The development of the devastating neurodegenerative condition, Alzheimer's disease, is strongly associated with amyloid-␤ (A␤) deposition, neuronal apoptosis, and cell loss. Here, we provide evidence that implicates these same mechanisms in the retinal disease glaucoma, a major cause of irreversible blindness worldwide, previously associated simply with the effects of intraocular pressure. We show that A␤ colocalizes with apoptotic retinal ganglion cells (RGC) in experimental glaucoma and induces significant RGC apoptosis in vivo in a dose-and time-dependent manner. We demonstrate that targeting different components of the A␤ formation and aggregation pathway can effectively reduce glaucomatous RGC apoptosis in vivo, and finally, that combining treatments (triple therapy) is more effective than monotherapy. Our work suggests that targeting the A␤ pathway provides a therapeutic avenue in glaucoma management. Furthermore, our work demonstrates that the combination of agents affecting multiple stages in the A␤ pathway may be the most effective strategy in A␤-related diseases.combination therapy ͉ neuroprotection ͉ retinal ganglion cell apoptosis A lthough glaucoma, a major cause of blindness worldwide (1), is commonly linked to raised intraocular pressure (IOP) (2), the precise means by which IOP may lead to the irreversible destruction of retinal ganglion cells (RGCs, which are the nerve cells that transfer visual information from the eye to the brain) is far from clear. Indeed, interpretation of the mechanism is further complicated by the fact that damage can also occur at low IOP. Thus, for example, recent evidence indicates progressive visual-field loss in patients despite normalization of IOP with pressure-lowering treatment strategies (3, 4), which means that an alternative approach to the treatment of glaucoma is urgently needed. The principal step leading to irreversible loss of vision in glaucoma is RGC apoptosis, and the question is what mechanisms precede this cell death. Raised IOP in experimental glaucoma models can clearly precipitate RGC apoptosis (5-7) whatever the actual intervening steps. However, the presence of progressive glaucomatous damage in patients with normalized IOP has focused a growing body of work on alternative strategies to those regulating IOP and especially approaches targeting the cellular mechanisms leading to apoptosis.Amyloid-␤ (A␤) is the major constituent of senile plaques in Alzheimer's disease (AD), the formation of which, caused by abnormal processing of amyloid precursor protein (APP), has been involved in AD neuropathology, although the proximate cause of the neurodegeneration responsible for cognitive impairment is not clear (8). A␤ has recently been reported to be implicated in the development of RGC apoptosis in glaucoma, with evidence of caspase-3-mediated abnormal APP processing and increased expression of A␤ in RGCs in experimental glaucoma (9) and decreased vitreous A␤ levels (consistent with retinal A␤ deposition) in patients with glaucoma (10). Further evidenc...

By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.

The glucocorticoid-inducible protein annexin (ANXA) 1 is an anti-inflammatory mediator that down-regulates the host response. Endogenously, ANXA1 is released in large amounts from adherent polymorphonuclear neutrophils (PMN) and binds to their cell surface to inhibit their extravasation into inflamed tissues. The present study determined the effects of exogenous ANXA1 on several functions of human PMN in vitro. Addition of 0.1-1 microM human recombinant ANXA1 to the PMN provoked rapid and transient changes in intracellular Ca2+ concentrations that were blocked by the Ca2+ channel inhibitor SKF-96365. Although ANXA1 did not affect oxidant production and only minimally affected PMN chemotactic properties, the ANXA1-promoted Ca2+ influx was associated with two important functional effects: shedding of L-selectin and acceleration of PMN apoptosis. The latter effect was confirmed using three distinct technical procedures, namely, cell cycle, Hoechst staining, and ANXA5 binding assay. ANXA1-induced PMN apoptosis was insensitive to inhibitors of L-selectin shedding, whereas it appeared to be associated with dephosphorylation of the proapoptotic intracellular mediator BAD. In conclusion, exogenous ANXA1 displayed selective actions on human PMN. We propose that the new proapoptotic effect reported here may be part of the spectrum of ANXA1-mediated events involved in the resolution of acute inflammation.

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5-6 cells per 100-m of vessel length) and emigration (maximal at 4 hr: 8-10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg͞kg s.c.) or its mimetic peptide Ac2-26 (13 mg͞kg s.c.) did not modify cell rolling but markedly reduced (>50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2-26 (13 mg͞kg) or recombinant human LC1 (0.7-2 mg͞kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ؎ 0.75 min and 2.36 ؎ 0.31 min, respectively (n ‫؍‬ 20-25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inf lammatory response.

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mitochondria; apoptosis; sepsis; heat-shock protein 70; proto-oncogene protein c-Bcl-2

Annexin 1 (ANX1), a calcium-binding protein, participates in the regulation of early inflammatory responses. Whereas some of its effects depend on intracellular interactions, a growing number of observations indicate that ANX1 may also act via autocrine/paracrine functions following externalization to the outer side of the plasma membrane. We studied the effects of ANX1 on leukocyte adhesion to endothelial cells using as a model system the monocytic cell line U937 and human bone marrow microvascular endothelial cells. Exogenous rANX1, as well as endogenous ANX1 externalized by U937 differentiated in vitro, inhibited monocyte firm adhesion to vascular endothelium. Both binding of ANX1 to U937 cells and ANX1-mediated inhibition of cell adhesion involved the short N-terminal domain of the ANX1 molecule. Under experimental conditions in which ANX1 inhibited U937 adhesion to human bone marrow microvascular endothelial cells, this protein specifically colocalized with the α4 integrin, and a direct interaction between ANX1 and the α4 integrin could be documented by immunoprecipitation experiments. Moreover, ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to α4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium.

We have used a transgenic animal model, which constitutively develops hepatocarcinoma (Antithrombin III SV40 T large Antigen: ASV), to study the involvement of Annexin 1 (ANX1) in liver regeneration and malignant transformation. Primary hepatocytes isolated from normal mice did not express ANX1. In contrast, ANX1 was strongly expressed in hepatocytes of transgenic mice during constitutive development of hepatocarcinoma. In ASV transgenic mice, an elevated ANX1 level preceded the appearance of the tumor, indicating that it could be a good marker in the diagnosis of cancer. One-third hepatectomy in normal mice resulted in stimulation of ANX1 synthesis and phosphorylation. This upregulation correlated with increased synthesis of EGF and consequently with increased phosphorylation of the EGF receptor (EGF-R). Stable transfection of a hepatocyte cell line derived from ASV transgenic mice (mhAT2) with antisense complementary DNA for ANX1 reduced the proliferation rate as well as cytosolic phospholipase A 2 (cPLA 2 ) activity. Thus, ANX1 expression and phosphorylation could be a factor implicated in liver regeneration and tumorigenesis, either through modulation of cPLA 2 activity or EGF-R function. (HEPATOLOGY 2000;31:371-380.)Annexin 1 (ANX1) is a member of a multigene family of Ca 2ϩ /phospholipid binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase. 1 We have shown that ANX1, is transcriptionally regulated by interleukin-6 (IL-6) and phorbol myristate acetate (PMA) in human lung adenocarcinoma cell (A 549) through the induction of C/EBP␤ transcription factor. Based on these data we proposed a dual role for ANX1 in cellular differentiation 2 and as a new acute-phase protein. 3 These results prompted us to investigate the role of ANX1 in the liver. While we were carrying these experiments, ANX1 was reported to be strongly expressed in human hepatocellular (hcc) carcinoma cells, suggesting that its induction in the liver could be associated with malignant transformation. 4,5 We therefore addressed the question of the role of ANX1 in liver transformation.Recently, transgenic mice (ASV) expressing the SV40 early region coding for large T antigen under the control of the liver-specific human antithrombin III gene promoter have been created in our laboratory to study oncogenic transformation on hepatocyte function. 6 Phenotypically, the animals present a hepatocellular carcinoma, with a neoplasia at 3 to 9 weeks and later (after tumor development) a severe dysplasia, an augmentation of hepatocytes in S phases, an important cytolysis (necrosis), and an early apoptosis (2.5%-5%). Up to 25 weeks no major difference was obvious between the liver weight of transgenic (ASV) and control mice. After that, liver weight progressively increased reaching sevenfold the normal liver weight at the terminal stage of hcc. Eight old mice die for a general disorder and an important cachexia. 6 Using this model as paradigm for our study of the role of ANX1 in liver, we now report that ANX1 is strongly ...