Fingolimod (FTY720-P) Does Not Stabilize the Blood–Brain Barrier under Inflammatory Conditions in an in Vitro Model

Breakdown of the blood-brain barrier (BBB) is an early hallmark of multiple sclerosis (MS), a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P), a signaling protein, via S1P receptors (S1P₁). Fingolimod phosphate (FTY720-P) a functional S1P₁ antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability—in particular, on the tight junction proteins occludin, claudin 5 and ZO-1—has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC) culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP)-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P₁ plays a dual role in vascular permeability, depending on its ligand. Thus, S1P₁ provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers—such as the blood-retinal barrier—which might result in macular edema.     

Kalkitoxin Reduces Osteoclast Formation and Resorption and Protects against Inflammatory Bone Loss

Osteoclasts, bone-specified multinucleated cells produced by monocyte/macrophage, are involved in numerous bone destructive diseases such as arthritis, osteoporosis, and inflammation-induced bone loss. The osteoclast differentiation mechanism suggests a possible strategy to treat bone diseases. In this regard, we recently examined the in vivo impact of kalkitoxin (KT), a marine product obtained from the marine cyanobacterium Moorena producens (previously Lyngbya majuscula), on the macrophage colony-stimulating factor (M-CSF) and on the receptor activator of nuclear factor κB ligand (RANKL)-stimulated in vitro osteoclastogenesis and inflammation-mediated bone loss. We have now examined the molecular mechanism of KT in greater detail. KT decreased RANKL-induced bone marrow-derived macrophages (BMMs) tartrate-resistant acid phosphatase (TRAP)-multinucleated cells at a late stage. Likewise, KT suppressed RANKL-induced pit area and actin ring formation in BMM cells. Additionally, KT inhibited several RANKL-induced genes such as cathepsin K, matrix metalloproteinase (MMP-9), TRAP, and dendritic cell-specific transmembrane protein (DC-STAMP). In line with these results, RANKL stimulated both genes and protein expression of c-Fos and nuclear factor of activated T cells (NFATc1), and this was also suppressed by KT. Moreover, KT markedly decreased RANKL-induced p-ERK1/2 and p-JNK pathways at different time points. As a result, KT prevented inflammatory bone loss in mice, such as bone mineral density (BMD) and osteoclast differentiation markers. These experiments demonstrated that KT markedly inhibited osteoclast formation and inflammatory bone loss through NFATc1 and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, KT may have potential as a treatment for destructive bone diseases.     

Reciprocal Roles of Angiotensin II and Angiotensin II Receptors Blockade (ARB) in Regulating Cbfa1/RANKL via cAMP Signaling Pathway: Possible Mechanism for Hypertension-Related Osteoporosis and Antagonistic Effect of ARB on Hypertension-Related Osteoporosis

Hypertension is a risk factor for osteoporosis. Animal and epidemiological studies demonstrate that high blood pressure is associated with increased calcium loss, elevated parathyroid hormone, and increased calcium movement from bone. However, the mechanism responsible for hypertension-related osteoporosis remains elusive. Recent epidemiological studies indicate the benefits of Angiotensin II Receptors Blockade (ARB) on decreasing fracture risks. Since receptors for angiotensin II, the targets of ARB, are expressed in both osteoblasts and osteoclasts, we postulated that angiotensin II plays an important role in hypertension-related osteoporosis. Cbfa1 and RANKL, the important factors for maintaining bone homeostasis and key mediators in controlling osteoblast and osteoclast differentiation, are both regulated by cAMP-dependent signaling. Angiotensin II along with factors such as LDL, HDL, NO and homocysteine that are commonly altered both in hypertension and osteoporosis, can down-regulate the expression of Cbfa1 but up-regulate RANKL expression via the cAMP signaling pathway. We thus hypothesized that, by altering the ratio of Cbfa1/RANKL expression via the cAMP-dependent pathway, angiotensin II differently regulates osteoblast and osteoclast differentiation leading to enhanced bone resorption and reduced bone formation. Since ARB can antagonize the adverse effect of angiotensin II on bone by lowering cAMP levels and modifying other downstream targets, including LDL, HDL, NO and Cbfa1/RANKL, we propose the hypothesis that the antagonistic effects of ARB may also be exerted via cAMP signaling pathway.     

FTY720对聚乙烯颗粒诱导的破骨细胞前体细胞RAW264.7分化的影响

目的观察FTY720对聚乙烯颗粒诱导的破骨细胞前体细胞RAW264.7分化的影响,探讨其防治人工关节无菌性松动的可能性。方法构建破骨细胞-成骨细胞(RAW264.7-MC3T3)小室共培养体系及破骨细胞-骨片体系,用FTY720干预受聚乙烯磨损颗粒刺激的RAW264.7细胞的分化,倒置显微镜观察分化细胞的形态;抗酒石酸酸性磷酸酶(Tartrate-resistant acid phosphatas,TRAP)染色法对破骨细胞进行计数;扫描电镜观察破骨细胞的一般形态及骨吸收效应;ELISA法检测共培养体系中肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和白细胞介素-6(Lnterleuk-in-6,IL-6)的分泌水平;RT-PCR检测破骨细胞表面核因子κB受体活化子(Receptor activator of NFκB,RANK)和TRAP基因mRNA的转录水平。结果聚乙烯颗粒组RAW264.7细胞体积增大,胞质丰富,胞体边缘不齐呈云雾状,细胞核较多;FTY720组TRAP(+)细胞数明显低于聚乙烯颗粒组(P<0.01);破骨细胞呈圆形,细胞间可通过纤维样足突连接,骨片上有破骨细胞附着生长,FTY720组骨吸收陷窝和骨吸收面积均明显低于聚乙烯颗粒组(P<0.01);聚乙烯颗粒组TNF-α和IL-6的分泌水平均明显增加(P<0.01),FTY720组TNF-α和IL-6的分泌受到抑制;FTY720组破骨细胞表面TRAP和RANK基因mRNA的转录水平均明显低于颗粒组(P<0.01)。结论 FTY720能有效抑制破骨细胞前体细胞RAW264.7分化成熟,减少破骨细胞的形成及对骨片的溶解吸收,减少TNF-α、IL-6等炎性因子的分泌,下调RANK、TRAP等破骨细胞特异细胞表型和功能基因mRNA的转录水平,有望成为防治人工关节无菌性松动的药物。     

BZM055, an iodinated radiotracer candidate for PET and SPECT imaging of myelin and FTY720 brain distribution

FTY720 (fingolimod, Gilenya?) is a sphingosine 1-phosphate (S1P) receptor modulator that shows significant therapeutic efficacy after oral administration to patients of multiple sclerosis. Because FTY720 does not contain any atom whose PET or SPECT radioisotope would have a half-life compatible with its pharmacokinetic properties, it cannot be used directly for imaging. Instead, we propose BZM055 as a surrogate tracer to study its pharmacokinetics and organ distribution in patients and, given that FTY720 accumulates in myelin sheaths, for myelin imaging. BZM055 (2?a, 2-iodo-FTY720) can be easily radiolabeled with 123I (for SPECT) or 12?I (for PET). Not only does it closely mimic the pharmacokinetics and organ distribution of FTY720, but also its affinity, selectivity for S1P receptors, phosphorylation kinetics, and overall physicochemical properties. [123I]BZM055 is currently under development for clinical imaging.     

Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.     

Sphingosine-1-Phosphate Receptor Type 4 (S1P4) Is Differentially Regulated in Peritoneal B1 B Cells upon TLR4 Stimulation and Facilitates the Egress of Peritoneal B1a B Cells and Subsequent Accumulation of Splenic IRA B Cells under Inflammatory Conditions

Background: Gram-negative infections of the peritoneal cavity result in profound modifications of peritoneal B cell populations and induce the migration of peritoneal B cells to distant secondary lymphoid organs. However, mechanisms controlling the egress of peritoneal B cells from the peritoneal cavity and their subsequent trafficking remain incompletely understood. Sphingosine-1-phosphate (S1P)-mediated signaling controls migratory processes in numerous immune cells. The present work investigates the role of S1P-mediated signaling in peritoneal B cell trafficking under inflammatory conditions. Methods: Differential S1P receptor expression after peritoneal B cell activation was assessed semi‑quantitatively using RT-PCR in vitro. The functional implications of differential S1P₁ and S1P₄ expression were assessed by transwell migration in vitro, by adoptive peritoneal B cell transfer in a model of sterile lipopolysaccharide (LPS)‑induced peritonitis and in the polymicrobial colon ascendens stent peritonitis (CASP) model. Results: The two sphingosine-1-phosphate receptors (S1PRs) expressed in peritoneal B cell subsets S1P₁ and S1P₄ are differentially regulated upon stimulation with the TLR4 agonist LPS, but not upon PMA/ionomycin or B cell receptor (BCR) crosslinking. S1P₄ deficiency affects both the trafficking of activated peritoneal B cells to secondary lymphoid organs and the positioning of these cells within the functional compartments of the targeted organ. S1P₄ deficiency in LPS-activated peritoneal B cells results in significantly reduced numbers of splenic innate response activator B cells. Conclusions: The S1P-S1PR system is implicated in the trafficking of LPS-activated peritoneal B cells. Given the protective role of peritoneal B1a B cells in peritoneal sepsis, further experiments to investigate the impact of S1P₄-mediated signaling on the severity and mortality of peritoneal sepsis are warranted.     

The Pathophysiology of Osteoporosis after Spinal Cord Injury

Spinal cord injury (SCI) affects approximately 300,000 people in the United States. Most individuals who sustain severe SCI also develop subsequent osteoporosis. However, beyond immobilization-related lack of long bone loading, multiple mechanisms of SCI-related bone density loss are incompletely understood. Recent findings suggest neuronal impairment and disability may lead to an upregulation of receptor activator of nuclear factor-κB ligand (RANKL), which promotes bone resorption. Disruption of Wnt signaling and dysregulation of RANKL may also contribute to the pathogenesis of SCI-related osteoporosis. Estrogenic effects may protect bones from resorption by decreasing the upregulation of RANKL. This review will discuss the current proposed physiological and cellular mechanisms explaining osteoporosis associated with SCI. In addition, we will discuss emerging pharmacological and physiological treatment strategies, including the promising effects of estrogen on cellular protection.     

Molecular Targeted Therapy for the Bone Loss Secondary to Pyogenic Spondylodiscitis Using Medications for Osteoporosis: A Literature Review

Pyogenic spondylodiscitis can cause severe osteolytic and destructive lesions in the spine. Elderly or immunocompromised individuals are particularly susceptible to infectious diseases; specifically, infections in the spine can impair the ability of the spine to support the trunk, causing patients to be bedridden, which can also severely affect the physical condition of patients. Although treatments for osteoporosis have been well studied, treatments for bone loss secondary to infection remain to be elucidated because they have pathological manifestations that are similar to but distinct from those of osteoporosis. Recently, we encountered a patient with severely osteolytic pyogenic spondylodiscitis who was treated with romosozumab and exhibited enhanced bone formation. Romosozumab stimulated canonical Wnt/β-catenin signaling, causing robust bone formation and the inhibition of bone resorption, which exceeded the bone loss secondary to infection. Bone loss due to infections involves the suppression of osteoblastogenesis by osteoblast apoptosis, which is induced by the nuclear factor-κB and mitogen-activated protein kinase pathways, and osteoclastogenesis with the receptor activator of the nuclear factor-κB ligand-receptor combination and subsequent activation of the nuclear factor of activated T cells cytoplasmic 1 and c-Fos. In this study, we review and discuss the molecular mechanisms of bone loss secondary to infection and analyze the efficacy of the medications for osteoporosis, focusing on romosozumab, teriparatide, denosumab, and bisphosphonates, in treating this pathological condition.     

Piezoelectric Microvibration Mitigates Estrogen Loss-Induced Osteoporosis and Promotes Piezo1, MicroRNA-29a,and Wnt3a Signaling in Osteoblasts

Biophysical stimulation alters bone-forming cell activity, bone formation and remodeling. The effect of piezoelectric microvibration stimulation (PMVS) intervention on osteoporosis development remains uncertain. We investigated whether 60 Hz, 120 Hz, and 180 Hz PMVS (0.05 g, 20 min/stimulation, 3 stimulations/week for 4 consecutive weeks) intervention affected bone integrity in ovariectomized (OVX) mice or osteoblastic activity. PMVS (120 Hz)-treated OVX mice developed fewer osteoporosis conditions, including bone mineral density loss and trabecular microstructure deterioration together with decreased serum resorption marker CTX-1 levels, as compared to control OVX animals. The biomechanical strength of skeletal tissue was improved upon 120 Hz PMVS intervention. This intervention compromised OVX-induced sparse trabecular bone morphology, osteoblast loss, osteoclast overburden, and osteoclast-promoting cytokine RANKL immunostaining and reversed osteoclast inhibitor OPG immunoreactivity. Osteoblasts in OVX mice upon PMVS intervention showed strong Wnt3a immunoreaction and weak Wnt inhibitor Dkk1 immunostaining. In vitro, PMVS reversed OVX-induced loss in von Kossa-stained mineralized nodule formation, Runx2, and osteocalcin expression in primary bone-marrow stromal cells. PMVS also promoted mechanoreceptor Piezo1 expression together with increased microRNA-29a and Wnt3a expression, whereas Dkk1 rather than SOST expression was repressed in MC3T3-E1 osteoblasts. Taken together, PMVS intervention promoted Piezo1, miR-29a, and Wnt signaling to upregulate osteogenic activity and repressed osteoclastic bone resorption, delaying estrogen deficiency-induced loss in bone mass and microstructure. This study highlights a new biophysical remedy for osteoporosis.     

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.     

FTY720 Inhibits Expansion of Breast Cancer Stem Cells via PP2A Activation

Growing evidence suggests that breast cancer originates from a minor population of cancer cells termed cancer stem cells (CSCs), which can be identified by aldehyde dehydrogenase (ALDH) activity-based flow cytometry analysis. However, novel therapeutic drugs for the eradication of CSCs have not been discovered yet. Recently, drug repositioning, which finds new medical uses from existing drugs, has been expected to facilitate drug discovery. We have previously reported that sphingosine kinase 1 (SphK1) induced proliferation of breast CSCs. In the present study, we focused on the immunosuppressive agent FTY720 (also known as fingolimod or Gilenya), since FTY720 is known to be an inhibitor of SphK1. We found that FTY720 blocked both proliferation of ALDH-positive cells and formation of mammospheres. In addition, we showed that FTY720 reduced the expression of stem cell markers such as Oct3/4, Sox2 and Nanog via upregulation of protein phosphatase 2A (PP2A). These results suggest that FTY720 is an effective drug for breast CSCs in vitro.     

Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1

The healthy skeleton requires a perfect coordination of the formation and degradation of bone. Metabolic bone disease like osteoporosis is resulted from the imbalance of bone formation and/or bone resorption. Osteoporosis also reflects lower level of bone matrix, which is contributed by up-regulated osteoclast-mediated bone resorption. It is reported that monocytes/macrophage progenitor cells or either hematopoietic stem cells (HSCs) gave rise to multinucleated osteoclasts. Thus, inhibition of osteoclastic bone resorption generally seems to be a predominant therapy for treating osteoporosis. Recently, more and more natural compounds have been discovered, which have the ability of inhibiting osteoclast differentiation and fusion. Alliin (S-allyl-l-cysteine sulfoxides, SACSO) is the major component of aged garlic extract (AGE), bearing broad-spectrum natural antioxidant properties. However, its effects on bone health have not yet been explored. Hence, we designed the current study to explore its effects and role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast fusion and differentiation. It was revealed that alliin had an inhibitory effect in osteoclasteogenesis with a dose-dependent manner via blocking the c-Fos-NFATc1 signaling pathway. In addition, alliin decreased the generation of reactive oxygen species (ROS) and down-regulated the expression of NADPH oxidase 1 (Nox1). The overall results revealed that alliin could be a potential therapeutic agent in the treatment of osteoporosis.     

Inhibitory Effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside on RANKL-Induced Osteoclast Differentiation and ROS Generation in Macrophages

In bone homeostasis, bone loss due to excessive osteoclasts and inflammation or osteolysis in the bone formation process cause bone diseases such as osteoporosis. Suppressing the accompanying oxidative stress such as ROS in this process is an important treatment strategy for bone disease. Therefore, in this study, the effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (BAG), an arylbutanoid glycoside isolated from Betula platyphylla var. japonica was investigated in RANKL-induced RAW264.7 cells and LPS-stimulated MC3E3-T1 cells. BAG inhibited the activity of TRAP, an important marker of osteoclast differentiation and F-actin ring formation, which has osteospecific structure. In addition, the protein and gene levels were suppressed of integrin β3 and CCL4, which play an important role in the osteoclast-induced bone resorption and migration of osteoclasts, and inhibited the production of ROS and restored the expression of antioxidant enzymes such as SOD and CAT lost by RANKL. The inhibitory effect of BAG on osteoclast differentiation and ROS production appears to be due to the inhibition of MAPKs phosphorylation and NF-κβ translocation, which play a major role in osteoclast differentiation. In addition, BAG inhibited ROS generated by LPS and effectively restores the mineralization of lost osteoblasts, thereby showing the effect of bone formation in the inflammatory situation accompanying bone loss by excessive osteoclasts, suggesting its potential as a new natural product-derived bone disease treatment.     

Extracellular Sphingosine-1-Phosphate Downstream of EGFR Increases Human Glioblastoma Cell Survival

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.     

An Overview of the Molecular Mechanisms Contributing to Musculoskeletal Disorders in Chronic Liver Disease: Osteoporosis,Sarcopenia, and Osteoporotic Sarcopenia

The prevalence of osteoporosis and sarcopenia is significantly higher in patients with liver disease than in those without liver disease and osteoporosis and sarcopenia negatively influence morbidity and mortality in liver disease, yet these musculoskeletal disorders are frequently overlooked in clinical practice for patients with chronic liver disease. The objective of this review is to provide a comprehensive understanding of the molecular mechanisms of musculoskeletal disorders accompanying the pathogenesis of liver disease. The increased bone resorption through the receptor activator of nuclear factor kappa (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) system and upregulation of inflammatory cytokines and decreased bone formation through increased bilirubin and sclerostin and lower insulin-like growth factor-1 are important mechanisms for osteoporosis in patients with liver disease. Sarcopenia is associated with insulin resistance and obesity in non-alcoholic fatty liver disease, whereas hyperammonemia, low amount of branched chain amino acids, and hypogonadism contributes to sarcopenia in liver cirrhosis. The bidirectional crosstalk between muscle and bone through myostatin, irisin, β-aminoisobutyric acid (BAIBA), osteocalcin, as well as the activation of the RANK and the Wnt/β-catenin pathways are associated with osteosarcopenia. The increased understandings for these musculoskeletal disorders would be contributes to the development of effective therapies targeting the pathophysiological mechanism involved.