[Abstract unavailable

The binding of advanced glycation end-products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. In the present study, we show that the cellular constituents of small vessels, endothelial cells (EC) and pericytes express novel splice variants of RAGE mRNA coding for the isoforms that lack the N-terminal V-type immunoglobulin-like domain (N-truncated) or the C-terminal transmembrane domain (C-truncated), as well as the known full-length mRNA. The ratio of the expression of the three variants was different between EC and pericytes; the content of the C-truncated form was highest in EC, whereas the full-length form was the most abundant in pericytes. Transfection experiments with COS-7 cells demonstrated that those variant mRNAs were translated into proteins as deduced; C-truncated RAGE was efficiently secreted into the culture media, and N-truncated RAGE was located mainly on the plasma membrane. The three isoforms were also detected in primary cultured human EC and pericytes. Further, full-length and C-truncated forms of RAGE bound to an AGE-conjugated column, whereas N-truncated RAGE did not. The AGE induction of extracellular-signal-related kinase phosphorylation and vascular endothelial growth factor in EC and of the growth and cord-like structure formation of EC was abolished completely by C-truncated RAGE, indicating that this endogenous secretory receptor (endogenous secretory RAGE) is cytoprotective against AGE. The results may contribute to our understanding of the molecular basis for the diversity of cellular responses to AGE and for individual variations in the susceptibility to diabetic vascular complications.

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.

We have shown previously that vascular endothelial growth factor (VEGF) synthesized by the cellular constituents of small vessels per se, viz. endothelial cells and pericytes, participates in the hypoxia-driven proliferation of both cell types (Nomura, M., Yamagishi, S.,

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to bcatenin-dependent formation of the blood-retina-barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger bcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12 À/À mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen-induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction.