Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1β, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1β knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1β has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

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(1) Patients with AAA have fewer atherosclerotic risk factors than do patients with AOD. (2) Patients with AAA and AOD have significantly different inflammatory activity. (3) The data supports the hypothesis that AAA and AOD are probably two different pathological entities.

Matrix metalloproteinases (MMPs) have recently been implicated in the pathogenesis of multiple sclerosis. Their suggested role includes the disruption of the blood-brain barrier, immune cell transmigration into the central nervous system, and myelin degradation. The present study characterized the mRNA level of a wide spectrum of MMPs and tissue inhibitors of metalloproteinases (TIMPs) expressed by peripheral blood leukocytes from relapsing-remitting (n = 16) and secondary-progressive (n = 12) multiple sclerosis patients. The expression of the same MMPs and TIMPs was evaluated also in a prospective 12-month follow-up of 6 patients randomly chosen from each of the 2 groups during interferon beta-1a treatment. Reverse transcription-polymerase chain reaction assessment demonstrated elevated levels of MT1-MMP and MMP-7 mRNA levels in both groups of patients, and no significant differences in MMP-9 levels, compared with healthy controls. Divergent expression of MMP-2 between relapsing-remitting and secondary-progressive patients compared with controls was observed. Interferon-beta treatment was associated with significant suppression of MMP-9 and MMP-7 mRNA in relapsing-remitting patients, though no significant changes were observed in the secondary-progressive group. These results contribute to the understanding of the IFN-beta-mediated immunomodulatory and therapeutic effects in multiple sclerosis patients and also support evidence for distinct immune mechanism(s) underlying relapsing-remitting versus secondary-progressive multiple sclerosis. The study also suggests that MMPs may be considered as potential biomarkers for response to treatment as well as targets for immunotherapy in multiple sclerosis.

Coeliac disease (CD), an inflammatory enteropathy, is believed to be caused by immune sensitivity to ingested gluten. T‐cell activation appears to be implicated in the disease although little is known regarding the role of T‐cell subsets, Th1/Th2, and the cytokines they secrete. Reverse transcription‐polymerase chain reaction was used to examine the mRNA expression of a wide profile of cytokines in intestinal and peripheral samples taken from active and inactive CD paediatric patients. Differential mRNA expression was observed for cytokines, between CD patients and controls, in both compartments. The percentage of samples expressing interleukin (IL)‐2, interferon (IFN)‐γ, tumor necrosis factor (TNF)‐β, IL‐10, IL‐1β, TNF‐α and transforming growth factor (TGF)‐β mRNA from active CD patients was higher than from controls. A prominent finding was the expression of both Th1 (IFN‐γ, IL‐2) and Th2 (IL‐4, IL‐10)‐associated cytokine transcripts in the same biopsies and peripheral blood cells from patients with active CD implying activation of Th0 cells. The expression of IL‐2 and IL‐4 mRNA was not observed in peripheral blood samples from inactive CD patients associating them with disease activity. These results are important to the understanding of the inflammatory process in CD while cytokine levels may prove to be relevant markers of disease activity.

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase-9 (MMP-9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor alpha (TNF-alpha)]. We show that hypoxia (<0.3% O2 for 48 h) reduced the output of TNF-alpha-induced proMMP-9 by threefold (P < 0.01) in the U937 monocytic cell line and in primary human monocytes. TNF-alpha induced MMP-9 transcription by threefold, but no significant difference was observed in MMP-9 mRNA steady-state between normoxia and hypoxia, which inhibited the trafficking of proMMP-9 via secretory vesicles and increased the intracellular accumulation of proMMP-9 in the cells by 47% and 62% compared with normoxia (P < 0.05), as evaluated by zymography of cellular extracts and confocal microscopy, respectively. Secretion of proMMP-9 was reduced by the addition of cytochalazin B or nocodazole, which inhibits the polymerization of actin and tubulin fibers, or by the addition of the Rho kinase inhibitor Y27632, suggesting the involvement of the cytoskeleton and the Rho GTPases in the process of enzyme secretion. Furthermore, attachment of proMMP-9 to the cell membrane increased after hypoxia via its interactions with surface molecules such as CD44. In addition, the reduced migration of monocytes in hypoxia was shown to be mediated, at least partially, by secreted MMP-9. Thus, hypoxia post-translationally reduced the secreted amounts of proMMP-9 by using two mutually nonexclusive mechanisms: mostly, inhibition of cellular trafficking and to a lesser extent, attachment to the membrane.

Exposure of endothelial cells (ECs) to hypoxia has separately been shown to induce their angiogenesis or death. Matrix metalloproteinase (MMP)-2 is associated with EC angiogenesis, although recent studies also implicate this molecule in EC death. We studied the effect of hypoxia in the absence or presence of TNF-alpha (characteristic of the inflammatory microenvironment accompanying hypoxia) on MMP-2 expression and its role in angiogenesis (proliferation, migration, and tube formation) and in the death of primary human umbilical vein endothelial cells (HUVECs). Hypoxia alone (24-48 h in 0.3% O(2) in the hypoxic chamber) and furthermore, when combined with TNF-alpha, significantly enhanced MMP-2 expression and activity. Hypoxia also led to a reduction in membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 mRNA and protein while enhancing the expression of alpha(v)beta(3) integrin and the cytoskeletal protein phosphopaxillin. Moreover, hypoxia led to colocalization of alpha(v)beta(3) and MMP-2, but not MT1-MMP, with phosphopaxillin in ECs. These results suggest MT1-MMP-independent activation of MMP-2 during hypoxia and support interactions between the ECM, integrins, and the cytoskeleton in hypoxia-induced MMP-2-related functions. Hypoxia enhanced EC migration in an MMP-2-dependent manner while leading to a reduction of cell number via their apoptosis, which was also dependent on MMP-2. In addition, hypoxia caused an aberrant tubelike formation on Matrigel that appeared to be unaffected by MMP-2. The hypoxia-induced, MMP-2-dependent migration of ECs is in accordance with the proangiogenic role ascribed to MMP-2, while the involvement of this protease in the hypoxia-related death of ECs supports an additional apoptotic role for this protease. Hence, in the hypoxic microenvironment, MMP-2 appears to have a dual autocrine role in determining the fate of ECs.