Aldose reductase mediates the mitogenic signals of cytokines
Introduction
Although the precise mechanisms by which diabetes accelerates atherogenesis are not well understood, the formation of advanced glycosylation end-products (AGE), increased oxidative stress and lipid peroxidation have been implicated as causative factors in the higher cardiovascular risk of diabetics [1], [2], [3]. Increased oxidative stress during diabetes could lead to the generation of reactive carbonyl species that display a variety of proatherogenic effects [4]. Several of these carbonyls are substrates of aldose reductase (AR), an aldo–keto-reductase, which has been implicated in the development of diabetic complications [2], [5]. Aldose reductase inhibitors (ARI) attenuate some of the prolonged complications of diabetes such as peripheral neuropathy, cataractogenesis, nephropathy, and retinopathy [6]. However, the role of AR in mediating the vascular complications of diabetes remains unknown. In contrast to the injurious role of AR in diabetes, which has been linked to the accumulation of osmotically active sorbitol derived from the reduction of glucose by AR, our recent studies show that AR may be a key component of cellular metabolism for the detoxification of aldehydes derived from lipid peroxidation [7], [8]. In vitro the enzyme is an efficient catalyst for the reduction of a wide range of aliphatic and aromatic aldehydes, particularly short- or medium-chain alkenals and 4-hydroxyalkenals, which are the most abundant and cytotoxic end products of lipid peroxidation [7]. We have demonstrated that AR is an efficient catalyst for reducing glutathione-alkenal conjugates [8], indicating that the enzyme may be important in preventing the cellular accumulation of lipid peroxidation-derived aldehydes or their metabolites.
Previous studies have shown that oxidative stress caused during hyperglycemia or atherosclerosis leads to the formation and accumulation of lipid peroxidation product such as 4-hydroxy-trans-2-nonenal (HNE) [9]. High titers of antibodies against protein-HNE adducts have been demonstrated in atherosclerotic humans and in animal models of atherosclerosis. Moreover, the formation of HNE and/or related aldehyde-modified protein or lipoprotein adducts in atherosclerotic lesions has been documented [10]. Free HNE is a mitogen for VSMCs, and inhibition of its metabolism by AR inhibitors prevents VSMC growth in culture and in balloon-injured arteries [11], [12]. These observations indicate that the formation and reactivity of HNE may be an important component of mitogenic signaling, which in VSMC is mediated and triggered by reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions. The ROS trigger a variety of responses in VSMC and stimulate several redox-sensitive kinases such as the MAP kinases, protein kinase-C (PKC) and enhance the expression of several genes such as, TNFα and IL-8 by activating specific transcription factors [13], [14]. Since both cytokines and growth factors generate ROS and presumably lipid-derived aldehydes that could potentially mediate their proliferative/apoptotic responses, we investigated the role of AR in these processes. Particularly significant in the signaling cascade may be the activation of the redox-sensitive transcription factor NF-κB, which is stimulated by both TNF-α and growth factors [15], [16].
The activation of NF-κB by ROS has been demonstrated during hyperglycemia, as well as during cytokine and growth factor stimulation, and the activators of NF-κB such as TNFα and IL-1 are enhanced during atherogenesis [17], [18]. It has been shown previously that TNF-α is the main mitogen responsible for the proliferation of VSMC in balloon-injured arteries [18]. The generation of TNF-α is also increased during diabetes, and the hyperproliferative responses of diabetic vessels have been linked to increased TNF-α activity [19]. As we have shown that AR efficiently reduces lipid-derived aldehydes and their glutathione conjugates, we tested the hypothesis that AR could be an important regulator of VSMC growth, and that the pro-mitogenic effects of AR are in part due to its ability to facilitate NF-κB signaling. We demonstrate here that inhibition of AR by sorbinil or tolrestat prevents TNF-α- induced cell growth and the activation of NF-κB, indicating that AR may be an important regulator of NF-κB-mediated signaling during inflammation, atherosclerosis, and diabetes.
Section snippets
Materials
Dulbecco's modified eagle's medium (DMEM), PBS, penicillin/streptomycin solution, trypsin and fetal bovine serum were purchased from Invitrogen. Antibodies against IκB-α and p50 were obtained from Santa Cruz Biotechnology. Phospho-IκB-α (Ser32) antibody was purchased from New England Biolabs. Consensus oligonucleotides for NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′) and AP1 (5′-CGCTTGATGAGTCAGCCGGAA-3′) transcription factors were obtained from Promega Corp. Sorbinil and tolrestat were gifts from
AR inhibitors attenuate TNF-α-induced VSMC proliferation
We examined the effect of the two structurally different AR inhibitors, sorbinil and tolrestat, on TNF-α-induced mitogenic signaling. Stimulation of VSMC for 24 h with TNF-α resulted in increased cell proliferation compared to non-stimulated cells (Fig. 1) as measured by cell counts using Trypan blue, thymidine incorporation, and MTT assay. Incubation of VSMC for 24 h with 10–20 μM sorbinil or tolrestat prior to stimulation with TNF-α prevented VSMC proliferation. In the absence of TNF-α, ARI
Discussion
Abnormal VSMC proliferation underlies the high propensity of diabetics for restenosis and could be an important factor in the increased incidence and severity of atherosclerosis due to diabetes. It has been reported that inhibition of AR prevents hyperproliferation and the hypertrophy of VSMC in high glucose medium, suggesting that AR may be an important mediator of glucose-induced VSMC growth [22]. However, subsequent studies have shown that even under normal glucose concentrations, inhibition
Acknowledgements
This work was supported in part by NIH grants DK36118 (to S.K.S.) and HL55477 (to A. Bhatnagar). We are grateful to Dr B.B. Aggarwal, University of Texas, MD Anderson Cancer Institute, Houston, TX, for providing recombinant TNF-α.
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