Rewiring Endothelial Sphingolipid Metabolism to Favor S1P Over Ceramide Protects From Coronary Atherosclerosis.

BACKGROUND: Growing evidence correlated changes in bioactive sphingolipids, particularly S1P (sphingosine-1-phosphate) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining lesion-prone areas plays a pivotal role in atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood. METHODS: We used an established model of coronary atherosclerosis in mice, combined with sphingolipidomics, RNA-sequencing, flow cytometry, and immunostaining to investigate the contribution of sphingolipid metabolism and signaling to endothelial cell (EC) activation and dysfunction. RESULTS: We demonstrated that hemodynamic stress induced an early metabolic rewiring towards endothelial sphingolipid de novo biosynthesis, favoring S1P signaling over ceramides as a protective response. This finding is a paradigm shift from the current belief that ceramide accrual contributes to endothelial dysfunction. The enzyme SPT (serine palmitoyltransferase) commences de novo biosynthesis of sphingolipids and is inhibited by NOGO-B (reticulon-4B), an ER membrane protein. Here, we showed that NOGO-B is upregulated by hemodynamic stress in myocardial EC of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and humans. We demonstrated that mice lacking NOGO-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of NOGO-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective endothelial transcriptional signature. CONCLUSIONS: These data demonstrated that hemodynamic stress induced a protective rewiring of sphingolipid metabolism, favoring S1P over ceramide. NOGO-B deletion sustained the rewiring of sphingolipid metabolism toward S1P protecting EC from activation under hemodynamic stress and refraining coronary atherosclerosis. These findings also set forth the foundation for sphingolipid-based therapeutics to limit atheroprogression.

BST1 regulates nicotinamide riboside metabolism via its glycohydrolase and base-exchange activities.

Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss-Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation.

Beneficial effect of STAT3 decoy oligodeoxynucleotide transfection on organ injury and mortality in mice with cecal ligation and puncture-induced sepsis.

Sepsis is a major clinical challenge with unacceptably high mortality. The signal transducers and activators of transcription (STAT) family of transcription factors is known to activate critical mediators of cytokine responses, and, among this family, STAT3 is implicated to be a key transcription factor in both immunity and inflammatory pathways. We investigated whether in vivo introduction of synthetic double-stranded STAT3 decoy oligodeoxynucleotides (ODNs) can provide benefits for reducing organ injury and mortality in mice with cecal ligation and puncture (CLP)-induced polymicrobial sepsis. We found that STAT3 was rapidly activated in major end-organ tissues following CLP, which was accompanied by activation of the upstream kinase JAK2. Transfection of STAT3 decoy ODNs downregulated pro-inflammatory cytokine/chemokine overproduction in CLP mice. Moreover, STAT3 decoy ODN transfection significantly reduced the increases in tissue mRNAs and proteins of high mobility group box 1 (HMGB1) and strongly suppressed the excessive elevation in serum HMGB1 levels in CLP mice. Finally, STAT3 decoy ODN administration minimized the development of sepsis-driven major end-organ injury and led to a significant survival advantage in mice after CLP. Our results suggest a critical role of STAT3 in the sepsis pathophysiology and the potential usefulness of STAT3 decoy ODNs for sepsis gene therapy.

Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells.

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous member of the GRK family that restrains cellular activation by G protein-coupled receptor (GPCR) phosphorylation leading to receptor desensitization and internalization, but has been identified to regulate a variety of signaling molecules, among which may be associated with inflammation. In this study, we attempted to establish the regulatory role of GRK2 in the Toll-like receptor (TLR) signaling pathway for inducible nitric oxide synthase (iNOS) expression in microglial cells. When mouse MG6 cells were stimulated with the TLR4 ligands lipopolysaccharide (LPS) and paclitaxel, we found that interferon regulatory factor 1 (IRF1) protein expression and activation was upregulated, transcription of interferon-ß (IFN-ß) was accelerated, induction/activation of STAT1 and activation of STAT3 were promoted, and subsequently iNOS expression was upregulated. The ablation of GRK2 by small interfering RNAs (siRNAs) not only eliminated TLR4-mediated upregulation of IRF1 protein expression and nuclear translocation but also suppressed the activation of the STAT pathway, resulting in negating the iNOS upregulation. The TLR3-mediated changes in IRF1 and STAT1/3, leading to iNOS induction, were also abrogated by siRNA knockdown of GRK2. Furthermore, transfection of GRK2 siRNA blocked the exogenous IFN-ß supplementation-induced increases in phosphorylation of STAT1 as well as STAT3 and abrogated the augmentation of iNOS expression in the presence of exogenous IFN-ß. Taken together, our results show that GRK2 regulates the activation of IRF1 as well as the activation of the STAT pathway, leading to upregulated transcription of iNOS in activated microglial cells. Modulation of the TLR signaling pathway via GRK2 in microglia may be a novel therapeutic target for treatment of neuroinflammatory disorders.

Different involvement of the MAPK family in inflammatory regulation in human pulmonary microvascular endothelial cells stimulated with LPS and IFN-γ.

Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.

Nucleic-acid based gene therapy approaches for sepsis.

Despite advances in overall medical care, sepsis and its sequelae continue to be an embarrassing clinical entity with an unacceptably high mortality rate. The central reason for high morbidity and high mortality of sepsis and its sequelae is the lack of an effective treatment. Previous clinical trials have largely failed to identify an effective therapeutic target to improve clinical outcomes in sepsis. Thus, the key goal favoring the outcome of septic patients is to devise innovative and evolutionary therapeutic strategies. Gene therapy can be considered as one of the most promising novel therapeutic approaches for nasty disorders. Since a number of transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), play a pivotal role in the pathophysiology of sepsis that can be characterized by the induction of multiple genes and their products, sepsis may be regarded as a gene-related disorder and gene therapy may be considered a promising novel therapeutic approach for treatment of sepsis. In this review article, we provide an up-to-date summary of the gene-targeting approaches, which have been developed in animal models of sepsis. Our review sheds light on the molecular basis of sepsis pathology for the development of novel gene therapy approaches and leads to the conclusion that future research efforts may fully take into account gene therapy for the treatment of sepsis.

Activator Protein-1 Decoy Oligodeoxynucleotide Transfection Is Beneficial in Reducing Organ Injury and Mortality in Septic Mice.

OBJECTIVES: Inflammation and apoptosis are decisive mechanisms for the development of end-organ injury in sepsis. Activator protein-1 may play a key role in regulating expression of harmful genes responsible for the pathophysiology of septic end-organ injury along with the major transcription factor nuclear factor-κB. We investigated whether in vivo introduction of circular dumbbell activator protein-1 decoy oligodeoxynucleotides can provide benefits for reducing septic end-organ injury. DESIGN: Laboratory and animal/cell research. SETTINGS: University research laboratory. SUBJECTS: Male BALB/c mice (8-10 wk old). INTERVENTIONS: Activator protein-1 decoy oligodeoxynucleotides were effectively delivered into tissues of septic mice in vivo by preparing into a complex with atelocollagen given 1 hour after surgery. MATERIALS AND MAIN RESULTS: Polymicrobial sepsis was induced by cecal ligation and puncture in mice. Activator protein-1 decoy oligodeoxynucleotide transfection inhibited abnormal production of proinflammatory and chemotactic cytokines after cecal ligation and puncture. Histopathologic changes in lung, liver, and kidney tissues after cecal ligation and puncture were improved by activator protein-1 decoy oligodeoxynucleotide administration. When activator protein-1 decoy oligodeoxynucleotides were given, apoptosis induction was strikingly suppressed in lungs, livers, kidneys, and spleens of cecal ligation and puncture mice. These beneficial effects of activator protein-1 decoy oligodeoxynucleotides led to a significant survival advantage in mice after cecal ligation and puncture. Apoptotic gene profiling indicated that activator protein-1 activation was involved in the up-regulation of many of proapoptotic and antiapoptotic genes in cecal ligation and puncture-induced sepsis. CONCLUSIONS: Our results indicate a detrimental role of activator protein-1 in the sepsis pathophysiology and the potential usefulness of activator protein-1 decoy oligodeoxynucleotides for the prevention and treatment of septic end-organ failure.

A Role of the ABCC4 Gene Polymorphism in Airway Inflammation of Asthmatics.

The ATP-binding cassette subfamily C member 4 gene encodes a transmembrane protein involved in the export of proinflammatory molecules, including leukotriene, prostaglandin, and sphingosine-1-phosphate across the plasma membrane. Those metabolites play important roles in asthma. We investigated the potential associations between ABCC4 gene polymorphisms and asthma phenotype. In total, 270 asthma patients and 120 normal healthy controls were enrolled for a genetic association study. Two polymorphisms (-1508A>G and -642C>G) in the ABCC4 promoter were genotyped. The functional variability of the promoter polymorphisms was analyzed by luciferase reporter assay. Inflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. Serum and urinary eicosanoid metabolites, sphingosine-1-phosphate, were evaluated by quadrupole time-of-flight mass spectrometry. Asthma patients carrying the G allele at -1508A>G had significantly higher serum levels of periostin, myeloperoxidase, and urinary levels of 15-hydroxyeicosatetraenoic acid and sphingosine-1-phosphate (P = 0.016, P = 0.027, P = 0.032, and P = 0.010, resp.) compared with noncarrier asthma patients. Luciferase activity was significantly enhanced in human epithelial A549 cells harboring a construct containing the -1508G allele (P < 0.01 for each) compared with a construct containing the -1508A allele. A functional polymorphism in the ABCC4 promoter, -1508A>G, may increase extracellular 15-hydroxyeicosatetraenoic acid, sphingosine-1-phosphate, and periostin levels, contributing to airway inflammation in asthmatics.

Diminished responsiveness to dobutamine as an inotrope in mice with cecal ligation and puncture-induced sepsis: attribution to phosphodiesterase 4 upregulation.

Dobutamine has been used in septic shock for many years as an only inotrope, but its benefit has been questioned. We weighed the effects of dobutamine and milrinone as inotropes in mice with cecal ligation and puncture (CLP)-induced polymicrobial sepsis. CLP-induced septic mice exhibited significant cardiac inflammation, as indicated by greatly increased mRNAs of proinflammatory cytokines and robust infiltration of inflammatory cells in the ventricular myocardium. Elevations of plasma cardiac troponin-I showed cardiac injury in CLP mice. Noninvasive echocardiographic assessment of cardiac function revealed that despite preserved left ventricular function in the presence of fluid replacement, the dobutamine inotropic response was significantly impaired in CLP mice compared with sham-operated controls. By contrast, milrinone exerted inotropic effects in sham-operated and CLP mice in an equally effective manner. Surface expression levels of ß1-adrenoceptors and α-subunits of three main G protein families in the myocardium were unaffected by CLP-induced sepsis. Plasma cAMP levels were significantly elevated in both sham-operated and CLP mice in response to milrinone but only in sham-operated controls in response to dobutamine. Of phosphodiesterase (PDE) isoforms, PDE4D, but not PDE3A, both of which are responsible for cardiac cAMP hydrolysis, was significantly upregulated in CLP mouse myocardium. We define a novel mechanism for the impaired responsiveness to dobutamine as an inotrope in sepsis, and understanding the role of PDE4D in modulating cardiac functional responsiveness in sepsis may open the potential of a PDE4D-targeted therapeutic option in septic patients with low cardiac output who have a need for inotropic support.NEW & NOTEWORTHY Advisability of the usefulness of dobutamine in septic shock management is limited. Here, we reveal that the effect of dobutamine as a positive inotrope is impaired in mice with cecal ligation and puncture-induced sepsis without changes in cardiac ß1-adrenoceptor signaling as a result of cAMP breakdown achieved by upregulated phosphodiesterase 4D.

Effects of MBL2 polymorphisms in patients with diisocyanate-induced occupational asthma.

Diisocyanate (DI) is the most common cause of occupational asthma (OA) in Korea. Mannose-binding lectin (MBL) initiates the lectin complement activation pathway following oxidative stress and plays an important role in the regulation of inflammatory processes. To determine whether there is a genetic association between MBL2 polymorphisms and DI-OA, 99 patients with DI-OA, 99 asymptomatic exposed controls (AECs) and 144 unexposed normal controls were enrolled in this study. Three polymorphisms (-554 G>C, -431A>C and -225 G>C) in the MBL2 promoter were genotyped, and serum MBL levels were determined by enzyme-linked immunosorbent assay. Functional variabilities in the promoter polymorphisms were analyzed by a luciferase reporter assay and electrophoretic mobility shift assay (EMSA). A significantly higher frequency of haplotype (ht) 2 [CAG] was noted in the DI-OA group compared with the AEC group (P=0.044). The patients with DI-OA carrying ht2 [CAG] had significantly lower PC20 methacholine levels (P<0.001) than the non-carriers. The serum MBL levels were significantly higher in the DI-exposed subjects (both the DI-OA patients and AECs) carrying ht1 [GAG] (P=0.028). Luciferase activity was significantly enhanced in ht1 [GAG] compared with ht2 [CAG] in human hepatocarcinoma cells (Hep3B) (P=0.002). The EMSA showed that a -554G probe produced a specific shifted band compared with the -554C probe. These findings suggest that decreased serum MBL levels due to polymorphisms of the MBL2 gene may increase susceptibility to the development of DI-OA in DI-exposed individuals.

Association Between PTPN22 Polymorphisms and IgE Responses to Staphylococcal Superantigens in Chronic Urticaria.

Protein tyrosine phosphatase-22 (PTPN22) gene encodes lymphoid-specific tyrosine phosphatase (Lyp), an inhibitor of T cell activation. A polymorphism of the PTPN22 gene has been found to be associated with chronic urticaria (CU). We investigated the associations between PTPN22 gene polymorphisms and CU characteristics, including serum specific IgE antibodies response to toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA). CU patients (n=409) and normal healthy controls (n=388) were enrolled in the present study. Serum specific IgE to TSST-1 and SEA were measured by ImmunoCAP®. Five PTPN22 single nucleotide polymorphisms, -1123G>C, 1858C>T, 13145A>G, 14943C>T, and 20628A>G, were genotyped. There were no significant differences in genotype or haplotype frequencies of these polymorphisms between the 2 groups. CU patients carrying the GG genotype at 20628A>G (P=0.035) or haplotype 3 [GGG] (P=0.047) had a significantly higher prevalence of serum specific IgE to TSST-1 compared to non-carriers. Similarly, CT/TT genotype at 14943C>T had a significantly higher prevalence of serum specific IgE to SEA (P=0.045). The findings suggest that the PTPN22 gene polymorphisms at 20628A>G and 14943C>T may enhance serum specific IgE responses to TSST-1 and SEA, which may contribute to CU pathogenesis.

Elevated platelet activation in patients with chronic urticaria: a comparison between aspirin-intolerant and aspirin-tolerant groups.

BACKGROUND: Platelets are actively involved in immune inflammatory processes that release inflammatory mediators. Platelet activation has been reported in various inflammatory diseases; however, few studies have described platelet involvement in chronic urticaria (CU). OBJECTIVE: To investigate platelet-activation markers, namely P2Y12 receptor and P-selectin expression, and soluble P-selectin level in patients with aspirin-intolerant CU (AICU) and aspirin-tolerant CU (ATCU). METHODS: Forty-eight patients with CU and 25 normal controls were enrolled in this study. Aspirin intolerance in patients with CU was confirmed by an oral provocation test. P2Y12 and P-selectin expressions on platelets were measured using flow cytometry; soluble P-selectin level in plasma was measured by enzyme-linked immunosorbent assay. To study the functional effects of aspirin, platelets were treated with aspirin (2 mmol/L) and the expressions of P2Y12 and P-selectin were compared between the AICU and ATCU groups. RESULTS: The expression of P2Y12 was significantly higher in patients with CU compared with controls, whereas no significant difference was noted in the expression of P-selectin level. The levels were not significantly different according to urticaria symptom score, symptom control status, and aspirin intolerance. Soluble P-selectin level was significantly higher in the AICU group than in the ATCU group compared with controls. Aspirin did not significantly suppress P2Y12 and P-selectin expressions on platelets in the AICU group, whereas significant suppression was noted in the ATCU group. CONCLUSION: These findings suggest that increased platelet activation contributes to skin inflammation in patients with AICU and those with ATCU. The functional difference of platelets in response to aspirin may contribute to persistent skin inflammation in patients with AICU.