Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.

Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting pro-inflammatory nitric oxide (NO) production by macrophages (Mϕs). Consistent with previous observations, we observed that although Mϕs stimulated in the presence of AqH expressed NO-synthase-2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of L-arginine (L-Arg) into L-citrulline, was augmented in lysates of Mϕs stimulated in the presence of AqH. These data suggested that intracellular L-arg may have been limited by AqH. However, we observed increased mRNA expression of the L-arg transporter, CAT2B, and increased L-arg uptake in Mϕs stimulated in the presence of AqH. Arginases were expressed by stimulated Mϕs but competition for L-arg with NOS2 was excluded. Expression of GTP cyclohydrolase which produces tetrahydrobiopterin (H4B), an essential cofactor for NOS2 homodimerization, increased after Mϕ stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together these data provided no evidence for inhibited NOS2 enzymatic activity by AqH suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in Mϕs via ascorbate which limited degradation of H4B. Therefore, AqH may augment NO production in macrophages by stabilizing H4B and increasing intracellular L-Arg.

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Classical cavernous sinus embolism is a rare clinical finding, presented most commonly by complaints of headache, diplopia, visual field defects, facial pain, and progressive neurological deficits. Many patients exhibit symptoms of III, IV, and VI nerve palsies. We hereby report a rare case of aseptic cavernous sinus embolism developed in a 75-year-old male with primary lung cancer who presented with binocular diplopia due to unilateral third and sixth cranial nerve palsies with pupil-sparing. The possibility of cavernous sinus cancer embolus should be considered if the routine examination excluded metastases, infiltration, carcinomatous meningitis, or the paraneoplastic process. 18F-FDG PET imaging may provide a promising diagnostic modality for the diagnosis of cancer embolus.

Objectives Peripheral arterial disease (PAD) is a systemic inflammatory disorder that affect millions of people worldwide. The secretion of Interleukin-1 beta (IL-1β) and Interleukin-18 (IL-18) is controlled by inflammasomes. Rencent study showed that Nlrp3 inflammasome activation was linked to mitochondrial dysfunction and damaged tissue could release mitochondrial DAMPs. The role of Nlrp3 inflammasome in PAD is still unknown. The present study investigated the role of Nlrp3 inflammasome in mouse hindlimb ischaemia and whether mitochondrial DAMP fMLP could activate Nlrp3 inflammasome. Methods Hindlimb ischaemia was induced in C56BL/6, Nlrp3 knockout and Caspase 1 knockout mice. Blood flow recovery was measured by Laser Doppler Perfusion Imaging (LDPI) each week. The mobilisation and function of endothelial progenitor cells (EPCs) of these mice were tested. Bone marrow derived macrophage cells (BMMCs) of these mice were treated with fMLP and hypoxia. IL-1β and IL-18 were examined in plasma and supernatant of cell culture medium after treatment. Results The blood flow recovery of Nlrp3 knockout mice and Caspase 1 knockout mice was retarded compared with wild type mice. Nlrp3 and Caspase1 deficiency dampened mobilisation and adhesion but not tube formation or migration capability of EPCs. Mitochondrial DAMP fMLP together with hypoxia activated Nlrp3 inflammasome in BMMCs and induced the release of IL-1β, which was Caspase 1 dependent. IL-1β and IL-18 subsequently promoted tube formation capability of EPCs. Conclusions The Mitochondrial DAMP fMLP activated the Nlrp3 Inflammasome, which promoted angiogenesis after hindlimb Ischaemia.