Annexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family with members of the family being expressed throughout animal and plant kingdoms. Structurally, annexins are characterized by a highly alpha-helical and tightly packed protein core domain considered to represent a Ca2+-regulated membrane binding module. Many of the annexin cores have been crystallized, and their molecular structures reveal interesting features that include the architecture of the annexin-type Ca2+ binding sites and a central hydrophilic pore proposed to function as a Ca2+ channel. In addition to the conserved core, all annexins contain a second principal domain. This domain, which NH2-terminally precedes the core, is unique for a given member of the family and most likely specifies individual annexin properties in vivo. Cellular and animal knock-out models as well as dominant-negative mutants have recently been established for a number of annexins, and the effects of such manipulations are strikingly different for different members of the family. At least for some annexins, it appears that they participate in the regulation of membrane organization and membrane traffic and the regulation of ion (Ca2+) currents across membranes or Ca2+ concentrations within cells. Although annexins lack signal sequences for secretion, some members of the family have also been identified extracellularly where they can act as receptors for serum proteases on the endothelium as well as inhibitors of neutrophil migration and blood coagulation. Finally, deregulations in annexin expression and activity have been correlated with human diseases, e.g., in acute promyelocytic leukemia and the antiphospholipid antibody syndrome, and the term annexinopathies has been coined.

Eukaryotic cells contain various Ca(2+)-effector proteins that mediate cellular responses to changes in intracellular Ca(2+) levels. A unique class of these proteins - annexins - can bind to certain membrane phospholipids in a Ca(2+)-dependent manner, providing a link between Ca(2+) signalling and membrane functions. By forming networks on the membrane surface, annexins can function as organizers of membrane domains and membrane-recruitment platforms for proteins with which they interact. These and related properties enable annexins to participate in several otherwise unrelated events that range from membrane dynamics to cell differentiation and migration.

Aberrant neovascularisation contributes to diseases such as cancer, blindness and atherosclerosis and is the consequence of inappropriate angiogenic signalling. While many regulators of pathogenic angiogenesis have been identified, our understanding of this process is incomplete. Here we explored the transcriptome of retinal microvessels isolated from mouse models of retinal disease that exhibit vascular pathology and uncovered an up-regulated gene, leucine-rich alpha-2-glycoprotein-1 (Lrg1), of previously unknown function. We show that in the presence of TGFβ1, LRG1 is mitogenic to endothelial cells and promotes angiogenesis. Mice lacking Lrg1 develop a mild retinal vascular phenotype but exhibit a significant reduction in pathological ocular angiogenesis. LRG1 binds directly to the TGFβ accessory receptor endoglin which, in the presence of TGFβ1, results in promotion of the pro-angiogenic Smad1/5/8 signalling pathway. LRG1 antibody blockade inhibits this switch and attenuates angiogenesis. These studies reveal a novel regulator of angiogenesis that mediates its effect through modulating TGFβ signalling.

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...

Apoptotic nerve cell death is implicated in the pathogenesis of several devastating neurodegenerative conditions, including glaucoma and Alzheimer's and Parkinson's diseases. We have devised a noninvasive real-time imaging technique using confocal laserscanning ophthalmoscopy to visualize single nerve cell apoptosis in vivo, which allows longitudinal study of disease processes that has not previously been possible. Our method utilizes the unique optical properties of the eye, which allow direct microscopic observation of nerve cells in the retina. We have been able to image changes occurring in nerve cell apoptosis over hours, days, and months and show that effects depend on the magnitude of the initial apoptotic inducer in several models of neurodegenerative disease in rat and primate. This technology enables the direct observation of single nerve cell apoptosis in experimental neurodegeneration, providing the opportunity for detailed investigation of fundamental disease mechanisms and the evaluation of interventions with potential clinical applications, together with the possibility of taking this method through to patients.A poptosis is an orchestrated form of cell ''death by suicide.'' It is essential in both the development and normal maintenance of tissue function. It is also implicated in the pathology of a number of severe neurodegenerative disorders such as glaucoma, motor neuron, and Alzheimer's, Parkinson's, and Huntington's diseases (1). There is evidence that a similar pathogenesis may contribute to neurodegenerative processes in these conditions (2), and that the extent of nerve cell loss is correlated with functional deficit (3-6). If we could directly visualize this process, it would facilitate a much more precise diagnosis and critically enable accurate tracking of the disease state and the action of therapy. However, until now, it has not been possible to detect nerve cell apoptosis in vivo (1, 7-10).Annexin 5 is a protein that, in the presence of Ca 2ϩ , has a high affinity for phosphatidylserine (PS), an anionic phospholipid that is enriched in the inner leaflet of plasma membranes. The externalization of PS from the inner leaflet to the outer layer of the cell membrane is an invariant early feature in the apoptotic process that occurs before DNA fragmentation and nuclear condensation. Because of its properties, FITC-annexin 5 has become widely used in the cytological detection of cells undergoing apoptosis (11). More recently, annexin 5 has been shown to be effective in the identification of apoptosis in vivo by using radiological and macroscopic fluorescent techniques (7,8,10,12,13).However, existing in vivo techniques using annexin 5 either have been unable to resolve the process to a single cellular level (7-9) or require an invasive method performed under terminal anesthesia (10). Imaging the eye, compared with the rest of the body, offers a unique opportunity because of the presence of clear optical media allowing direct visualization of labeled disease processes as they occur. This mea...

Here we show that EGF and EGF receptor (EGFR) are trafficked through a subpopulation of multivesicular endosomes/bodies (MVBs) that are distinct from morphologically identical vacuoles that label for the late endosomal marker lyso-bisphosphatidic acid (LBPA). EGF stimulation increases both MVB biogenesis and inward vesiculation within EGFR-containing MVBs. Deletion of annexin 1, a substrate of EGFR tyrosine kinase, abolishes the effect of EGF stimulation on inward vesiculation. This phenotype is reversible by transfection with wild-type but not Y21F phosphorylation mutant annexin 1. Deletion of annexin 1 has no effect on EGF-stimulated MVB biogenesis, suggesting that MVB biogenesis and inward vesiculation within MVB are mediated by separate mechanisms. Loss or depletion of annexin 1 has no effect on EGF degradation and causes only a small delay in EGFR degradation, indicating that annexin 1 operates downstream of Hrs- and ESCRT-mediated sorting and is required solely for EGF-stimulated inward vesiculation. Annexin 1 accumulates on internal vesicles of MVB after EGF-stimulated inward vesiculation, suggesting that it may be required for a late stage in inward vesiculation.

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The results demonstrated that RGC apoptosis in glaucoma correlates strongly with elevated IOP and is significantly associated with IOP-induced changes in specific ECM components in the RGC layer. The study shows for the first time a link between MMP-9, laminin degradation, RGC apoptosis, and IOP exposure in glaucoma. The findings suggest that abnormal ECM remodeling in the glaucomatous retina may relate to RGC death and support the notion that the retina is a primary site of injury in glaucoma.