Selectivity and Specificity of Sphingosine-1-Phosphate Receptor Ligands: Caveats and Critical Thinking in Characterizing Receptor-Mediated Effects

Receptors for sphingosine-1-phosphate (S1P) have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444), used extensively as specific S1P2 and S1P3 receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P2 receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca2+ concentration via P2 receptor or α1A-adrenoceptor stimulation and α1A-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P3-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P1/3 receptor antagonist, VPC23019, does not inhibit S1P3-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes

Immunomodulatory therapeutic strategies in stroke

The role of immunity in all stages of stroke is increasingly being recognised, from the pathogenesis of risk factors to tissue repair, leading to the investigation of a range of immunomodulatory therapies. In the acute phase of stroke, proposed therapies include drugs targeting pro-inflammatory cytokines, matrix metalloproteinases, and leukocyte infiltration, with a key objective to reduce initial brain cell toxicity. Systemically, the early stages of stroke are also characterised by stroke-induced immunosuppression, where downregulation of host defences predisposes patients to infection. Therefore, strategies to modulate innate immunity post-stroke have garnered greater attention. A complementary objective is to reduce longer term sequelae, by focusing on adaptive immunity. Following stroke onset, the integrity of the blood-brain barrier is compromised, exposing central nervous system (CNS) antigens to systemic adaptive immune recognition, potentially inducing autoimmunity. Some pre-clinical efforts have been made to tolerise the immune system to CNS antigens pre-stroke. Separately, immune cell populations which exhibit a regulatory phenotype (T and B regulatory cells) have been shown to ameliorate post-stroke inflammation and contribute to tissue repair. Cell-based therapies, established in oncology and transplantation, could become a strategy to treat the acute and chronic stages of stroke. Furthermore, a role for the gut microbiota in ischemic injury has received attention. Finally, the immune system may play a role in remote ischemic preconditioning-mediated neuroprotection against stroke. The development of stroke therapies involving organs distant to the infarct site, therefore, should not be overlooked. This review will discuss the immune mechanisms of various therapeutic strategies, surveying published data and discussing more theoretical mechanisms of action that have yet to be exploited

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-β-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosi

Bone regenerative potential of the selective sphingosine 1-phosphate receptor modulator siponimod: In vitro characterisation using osteoblast and endothelial cells

The repair of critical bone defects remains a significant therapeutic challenge. While the implantation of drug-eluting scaffolds is an option, a drug with the optimal pharmacological properties has not yet been identified. Agents acting at sphingosine 1-phosphate (S1P) receptors have been considered, but those investigated so far do not discriminate between the five known S1P receptors. This work was undertaken to investigate the potential of the specific S1P1/5 modulator siponimod as a bone regenerative agent, by testing in vitro its effect on cell types critical to the bone regeneration process. hFOB osteoblasts and HUVEC endothelial cells were treated with siponimod and other S1P receptor modulators and investigated for changes in intracellular cyclic AMP content, viability, proliferation, differentiation, attachment and cellular motility. Siponimod showed no effect on the viability and proliferation of osteoblasts and endothelial cells, but increased osteoblast differentiation (as shown by increased alkaline phosphatase activity). Furthermore, siponimod significantly increased endothelial cell motility in scratch and transwell migration assays. These effects on osteoblast differentiation and endothelial cell migration suggest that siponimod may be a potential agent for the stimulation of localised differentiation of osteoblasts in critical bone defects

Sphingosine 1-phosphate, a potential target in neovascular retinal disease

Neovascular ocular diseases (such as age-related macular degeneration, diabetic retinopathy and retinal vein occlusion) are characterised by common pathological processes that contribute to disease progression. These include angiogenesis, oedema, inflammation, cell death and fibrosis. Currently available therapies target the effects of vascular endothelial growth factor (VEGF), the main mediator of pathological angiogenesis. Unfortunately, VEGF blockers are expensive biological therapeutics that necessitate frequent intravitreal administration and are associated with multiple adverse effects. Thus, alternative treatment options associated with fewer side effects are required for disease management. This review introduces sphingosine 1-phosphate (S1P) as a potential pharmacological target for the treatment of neovascular ocular pathologies. S1P is a sphingolipid mediator that controls cellular growth, differentiation, survival and death. S1P actions are mediated by five G protein-coupled receptors (S1P1-5 receptors) which are abundantly expressed in all retinal and subretinal structures. The action of S1P on S1P1 receptors can reduce angiogenesis, increase endothelium integrity, reduce photoreceptor apoptosis and protect the retina against neurodegeneration. Conversely, S1P2 receptor signalling can increase neovascularisation, disrupt endothelial junctions, stimulate VEGF release, and induce retinal cell apoptosis and degeneration of neural retina. The aim of this review is to thoroughly discuss the role of S1P and its different receptor subtypes in angiogenesis, inflammation, apoptosis and fibrosis in order to determine which of these S1P-mediated processes may be targeted therapeutically

Sphingosine 1-phosphate (S1P) signalling: role in bone biology and potential therapeutic target for bone repair

The lipid mediator sphingosine 1-phosphate (S1P) affects cellular functions in most systems. Interest in its therapeutic potential has increased following the discovery of its G protein-coupled receptors and the recent availability of agents that can be safely administered in humans. Although the role of S1P in bone biology has been the focus of much less research than its role in the nervous, cardiovascular and immune systems, it is becoming clear that this lipid influences many of the functions, pathways and cell types that play a key role in bone maintenance and repair. Indeed, S1P is implicated in many osteogenesis-related processes including stem cell recruitment and subsequent differentiation, differentiation and survival of osteoblasts, and coupling of the latter cell type with osteoclasts. In addition, S1P’s role in promoting angiogenesis is well-established. The pleiotropic effects of S1P on bone and blood vessels have significant potential therapeutic implications, as current therapeutic approaches for critical bone defects show significant limitations. Because of the complex effects of S1P on bone, the pharmacology of S1P-like agents and their physico-chemical properties, it is likely that therapeutic delivery of S1P agents will offer significant advantages compared to larger molecular weight factors. Hence, it is important to explore novel methods of utilizing S1P agents therapeutically, and improve our understanding of how S1P and its receptors modulate bone physiology and repair

The immune system and stroke: from current targets to future therapy

Stroke is a major cause of morbidity and mortality worldwide. Despite the intensive search for new therapies, hundreds of agents targeting various pathophysiological mechanisms have failed clinical trials, and the thrombolytic agent tissue plasminogen activator is currently the only FDA-approved medication for the treatment of acute ischaemic stroke (AIS). The immune system is involved in all stages of stroke, from the pathogenesis of risk factors to neurotoxicity, to tissue remodelling and repair. There is a bi-directional interaction between the brain and the immune system, with stroke-induced immunosuppression and subsequent infection a principal source of patient mortality. Newer work also points to a role for the gut microbiota in the immune response to stroke, while clinical sequelae such as dementia might now also be explained in immune terms. However, the exact roles of innate and adaptive components have not been fully elucidated, with studies reporting both detrimental and beneficial functions. Time is a key determinant in defining whether immunity and inflammation are neuroprotective or neurotoxic. The local inflammatory milieu also has a clear influence on many proposed treatments. This review examines the individual components of the immune response to stroke, highlighting the most promising future stroke immunotherapies

Sphingosine kinase 2 activates autophagy and protects neurons against ischemic injury through interaction with Bcl-2 via its putative BH3 domain

Our previous findings suggest that sphingosine kinase 2 (SPK2) mediates ischemic tolerance and autophagy in cerebral preconditioning. The aim of this study was to determine by which mechanism SPK2 activates autophagy in neural cells. In both primary murine cortical neurons and HT22 hippocampal neuronal cells, overexpression of SPK2 increased LC3II and enhanced the autophagy flux. SPK2 overexpression protected cortical neurons against oxygen glucose deprivation (OGD) injury, as evidenced by improvement of neuronal morphology, increased cell viability and reduced lactate dehydrogenase release. The inhibition of autophagy effectively suppressed the neuroprotective effect of SPK2. SPK2 overexpression reduced the co-immunoprecipitation of Beclin-1 and Bcl-2, while Beclin-1 knockdown inhibited SPK2-induced autophagy. Both co-immunoprecipitation and GST pull-down analysis suggest that SPK2 directly interacts with Bcl-2. SPK2 might interact to Bcl-2 in the cytoplasm. Notably, an SPK2 mutant with L219A substitution in its putative BH3 domain was not able to activate autophagy. A Tat peptide fused to an 18-amino acid peptide encompassing the native, but not the L219A mutated BH3 domain of SPK2 activated autophagy in neural cells. The Tat-SPK2 peptide also protected neurons against OGD injury through autophagy activation. These results suggest that SPK2 interacts with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1 and activating autophagy. The observation that Tat-SPK2 peptide designed from the BH3 domain of SPK2 activates autophagy and protects neural cells against OGD injury suggest that this structure may provide the basis for a novel class of therapeutic agents against ischemic stroke

Glutamate Excitoxicity Is the Key Molecular Mechanism Which Is Influenced by Body Temperature during the Acute Phase of Brain Stroke

Glutamate excitotoxicity, metabolic rate and inflammatory response have been associated to the deleterious effects of temperature during the acute phase of stroke. So far, the association of temperature with these mechanisms has been studied individually. However, the simultaneous study of the influence of temperature on these mechanisms is necessary to clarify their contributions to temperature-mediated ischemic damage. We used non-invasive Magnetic Resonance Spectroscopy to simultaneously measure temperature, glutamate excitotoxicity and metabolic rate in the brain in animal models of ischemia. The immune response to ischemia was measured through molecular serum markers in peripheral blood. We submitted groups of animals to different experimental conditions (hypothermia at 33°C, normothermia at 37°C and hyperthermia at 39°C), and combined these conditions with pharmacological modulation of glutamate levels in the brain through systemic injections of glutamate and oxaloacetate. We show that pharmacological modulation of glutamate levels can neutralize the deleterious effects of hyperthermia and the beneficial effects of hypothermia, however the analysis of the inflammatory response and metabolic rate, demonstrated that their effects on ischemic damage are less critical than glutamate excitotoxity. We conclude that glutamate excitotoxicity is the key molecular mechanism which is influenced by body temperature during the acute phase of brain stroke

The effect of fingolimod on regulatory T cells in a mouse model of brain ischaemia

Background: The role of the immune system in stroke is well-recognised. Fingolimod, an immunomodulatory agent licensed for the management of relapsing-remitting multiple sclerosis, has been shown to provide benefit in rodent models of stroke. Its mechanism of action, however, remains unclear. We hypothesised fingolimod increases the number and/or function of regulatory T cells (Treg), a lymphocyte population which promotes stroke recovery. The primary aim of this study was to rigorously investigate the effect of fingolimod on Tregs in a mouse model of brain ischaemia. The effect of fingolimod in mice with common stroke-related comorbidities (ageing and hypercholesteremia) was also investigated. Methods: Young (15–17 weeks), aged C57BL/6 mice (72–73 weeks), and ApoE?/? mice fed a high-fat diet (20–21 weeks) underwent permanent electrocoagulation of the left middle cerebral artery. Mice received either saline or fingolimod (0.5 mg/kg or 1 mg/kg) at 2, 24, and 48 h post-ischaemia via intraperitoneal injection. Another cohort of young mice (8–9, 17–19 weeks) received short-term (5 days) or long-term (10 days) fingolimod (0.5 mg/kg) treatment. Flow cytometry was used to quantify Tregs in blood, spleen, and lymph nodes. Immunohistochemistry was used to quantify FoxP3+ cell infiltration into the ischaemic brain. Results: Fingolimod significantly increased the frequency of Tregs within the CD4+ T cell population in blood and spleen post-ischaemia in all three mouse cohorts compared to untreated ischemic mice. The highest splenic Treg frequency in fingolimod-treated mice was observed in ApoE?/? mice (9.32 ± 1.73% vs. 7.8 ± 3.01% in young, 6.09 ± 1.64% in aged mice). The highest circulating Treg frequency was also noted in ApoE?/? mice (8.39 ± 3.26% vs. 5.43 ± 2.74% in young, 4.56 ± 1.60% in aged mice). Fingolimod significantly increased the number of FoxP3+ cells in the infarct core of all mice. The most pronounced effects were seen when mice were treated for 10 days post-ischaemia. Conclusions: Fingolimod increases Treg frequency in spleen and blood post-ischaemia and enhances the number of FoxP3+ cells in the ischaemic brain. The effect of fingolimod on this regulatory cell population may underlie its neuroprotective activity and could be exploited as part of future stroke therapy