Upregulation of inducible NO synthase by exogenous adenosine in vascular smooth muscle cells activated by inflammatory stimuli in experimental diabetes

BACKGROUND: Adenosine has been shown to induce nitric oxide (NO) production via inducible NO synthase (iNOS) activation in vascular smooth muscle cells (VSMCs). Although this is interpreted as a beneficial vasodilating pathway in vaso-occlusive disorders, iNOS is also involved in diabetic vascular dysfunction. Because the turnover of and the potential to modulate iNOS by adenosine in experimental diabetes have not been explored, we hypothesized that both the adenosine system and control of iNOS function are impaired in VSMCs from streptozotocin-diabetic rats. METHODS: Male Sprague-Dawley rats were injected with streptozotocin once to induce diabetes. Aortic VSMCs from diabetic and nondiabetic rats were isolated, cultured and exposed to lipopolysaccharide (LPS) plus a cytokine mix for 24 h in the presence or absence of (1) exogenous adenosine and related compounds, and/or (2) pharmacological agents affecting adenosine turnover. iNOS functional expression was determined by immunoblotting and NO metabolite assays. Concentrations of adenosine, related compounds and metabolites thereof were assayed by HPLC. Vasomotor responses to adenosine were determined in endothelium-deprived aortic rings. RESULTS: Treatment with adenosine-degrading enzymes or receptor antagonists increased iNOS formation in activated VSMCs from nondiabetic and diabetic rats. Following treatment with the adenosine transport inhibitor NBTI, iNOS levels increased in nondiabetic but decreased in diabetic VSMCs. The amount of secreted NO metabolites was uncoupled from iNOS levels in diabetic VSMCs. Addition of high concentrations of adenosine and its precursors or analogues enhanced iNOS formation solely in diabetic VSMCs. Exogenous adenosine and AMP were completely removed from the culture medium and converted into metabolites. A tendency towards elevated inosine generation was observed in diabetic VSMCs, which were also less sensitive to CD73 inhibition, but inosine supplementation did not affect iNOS levels. Pharmacological inhibition of NOS abolished adenosine-induced vasorelaxation in aortic tissues from diabetic but not nondiabetic animals. CONCLUSIONS: Endogenous adenosine prevented cytokine- and LPS-induced iNOS activation in VSMCs. By contrast, supplementation with adenosine and its precursors or analogues enhanced iNOS levels in diabetic VSMCs. This effect was associated with alterations in exogenous adenosine turnover. Thus, overactivation of the adenosine system may foster iNOS-mediated diabetic vascular dysfunction

Antiplatelet and anticoagulant therapy for non-ST-elevation acute coronary syndromes in a general hospital

Aim: To audit compliance with the 2011 European Society of Cardiology (ESC) guidelines for prescribing antiplatelet and anticoagulant therapy in patients diagnosed with non-ST-elevation acute coronary syndromes (NSTE-ACS). Methods: Five-month audit at Cardiac Catheterisation Suite, Mater Dei Hospital, Malta. A data collection form for inpatients diagnosed with NSTE-ACS was developed, psychometrically evaluated and completed for each patient by the investigator. Comparative analysis to assess whether these patients were being managed according to the guidelines was undertaken. Results: 165 patients were recruited; 73% were male, 37% were between 66 and 75 years and 44% were active smokers. The most common risk factors for cardiovascular disease in these patients were hypertension (27%), dyslipidemia and obesity (both 16%). 41% of the patients underwent a percutaneous coronary intervention, either ad hoc (34%) or elective (7%). Compliance to guidelines for prescription of aspirin and clopidogrel 300mg loading dose was 95% and 88% respectively. Compliance to guidelines for prescription of aspirin and clopidogrel 75mg daily maintenance dose was 97% and 96% respectively. Compliance to guidelines for prescription of proton pump inhibitors (PPIs) in patients receiving dual antiplatelet therapy (DAPT) was only 19%; 81% of the patients were prescribed omeprazole against guideline recommendations. Compliance to guidelines for prescription of anticoagulation therapy was 95% with respect to prescription of heparin treatment in combination with DAPT. Compliance to guidelines for prescription of enoxaparin was 81%. Compliance to guidelines for other antiplatelet and anticoagulant drugs recommended in the guidelines, including ticagrelor, prasugrel, glycoprotein IIb/IIIa receptor antagonists, fondaparinux and bivalirudin, was not measured. Conclusions: Prescription of antiplatelet and anticoagulant therapy for NSTE-ACS in Malta is predominantly in accordance with ESC guidelines. Alternative antiplatelet and anticoagulant drugs and PPIs should be included in the Maltese hospital formulary to improve NSTE-ACS management in line with guideline recommendations.peer-reviewe

TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are critical for the clearance of myelin debris in areas of demyelination, a key step to allow remyelination. TREM2 is expressed by microglia and promotes microglial survival, proliferation, and phagocytic activity. Herein we demonstrate that TREM2 was highly expressed on myelin-laden phagocytes in active demyelinating lesions in the CNS of subjects with MS. In gene expression studies, macrophages from subjects with TREM2 genetic deficiency displayed a defect in phagocytic pathways. Treatment with a new TREM2 agonistic antibody promoted the clearance of myelin debris in the cuprizone model of CNS demyelination. Effects included enhancement of myelin uptake and degradation, resulting in accelerated myelin debris removal by microglia. Most importantly, antibody-dependent TREM2 activation on microglia increased density of oligodendrocyte precursors in areas of demyelination, as well as the formation of mature oligodendrocytes thus enhancing remyelination and axonal integrity. These results are relevant as they propose TREM2 on microglia as a potential new target to promote remyelination

The MS4A gene cluster is a key modulator of soluble TREM2 and Alzheimer's disease risk

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in cerebrospinal fluid (CSF) has been associated with Alzheimer's disease (AD). TREM2 plays a critical role in microglial activation, survival, and phagocytosis;however, the pathophysiological role of sTREM2 in AD is not well understood. Understanding the role of sTREM2 in AD may reveal new pathological mechanisms and lead to the identification of therapeutic targets. We performed a genome-wide association study (GWAS) to identify genetic modifiers of CSF sTREM2 obtained from the Alzheimer's Disease Neuroimaging Initiative. Common variants in the membrane-spanning 4-domains subfamily A (MS4A) gene region were associated with CSF sTREM2 concentrations (rs1582763;P = 1.15 x 10(-15));this was replicated in independent datasets. The variants associated with increased CSF sTREM2 concentrations were associated with reduced AD risk and delayed age at onset of disease. The single-nucleotide polymorphism rs1582763 modified expression of the MS4A4A and MS4A6A genes in multiple tissues, suggesting that one or both of these genes are important for modulating sTREM2 production. Using human macrophages as a proxy for microglia, we found that MS4A4A and TREM2 colocalized on lipid rafts at the plasma membrane, that sTREM2 increased with MS4A4A overexpression, and that silencing of MS4A4A reduced sTREM2 production. These genetic, molecular, and cellular findings suggest that MS4A4A modulates sTREM2. These findings also provide a mechanistic explanation for the original GWAS signal in the MS4A locus for AD risk and indicate that TREM2 may be involved in AD pathogenesis not only in TREM2 risk-variant carriers but also in those with sporadic disease

Gender differences and pharmacological regulation of angiogenesis induced by synovial fluids in inflammatory arthritis

Several mediators including cytokines, growth factors and metalloproteinases (MMP) modulate pathological angiogenesis associated with inflammatory arthritis. The biological factors underlying sex disparities in the incidence and severity of rheumatic musculoskeletal diseases are only partially understood. We hypothesized that synovial fluids (SFs) from rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients would impact on endothelial biology in a sexually dimorphic fashion. Immune cell counts and levels of pro-angiogenic cytokines found in SFs from RA and PsA patients (n = 17) were higher than in osteoarthritis patients (n = 6). Synovial VEGF concentration was significantly higher in male than in female RA patients. Zymography revealed that SFs comprised solely MMP-9 and MMP-2, with significantly higher MMP-9 levels in male than female RA patients. Using in vitro approaches that mimic the major steps of the angiogenic process, SFs from RA and PsA patients induced endothelial migration and formation of capillary-like structures compared to control. Notably, endothelial cells from female donors displayed enhanced angiogenic response to SFs with respect to males. Treatment with the established anti-angiogenic agent digitoxin prevented activation of focal adhesion kinase and SF-induced in vitro angiogenesis. Thus, despite higher synovial VEGF and MMP-9 levels in male patients, the responsiveness of vascular endothelium to SF priming was higher in females, suggesting that gender differences in angiogenic responses were mainly related to the endothelial genotype. These findings may have implications for pathogenesis and targeted therapies of inflammatory arthritis

Dimethyl fumarate induces changes in B- and T-lymphocyte function independent of the effects on absolute lymphocyte count

BACKGROUND: Dimethyl fumarate (DMF) is used to treat relapsing multiple sclerosis and causes lymphopenia in a subpopulation of treated individuals. Much remains to be learned about how the drug affects B- and T-lymphocytes. OBJECTIVES: To characterize changes in B- and T-cell phenotype and function induced by DMF and to investigate whether low absolute lymphocyte count (ALC) is associated with unique functional changes. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from DMF-treated patients, untreated patients, and healthy controls. A subset of DMF-treated patients was lymphopenic (ALC \u3c 800). Multiparametric flow cytometry was used to evaluate cellular phenotypes. Functional response to non-specific and viral peptide stimulation was assessed. RESULTS: DMF reduced circulating memory B-cells regardless of ALC. Follicular T-helper cells (CD4 CXCR5) and mucosal invariant T-cells (CD8 CD161) were also reduced. DMF reduced T-cell production of pro-inflammatory cytokines in response to polyclonal (PMA/ionomycin) and viral peptide stimulation, regardless of ALC. No differences in activation-induced cell death or circulating progenitors were observed between lymphopenic and non-lymphopenic DMF-treated patients. CONCLUSION: These data implicate DMF-induced changes in lymphocytes as an important component of the drug\u27s efficacy and expand our understanding of the functional significance of DMF-induced lymphopenia

Thienocycloheptapyridazines as new muscarinic agents

A series of thienocycloheptapyridazines (3aa-dd), structurally related to Minaprine, was synthesized and compounds tested for their affinity towards muscarinic receptors. All of them showed Ki values in the micromolar range towards both the antagonist 3H-QNB and the agonist 3H-OXO-M, thus indicating that they act as antagonists at the muscarinic receptors. Moreover a theoretical study was performed on their interaction behaviour with a three dimensional (3-D) model of the human m1 muscarinic receptor