Deepak Chandra

Aldose reductase mediates mitogenic signaling in vascular smooth muscle cells

Abnormal vascular smooth muscle cell (VSMC) proliferation is a key feature of atherosclerosis and restenosis; however, the mechanisms regulating growth remain unclear. Herein we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR) inhibits NF-κB activation during restenosis of balloon-injured rat carotid arteries as well as VSMC proliferation due to tumor necrosis factor α (TNF-α) stimulation. Inhibition of VSMC growth by AR inhibitors was not accompanied by increase in cell death or apoptosis. Inhibition of AR led to a decrease in the activity of the transcription factor NF-κB in culture and in the neointima of rat carotid arteries after balloon injury. Inhibition of AR in VSMC also prevented the activation of NF-κB by basic fibroblast growth factor (bFGF), angiotensin-II (Ang-II), and platelet-derived growth factor (PDGF-AB). The VSMC treated with AR inhibitors showed decreased nuclear translocation of NF-κB and diminished phosphorylation and proteolytic degradation of IκB-α. Under identical conditions, treatment with AR inhibitors also prevented the activation of protein kinase C (PKC) by TNF-α, bFGF, Ang-II, and PDGF-AB but not phorbol esters, indicating that AR inhibitors prevent PKC stimulation or the availability of its activator but not PKC itself. Treatment with antisense AR, which decreased the AR activity by >80%, attenuated PKC activation in TNF-α, bFGF, Ang-II, and PDGF-AB-stimulated VSMC and prevented TNF-α-induced proliferation. Collectively, these data suggest that inhibition of NF-κB may be a significant cause of the antimitogenic effects of AR inhibition and that this may be related to disruption of PKC-associated signaling in the AR-inhibited cells.

Nitric oxide regulates the polyol pathway of glucose metabolism in vascular smooth muscle cells

Increased reduction of glucose via the polyol pathway enzyme aldose reductase (AR) has been linked to the development of secondary diabetic complications. Because AR is a redox‐sensitive protein, which in vitro is readily modified by NO donors, we tested the hypothesis that NO may be a physiological regulator of AR. We found that administration of the NO synthase (NOS) inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) increased sorbitol accumulation in the aorta of nondiabetic and diabetic rats, whereas treatment with L‐arginine (a precursor of NO) or nitroglycerine patches prevented sorbitol accumulation. When incubated ex vivo with high glucose, sorbitol accumulation was increased by L‐NAME and prevented by L‐arginine in strips of aorta from rats or wild‐type, but not eNOS‐deficient, mice. Exposure to NO donors also inhibited AR and prevented sorbitol accumulation in rat aortic vascular smooth muscle cells (VSMC) in culture. The NO donors also increased the incorporation of radioactivity in the AR protein immunoprecipitated from VSMC in which the glutathione pool was prelabeled with [35S]‐cysteine. Based on these observations, we suggest that NO regulates the vascular synthesis of polyols by S‐thiolating AR;therefore, increasing NO synthesis or bioavailability may be useful in preventing diabetes‐induced changes in the polyol pathway.—Ramana, K. V., Chandra, D., Srivastava, S., Bhatnagar, A., Srivastava, S. K. Nitric oxide regulates the polyol pathway of glucose metabolism in vascular smooth muscle cells. FASEB J. 17, 417–425 (2003)

Structural and kinetic modifications of aldose reductase by S-nitrosothiols.

Modification of aldose reductase (AR) by the nitrosothiols S-nitroso-N-acetyl penicillamine (SNAP) and N-(beta-glucopyranosyl)-N(2)-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3-7-fold increase in its k(cat) and a 25-40-fold increase in its K(m) for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO(2-)(4) anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by P(i) or O(2), indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-K(m)-high-k(cat) form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.

Regulation of aldose reductase and the polyol pathway activity by nitric oxide.

Increased flux of glucose through the polyol pathway has been implicated in the pathophysiology of secondary diabetic complications. The first step of this pathway, which generates sorbitol from glucose, is catalyzed by aldose reductase (AR) (AKR1B). In vitro, the binding of substrates and inhibitors to AR is highly sensitive to the oxidation state of the enzyme due to the presence of a hyper-reactive cysteine residue at the active site of the enzyme. This residue (Cys-298) can be readily modified in air or, by thiol-modifying reagents, nitric oxide (NO) donors and nitrosothiols. We show that exposure of rat erythrocytes to NO donors inhibits AR activity and AR mediated accumulation of sorbitol, possibly by S-glutathiolation of Cys-298. Both glutathiolation and inhibition of AR are reversible upon subsequent incubation of the cells with fresh media without NO donors. These observations suggest that NO regulates the cellular activity of AR and in turn the flux of glucose via the polyol pathway. The inhibition of AR by exogenous or endogenous NO appears to be related to reversible S-glutathiolation of the AR protein. Because hyperglycemic states are associated with a decrease in NO generation, the loss of NO-mediated repression of AR may be a significant factor in the activation of the polyol pathway and the development of secondary diabetic complications.

Aldose reductase mediates the mitogenic signals of cytokines

Chronic hyperglycemia is associated with the activation of aldose reductase (AR), an increase in cytokines such as TNF-alpha and IL-8 and oxidative stress. Alterations in this interdependent cascade of signals may be responsible for the diabetes-induced increase in the incidence and severity of cardiovascular diseases such as atherosclerosis and hypertension. We have previously shown that inhibition of AR prevents cultured vascular smooth muscle cell (VSMC) growth and restenosis of balloon-injured carotid arteries. To identify the mechanisms by which inhibition of AR prevents cell growth, we examined the effects of AR inhibition on mitogenic signaling by cytokines. Stimulation with TNF-alpha led to the activation of the transcription factor NF-kappaB and enhanced VSMC growth. Treatment with the AR inhibitors sorbinil or tolrestat, attenuated mitogen-induced activation of NF-kappaB and VSMC proliferation. In cultured VSMC, AR inhibitors prevented signaling events upstream of NF-kappaB activation, i.e. IkappaB-alpha phosphorylation and IkappaB-alpha degradation. Inhibition of AR also prevented protein kinase C (PKC) activation by TNF-alpha, but did not affect PKC activation by phorbol esters, indicating that inhibition of AR interrupts mitogenic signaling upstream of PKC. Together, these results indicate a pivotal role of AR or its reaction product(s) in the mitogenic signals initiated by cytokines that are elevated in diabetes and its cardiovascular complications such as atherosclerosis. These observations suggest a possible therapeutic use of AR inhibitors in these pathological conditions.

Role of aldose reductase in TNF-α-induced apoptosis of vascular endothelial cells

Abstract Apoptosis of vascular endothelial cells (VECs) and concomitant proliferation of the underlying vascular smooth muscle cells (VSMCs) in large arteries are the key features of atherosclerosis and restenosis. However, the mechanisms underlying endothelial cell death and abnormal smooth muscle cell proliferation during the development of vascular lesions remain unclear. We have previously demonstrated that treatment with inhibitors of the aldehyde-metabolizing enzyme and aldose reductase (AR) attenuates restenosis of balloon-injured rat carotid arteries. The inhibition of AR also prevents the apoptosis of VECs induced by the tumor necrosis factor-alpha (TNF-α). Apoptosis of the VECs was determined by the incorporation of [3H]-thymidine and the activation of caspase-3. Stimulation of the VECs with TNF-α led to an increase in the DNA-binding activity of the transcription factor, nuclear factor-kappa binding protein (NF-κB) and the induction of the adhesion molecule (ICAM)-1. Treatment of VECs with the AR inhibitor, tolrestat, prevented the activation of NF-κB and diminished ICAM-1 induction stimulated by TNF-α. These results indicate an obligatory requirement of AR activity in the transduction of intracellular signaling initiated by the ligation of the TNF-α receptors leading to the activation of NF-κB. Although the specific signaling events interrupted by AR inhibition remain unknown, our results suggest that product(s) of AR catalysis may be essential for NF-κB activation. These observations could form the basis of future investigations into the therapeutic utility of AR inhibitors in preserving endothelial function and integrity during atherosclerosis and diabetes.

Metabolic regulation of aldose reductase activity by nitric oxide donors

Regulation of aldose reductase (AR), a member of the aldo-keto reductase superfamily, by nitric oxide (NO) donors was examined. Incubation of human recombinant AR with S-nitrosoglutathione (GSNO) led to inactivation of the enzyme and the formation of an AR-glutathione adduct. In contrast, incubation with S-nitroso-N-acetyl penicillamine (SNAP) or N-(beta-D-glucopyranosyl)-SNAP (GlycoSNAP) led to an increase in enzyme activity which was accompanied by the direct nitrosation of the enzyme and the formation of a mixed disulfide with the NO-donor. To examine in vivo modification, red blood cells (RBC) and rat aortic vascular smooth muscle cells (VSMC) were incubated with 1 mM GSNO or SNAP. Exposure of VSMC to SNAP and GSNO for 2 h at 37 degrees C led to approximately 71% decrease in the enzyme activity with DL-glyceraldehyde as the substrate. Similarly, exposure of RBC in 5 mM glucose to NO-donors for 30 min at room temperature, followed by increasing the glucose concentration to 40 mM, resulted in >75% decrease in the formation of sorbitol. These investigations indicate that NO and/or its bioactive metabolites can regulate cellular AR, leading to either activation (by nitrosation) or inactivation (by S-thiolation).

Oxidative stress-induced up-regulation of the chloride channel and Na+/Ca2+ exchanger during cataractogenesis in diabetic rats

We have determined the abundance of the chloride channel, ClC-3, and Na(+)/Ca(2+) exchanger proteins in isolated rat lens cortex fiber cells by immunofluorescence method using polyclonal anti-ClC-3 antibodies and monoclonal antibodies against the canine cardiac Na(+)/Ca(2+) exchanger protein. These proteins were also quantified in the lens cortex of streptozotocin-injected rats by Western blots. Also, mRNA for ClC-3 was determined by Northern blot analysis. The isolated rat lens cortical fibers expressed basal levels of ClC-3 and Na(+)/Ca(2+) exchanger proteins. As compared to controls, the ClC-3 protein in the lens cortex of diabetic rats (blood glucose>400 mg%) increased by 2.5-fold in 7 days and 4.5-fold in 14 days. However, the ClC-3 protein decreased to near-normal values in 40 days. The changes in ClC-3 mRNA closely followed the protein levels. Similarly, as compared to controls, on Day 7, the Na(+)/Ca(2+) exchanger protein in the diabetic rat lens cortex increased by 3.5-fold and on Day14 by 5.5-fold. Subsequently, it decreased to control levels on Day 40. Treatment with the antioxidant, Trolox (2 mg/kg body weight), prevented the initial increase in ClC-3 and Na(+)/Ca(2+) exchanger proteins. The up-regulation of ClC-3 and Na(+)/Ca(2+) exchanger proteins during the early stages of diabetes and its prevention by antioxidants suggests that the proteins regulating ion transport may have a pathophysiological role in the development of diabetic cataracts.