A re-evaluation of the use of glyceryl trinitrate to assess the degree of adrenergic beta receptor blockade

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Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy:effects of laser photocoagulation and angiotensin receptor blockade

Background: Proliferative diabetic retinopathy (PDR) may be a response to abnormal angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and the soluble angiopoietin receptor tie-2. The authors hypothesised the following: (a) there are differences in plasma levels of these growth factors in different grades of diabetic retinopathy; and (b) that the effects of intervention with panretinal laser photocoagulation (PRP) for PDR, and angiotensin receptor blockade (using eprosartan) for patients with other grades of diabetic retinopathy will be to reduce levels of the growth factors. Methods: Cross sectional and interventional study (using PRP and eprosartan) in diabetic patients. VEGF, Ang-2, and tie-2 were measured by ELISA. Results: VEGF (p<0.001) and Ang-2 levels (p<0.001) were significantly higher in 93 diabetic patients compared to 20 healthy controls, with the highest levels in grade 2 and grade 3 diabetic retinopathy (p<0.05). Tie-2 was lower in diabetics compared to controls (p = 0.008), with no significant differences between the diabetic subgroups. Overall, VEGF significantly correlated with Ang-2 (p<0.001) and tie-2 (p = 0.004) but the correlation between Ang-2 and tie-2 levels was not significant (p = 0.065). Among diabetic patients only, VEGF levels were significantly correlated with Ang-2 (p<0.001) and tie-2 (p<0.001); the correlation between Ang-2 and tie-2 levels was also significant (p<0.001). There were no statistically significant effects of laser photocoagulation on plasma VEGF, Ang-2, and tie-2 in the 19 patients with PDR, or any effects of eprosartan in the 28 patients with non-proliferative diabetic retinopathy. Conclusion: Increased plasma levels of VEGF and Ang-2, as well as lower soluble tie-2, were found in diabetic patients. The highest VEGF and Ang-2 levels were seen among patients with pre-proliferative and proliferative retinopathy, but there was no relation of tie-2 to the severity of retinopathy. As the majority of previous research into Ang-2 and tie-2 has been in relation to angiogenesis and malignancy, the present study would suggest that Ang-2 and tie-2 may be used as potential indices of angiogenesis in diabetes mellitus (in addition to VEGF) and may help elucidate the role of the angiopoietin/tie-2 system in this condition.

Pharmacologically induced and stimulus evoked rhythmic neuronal oscillatory activity in the primary motor cortex in vitro

Parkinson's disease (PD) is associated with enhanced synchronization of neuronal network activity in the beta (15-30 Hz) frequency band across several nuclei of the basal ganglia (BG). Deep brain stimulation of the subthalamic nucleus (STN) appears to reduce this pathological oscillation, thereby alleviating PD symptoms. However, direct stimulation of primary motor cortex (M1) has recently been shown to be effective in reducing symptoms in PD, suggesting a role for cortex in patterning pathological rhythms. Here, we examine the properties of M1 network oscillations in coronal slices taken from rat brain. Oscillations in the high beta frequency range (layer 5, 27.8 +/- 1.1 Hz, n=6) were elicited by co-application of the glutamate receptor agonist kainic acid (400 nM) and muscarinic receptor agonist carbachol (50 mu M). Dual extracellular recordings, local application of tetrodotoxin and recordings in M1 micro-sections indicate that the activity originates within deep layers V/VI. Beta oscillations were unaffected by specific AMPA receptor blockade, abolished by the GABA type A receptor (GABAAR) antagonist picrotoxin and the gap-junction blocker carbenoxolone, and modulated by pentobarbital and zolpidem indicating dependence on networks of GABAergic interneurons and electrical coupling. High frequency stimulation (HFS) at 125 Hz in superficial layers, designed to mimic transdural/transcranial stimulation, generated gamma oscillations in layers 11 and V (incidence 95%, 69.2 +/- 7.3 Hz, n=17) with very fast oscillatory components (VFO; 100-250 Hz). Stimulation at 4 Hz, however, preferentially promoted theta activity (incidence 62.5%, 5.1 +/- 0.6 Hz, n=15) that effected strong amplitude modulation of ongoing beta activity. Stimulation at 20 Hz evoked mixed theta and gamma responses. These data suggest that within M1, evoked theta, gamma and fast oscillations may coexist with and in some cases modulate pharmacologically induced beta oscillations.

Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Blockade of interleukin 6 signaling improves the survival rate of transplanted bone marrow stromal cells and increases locomotor function in mice with spinal cord injury

Bone marrow stromal cells (BMSCs) have the potential to improve functional recovery in patients with spinal cord injury (SCI); however, they are limited by low survival rates after transplantation in the injured tissue. Our objective was to clarify the effects of a temporal blockade of interleukin 6 (IL-6)/IL-6 receptor (IL-6R) engagement using an anti-mouse IL-6R monoclonal antibody (MR16-1) on the survival rate of BMSCs after their transplantation in a mouse model of contusion SCI. MR16-1 cotreatment improved the survival rate of transplanted BMSCs, allowing some BMSCs to differentiate into neurons and astrocytes, and improved locomotor function recovery compared with BMSC transplantation or MR16-1 treatment alone. The death of transplanted BMSCs could be mainly related to apoptosis rather than necrosis. Transplantation of BMSC with cotreatment of MR16-1 was associated with a decrease of some proinflammatory cytokines, an increase of neurotrophic factors, decreased apoptosis rates of transplanted BMSCs, and enhanced expression of survival factors Akt and extracellular signal-regulated protein kinases 1/2. We conclude that MR16-1 treatment combined with BMSC transplants helped rescue neuronal cells and axons after contusion SCI better than BMSCs alone by modulating the inflammatory/immune responses and decreasing apoptosis. © 2013 by the American Association of Neuropathologists, Inc.

25-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages

Recent studies have shown that Toll-like receptor (TLR)- signalling contributes significantly to the inflammatory events of atherosclerosis. As products of cholesterol oxidation (oxysterols) accumulate within atherosclerotic plaque and have been proposed to contribute to inflammatory signalling in the diseased artery, we investigated the potential of 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-HC) and 25-hydroxycholesterol (25-HC) to stimulate inflammatory signalling via the lipid-recognising TLRs 1, 2, 4 and 6. Each oxysterol stimulated secretion of the inflammatory chemokine interleukin-8 (IL-8), but not I?B degradation or tumour necrosis factor- release from monocytic THP-1 cells. Transfection of TLR-deficient HEK-293 cells with TLRs 1, 2, 4 or 6 did not increase sensitivity to the tested oxysterols. Moreover, blockade of TLR2 or TLR4 with specific inhibitors did not reduce 25-hydroxycholesterol (25-HC) induced IL-8 release from THP-1 cells. We conclude that although the oxysterols examined in this study may contribute to increased expression of certain inflammatory genes, this occurs by mechanisms independent of TLR signalling.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2

To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

1 Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. 2 In the presence of indomethacin (3μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC50 8.0 ± 0.1, n = 5). 3 Establishment of a rank order of potency using the EP4 > EP2 agonist 11-deoxy PGE1, and the EP2 > EP4 agonist PGE1-OH (mean pEC 50 of 7.6 ± 0.1 (n = 6) and 6.4 ± 0.1 (n = 4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations < 1 μM failed to relax the tissues. 4 Blockade of EP 4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration-response curves, exhibiting pA2 and pKB values of 5.7 ± 0.1, n = 3, and 8.4, n = 3, respectively. 5 The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3 ± 0.1 (n = 4) and 8.1 ± 0.1 (n = 9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGFα2 failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC50 7.4 ± 0.3, n = 3). 6 These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

Blockade of interleukin-6 effects on cytokine profiles and macrophage activation after spinal cord injury in mice

The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. MR16-1 antibodies versus isotype control antibodies or saline alone was administered immediately after thoracic SCI in mice. MR16-1-treated group samples showed increased neuronal regeneration and locomotor recovery compared with controls. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. MR16-1 treatment promoted arginase-1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site and enhanced positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.