Discussion #1: AGE, RAGE, Diseases, Inhibitions ….

yamamoto-135x149RAGE was originally identified and named for its ability to bind AGE. RAGE consists of an immunoglobulin-like extracellular region, a transmembrane domain and a short cytoplasmic tail which is essential for intracellular signal transduction. Since other proinflammatory ligands of RAGE have been identified; e.g. high-mobility group B protein 1 (HMGB1), S100-calcium binding (S100) proteins CD11b (Mac-1), amyloid b-proteins, and complement C3a, RAGE is considered a pattern-recognition receptor (PRR), participating in a wide variety of pathological processes, such as diabetic complications, atherosclerosis, cancer, neurodegenerative disorders, and inflammation.

Even then, RAGE can be classified into two isoforms, signal-transducible membrane-bound RAGE (above-mentioned RAGE) and soluble RAGE (sRAGE). sRAGE is generated from membrane-bound RAGE by ectodomain shedding and from alternative splicing of RAGE gene transcripts as endogenous secretory RAGE (esRAGE). The sRAGE is thought to act locally and systemically as a decoying ligand. The administration of sRAGE has been shown to attenuate experimental animal models of various RAGE-related diseases. When the ligands are not in excess, sRAGE is reported to mediate inflammation by directly binding to monocytes. However, the molecular mechanism is unknown. Recent clinical studies have focused on the significance of circulating sRAGE in a variety of pathophysiological conditions. Above all, findings in both type 1 and type 2 diabetic patients are quite confusing and have been reported to be both increased and decreased. The presence of renal insufficiency can affect circulating sRAGE level, which may explain controversial findings of sRAGE in diabetes.

These leave us with more questions than answers. The key question for the future is whether complete or slight inhibition of RAGE, which may also inhibit sRAGE function, will be of some benefit in RAGE-associated diseases. What cell type has to be targeted? What time course of inhibition is best? And finally, will this approach work in patients suffering from AGE-RAGE-associated diseases?

Yasuhiko Yamamoto, MD, PhD
Department of Biochemistry & Molecular Vascular Biology,
Kanazawa University Graduate School of Medical Science
Japan

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gugliucci1Dr. Yamamoto opens a very interesting topic for discussion as well as suggesting several avenues for future research on this area.

Another important aspect that has not been addressed so far, and may provide important clues to the normal physiology and the role in disease of sRAGE are the kinetic aspects of its presence in plasma, namely the half life of the sRAGE  and their site and rate of production.

In particular, we believe that attention should be paid to the following aspects:

  1. What are the tissues that produce sRAGE?
  2. What is the half life of sRAGE in the circulation?
  3. If sRAGE acts as a decoy:
    1. Does its production increase when the load of ligands (AGEs coming from the diet, renal failure, diabetes; beta amyloid, calgranulins, etc) is increased?
    2. Does the kinetic of the removal of the sRAGE plasma ligands increase when the load of ligands increases?

The answer to those questions may provide insights into the interpretation of somewhat controversial results that have been recently published. In several papers  published  lately the concentration of sRAGE are measured in a static way, without having any notion about the speed of production and of all removal of this receptor in plasma; which then complicates the interpretation of the  results.

A parallel or an analogy can be made with another plasma protein that acts as a ligand to avoid oxidative stress and renal damage: haptoglobin. Haptoglobin in plasma is measured as a an index of the presence of intravascular hemolysis. Serum haptoglobin binds to hemoglobin released by intravascular hemolysis preventing it from to be released free in the plasma, and therefore, preventing oxidative stress from free iron, hemin, and tubular damage by alphabeta diamers in the kidney. The steady-state concentrations of haptoglobin fall dramatically when there is an increase in the load of hemoglobin produced by hemolysis. That produces the seemingly paradoxical result in which an increased hemolysis produces a dramatic decrease in haptoglobin levels showing that haptoglobin has been consumed by the flux of ligands and that the rate of disappearance from the plasma is higher than the rate production. While the flux of haptoglobin had dramatically increased, the production has not compensated for the larger increase in the removal of the ligand-binding protein from the plasma.

Something similar maybe occuring in the case of sRAGE that could explain some of the seemingly paradoxical results showing that in conditions in which the sRAGE and the AGE load is increased are accompanied by a decrease in soluble RAGEs.

Therefore, we believe that further research should be conducted not only on steady state static measurements of this product in plasma, but mainly on the kinetics of its production, sites of origin, and rate of removal and the interaction of those with an increased in AGEs and other ligand loads related to inflammatory conditions.

For instance, nowadays, state of the art stable isotope measurements of 13 C  leucine can be employed to measure the kinetics of sRAGE production and removal, in physiological and pathological states, by following up a load of an IV or oral load of 13C leucine in a patient in a period of hours. After purification of sRAGE from plasma by immunoprecipitation followed by  hydrolysis and a GCMS/MS to determine the presence of heavy leucine in the sRAGE extracted from plasma and calculate its kinetics.

Alejandro Gugliucci, MD, PhD
Professor and Associate Dean for Research
Touro University, California
USA

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monnier1I applaud the opening of the IMARS website for scientific discussion.

Dr. Yamamoto has nicely summarized the RAGE field and has formulated very pertinent questions concerning anti-RAGE therapy. I have followed the RAGE field only from a distance so I hope my questions are not trivial.

First, do we really know which are the most important RAGE ligands in vivo? Establishment of a ligand hierarchy for each disease condition would seem important. Together with that, which compartment (lung, liver, GI tract, brain) contributes which ligand ? Has anyone done the sRAGE immunoprecipitation proteomics to see what ligands are bound by sRAGE?

Second, is it possible to selectively inactivate sRAGE with i.v. infusion of an anti-sRAGE antibody that does not bind/activate membrane-bound RAGE?. Finally, does RAGE deletion protect against TLR mediated signaling? I am sure somebody must have done this research.

Vincent Monnier
Prof. Pathology/Biochem
Case Western Reserve University, Department of Pathology
Cleveland Ohio
USA

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ft-135x149In the IMARS Highlights of May 15, 2008, John Baynes discussed the origin of CML not only in vivo but also in foods.

The question was: is CML formed primarily as a result of lipoxidation reactions or as a result of the classical Maillard reaction with reducing sugars?

Januszewski et al. (2003) suggested that CML was formed in vivo mainly from lipid peroxidation products. However do we have enough evidence on the relative contribution of lipoperoxides versus reducing sugars in the formation of CML in biological systems?

In foods, where CML can also be formed, different data have been used to answer the question of the origin of CML. First of all a food and CML database based on an Elisa assay indicates that the more fat is reported in food the more CML is also found. However the ELISA method used to quantify CML in food is now coming into question and a more accurate method (LC-MS/MS) used by Assar et al. (2008) and Tessier et al. (2010) does not confirm this trend. For instance Assar et al. did not find any significant amount of CML in butter and Tessier et al. found a positive correlation between carbohydrate content and CML content in chocolate-flavored drink powders, but no correlation between fat and CML contents.

Does the formation of CML in food follow the same pathway as its formation in vivo? Can the evidence found in vivo be applied to the field of food chemistry and inversely? What further data are needed to answer the question of the origin of CML?

Frédéric J TESSIER, PhD
Associate professor, Analytical Chemistry and Nutrition
Institut Polytechnique LaSalle Beauvais
19 rue Pierre Waguet, 60026 Beauvais
France

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mp-135x150I think the low CML content in butter does not contradict the hypothesis of lipid derived CML in food. It is not surprising that butter contains low concentrations of CML despite of the high fat content:
1. The lipids in butter are mostly saturated and cannot generate very well CML-precursors.
2. Thermal processing of butter is relatively low
3. Protein and amino acid content in butter is relatively low.

Independent of the question if carbohydrates or lipids are better CML-precursors: lysine is always required to generate CML.

I suggest to test the contribution of lipids and carbohydrates to CML formation using infant formulas: They contain many unsaturated fatty acids as well as lactose and are processed in a way promoting both thermal reaction as well as oxidation.

Prof. Dr. Monika Pischetsrieder
Henriette Schmidt-Burkhardt Chair of Food Chemistry
Department of Chemistry and Pharmacy – Emil Fischer Center
University of Erlangen-Nuremberg
Schuhstr. 19
91052 Erlangen – Germany

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