Synergistic actions of blocking angiopoietin-2 and tumor necrosis factor-α in suppressing remodeling of blood vessels and lymphatics in airway inflammation.

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.

Angiopoietin-2 promotes inflammatory activation of human macrophages and is essential for murine experimental arthritis.

BACKGROUND: Angiopoietin (Ang)-1 and Ang-2, and their shared receptor Tie2, are expressed in rheumatoid arthritis (RA) synovial tissue, but the cellular targets of Ang signalling and the relative contributions of Ang-1 and Ang-2 to arthritis are poorly understood. OBJECTIVES: To determine the cellular targets of Ang signalling in RA synovial tissue, and the effects of Ang-2 neutralisation in murine collagen-induced arthritis (CIA). METHODS: RA and psoriatic arthritis (PsA) synovial biopsies were examined for expression of Tie2 and activated phospho (p)-Tie2 by quantitative immunohistochemistry and immunofluorescent double staining. Human monocyte and macrophage Tie2 expression was determined by flow cytometry and quantitative PCR. Regulation of macrophage intracellular signalling pathways and gene expression were examined by immunoblotting and ELISA. CIA was assessed in mice treated with saline, control antibody, prednisolone or neutralising anti-Ang-2 antibody. RESULTS: Expression of synovial Tie2 and p-Tie2 was similar in RA and PsA. Tie2 activation in RA patient synovial tissue was predominantly localised in synovial macrophages and was expressed by human macrophage. Ang-1 and Ang-2 stimulated activation of multiple intracellular signalling pathways, and cooperated with tumour necrosis factor to induce macrophage interleukin 6 and macrophage inflammatory protein 1α production. Ang-2 selectively suppressed macrophage thrombospondin-2 production. Ang-2 neutralisation significantly decreased disease severity, synovial inflammation, neo-vascularisation and joint destruction in established CIA. CONCLUSIONS: The authors identify synovial macrophages as primary targets of Ang signalling in RA, and demonstrate that Ang-2 promotes the pro-inflammatory activation of human macrophages. Ang-2 makes requisite contributions to pathology in CIA, indicating that targeting Ang-2 may be of therapeutic benefit in the treatment of RA.

Chemopreventive properties of a selective inducible nitric oxide synthase inhibitor in colon carcinogenesis, administered alone or in combination with celecoxib, a selective cyclooxygenase-2 inhibitor.

The inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) are overexpressed in colonic tumors of humans, as well as in colon tumors that develop in rats after the administration of the colon-specific carcinogen, azoxymethane (AOM). iNOS may regulate COX-2 production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to assess the potential chemopreventive properties of highly selective iNOS inhibitors, administered individually and in combination with a selective COX-2 inhibitor, on the development of AOM-induced colonic aberrant crypt foci (ACF). F344 rats were fed experimental diets containing one of the following: 0, 10, 30, or 100 parts/million (ppm) of the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); 1800 ppm of the less potent, selective iNOS inhibitor aminoguanidine (AG); 500 ppm of the COX-2 inhibitor celecoxib; 320 ppm of the nonsteroidal anti-inflammatory sulindac (positive control); or 30 ppm of SC-51 with 500 ppm of celecoxib, and 100 ppm of SC-51 with 500 ppm of celecoxib. One and 2 weeks later, rats received s.c. injections of AOM at a dose of 15 mg/kg of body weight. At 17 weeks of age, all rats were sacrificed. Colons were evaluated for ACF, and colonic mucosae were assayed for COX and NOS isoform enzyme activities. Samples of venous blood, collected at various time points, were analyzed for these agents. SC-51, administered alone, demonstrated dose-dependent inhibition of the incidence of colonic ACF. The highest doses of SC-51 (100 ppm) and AG (1800 ppm) significantly suppressed the incidence of colonic ACF (P < 0.01 and < 0.001, respectively) and crypt multiplicity in terms of numbers of aberrant crypts/focus (P < 0.0001). Importantly, the combination of either low or high effective doses of SC-51 (30 or 100 ppm) and celecoxib (500 ppm) suppressed AOM-induced colonic ACF formation (P < 0.05 and < 0.001, respectively) and reduced multiplicity of four or more aberrant crypts/focus (P < 0.0001) to a greater extent than did these agents administered individually. As expected, sulindac inhibited colonic ACF formation (P < 0.001) and reduced the multiplicity of four or more aberrant crypts (P < 0.0001) to approximately 45%. The enzymatic activities of COX-2 and iNOS were significantly induced in the AOM-treated animals, and administration of the iNOS inhibitors, SC-51 and AG, significantly inhibited the activities of both iNOS and COX-2 in the colonic mucosa. The combined administration of SC-51 and celecoxib inhibited the COX-2 activity to a greater extent than did either of these agents administered alone. These findings support the hypothesis that selective iNOS inhibitors may have chemopreventive properties and that coadministration with a selective COX-2 inhibitor may have additional chemopreventive potential.

Regulation of the early subnormal retinal oxygenation response in experimental diabetes by inducible nitric oxide synthase.

We aimed to test the hypothesis that the inducible form of nitric oxide synthase (iNOS) contributes to the development of an early subnormal retinal oxygenation response in preclinical models of diabetic retinopathy. In urethane anesthetized Sprague Dawley rats or C57BL/6 mice, functional magnetic resonance imaging was used to noninvasively measure the change in retinal oxygen tension (Delta PO(2)) during a carbogen-inhalation challenge. In the rat experiments, the retinal Delta PO(2) of the following groups were compared: control rats (n = 9), 3-month diabetic rats (n = 5), and 3-month diabetic rats treated orally with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of an inhibitor of iNOS (n = 6). In addition, the retinal Delta PO(2) of the following mouse groups were compared: C57BL/6 mice (n = 20), C57BL/6-Nos2(tm1 Lau) mice (n = 10), 4-month diabetic mice (n = 13), and 4-month diabetic knockout mice (n = 6). Only the Delta PO(2) of the superior hemiretina of the diabetic rat and mice groups were significantly subnormal (P < 0.05). The superior Delta PO(2) of the diabetic rats treated with the prodrug was not significantly (P > 0.05) different from their respective normal controls. In the mice experiments, the superior retinal Delta PO(2) of the iNOS null mice was not statistically different (P > 0.05) from that of normal control mice. iNOS is required for the development of an early subnormal Delta PO(2) in experimental diabetic retinopathy.

Drug intervention can correct subnormal retinal oxygenation response in experimental diabetic retinopathy.

PURPOSE: Early subnormal retinal oxygenation response to a hyperoxic provocation (DeltaPo2) is strongly associated with subsequent experimental diabetic retinopathy and can be reversed by drug treatments started with the induction of diabetes. It is not yet known whether drug treatment can reverse an established subnormal DeltaPo2. METHODS: Retinal DeltaPo2 was measured in two separate experimental paradigms in streptozotocin-induced diabetic rats. In a prevention study, measurements were performed in untreated diabetic rats, 3 months after the initiation of hyperglycemia (D3mo), in age-matched nondiabetic rats (C3mo), and in diabetic rats treated orally for 3 months with celecoxib, a cyclooxygenase (COX)-2-selective inhibitor, (D3mo+COX2i). In an intervention study, measurements were performed in untreated diabetic rats 4 months after the initiation of diabetes (D4mo), in age-matched nondiabetic rats (C4mo), and in diabetic rats that were untreated for 3 months and then were orally treated for an additional month with either celecoxib (D4mo+COX2i) or l-N (6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of an inhibitor of inducible nitric oxide synthase (iNOS, D4mo+ iNOSi). RESULTS: In the prevention arm, subnormal (P < 0.05) retinal DeltaPo2 was found in the D3mo group, but not in the D3mo+COX2i group (P > 0.05). In a previous study, it was reported that retinal DeltaPo2 also corrected in a D3mo+iNOSi group. In the intervention arm, retinal DeltaPo2 levels in the D4mo and D4mo+iNOSi, but not the D4mo+COX2i, groups were (P < 0.05) subnormal. CONCLUSIONS: These results demonstrate, for the first time, that drug treatment can reverse an established subnormal DeltaPo2. Furthermore, this effect could not be predicted by a drugs' ability to prevent the development of subnormal DeltaPo2.

Synthesis and biological characterization of L-N(6)-(1-iminoethyl)lysine 5-tetrazole-amide, a prodrug of a selective iNOS inhibitor.

The 5-tetrazole amide of L-N(6)-(1-iminoethyl)lysine (L-NIL), L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide (1), has been prepared and evaluated. In contrast to L-NIL, 1 is a stable, nonhygroscopic, crystalline solid. Unlike L-NIL, 1 has minimal inhibitory activity in vitro on human inducible nitric oxide synthase (iNOS). However, it is rapidly converted in vivo to L-NIL and produces dose-dependent inhibition of iNOS in acute and chronic models of inflammation in the rodent with efficacy comparable to L-NIL. In addition, both 1 and L-NIL exhibit significant and comparable in vivo selectivity for the inhibition of iNOS vs endothelial NOS. Doses approximately 80-fold greater than those that inhibited inflammation do not elevate systemic blood pressure. In summary, both the physical properties and the pharmacological profile of 1 make it an ideal molecule for preclinical and clinical studies on the role of selective iNOS inhibitors in mediating inflammatory disease processes.

A prodrug of a selective inhibitor of inducible nitric oxide synthase is neuroprotective in the rat model of glaucoma.

PURPOSE: To test the hypothesis that nitric oxide, synthesized by inducible nitric oxide synthase, causes degeneration of retinal ganglion cells in an animal model of glaucoma. METHODS: Rats with unilateral, chronic, moderately elevated intraocular pressure were treated orally with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of an inhibitor of inducible nitric oxide synthase. The loss of retinal ganglion cells was quantitated as an indicator of glaucomatous damage. RESULTS: At the end of seven months, rat eyes with chronic, moderately elevated intraocular pressure lost approximately 20,000 retinal ganglion cells. Treatment with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide for seven months completely prevented the loss of retinal ganglion cells in eyes with chronic, moderately elevated intraocular pressure. When treatment with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide was delayed and started after three months of chronic, moderately elevated intraocular pressure, further loss of retinal ganglion cells was prevented. CONCLUSION: Pharmacological neuroprotection with a selective inhibitor of inducible nitric oxide synthase may be useful for the treatment of glaucoma.

Loss of retinal ganglion cells following retinal ischemia: the role of inducible nitric oxide synthase.

Following experimental, transient, retinal ischemia in the rat, there is loss of retinal neurons, which occurs over several weeks. Retinal ganglion cells (RGCs) are particularly susceptible and there is early, massive degeneration of these neurons after ischemia. We have determined the early mechanisms by which RGCs are killed following ischemia. Retinal ischemia/reperfusion was produced in rats by transient unilateral elevation of intraocular pressure above systolic blood pressure. Retinas were studied by immunohistochemistry for the presence of inducible nitric oxide synthase (NOS-2) at several time points post-ischemia and specific cell types were identified. Rats were also treated orally with L -N(6) -(1-iminoethyl)lysine 5-tetrazole amide (SC-51), a prodrug of an inhibitor of NOS-2 or with aminoguanidine (AG) for a period of 14 days. Retrograde labelling with Fluoro-Gold quantitated the loss of RGCs. NOS-2 was not present in the normal retina and was not present in the eyes that were contralateral to the ischemic eyes. Within 24hr after ischemia, polymorphonuclear leukocytes containing NOS-2 had entered the ganglion cell layer and surrounded RGCs. Within 5 days after ischemia, NOS-2 was present in many inner retina cells and in invading monocytes in the vitreous. Between 7 and 14 days post-ischemia, there were few hematogenous cells in the retina but NOS-2 was sparsely detectable in microglia and other cells of the inner retina. Two weeks after ischemia, rat eyes lost approximately 50% of the RGCs. Treatment with AG for 14 days following ischemia was partially neuroprotective; approximately 28% of the RGCs were lost. Treatment with SC-51 for 14 days following ischemia almost completely prevented the loss of RGCs. Thus, within 24hr following ischemia, polymorphonuclear leukocytes containing NOS-2 attack and kill neurons in the ganglion cell layer. For 2 weeks after ischemia, NOS-2 appears transiently in the retina in several different cell types at different times. Continuous pharmacological treatment with inhibitors of NOS-2 activity during the 2 weeks post-ischemia period provides significant neuroprotection against the loss of RGCs.

Quantitative ex vivo detection of rodent retinal ganglion cells by immunolabeling Brn-3b.

To evaluate the neuroprotective potential of drug candidates to treat human glaucoma, a short-term rodent model of retinal ganglion cell death was employed. Transient ischemia applied to the rodent retina, with subsequent reperfusion for 1-4 weeks, produces an experimental retinal ganglion cell death that is quantifiable. A widely used method to detect viable retinal ganglion cells involves surgical injection of labeling compounds into the superior colliculus of the rodent brain, the retrograde transport of the compounds along the axons to the retina, and subsequent microscopic evaluation of the retina. In order to circumvent the labor intensive and invasive surgery of this method, we sought an alternative means of assessing retinal ganglion cell survival that would be more suitable for high-throughput analysis. We therefore developed a method of immunolabeling whole retinas ex vivo with an antibody to Brn-3b, an antigen expressed in a subpopulation of retinal ganglion cells, that allows for detection of a representative retinal ganglion cell population. Fluorescently tagged Brn-3b immunolabeled retinas were flat-mounted, digitally imaged, and assessed using image analysis software. We determined that 60 min of ischemia caused a 49% and a 32% decrease in Brn-3b positive retinal ganglion cells in Lewis rats after 4 weeks reperfusion, and Sprague-Dawley rats after 2 weeks reperfusion, respectively. In Swiss Webster ND4 mouse retinas subjected to 45 min ischemia and 7 days reperfusion, we found a 70% decrease in Brn-3b positive cells. Thus, ex vivo immunolabeling of retinal ganglion cells using antibody to Brn-3b provides an alternative to other methods of quantifying retinal ganglion cells.

3-Hydroxy-4-methyl-5-pentyl-2-iminopyrrolidine: a potent and highly selective inducible nitric oxide synthase inhibitor.

(3S,4S,5R)-2-Imino-4-methyl-5-pentyl-3-pyrrolidinol hydrochloride (1) is a potent inducible nitric oxide synthase (i-NOS) inhibitor that has three times the selectivity of its parent, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine hydrochloride (2).