors in BPD

Lysophosphatidic acid receptor 1 (LPAR1) is the first identified LPA receptor, which is expressed in most organs and tissues [118]. LPAR1 acts through Gα12/13, Gαq/11, and Gαi/o. LPA signalling through its receptor is required for normal lung alveolarisation during development [10]. Defective alveolarisation was observed in LPAR1-deficient mice at 3weeks as compared to the wild type mice. The lungs of knock out mice exhibited decreased alveolar numbers, septal tissue volumes/surface areas, increased volumes of the distal airspaces, and less organized elastic fibres critical to the development of alveolar septa [119][9]. In another study of hyperoxia-induced lung injury in neonatal rats, LPAR1 deficiency reduced pulmonary injury by reducing pulmonary inflammation and fibrosis [120]. However, hyperoxia-induced alveolar simplification was not affected by LPAR1 deficiency. Treatment with Ki16425, a LPA1 and LPA3 receptor antagonist, protected against pulmonary artery hypertension and right ventricular hypertrophy. In addition to LPAR1, the expression of LPAR3 was also increased at 72 and 120h post-hyperoxia with 85% O2. The study also shows the importance of LPAR1 and 3 receptors during development [117]. In a neonatal rat model LPA-LPAR1-dependent signalling was found to have a role in mediating the impact of a ‘second hit’ with LPS in adult mice, exposed to hyperoxia as neonates [121]. LPAR1- deficiency protected against the LPS-induced lung injury. Adult controls with BPD exhibited an exacerbated response toward LPS with an increased expression of pro-inflammatory mRNAs, whereas LPAR1-deficient rats with BPD were less sensitive to this ‘second hit’ with a decreased pulmonary influx of macrophages and neutrophils, IL-6 production, and mRNA expression of IL-6, monocyte chemoattractant protein-1, cytokine-induced neutrophil chemoattractant 1, plasminogen activator inhibitor-1, and tissue factor [121]. Genome-wide gene expression profiling of mouse lung tissue subjected to hyperoxia showed significant upregulation of LPAR-2 and 3 as compared to room air controls whereas there was downregulation of LPAR-4 [77]. The LPA signaling in BPD is depicted in Figure 4.

Figure 4. Lysophosphatidic acid (LPA) signalling in BPD.

The LPA produced extracellularly from LPC through ATX acts via LPAR, the G-protein coupled receptors to activate a series of signalling molecules, causing cellular responses. In BPD, this signalling is altered due to increased LPA and result in various lung pathologies. LPC: Lysophosphatidyl choline; ATX: Autotaxin; LPAR: Lysophosphatidic acid receptor.

Conclusion and future prospects

Various studies have shown the importance of S1P and LPA signalling in maintaining diverse biological processes including survival, apoptosis, proliferation, inflammation and vascular barrier integrity. S1P and LPA affect the disease conditions differently based on the type of receptor involved. Ceramide promotes cell death but affects vascular integrity in a variable manner, which has been shown in a number of lung disease conditions. The major challenge of receptor-mediated interventions is to understand the physiological and pathophysiological roles played by a single S1P or LPA receptor subtype, as well as combinations of receptors and the downstream signalling pathways activated by each of the receptor. With the continued development of receptor-specific mutants as well as receptor-specific agonists and antagonists that have favourable properties, this issue can be resolved to a large extent. Recent animal studies using small molecule inhibitors for lung diseases suggests that SPHKs, S1P and S1PR signalling axis could be potential targets for treating respiratory diseases. However, decreasing S1P levels in circulation or lung tissue could have an adverse impact on developing lung vasculature and its barrier integrity. Targeting S1P lyases by developing agonists/inhibitors to increase or decrease the concentration of sphingosine would also be a potential therapeutic approach for BPD. Developing small molecules that can act on specific isoforms of ceramide synthases would also be of potential therapeutic use. More work on the role and molecular mechanisms of the ATX-LPA receptor axis mediated changes in the lung will shed novel light on the treatment of pulmonary diseases. Pharmaceutical companies are already developing ATX inhibitors for future clinical use. More translational research on the pharmacological interventions to establish the effects by restoring the lipid mediators on BPD is needed. In view of the limited options that are available for treating lung diseases including BPD, the clinical use of lipid inhibitors, which could offer better protection and benefits to the patients need be pursued.

Highlights.

• Tracheal aspirates of neonates going on to develop bronchopulmonary dysplasia showed elevated levels of sphingosine 1 phosphate (S1P) and ceramides.

• Neonatal murine model of BPD showed protection against Sphk1 gene was knocked out.

• In murine model, Autotaxin, Lysophosphatidic acid (LPA) receptors, Sphingosine kinase 1 (SPHK1) and S1P showed an increase in the lungs following hyperoxia.

• LPA and S1P pathway could serve as excellent therapeutic targets in BPD.