Barrier maintenance by S1P during inflammation and sepsis

ABSTRACT

Sphingosine 1-phosphate (S1P) is a multifaceted lipid signaling molecule that activates five specific G protein-coupled S1P receptors. Despite the fact that S1P is known as one of the strongest barrier-enhancing molecules for two decades, no medical application is available yet. The reason for this lack of translation into clinical practice may be the complex regulatory network of S1P signaling, metabolism and transportation.

In this review, we will provide an overview about the physiology and the network of S1P signaling with the focus on endothelial barrier maintenance in inflammation. We briefly describe the physiological role of S1P and the underlying S1P signaling in barrier maintenance, outline differences of S1P signaling and metabolism in inflammatory diseases, discuss potential targets and compounds for medical intervention, and summarize our current knowledge regarding the role of S1P in the maintenance of specialized barriers like the blood-brain barrier and the placenta.

KEYWORDS:

• Endothelial cell
• sphingosine 1-phosphate
• sphingosine kinase
• S1P lyase
• S1P receptor
• apolipoprotein M

1.

S1P in embryonic development and physiology

Blood vessels are lined by a layer of endothelial cells (ECs), which are responsible for the movement of fluid, proteins or circulating cells between the blood and the interstitium. The EC barrier is attached to the basement membrane, and via focal adhesions (FAs), ECs are tethered to the underlying extracellular matrix (ECM).^{1, 2} Adjacent ECs are joined by interendothelial junctions, namely adherens junctions (AJs), tight junctions (TJs) and gap junctions (GJs). Next to the regulation of the paracellular passage of fluid and solutes into the interstitium, cell-cell junctions are also involved in cell-growth or cell-cell interactions. Alterations in expression of cell-cell junctions lead to changes in permeability of the EC barrier.^1,2 Macromolecules greater than 3 nm in size are transported via the transcellular pathway. This includes mainly the transport by vesicles (Figure 1).^1–3 Inflammation and inflammation associated diseases like sepsis are often marked by an increase of vascular permeability. Due to changes in interendothelial junctions, the EC barrier is disrupted and water and proteins infiltrate the extravascular compartment, causing damage to tissue and organs, and breakdown of the mean arterial pressure due to fluid leakage. Despite resuscitation and vasopression, one additional strategy for treatment includes the enhancement of the EC barrier to restrict the movement of fluid or proteins and maintain tissue homeostasis. The sphingolipid sphingosine 1-phosphate (S1P) has the ability to regulate the integrity of the vascular endothelium⁴ and is an interesting candidate to protect the function of the EC barrier.

Figure 1. Schematic picture of the vascular EC barrier. ECs line blood vessels and restrict the movement of water and solutes between the blood and interstitium. Attached to the basement membrane, ECs are tethered to the ECM by FAs. Interendothelial junctions connect neighboring ECs and regulate the paracellular passage of water and solutes. Macromolecules are transported via the transcellular pathway by the formation of vesicles.^1–3 ECM, extracellular matrix; FA, focal adhesion; S1PR, sphingosine 1-phosphate receptor; Spns2, spinster homolog 2

In its function as extra- and intracellular messenger S1P regulates processes relevant for the progression of inflammatory diseases like sepsis. S1P for instance is involved in the maintenance of the vasculature, the inflammatory response and the circulation of lymphocytes.^5–8 Produced intracellularly by sphingosine kinases 1 or 2 (SphK1/2),^9–12 S1P is either exported by spinster homolog 2 (Spns2)^13,14 or major facilitator superfamily transporter 2b (Mfsd2b),¹⁵ cleaved by the S1P degrading enzyme S1P lyase (SGPL1)¹⁶ or dephosphorylated to sphingosine (Sph) by S1P phosphatases.¹⁷ Outside the cells high concentrations of S1P are found in plasma, where it is bound to high-density lipoprotein (HDL) or albumin for transport to its specific G-protein coupled receptors S1P_1-5¹⁸ (Figure 2).

Figure 2. Network of S1P metabolism, distribution and signaling. S1P is produced by sphingosine kinases 1 and 2 (SphK1/2) and irreversibly degraded by the S1P-lyase. The specific S1P transporters spinster homolog 2 (Spns2) and major facilitator superfamily transporter 2b (Mfsd2b) release S1P out of cells, where it is associated with its transporters serum albumin and apolipoprotein M (apoM) containing high-density lipoprotein (HDL). Five specific G protein-coupled S1P receptors (S1P_1-5) can be activated by S1P and transduce signals inside cells via coupling to different trimeric G proteins. Synthesis, degradation, release, transportation and receptor signaling of S1P can be pharmacologically modified. Further details are provided in the text. HGF, hepatocyte growth factor; DOP, 4-deoxypyridoxine; THI, 2-acetyl-4-tetrahydroxybutyl imidazole; Sph, sphingosine; FTY720-P, FTY720-phosphate

The importance of S1P signaling for the integrity of the vascular system was first demonstrated in S1P₁^−/- mice, which showed a major defect in the formation of blood vessels during embryogenesis.¹⁹ Vasculogenesis and angiogenesis are essential for blood vessel formation and maintenance. In vasculogenesis, new blood vessels are formed out of differentiated ECs. In angiogenesis, the new vascular system is stabilized by the differentiation of vascular smooth muscle cells (VSMCs) to cover new vessels.^20–23 S1P₁^−/- mouse embryos died between week 12.5 and 14.5 because of severe bleeding,¹⁹ which occurred due to the incomplete coverage of the developed blood vessels by smooth muscle cells, leading to their instability and death of the embryos.²⁰ Besides S1P₁, additional deletion of S1P₂ and S1P₃ in double and triple knock-out mice increased the severity of the vascular phenotype, indicating cooperative functions of S1P receptors in vascular development.²⁴

Mice lacking S1P in plasma (pS1Pless) displayed vascular leakages and a higher sensitivity to leak-inducing agents compared to wild-type mice. These effects were counteracted either by transfusion of erythrocytes to replenish missing S1P, or by the application of S1P₁ agonists, which indicates the important role of S1P and its receptor S1P₁ to maintain the vascular integrity.^2,6 Important for the generation of S1P are the sphingosine kinases SphK1 and SphK2. Genetic deletion of one of the enzyme isoforms showed no obvious phenotype in mice, whereas the knock-out of both enzymes simultaneously led to embryonic lethality.²⁵ This suggests that both isoforms compensate for each other, and that S1P produced by them is needed for embryonic development. Deletion of both sphingosine kinases in hematopoietic cells and ECs led to increased vascular leakage and impaired survival after anaphylaxis.⁶

2. Barrier maintenance via the S1P-S1PR signaling pathway

Of great interest are the mechanisms behind S1P promoted barrier protection. Under physiological conditions the barrier function is maintained by the balance of S1P receptors on ECs. ECs express predominantly S1P₁ and S1P₃,^26,27 expression of S1P₂ was also reported, and the expression of S1P₅ is restricted to ECs of the blood-brain barrier.^28–30 All receptors are accessible for S1P and enable different signal transduction pathways.³¹

Measurements of transendothelial electrical resistance (TEER) in human and bovine ECs showed the barrier enhancing properties of S1P. Treatment of ECs with up to 1 µM S1P led to increased EC barrier stability and even after barrier disruption by edemagenic agents, S1P was able to stabilize the endothelial monolayer.⁴ Early studies identified S1P signaling via S1P₁ and G_αi-protein as primary source of the regulation of EC barrier function, and an involvement of the family of Rho-GTPases in the S1P-S1PR mediated cytoskeletal rearrangement and barrier enhancement. The maintenance of barrier integrity via the S1P/S1P₁ axis was conducted by protein kinase B (Akt) signaling. Due to the activation of Akt, activity of Rac and Cdc42 was forwarded, leading to the stimulation and formation of TJs in ECs including the redistribution of zona occludens-1 (ZO-1) and claudin-5 as well as the enrichment of platelet-endothelial cell adhesion molecule-1 (PECAM) and junctional adhesion molecule (JAM) at cell contact sides to promote barrier stability.^32,33

Ras-related C3 botulinum toxin substrate 1 guanosine 5ʹ-triphosphatase (Rac-GTPase) was activated and in turn activated p21-associated Ser/Thr kinase (PAK-1) and the LIM kinase. After the phosphorylation of cofilin, actin severing was reduced, leading to lamellipodia formation and thickening of cortical actin.^4,34 Furthermore, Singleton et al. identified the recruitment of S1P₁ to caveolin-enriched microdomains (CEMs) as an essential step to maintain the EC barrier.³⁵ Activation of S1P₁ by S1P in CEMs induced the formation of phosphatidylinositol-3,4,5-triphosphate (PIP3) by the PI3 Kinase, leading to the recruitment of T-lymphoma invasion and metastasis gene 1 (Tiam1) to the CEM. Tiam1 on the one hand modulated the translocation of α-actinin 1 and 4 and, on the other hand, catalyzed the formation of Rac1-GTP, which was an important factor in S1P-S1P₁ mediated barrier enhancement and reduced the barrier permeability by cortical actin cytoskeletal rearrangement.³⁵ The enhancement of EC barriers by oxidized phospholipids like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) in CEMs was mediated via Src/Akt/S1P₁ signaling. OxPAPC facilitated the activation of Src/Fyn, which in turn partially phosphorylated Akt, leading to the phosphorylation and activation of S1P₁. S1P₁ set of signaling cascades via the mammalian target of rapamycin (mTOR) and the phosphoinositide 3-kinase (PI3K) and promoted further phosphorylation of Akt and the cytoskeletal rearrangement and barrier enhancement by Rac1 activation.³⁶

An interesting feature of S1P in the maintenance of EC barrier was the remodeling of FAs. In comparison to thrombin, which caused intercellular gaps and promoted stress fiber formation, S1P showed barrier enhancing properties. Acting via the receptor S1P₁ and the activation of the Rac-GTPase, S1P expedited the rearrangement of FAs by FA Kinase (FAK), paxillin and G protein-coupled receptor kinase-interacting proteins (GITs). Existing FAs were disassembled by S1P, and FAKs and paxillin translocated to the cell periphery, mediated by GITs. Finally, new FAs were formed and linked to the cortical actin ring, stabilizing and protecting the EC barrier.^4,37,38 Additionally, Rac was also able to promote the activation of Src, which resulted in the activation of FAK in the cytosol. On the one hand, this increased the association of FAK with GIT1 during FA assembly and led to the formation of new focal contacts by FAK, paxillin and GIT2. On the other hand, activated FAK, either by Rac or by Src, promoted the release of Arp-3 and the formation of lamellipodia.^37–39 Furthermore, Rac activated c-Abl, a cytoskeletal effector that forwarded the recruitment of the myosin light chain kinase (MLCK) and cortactin to form lamellipodia.^39,40 Interestingly, Akt and Src were associated and maintained a mutual connection to regulate barrier integrity. Gao et al. showed that due to the long-term activation of Akt by the vascular endothelial growth factor (VEGF) or angiopoietin-1 (Ang-1), the activity of Src was modulated by Akt to stabilize TJs and the EC barrier. In opposition to the activation of Akt, its long-term inhibition led to uncontrolled activation of Src and the breakdown of EC barriers.⁴¹ This was further confirmed by a recent study showing that the short-time activation of Src led to reorganization of AJs and transient accumulation of VE-cadherin and, thus, promoted barrier integrity.⁴²

The S1P-S1P₁ axis was not only responsible for the rearrangement of actin in ECs, but also for the expression of surface molecules like PECAM-1 and vascular endothelial (VE)-cadherin, which were important for cell adhesion and cell-cell contacts. Long-term down-regulation of S1P₁ led to diminished expression of PECAM-1 and VE-cadherin on ECs, influencing the cell-cell-interactions to strengthen EC barriers. Additionally, the expression of the adhesion molecule E-selectin, which was part of the immune response after the exposure to cytokines like tumor necrosis factor α (TNFα), was reduced in S1P₁ knock-down cells.⁴³

S1P₁ is the dominant receptor on ECs, and recent studies indicate that under pathophysiological conditions vascular permeability and inflammation were promoted by a shift of receptor expression toward S1P₂. S1P₂ and its counteractive relation to S1P₁ are currently in focus to explain the mechanisms behind disruption of the EC barrier.⁴⁴

The barrier stabilizing and anti-inflammatory effects of S1P₁ are mediated via the G_αi protein, whereas the antagonistic effects of S1P₂ are regulated by G_α12/13. Key players in the S1P₂ mediated signaling cascade interfere at several points with S1P₁ regulated signaling. Sanchez et al. investigated the S1P₂ downstream effectors Ras homolog gene family, member A (RhoA), Rho-associated protein kinase (ROCK) and phosphatase and tensin homolog (PTEN).⁴⁴ Rho and PTEN were able to disrupt AJs, promoted stress fiber formation and endothelial permeability. By inhibiting Rac1 signaling, RhoA prevented AJ assembly, which was further detained by the phosphorylation of VE-cadherin by PTEN (Figure 3).⁴⁴ Under inflammatory conditions, inflammation was also promoted by S1P₂ signaling. Due to the activation of ROCK/nuclear factor kappa B (NFкB) and stress-activated protein kinase (SAPK), pro-inflammatory responses were evoked.⁴⁵ The inhibition of S1P₂ by specific antagonists was able to rescind the adverse effects. Additionally, the simultaneous stimulation of S1P₁ by S1P acted like a feedback mechanism to attenuate the effects of S1P₂ and promoted the maintenance of the EC barrier.^44,45 This hypothesis was further confirmed by Reinhard et al.⁴⁶ Under healthy conditions, S1P bound to its receptors S1P₁ and S1P₂ and activated the distinct G proteins. Furthermore, S1P activated Rac1, followed by S1P₂ mediated activation of RhoA. The latter induced local contractions in cells and suppressed Rac1. But in addition to Rac, S1P₁- G_αi signaling also led to activation of cell division control protein 42 (Cdc42), which abrogated the effects of RhoA and stabilized cell junctions (Figure 3).⁴⁶

Figure 3. Influence of S1P₁ and S1P₂ on endothelial barrier integrity. Signaling via S1P₁ promotes the stability of the endothelial barrier, whereas signaling transduced by S1P₂ enhances vascular permeability. Under physiological conditions, S1P binds equally well both receptors, of which S1P₁ is broader expressed. Subsequently, distinct sets of G proteins are activated. Firstly, Rac is activated, secondly, RhoA, which suppresses Rac. Downstream effectors of RhoA further inhibit S1P₁ signaling, leading to cell contraction and permeability. S1P₁-G_αi signaling also leads to the activation of Cdc42, which reverses the effects of RhoA and promotes barrier stability.^44–46 Akt, protein kinase B; Cdc42, cell division control protein 42; Rac, Ras-related C3 botulinum toxin substrate 1; RhoA, Ras homolog gene family, member A; ROCK, Rho-associated protein kinase; PTEN, phosphatase and tensin homolog; S1P, sphingosine 1-phosphate

3. S1P signaling and metabolism in inflammatory diseases

Triggered by infection or tissue injury, inflammation is an adaptive response of the body to restore homeostasis.⁴⁷ A dysregulated host response, however, may cause inflammatory tissue damage, sepsis, organ dysfunction, and acute respiratory distress syndrome (ARDS). Even in the novel coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), 29% of infected patients with a severe course of the disease develop ARDS.⁴⁸ COVID-19 patients had decreased serum and plasma levels of S1P compared to healthy controls with no significant changes of S1P levels in red blood cells.^49–51 The SphK2 inhibitor Opaganib is currently tested in a clinical trial for the treatment of COVID-19 patients (NCT04467840, Table 1), and the sphingosine analog and immune modulator FTY720 was planned for a clinical study in COVID-19 patients as well, but the trial was withdrawn (NCT04280588, Table 1). A main cause of mortality is a hyperinflammatory response of the body leading to septic shock, which is additionally facilitated by the viral infection of ECs and the following dysfunction of the EC barrier.^52–55 Breakdown of the EC barrier is a common trait in inflammatory diseases. S1P signaling and metabolism could be a valuable target to treat inflammation and to promote barrier stability.

Table 1. Clinical status of current S1P-related drug candidates

Drug		Target	Mechanism	Indication	Trial phase	CT ID
ABC294640	Opaganib	SphK2	Inhibitor	COVID-19	3 (active)	NCT04467840
				Cholangiocarcinoma	2 (active)	NCT03377179
ACT-128800	Ponesimod	S1P1,3.5	Agonist	Plaque psoriasis	2 (completed)	NCT00852670, NCT01208090
				Relapsing multiple sclerosis	Approved
ACT-334441	Cerenimod	S1P1,5	Agonist	Systemic lupus erythematosus	2 (completed)	NCT02472795
APD334	Etrasimod	S1P1,4,5	Agonist	Crohn’s disease	3 (active)	NCT04173273
				Ulcerative Colitis	3 (active)	NCT03945188, NCT03996369, NCT04706793, NCT04176588, NCT03950232
				Eosinophilic Esophagitis	2 (active)	NCT04682639
				Atopic Dermatitis	2 (active)	NCT04162769
				Alopecia Areata	2 (active)	NCT04556734
				Primary Biliary Cholangitis	2 (terminated)	NCT03155932
				Inflammatory Bowel Diseases	2 (terminated)	NCT03139032
				Pyoderma Gangrenosum	2 (terminated)	NCT03072953
BAF312	Siponimod	S1P1,5	Agonist	Relapsing multiple sclerosis	Approved
BMS-986104		S1P1	Agonist	Rheumatoid Arthritis	1 (completed)	NCT02211469
CS-0777		S1P1	Agonist	Multiple sclerosis	1 (completed)	NCT00616733
FTY720	Fingolimod, Gilenya	S1P1,3,4,5	Agonist (after phospho- rylation)	Relapsing-remitting multiple sclerosis	Approved
				Primary progressive multiple sclerosis	3 (terminated)	NCT00731692
				COVID-19	2 (withdrawn)	NCT04280588
				Neuropathy	1 (active)	NCT03943498, NCT03941743
				Cerebral edema	1 (active)	NCT04088630
				Stroke	2 (active)	NCT04675762, NCT04629872
GSK2018682		S1P1,5	Agonist	Relapsing-remitting multiple sclerosis	1 (completed)	NCT01387217, NCT01466322, NCT01431937
KRP-203	Mocravimod	S1P1	Agonist	Subacute cutaneous lupus erythematosus	2 (completed)	NCT01294774
				Ulcerative Colitis	2 (terminated)	NCT01375179
LX3305		SGPL1	Inhibitor	Rheumatoid Arthritis	2 (completed)	NCT00903383
MT-1303, AKP11	Amiselimod	S1P1,4,5	Agonist	Relapsing-remitting multiple sclerosis	2 (completed)	NCT01742052, NCT01890655
				Chronic plaque psoriasis	2 (completed)	NCT01987843
				Crohn’s disease	2 (completed)	NCT02389790, NCT02378688
				Systemic lupus erythematosus	1 (completed)	NCT02307643
				Inflammatory bowel disease	1 (completed)	NCT01666327
				Ulcerative Colitis	2 (active)	NCT04857112
ONO-4641	Ceralifimod	S1P1,5	Agonist	Relapsing-remitting multiple sclerosis	2 (terminated)	NCT01226745
RPC1063	Ozanimod	S1P1,5	Agonist	Relapsing multiple sclerosis	Approved
				Ulcerative Colitis	3 (completed)	NCT03915769, NCT02531126, NCT02435992
				Crohn’s disease	3 (active)	NCT03467958, NCT03464097, NCT03440385, NCT03440372
				COVID-19	2 (active)	NCT04405102

3.1 Sepsis

Sepsis is characterized by systemic inflammation, which affects the whole organ system, often ultimately leading to organ failure and death. In prospective studies, septic patients or patients suffering from ARDS showed decreased concentrations of S1P in serum. The loss of S1P in serum correlated with the severity of the disease.^56,57 Since disruption of the EC barrier and an uncontrollable inflammatory response are hallmarks of sepsis, the involvement of S1P metabolism and signaling in the progression of sepsis and other inflammatory diseases is likely. The Albumin Italian Outcome Sepsis Trial (ALBIOS) investigated the concentrations of S1P in plasma in correlation with the proteoglycan syndecan-1 (SYN-1), which covers ECs and stabilizes the EC barrier. The authors demonstrated an inverse relation between plasma S1P and SYN-1 and observed decreased levels of S1P in plasma of patients with septic shock with concomitantly elevated levels of SYN-1 and VE-cadherin. This correlation supported the hypothesis of an involvement of S1P in EC barrier stabilization since S1P inhibited the degradation of SYN-1 by metalloproteases.⁵⁸

Hemdan et al. examined the effects of pharmacologically modulated S1P signaling in a mouse model of sepsis.⁵⁹ First, administration of 4-deoxypyridoxine (DOP), an inhibitor of the S1P-degrading enzyme S1P-lyase, resulted in elevated levels of S1P. The second approach was the treatment with the sphingosine analog FTY720, which is phosphorylated by cells to the S1P bioequivalent FTY720-phosphate and able to activate all S1P receptors except S1P₂. Mice were pre-treated with either DOP or FTY720, and after sepsis induction both compounds were able to reduce the disruption of the vascular barrier in lung and liver.⁵⁹ Furthermore, those mice showed declined levels of the barrier disruptive molecule VEGF-A in plasma, and the infiltration of immune cells in the liver was significantly reduced, proposing a beneficial effect of S1P modulating agents on the EC barrier by improving its stability, which ultimately led to improved survival.⁵⁹

Next to indirect modulation of S1P signaling via the inhibition of S1P-degradation, the application of agonists and/or antagonists provides the opportunity to target specific signaling pathways. The activation of S1P₁ by its specific agonist SEW2871 and the simultaneous antagonization of S1P₂ by JTE013 showed beneficial effects in a mouse model of sepsis-induced acute kidney injury (AKI).⁶⁰ Pre-treatment of mice with both compounds led to a dose-dependent reduction of microvascular permeability. To depict close-to-clinic conditions, the compounds were administered 6 h after sepsis induction. At that time of sepsis and AKI, vascular permeability was already increased. Interestingly, SEW2871 was able to diminish vascular permeability and to improve kidney function, showing preventive as well as curative effects, whereas JTE013 showed only preventive effects.⁶⁰ But the administration of SEW2871 to amplify signaling via S1P₁ and therefore to promote barrier stability in sepsis is controversial. In a clinically relevant sepsis model, Flemming et al. treated mice 12 h after sepsis induction with SEW2871.⁶¹ Septic mice, but not the healthy control group, suffered from severe cardiac side effects, leading to a higher lethality due to prolonged administration of SEW2871. Additionally, no beneficial effects on barrier stability were detected. Since SEW2871 showed barrier promoting effects at lower dosages and when applied 1 h after sepsis induction, the authors suspected that the effects of SEW2871 depended on the dosage and time point of application. Another possibility could also be functional antagonism of SEW2871 toward S1P₁ after repeated administration, leading to the inhibition of S1P₁ signaling.⁶¹

3.2 ARDS/ALI

ARDS or acute lung injury (ALI) is often experienced by patients suffering from sepsis. ALI develops upon failure of the respiratory system, which is marked by a breakdown of vascular barriers and accumulation of fluid in the lungs.⁶² As a consequence, patients with ALI are mechanically ventilated, causing in turn ventilator-induced lung injury (VILI) due to mechanical stress.^63–65 Mechanical ventilation (MV) stretches endothelial and epithelial cells, induces vascular leakage and triggers an inflammatory response.^64,65

Based on the beneficial effects of S1P on EC barrier integrity in in vitro studies, Peng et al. conducted experiments in an in vivo model of ALI.⁶² Mice suffering from lipopolysaccharide (LPS)-induced sub-lethal lung inflammation were treated intravenously with S1P one hour after LPS challenge. S1P’s barrier modulating properties became apparent in the reduction of vascular leakages, in the prevention of phagocyte infiltration in the lung and in the increase of neutrophils in bronchoalveolar lavage fluid (BALF). Furthermore, intraperitoneal administration of the sphingosine analogue FTY720 reduced systemic inflammation and displayed the same beneficial effects as S1P.⁶² Besides rodents, the positive effects of S1P in inflammatory lung injuries were shown in a canine model of ALI. Here, intravenous S1P was able to maintain oxygenation of the lung and reduced lung edema formation. Additionally, it prevented an increase in lung tissue volume following accumulation of fluid.⁶³ Those studies showed the therapeutic potential of S1P and the sphingosine analogue FTY720 in in vivo models of ALI. But the way of administration also turned out to be important. Intratracheal administration was tested next to intravenous injection in a rodent model of ALI. S1P was either beneficial or harmful in ALI dependent on the dosage. Physiological concentrations of S1P and also of the S1P₁ agonist SEW2871 (< 0.3 mg/kg), applied intravenously or intratracheally, were able to reduce the permeability of lung tissue and inflammation. Higher dosages (0.5 mg/kg) of S1P and SEW2871 turned out to be barrier disruptive when given intratracheally, and S1P induced pulmonary edema and led to increased protein levels. Two mg/kg S1P given intratracheally turned out to be lethal due to pulmonary edema.⁶⁶ Next to the administration of S1P to treat ALI and barrier disruption, Zhao et al. suggested the inhibition of SGPL1 as a therapeutic option. In LPS induced murine ALI, genetic deletion of SGPL1 (S1PL^± mice) and the inhibition of SGPL1 by 2-acetyl-4-tetrahydroxybutyl imidazole (THI) led to increased intracellular S1P levels and attenuated inflammation in the lung. The elevated intracellular S1P levels protected the EC barrier from LPS-induced disruption, which was regulated by signaling via S1P₁ and the activation of Rac1.⁶⁷

3.3. Diabetes and Obesity

Around 463 million people worldwide suffer from diabetes. 90% of those cases are afflicted with type 2 diabetes mellitus (T2D), which is caused mainly by obesity.^68,69 Insulin, a hormone responsible for the maintenance of blood glucose levels, regulation of lipogenesis and protein synthesis, is secreted by β-cells of Langerhans islets in the pancreas. In T2D, the resistance of cells against insulin disturbed normal metabolism and led to endothelial dysfunction, amongst others.^69,70 Even before the onset of diabetes, patients suffering from metabolic syndrome (MetS) showed signs of endothelial dysfunction, heightened activity of RhoA/ROCK and elevated levels of markers of oxidative stress and inflammation.^71,72 In T2D, oxidative stress was responsible for the breakdown of vascular barriers, facilitated by mitochondrial collapse and an increased production of reactive oxygen species (ROS) as well as enhanced inflammatory stress response.^73,74 The subsequent change in glucose metabolism was responsible for altered expression of TJ proteins like occludin, ZO-1 or VE-cadherin. Furthermore, it caused the expression of inflammatory markers like vascular cell adhesion molecule-1 (VCAM-1) and led to the breakdown of EC barriers in brain or retina.^74–76 The current research provides a mixed picture about the role of S1P in obesity and diabetes. The generation of extracellular S1P by SphK1 provided a protective role and promoted glucose tolerance, insulin secretion and the survival of pancreatic β-cells.^69,77–80 SphK2 and SphK2 generated intracellular S1P possess a more complex role. On the one hand, SphK2 promoted insulin resistance, lipotoxicity and β-cell dysfunction and death via the activation of the mitochondrial apoptotic pathway.^69,81,82 On the other hand, SphK2 preserved insulin signaling in the liver via phosphorylation of Sph to S1P and consequently prevented the inhibition of the PI3K pathway by Sph.⁸³ Extracellular S1P occupies a dual role. Depending on its transport in plasma and its signaling pathway, S1P either promoted or prevented insulin resistance. A recent study showed that signaling via S1P_1/3 diminished insulin resistance by the activation of the Akt pathway and the maintenance of mitochondrial functions. Furthermore, this effect was sustained by ApoM, which facilitated the transport of S1P to its receptors, and by FTY720, which promoted the Akt pathway by activating S1P_1/3 signaling cascades.^84–87 However, S1P signaling via S1P₂ impaired insulin signaling by inhibition of Akt phosphorylation, which was abrogated by the S1P₂ antagonist JTE-013.⁸⁷ Given the prevalence of MetS and T2D, strategies to prevent and/or treat those diseases are needed. Despite the controversial role of S1P metabolism and signaling in obesity and diabetes, it may nonetheless be an interesting and important target to affect insulin signaling and to attenuate the adverse effects of obesity on EC barriers.

4. Potential targets and treatment strategies

4.1 Activation of S1P₁ with FTY720 analogs

Knowing the influence of S1P₁ on important functions of ECs, the S1P receptor agonist FTY720-phosphate might show unwanted side effects regarding the stability of cell barriers. FTY720-phosphate was beneficial under VEGF-induced conditions with enhanced vascular permeability,⁸⁸ and several studies evaluated the immunosuppressive effects of FTY720 due to the down-regulation of S1P₁ expression and signaling in lymphocytes, which proved beneficial for the treatment of autoimmune diseases like multiple sclerosis.^89,90

In the central nervous system, FTY720 and its active form FTY720-phosphate displayed beneficial effects. Pre-treatment of brain microvascular ECs (BMECs) with FTY720-phosphate increased the expression of claudin-5 and led to enhanced TEER values. Furthermore, FTY720-phosphate was able to diminish the VCAM-1 expression and resultant barrier disruption caused by sera from MS patients, which pointed to a positive modification of the BBB by FTY720-phosphate to prevent the passage of lymphocytes.⁹¹ Also, the blood-nerve barrier (BNB) was positively affected by FTY720, which was shown after the pre-treatment of human peripheral nerve microvascular ECs (HPnMECs) with FTY720-phosphate. FTY720-phosphate decreased the barrier permeability in a dose-dependent manner. Barrier disruption was prevented by up-regulating claudin-5 expression, which also led to increased TEER values.⁹² In vivo, the effects of FTY720 on the BBB were investigated in 20 patients with MS. It reduced the passage of immunocompetent and autoreactive cells across the BBB by enhancing the functional activity of the BBB, which was demonstrated by decreased levels of circulating TJ proteins and reduced expression of S1P₁.⁹³ A recent study by Wang et al. indicated that the barrier enhancing effects of FTY720 were not caused by an alteration in the expression of AJ or TJ proteins, but rather by the activation of the nonreceptor tyrosine kinase c-Abl and the subsequent change in cytoskeletal structure.⁹⁴

In ECs, therapeutic concentrations of phosphorylated FTY720 compete with S1P for the binding to S1P₁. FTY720 is phosphorylated to FTY720-phosphate by SphK2. Short-term exposure to FTY720 disrupted the assembly of cortical actin and the migration of ECs. Long time exposure to FTY720 led to a reduced expression of S1P₁ and inhibited angiogenesis and wound healing.⁹⁵ Those detrimental effects of long-term exposure to FTY720 were also observed in a mouse model of bleomycin induced lung injury. The sustained activation of S1P₁ not only by FTY720-phosphate, but also by other agonists like AUY954, SEW2871 and even exogenously added S1P inhibited signaling via S1P₁ and led to vascular leakage and exacerbated lung injury.^96,97 Depending on the dosage and time of exposure, S1P₁ agonists applied to support the maintenance of EC barrier might develop long-term opposing effects and act as functional antagonists.⁹⁶ The natural feedback regulatory mechanism of receptor activation, internalization and proteasomal degradation was intensified by functional antagonists, resulting in reduced S1P₁ expression and signaling, which resulted in EC barrier destabilization.^97,98 To activate S1P₁ without mounting the devastating proteolytic cascade, which ultimately leads to enhanced EC barrier permeability, several analogs of FTY720 were screened and tested in vitro and in vivo for their effects on S1P₁ mediated cellular response: The analog (S)-FTY720-phosphonate displayed promising results and showed EC barrier-promoting properties.⁹⁹ Furthermore (S)-FTY720-phosphonate was able to maintain the S1P₁ expression, to reduce vascular leakage and to blunt the inflammatory reaction in bleomycin induced ALI. Activation of S1P₁ without induction of the β-arrestin pathway exerted barrier promoting effects and prevented degradation of S1P₁.⁹⁷ Additional studies revealed barrier enhancing properties of the FTY720 analogs (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F) and beta-glucuronide-FTY720 (FTY-G) in vitro.¹⁰⁰ According to TEER measurements after stimulation with S1P₁ agonists and inhibitors, FTY720-phosphate and its analogs induce S1P₁- G_αi signaling and barrier protection.¹⁰⁰ FTY720 analogs are therefore a promising approach for the treatment of inflammatory diseases like sepsis or ARDS/ALI and are able to prevent unwanted side effects like inhibition of S1P₁ signaling due to prolonged treatment.

4.2 Maintenance of plasma S1P concentrations by SphKs

S1P metabolism involves not only its degrading enzyme S1P-lyase, but also the SphK1/2, which help to maintain the concentration of S1P in blood and tissues by generating S1P. The S1P-lyase reinforces inflammation and tissue damage, whereas the SphK1 acts protective. In mechanical stress-induced VILI, SphK1^−/- mice had elevated levels of cytokines, proteins and cells in BALF and were more susceptible to lung injuries compared to WT mice. In Sgpl^± mice those effects were attenuated.⁶⁴ The importance of the SphK1 in maintaining EC barriers was demonstrated in Sphk1^−/- mice, which showed increased barrier permeability.^101,102 SphK1 was activated by several different stimuli. On the one hand VEGF and TNFα activated SphK1 and induced pro-inflammatory responses, whereas on the other hand Ang-1 stimulated anti-inflammatory pathways.¹⁰² The activation of SphK1 by Ang-1 proved to be important for the maintenance of the EC barrier. It resulted in increased levels of S1P, but inhibition of the S1P receptors 1 to 3 did not diminish the positive effects of Ang-1 on cell permeability, suggesting that Ang-1 mediated its effects via intracellular S1P.¹⁰² Disruption of EC barriers can either be achieved via thrombin induced protease-activated receptor 1 (PAR-1) activation or by LPS. In both cases wild-type mice were able to recover from the impact after two hours, whereas the damage to the endothelium in SphK1^−/- mice was persistent.¹⁰² Furthermore, in vitro studies with small interfering RNA (siRNA) to inhibit the expression of SphK1 demonstrated the importance of SphK1-derived S1P to recover from EC barrier disruption and the involvement of S1P₁ signaling to maintain EC barrier function.¹⁰¹ Normal levels of SphK2 in those mice had no effect, suggesting that the EC barrier was mainly maintained by S1P that was generated by SphK1, which may be due to its location at the plasma membrane.^9,101 Both SphK1 and SphK2 proved to be essential for the inflammatory response. The down-regulation as well as the inhibition of both enzymes caused elevated levels of the pro-inflammatory cytokine IL-6 in the cells.⁶⁷ Studies regarding the knock-out or inhibition of SphK2 are often contradictory since the loss or inhibition of SphK2 may be compensated by increased expression and activity of SphK1.^103,104 A discrepancy between in vivo and in vitro studies regarding the knock-out of SphK2 was also observed by Dimasi et al.¹⁰⁴ In vitro, the inhibition of SphK2 caused a disruption of the EC barrier. On the other hand, SphK2^−/- mice showed elevated levels of S1P in plasma and were able to maintain the EC barrier integrity in vivo. This was in contrast to the SphK1^−/- mice that showed significantly reduced plasma S1P levels and suffered from vascular leakage.¹⁰⁴ Application of SphK2 inhibitors like SLR080811 demonstrated increased levels of S1P in blood, serum and lymphatic fluids. The authors proposed an increased activity of SphK1 to maintain S1P levels.¹⁰⁵

4.3 The role of S1P transporters for barrier protection

Zhao et al. showed that intracellular S1P is involved in the protection of the EC barrier under inflammatory conditions.⁶⁷ Their findings support the hypothesis that ECs produce and transport S1P autonomously via the Spns2 transporter to maintain vascular barrier integrity.⁶⁷ The transport of intracellular S1P out of cells is conducted by two transporters: Spns2 and Mfsd2b. Located on ECs, Spns2 is involved in the establishment of the S1P gradient between tissue and circulation. Spns2-deficient mice showed decreased levels of S1P in plasma and impaired egress of T and B cells from lymphoid organs,^14,106 suggesting an involvement of Spns2 in inflammatory and autoimmune diseases. Furthermore, Spns2 was not only necessary for the transport of S1P, but also for the export of its analog FTY720-phosphate, and thus, it may play an important role in the treatment of diseases with S1P analogues.¹³ Since many inflammatory diseases are associated with the disruption of the EC barrier, the release of S1P and therefore the preservation of the S1P gradient between plasma and tissues by Spns2 may be involved in the regulation of the vascular barrier integrity. The down-regulation of Spns2 resulted in defective blood vessel formation and disrupted EC barriers, confirming the importance of Spns2 in the maintenance of vascular integrity.^26,107–109

The hepatocyte growth factor (HGF) promoted EC barrier function.¹¹⁰ Ephstein et al. demonstrated that the HGF/tyrosine-protein kinase Met (c-Met) and S1P₁ formed a signaling complex, which was involved in the maintenance of vascular barriers.¹¹¹ Due to the activation of c-Met by HGF, the S1P₁ receptor was trans-activated and led to the transduction of important signals for barrier protection.¹¹¹ Subsequent studies were able to connect the maintenance of vascular barrier by HGF with S1P metabolism and signaling. HGF/c-Met stimulated extracellular signal-regulated protein kinase 1/2 (ERK1/2), which promoted the activity of SphK1 and led to elevated levels of S1P.^107,112 Furthermore, the PI3K/Akt pathway was also activated by HGF/c-Met, which stimulated the transporter Spns2 and promoted the secretion of intracellular S1P to stimulate S1P₁ and enhanced barrier stability via the formation of lamellipodia (Figure 4).^107,113 Interestingly, not only HGF, but also apelin, a peptide involved in angiogenesis and the maintenance of cardiac lymphatic vasculature, interfered with S1P metabolism and transport. It stimulated the expression of SphK2 and Spns2 on lymphatic ECs (LECs) and caused elevated levels and secretion of S1P to maintain EC barrier integrity of the lymphatic endothelium.¹⁰⁸

Figure 4. Transactivation of S1P₁ and autonomous production of S1P. Expression of Spns2 on ECs enables the cells to transport S1P outside the cells, where it can act in an autocrine or paracrine manner to activate signaling via S1P₁. Furthermore, HGF binding to c-Met facilitates the production of S1P by stimulating SphK1 and promotes the autonomous sustenance of ECs with S1P by stimulating its transport via Spns2. This leads to the activation of S1P₁ by S1P and the enhancement of barrier stability.^26,107,111 Akt, protein kinase B; c-Met, tyrosine-protein kinase Met; Cer, ceramide; ERK, extracellular signal-regulated protein kinase; HGF, hepatocyte growth factor; PI3K, phosphoinositide 3-kinase; S1P, sphingosine 1-phosphate; Sph, sphingosine; Spns2, spinster homolog 2

Spns2 was particularly expressed in vascular ECs, which led to the hypothesis that ECs might be able to produce barrier stabilizing S1P autonomously. This hypothesis was supported by the findings of Jeya Paul et al.²⁶ Cell culture experiments with primary human umbilical vein ECs (HUVECs) showed that HUVECs depend on S1P₁ and its continuous stimulation by S1P for the integrity of the EC barrier. Moreover, HUVECs expressed the transporter Spns2 to ensure constant export of S1P for the maintenance of extracellular S1P levels and EC barrier integrity. The expression of Spns2 was attenuated under inflammatory conditions, which were mimicked by cytokines and LPS. Inflammatory conditions led to barrier disruption due to declined concentrations of extracellular S1P and resulted in EC barrier disruption. Interestingly the expression of the S1P₁ receptor was not affected by cytokine and LPS treatment, leaving the option to increase barrier stability under inflammatory conditions via the stimulation of S1P₁.²⁶

Mfsd2b is the second S1P transporter helping to maintain S1P concentration in plasma. Mfsd2b is expressed on erythrocytes and platelets.^15,114 Similar to the knock-out of Spns2, the knock-out of Mfsd2b led to significantly reduced levels of S1P in plasma. But unlike the deletion of Spns2, deletion of Mfsd2b did not affect egress and trafficking of lymphocytes. Furthermore, the integrity of blood vessels was not affected by the loss of Mfsd2b, yet the transporter seemed to be involved in protection against anaphylactic shock.¹⁵ This led to the suggestion that not the overall amount of S1P in plasma might be important for the protective effect of S1P in inflammatory diseases, but the environment in which S1P is released by Spns2 or Mfsd2b.¹⁵

4.4 The role of apoM associated S1P for barrier protection

As a hydrophobic molecule, S1P depends on carrier proteins for efficient transport. More than 60% of S1P in plasma was bound to HDL and the remaining S1P was transported by albumin.¹⁸ A recent study investigated the connection between the severity of sepsis and S1P in plasma either bound to albumin or to HDL.¹¹⁵ Interestingly, a shift of S1P from albumin to HDL was observed in patients with systemic inflammation, which was presumably an adaptive response of the body to maintain S1P levels in plasma. Further loss of S1P in septic shock correlated with the loss of HDL and vice versa. Elevated levels of HDL led to increased levels of HDL-S1P, supporting the therapeutic strategy of HDL administration to treat patients with septic shock.¹¹⁵ Compared to albumin-S1P, HDL-S1P was able to preserve the stability of the EC barrier longer, which was facilitated by S1P₁ and the activation of the PI3K-Akt-endothelial nitric oxide synthase (eNOS) pathway.^116,117 Additionally the S1P₁ receptor was recycled after the binding of HDL-S1P and expressed at higher numbers on the cell surface, promoting the barrier enhancing effects of HDL-S1P.^116,117 Further studies revealed the necessity of apolipoprotein M (apoM) as a chaperone for S1P for its binding to HDL.^118,119

The importance of apoM for S1P transport and its function as effector molecule became apparent in experiments with apoM^−/- mice. Those mice had approximately 60% less S1P in plasma, namely S1P bound to HDL, and only albumin-bound S1P was circulating.¹¹⁸ Moreover, apoM^−/- mice displayed decreased EC barrier function in the lung. This result suggested that apoM was necessary for the EC barrier stabilizing function of S1P, and that the vasoprotective effects of apoM-bound S1P were mediated via the activation and internalization of S1P₁.^118,119 Christoffersen et al. even hypothesized that the vascular EC barrier could be protected from injury via an increase of apoM⁺ HDL.¹¹⁸ Also, the anti-inflammatory properties of S1P were reinforced when it was bound to HDL with apoM. This apoM⁺-HDL suppressed inflammation through the activation of S1P₁ via the inhibition of TNFα induced NFкB activation and via the reduction of VCAM-1 to promote barrier stability.^120,121 These results were confirmed in an animal model of LPS induced ALI. Mice lacking apoM had elevated concentrations of pro-inflammatory cytokines in serum as well as more severe lung injury compared to wild-type mice. Treatment with the S1P₁ antagonist W146 under inflammatory conditions interfered with the apoM-S1P mediated improvement of lung injury and pointed to a beneficial role of S1P-S1P₁ signaling in ALI.¹²² Septic rats showed decreased levels of HDL-S1P and apoM in plasma according to the severity of sepsis. The treatment of those animals with HDL-S1P was able to prevent LPS induced ALI via reduction of pulmonary edema, vascular leakage and the expression of inflammatory factors.¹²³ Furthermore, the administration of HDL-S1P and the S1P₁ agonist SEW2871 enhanced the activation of eNOS and protected the EC barrier by supporting the proliferation and migration of ECs. Those beneficial effects were counteracted by the administration of the S1P₁ inhibitor W146, again confirming the important role of the transport of S1P by HDL and the signaling via S1P₁ under inflammatory conditions.¹²³

Treatment strategies to enhance the barrier stability in inflammatory diseases like sepsis are needed. Swendeman et al. engineered a soluble carrier for S1P, apoM-Fc, to transport S1P, activate S1P₁ signaling and to promote EC barrier stability.^119,124 In vitro experiments confirmed the impact of apoM-Fc on barrier stability due to the activation of S1P₁ and S1P₃. In vivo experiments were able to prove that administration of apoM-Fc could improve S1P levels in plasma and activate S1P₁ without causing lymphopenia, which led to the conclusion that S1P₁ in lymphatic tissues is not accessible for apoM-Fc. apoM-Fc-S1P revealed anti-hypotensive effects via S1P₁. After ischemia or reperfusion injuries, neutrophil accumulation at the infarcted site was attenuated, and endothelial homeostasis was maintained. Activation of S1P₁ by apoM-Fc also promoted the integrity of the blood-brain-barrier and suppressed neuronal damage after stroke in mice.¹²⁴

Inflammatory diseases like systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are also characterized by EC barrier dysfunction due to an abnormal immune reaction. The stimulation of S1P₁ with apoM-Fc and agonists showed beneficial effects under inflammatory conditions due to the enhancement of the EC barrier. Stimulation of S1P₁ prevented the degradation of VE-cadherin as well as the phosphorylation of myosin light chain 2 (MLC-2), leading to less cell contraction and the stabilization of adherent junctions.¹²⁵ In comparison to receptor modulators like FTY720-phosphate, the engineered carrier apoM-Fc proved to be beneficial. Depending on the dosage and frequency of administration, FTY720-phosphate could enhance leakage of the EC barrier, probably due to binding to S1P₁ and subsequent internalization and proteasomal degradation of the receptor.^96,98 The carrier apoM-Fc on the other hand transported S1P to its receptor and prevented excessive internalization of S1P₁. Furthermore, the half-life of apoM-Fc was about four days, providing another advantage compared to short-lived agonists like SEW2871. In conclusion, apoM-Fc proved to be a useful tool for enhancing S1P in plasma and to enforce its transport and binding to its receptors, mainly S1P₁, to stabilize vascular EC barrier integrity without causing negative side-effects like lymphopenia.^124,125

5. S1P in specialized endothelial barriers

5.1 Blood-brain barrier

The blood-brain barrier (BBB) is a specialized vascular structure, which seals the central nervous system (CNS) and maintains homeostasis in the brain by controlling the exchange of substances between the blood and CNS. This physiological barrier consists of specialized ECs, which form a monolayer connected via tight junctions to restrict the transport of substances to transcellular pathways^126,127 (Figure 1). Several diseases like amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) or epilepsy are marked by a breakdown of the BBB. Increased permeability of the EC barrier and abnormal flux of molecules and cells of the immune system arise from its degradation. As a consequence, neuroinflammation, dysfunction and degeneration occur.¹²⁷

Van Doorn et al. identified S1P₅ as a key player to maintain the stability of the EC barrier and immunoquiescence.¹²⁸ ECs of the BBB expressed S1P_1-3,5 at different levels, while S1P₁ and S1P₅ dominated. Moreover, the expression of S1P₅ was restricted to the brain.^28–30 As a consequence of a knock-down of S1P₅ in brain ECs (hCMEC/D3), the expression of S1P_1-3 was significantly reduced and the integrity of the EC barrier was compromised. This led to a pro-inflammatory condition of the brain, which was characterized by increased migration of monocytes, adhesion of leukocytes via VCAM-1 and intercellular adhesion molecule-1 (ICAM-1), and production of pro-inflammatory cytokines and chemokines. This state could be attenuated by the activation of S1P₅ via a specific agonist or FTY720-phosphate, emphasizing the important role of S1P₅ to maintain the BBB and modulate inflammatory processes.¹²⁸ S1P₁ was prominently expressed in brain endothelial cells,¹²⁸ and targeting S1P₁ in acute ischemic stroke (AIS) prevented neuroinflammation and breakdown of the BBB.¹²⁹ S1pr1^iECKO mice, which lack S1P₁ in ECs of the brain, suffered from BBB leakiness for small molecules. The permeability of the BBB was regulated by S1P₁, and leakage of the barrier was probably due to a changed localization of tight junction proteins as well as a change in the structure and function of AJ proteins.

The modulation of EC barrier integrity by S1P₁ is a new approach to target diseases of the CNS. By pharmacological modulation of S1P₁, a reversible opening of the BBB could be achieved and provided the possibility to deliver drugs to the CNS.¹³⁰ Inflammatory conditions reduced the integrity of EC barriers, which could be measured by a reduction of TEER. In an in vitro BBB model, Siponimod (BAF-312) was able to activate S1P₁ and S1P₅, and the maintenance of EC barrier integrity by both receptors was enhanced under inflammatory conditions. Depending on the duration of Siponimod treatment, the interaction of both receptors enhanced and promoted barrier stabilization by maintaining ZO-1 and claudin-5 expression.¹³¹

Next to the modulation of S1P signaling, the modulation of S1P metabolism is an interesting approach to strengthen and modify the BBB. The investigation of the role of intracellular S1P (iS1P) in hCMEC/D3 cells revealed opposing effects of the knock-down of SGPL1 (SPL-kd). SPL-kd enhanced the levels of iS1P in hCMEC/D3, and a barrier stabilizing or disruptive effect was observed depending on the conditions. Under inflammatory conditions, enhanced levels of iS1P were beneficial and led to the stabilization of the EC barrier, whereas, under normal conditions, a knock-down of the SGPL1 promoted the disruption of the barrier.¹³² Especially the beneficial effects of iS1P on the expression of AJ proteins like VE-cadherin, PECAM1 or β-catenin promoted the stabilization of EC barriers under inflammatory conditions. Furthermore, upregulation of signaling molecules like protein kinase C (PKC), adenosine monophosphate-activated protein kinase (AMPK) and p38-mitogen-activated protein kinase (MAPK) were observed together with downregulation of pro-inflammatory cytokines and VCAM1.¹³²

5.2 Placenta

The placenta is a complex and specialized organ and composed of fetal and maternal tissue. The placenta plays a crucial role in the development of the fetus and is responsible for the sustenance of the fetus with nutrients, oxygen, vitamins and other important metabolites, and for the removal of fetal waste products.¹³³ The maternal circulation is separated from the fetal by the placental membrane, which initially consists of four layers. One layer of the placental membrane is the feto-placental endothelium, and disruption and dysfunction of the vasculature peaks in diseases like preeclampsia (PE), intrauterine growth restriction (IUGR) and other inflammatory diseases, posing a risk to mother and fetus.^133,134

So far, the influence of S1P on placental development and EC barrier maintenance was not thoroughly investigated. Many pregnancy-related and inflammatory diseases are marked by a reduction of barrier function. S1P is involved in maintaining EC function and presents a new approach to target placental dysfunction. Placental arteries expressed the receptors S1P_1-3,5. S1P was involved in regulating the vascular tone of the placenta by Rho-associated kinases and Ca²⁺ sensitization. Additionally, S1P activated NOS3, which was responsible for vasoconstriction.¹³⁵ Recently, Del Gaudio et al. examined the sphingolipid metabolism in the feto-placental vasculature in PE. Alterations in the sphingolipid metabolism occurred on several sites. On the one hand, serine palmitoyltransferase (SPT), S1P phosphatase (SPP) and SGPL1 expression and activity were increased and caused decreased levels of S1P and enhanced levels of dihydrosphingosine (dhSph). Furthermore, expression of S1P₁ was significantly reduced, whereas expression of S1P₂ was upregulated, which resulted in endothelial dysfunction.^136,137 Decreased levels of endothelial S1P together with decreased expression of S1P₁ were one possibility for placental endothelial dysfunction. Therefore, optimization and support of signaling via the S1P-S1P₁ axis might be an option to maintain stability and integrity of the placental EC barrier. As mentioned previously, S1P-apoM bound to HDL displayed barrier enhancing effects. Neonatal HDL (nHDL) was also able to carry the S1P-apoM complex. In vitro, nHDL delivered S1P was able to induce signaling via phospholipase C (PLC) and extracellular signal-regulated protein kinases (ERK) and strengthened the EC barrier function. Furthermore, upregulation of eNOS was observed and pointed to the involvement of S1P in placental vasorelaxation and – dilation.¹³⁴ In an in vitro model of PE, apoM-HDL was reduced and led to degradation of the EC barrier. Vascular inflammation was diminished following administration of nHDL-S1P, depicted by reduced expression of inflammatory markers and surface molecules like ICAM and VCAM. Additionally, nHDL-S1P provided protection from oxidative stress by negatively regulating angiotensin II induced reactive oxygen species (ROS) production and decreasing the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1.¹³⁸ Although our knowledge about the maintenance of the placental EC barrier is still scarce, the contribution of S1P to EC barrier maintenance in the placenta may offer the opportunity to treat placental dysfunction and other inflammatory diseases.

6. Conclusion

While barrier maintenance is a complex process that requires organ-specific approaches for fine-tuning and stabilization, data from the literature clearly indicate the high potential of S1P for medical intervention. Some compounds like FTY720 or apoM-Fc are already available and tested in different disease entities, additional applications such as inhibition of the S1P lyase or modulation of SphKs and S1P transporters may lead to even more options in the future. Although targeting signaling and metabolic pathways of S1P is a challenging task, the herein presented data from the literature demonstrate that continuous research in this field could result in highly valuable treatment options in the future. S1P is probably the molecule with the highest potential for EC barrier stabilizing applications in the future.

Acknowledgments

We thank Ralf A. Claus from the Department of Anesthesiology and Intensive Care Medicine of the Jena University Hospital for critical reading.

Disclosure statement

The authors declare no conflicts of interest.

Additional information

Funding

This work was supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) within the Center for Sepsis Control and Care (CSCC) and the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) within the RTG 1715.