Original contributionInduction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products
Introduction
Hyperglycemia is the major causal factor for the development of various diabetic complications, although how hyperglycemia provokes these complications has not been fully elucidated yet. Several biochemical mechanisms are postulated to explain the adverse effects of hyperglycemia and ensuing tissue damage. They are increased flux of glucose through the polyol pathway [1], enhanced nonenzymatic glycation [2], accelerated generation of reactive oxygen species [3], and activation of the diacylglycerol-protein kinase C pathway [4].
Aldose reductase (AR), the first and rate-limiting enzyme in the polyol pathway, is a member of the monomeric NADPH-dependent aldo-keto reductase family that catalyzes reduction of glucose to the corresponding sugar alcohol, sorbitol. It has been generally accepted that expression of AR gene is osmotically regulated: increased AR activity under hyperosmotic conditions supplies sorbitol as one of the physiological osmolytes in the inner medullary cells of the kidney [5]. In the lens, excessive accumulation of sorbitol by increased flux through this pathway was indicated to play a key role in the development of diabetic cataract [6]. In other target organs of diabetic complications, on the other hand, activation of polyol pathway alleviate glutathione reductase and nitric oxide synthase activities due to the depletion of the cofactor NADPH. This may result in the increased susceptibility to oxidative stress and vascular dysfunction, leading to the early stage of diabetic complications [1]. Under hyperglycemic conditions, elevated expression of AR was reported in a variety of tissues including the target organs of diabetic complications, and in cultured cells originated from humans and experimental animals [7], [8], [9]. Molecular mechanisms underlying such augmented expression of AR under hyperglycemia remain unresolved; however, a recent study indicated that oxidative stress may induce AR mRNA expression in a cell line derived from rat vascular smooth muscle cells [10].
Glucose-induced oxidative stress is one of the biochemical mechanisms postulated to precipitate the development of diabetic complications. Among multiple pathways by which hyperglycemia may accelerate the generation of free radicals, it has been demonstrated that advanced glycation itself participates in the production of oxygen free radicals [11]. Upon exposure to reducing sugars such as glucose, proteins undergo nonenzymatic glycation and oxidation. In the early stages of this reaction, reversible Schiff bases and Amadori products are formed within protein molecules. Amadori products undergo a series of oxidation, dehydration, and fragmentation reactions to irreversibly form yellow-brown and fluorescent adducts called advanced glycation end products (AGEs) [2]. Excessive formation and accumulation of AGEs in tissues, serum, and erythrocytes could lead to tissue damage through a variety of mechanisms: alteration of structure and function of tissue proteins [2]; stimulation of cellular responses by binding to specific cell surface receptors [12], [13], [14], [15], [16]; and generation of reactive oxygen intermediates [17]. Hence, we investigated the effect of AGEs on AR mRNA expression and the level of AR protein in human microvascular endothelial cells. To our knowledge, this is the first report indicating that AGEs may augment the expression of AR in the vascular tissue.
Section snippets
Preparation of AGEs-modified bovine serum albumin (BSA)
AGEs-modified BSA was prepared by incubating BSA (fraction V, very low endotoxin; Miles, Kankakee, IL, USA) in phosphate-buffered saline (PBS; 10 mM, pH 7.4) with 0.4 M glucose at 37°C for 6 weeks under sterile conditions. Unincorporated sugar was removed by dialysis against PBS. Unmodified BSA was prepared under the same conditions without glucose. The amount of AGEs present in AGEs-modified BSA was assessed by measuring crossline, an AGE that has cross-linking and fluorescence characteristics
Induction of AR by AGEs in endothelial cells
We first assessed the effect of AGEs on AR mRNA and AR protein levels in cultured endothelial cells. Cells were incubated for 16 h with 5 or 500 μg/ml AGEs-BSA; 50 μM H2O2; or in hyperosmotic medium (500 mOsm/kg). As shown in Fig. 1A and B , both AR mRNA and AR protein were markedly increased by addition of 500 μg/ml AGEs-BSA (mRNA 1.92-fold, protein 1.68-fold, respectively) compared with a control group incubated in serum-free medium. The extent of increased mRNA and protein levels in the
Discussion
Our present findings indicate for the first time that high concentrations of AGEs induce the expression of AR mRNA and protein in HDMVEC. Moreover, increased levels of AR protein were depicted in HDMVEC exposed to sera from diabetic patients with ESRD, even though the presence of unknown oxidant in the serum from the patients of chronic renal failure has been reported and it might contribute to increased AR expression [27]. Previously we found significant increments in AGEs in the membranes of
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