The extended interaction of aldoses with proteins or lipids results in nonenzymatic glycation and oxidation, ultimately forming AGEs, the presence of which in the plasma and vessel wall is associated with diabetic vascular complications. We show here that AGE albumin in the intravascular space interacts with the vessel wall via binding to an integral membrane protein, receptor for AGE (RAGE), a member of the immunoglobulin superfamily, resulting in clearance from the plasma and induction of interleukin 6 mRNA. Intravenously infused 2SI-AGE albumin showed a rapid phase of plasma clearance with deposition in several organs. Rapid removal of 125I-AGE albumin from the plasma was prevented by administration ofa soluble, truncated form ofRAGE, which blocked binding of 12I-labeled AGE albumin to cultured endothelial cells and mononuclear phagocytes, as well as by pretreatment with anti-RAGE IgG. Ultrastructural studies with AGE albumin-colloidal gold conjugates perfused in situ showed that in murine coronary vasculature this probe was taken up by endothelial plasmalemmal vesicles followed by transport either to the abluminal surface or by accumulation in intracellular vesicular structures reminint ofendosomes and lysosomes. Consequences of AGE-RAGE interaction included induction of interleukin 6 mRNA expression in mice. These data indicate that RAGE mediates the interaction ofAGEs with the vessel wall, both for removal ofthese glycated proteins from the plasma and for changes in gene expression.When proteins or lipids are exposed to aldoses, they undergo nonenzymatic glycation and oxidation (1-8), ultimately forming AGEs, whose formation occurs during normal aging and is accelerated in diabetics (1-7). The presence of AGEs in the plasma and vessel wall has been linked to the pathogenesis of diabetic complications, stimulating investigations to determine mechanisms through which AGEs exert their pathologic effects.An important mechanism through which AGEs interact with cells is through specific receptors (9-13). We thus evaluated the role of the receptor for AGE (RAGE), which specifically binds AGEs (10-12), in mediating the interactions of these glycated molecules with target cells such as endothelial cells (ECs) and mononuclear phagocytes (MPs) (10,11,13). Previous studies have identified the presence of RAGE in bovine cardiac vasculature (13). We have now identified RAGE in murine coronary vasculature both in vivo and in vitro and employed this model to demonstrate that RAGE has a central role in uptake by the endothelium and in gene expression following AGE infusion. MATERIALS AND METHODSPreparation of AGE Albumin, RAGE, and Anti-RAGE Antibody. Mouse and bovine AGE albumin were prepared and characterized as described (2, 10, 11). Radiolabeling of AGE and native albumin was performed by the lactoperoxidase method (14); the tracers had specific radioactivities of -1.5 x 104 cpm/ng (10, 11). For AGE albumin-gold conjugates, colloidal gold particles (5 nm in diameter) were prepared as described (15,16). Bovine R...

By in vitro translation of mRNA's isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, ß-glucuronidase of the rat preputial gland and cathepsin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA's for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen . Polypeptides synthesized in the absence of microsomal membranes were 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes .In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; -60% of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing . As previously observed by other investigations in cultured fibroblasts (A . Gonzalez-Noriega, J . H . Grubbs, V. Talkad, and W. S. Sly, 1980, J. Cell Biol. 85 : 839-852; A. Hasilik and E. F. Neufeld, 1980, /. Biol. Chem ., 255:4937-4945 .) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their secretion . In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing . These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors takes place within the lysosome itself .Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathepsin D were detected from their differential sensitivity to digestion with endoglycosidases H and D . These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.

The visionaries predicted the existence of transcytosis in endothelial cells; the cell biologists deciphered its mechanisms and (in part) the molecules involved in the process; the cell pathologists unravelled the presence of defective transcytosis in some diseases. The optimistic perspective is that transcytosis, in general, and receptor-mediated transcytosis, in particular, will be greatly exploited in order to target drugs and genes to exclusive sites in and on endothelial cells (EC) or underlying cells. The current recognition that plasmalemmal vesicles (caveolae) are the vehicles involved in EC transcytosis has moved through various phases from initial considerations of caveolae as unmovable sessile non-functional plasmalemma invaginations to the present identification of a multitude of molecules and a crowd of functions associated with these ubiquitous structures of endothelial and epithelial cells. Further understanding of the molecular machinery that precisely guides caveolae through the cells so as to reach the target membrane (fission, docking, and fusion), to avoid lysosomes, or on the contrary, to reach the lysosomes, and discharge the cargo molecules will assist in the design of pathways that, by manipulating the physiological route of caveolae, will carry molecules of choice (drugs, genes) at controlled concentrations to precise destinations.

Circulating microparticles and endothelial progenitor cells in atherosclerosis: pharmacological effects of irbesartan. J Thromb Haemost 2012; 10: 680-91.Summary. Aims: This study aimed to (i) employ our newly designed model, the hypertensive-hypercholesterolemic hamster (HH), in order to find out whether a correlation exists between circulating microparticles (MPs), endothelial progenitor cells (EPCs) and their contribution to vascular dysfunction and (ii) to assess the effect of irbesartan treatment on HH animals (HHI). Methods and Results: The results showed that compared with the control (C) group, HH displayed: (i) a significant increase in plasma cholesterol and triglyceride concentration, and an augmentation of systolic and diastolic arterial blood pressure, and of heart rate; (ii) a marked elevation of MPs and a significant decrease in EPCs; (iii) structural modifications of the arterial wall correlated with altered protein expression of MMP2, MMP9, MMP12, TIMP1, TIMP2 and collagen type I and III; (iv) a considerably altered reactivity of the arterial wall closely correlated with MPs and EPC adherence; and (v) an inflammatory process characterized by augmented expression of P-Selectin, E-Selectin, von Willebrand factor, tissue factor, IL-6, MCP-1 and RANTES. Additionally, the experiments showed the potential of irbesartan to correct all altered parameters in HH and to mobilize EPCs by NO, chemokines and adhesion molecule-dependent mechanisms. Conclusions: Hypertension associated with hypercholesterolemia is accompanied by structural modifications and expression of pro-inflammatory molecules by the vessel wall, the alteration of vascular tone, enhanced release of MPs and reduced EPCs; the ratio between the latter two may be considered as a marker of vascular dysfunction. Irbesartan, which exhibits a pharmacological control on the levels of MPs and EPCs, has the potential to restore homeostasis of the arterial wall.

No abstract