The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1α(βS*, δS*), 2α, 6α, 8β-(R*), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1∝(ßS*,dS*),2∝,6a∝,8ß-(R*),8a∝]]-1,2,6,7,8,8a-hexahydro-ß,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 ± 1.9% versus 16.6 ± 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 ± 2.9% versus 23.4 ± 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC50, 6.37 ± 0.37) compared with both vehicle-treated (-logEC50, 5.81 ± 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 ± 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 ± 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries

Background: Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. The existence of single or multiple VEGF isoforms and receptors suggests that these proteins may have overlapping but distinct functions, which may be reflected in their cell expression and distribution. Methods: The localisation of VEGFs A–C and their receptors (VEGFRs 1–3, respectively) in 30 fresh human atherosclerotic arteries, 15 normal uterine arteries, and 15 saphenous veins using immunohistochemistry and western blotting. Results: Saphenous veins showed no staining for VEGF-B or VEGFR-2. Smooth muscle cells (SMCs) showed the strongest staining for VEGF-A, VEGF-B, VEGFR-1, and VEGFR-2 in all specimens. Conversely, VEGFR-3 and VEGF-C were predominately localised to the endothelial vasa vasorum in normal arteries, whereas medial SMCs showed the strongest staining in atherosclerotic arteries. Western blotting showed variations in VEGF protein localisation, with lower amounts of VEGF-B and VEGF-C in saphenous veins, compared with arterial tissue. Amounts of VEGF-C were lower than those of VEGF-A and VEGF-B in all specimens. Conclusion: This study provides direct evidence of the presence of VEGF proteins and receptors in human physiology and pathology, with variations in both the amounts of VEGF proteins expressed and their cellular distribution in normal arteries compared with atherosclerotic arteries. The presence of VEGFs A–C and their receptors in normal arterial tissue implies that VEGF functions may extend beyond endothelial cell proliferation. Reduced VEGFR-2 staining in atherosclerotic arteries may have implications for the atherosclerosis process and the development of vascular disease and its complications.

Synthesis of substituted 3-anilino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepine-2-ones and their evaluation as cholecystokinin-ligands

3-Amino-1,4-benzodiazepines as well as chemically related diverse amines were prepared from oxazepam and subsequently screened on the cholecystokinin receptor in a radiolabel binding assay. Oxazepam 2 was activated via its 3-chloro-1,4-benzodiazepine intermediate 3 and was reacted with a large series of aliphatic and aromatic amines. The substituted 3-anilino-1,4-benzodiazepine structure was identified as lead structure in a diverse series of 3-amino-1,4-benzodiazepines 4-38 and the full SAR (structure-activity relationship) optimisation provided 3-anilinobenzodiazepines 16-38 with CCK 1 receptor selectivity to CCK 2. The compounds 18, 24, 28 and 33 have shown affinities at the CCK 1 receptor of 11, 10, 11 and 9 nM, respectively. These equipotent CCK 1 ligands were fully evaluated in behaviour pharmacological essays. An antidepressant effect was identified in the tail suspension- and the Porsolt swimming-test. The ED 50 values for 24 and 28 were determined in these assays as 0.46 and 0.49 mg/kg. The mixed antagonist 37 showed in addition to the antidepressant effects anxiolytic properties. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Greater CD8+ TCR heterogeneity and functional flexibility in HIV-2 compared to HIV-1 infection

Virus-specific CD8+ T cells are known to play an important role in the control of HIV infection. In this study we investigated whether there may be qualitative differences in the CD8+ T cell response in HIV-1- and HIV-2-infected individuals that contribute to the relatively efficient control of the latter infection. A molecular comparison of global TCR heterogeneity showed a more oligoclonal pattern of CD8 cells in HIV-1- than HIV-2-infected patients. This was reflected in restricted and conserved TCR usage by CD8+ T cells recognizing individual HLA-A2- and HLA-B57-restricted viral epitopes in HIV-1, with limited plasticity in their response to amino acid substitutions within these epitopes. The more diverse TCR usage observed for HIV-2-specific CD8 T cells was associated with an enhanced potential for CD8+ expansion and IFN- production on cross-recognition of variant epitopes. Our data suggest a mechanism that could account for any possible cross-protection that may be mediated by HIV-2-specific CD8+ T cells against HIV-1 infection. Furthermore, they have implications for HIV vaccine development, demonstrating an association between a polyclonal, virus-specific CD8+ T cell response and an enhanced capacity to tolerate substitutions within T cell epitopes.

Antidepressant/anxiolytic and anti-nociceptive effects of novel 2-substituted 1,4-benzodiazepine-2-ones

Oxazepam (4a) has been used as overall starting material in the synthesis of novel 2-substituted 1,4-benzodiazepines. By reacting Oxazepam 4a with commercially available hydrazines, hydrazides, semicarbazide, aminoguanidine and N,N-dimethylamino aniline in ethanol under acetic conditions, a series of diazenyl-1,4-benzodiazepines 5a-5i and 2-amino- 1,4-benzodiazepine 5k were obtained in good yields. These novel compounds served as new chemical entities (NCE) for testing in mice. The diazo-benzodiazepine 5d has shown a promising antidepressant effect in initial experiments in vivo at a dose of 5 mg/kg. The highly coloured 2-aminobenzodiazepine derivative 5k showed over a dose range from 5-50 mg/kg an analgesic effect in mice. © Singh et al.