Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (HO) or overexpression of Nox4, which produces HO, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived HO to promote mtROS production. Mechanistically, HO increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived HO in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.
Epalrestat (EPS), approved in Japan, is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Here we report that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH), which is important for protection against oxidative injury, through transcription regulation. Treatment of Schwann cells with EPS caused a dramatic increase in intracellular GSH levels. EPS increased the mRNA levels of γ-glutamylcysteine synthetase (γ-GCS), the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a central role in regulating the expression of γ-GCS. ELISA revealed that EPS increased nuclear Nrf2 levels. Knockdown of Nrf2 by siRNA suppressed the EPS-induced GSH biosynthesis. Furthermore, pretreatment with EPS reduced the cytotoxicity induced by H2O2, tert-butylhydroperoxide, 2,2'-azobis (2-amidinopropane) dihydrochloride, and menadione, indicating that EPS plays a role in protecting against oxidative stress. This is the first study to show that EPS induces GSH biosynthesis via the activation of Nrf2. We suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by oxidative stress.
Heme oxygenase (HO)-1 has potent antioxidant and anti-inflammatory functions. Recent studies have shown that the upregulation of HO-1 is beneficial to counteract neuroinflammation, making HO-1 a new therapeutic target for neurological diseases. We have reported that epalrestat (EPS), which is currently used for the treatment of diabetic neuropathy, increases HO-1 levels through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) in bovine aortic endothelial cells. In this study, we tested the hypothesis that EPS upregulates HO-1 via Nrf2 activation in the component cells of the nervous system, by using rat Schwann cells and human SH-SY5Y cells. Treatment of Schwann cells with EPS at near-plasma concentration led to a dramatic increase in HO-1 levels. Nrf2 knockdown by small interfering RNA (siRNA) suppressed the EPS-induced HO-1 expression. EPS did not promote the intracellular accumulation of free ferrous ion and reactive oxygen species, by increasing ferritin via Nrf2 during HO-1 induction. Moreover, EPS stimulated the expression of superoxide dismutase 1 and catalase, which also are Nrf2 target gene products. It also markedly increased HO-1 levels in SH-SY5Y cells through the activation of Nrf2. We demonstrated for the first time that EPS upregulates HO-1, superoxide dismutase, and catalase by activating Nrf2. We suggest that EPS has the potential to prevent several neurological diseases. Key words epalrestat; heme oxygenase (HO)-1; superoxide dismutase (SOD); catalaseHeme oxygenase (HO)-1 is a stress-responsive enzyme that has anti-inflammatory, antioxidant, and cytoprotective functions. Expectations are high that the regulation and amplification of HO-1 by pharmacological approaches would lead to the discovery of novel drugs for the treatment of a variety of diseases.1) HO-1 has emerged as an anti-inflammatory therapeutic target.2) In particular, targeting HO-1 in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, has been reported.3) As HO-1 expression is involved in neuropathological changes, its regulation is essential for the development of new therapeutic approaches.3) However, such metalloporphyrins as cobalt protoporphyrin IX, which are prototypical inducers of HO-1 and commonly used in experimental cell culture and animal models, are not applicable to clinical interventions because of their high toxicity.2) It is expected that the upregulation of HO-1 by currently available pharmacological agents, whose safety and pharmacokinetics have already been confirmed clinically, would be useful for the treatment of a variety of diseases.The biochemical activities of heme degradation products and their metabolic derivatives contribute to the cytoprotective function of HO-1. HO-1 catalyzes the degradation of heme to produce ferrous iron, carbon monoxide, and biliverdin, the latter of which is subsequently converted into bilirubin. Carbon monoxide is involved in inflammation regulation.4) Bilirubin and biliverdin, which can scavenge peroxyl radicals, are cytoprotec...
Epalrestat (EPS) is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs), an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.
Schwann cell injury is observed in diabetic neuropathy. It is speculated that glycolaldehyde (GA), a precursor of advanced glycation end products (AGEs), contributes to the pathogenesis and development of diabetic neuropathy. Here, we demonstrated for the first time that GA at near-physiological concentration decreased the viability of rat Schwann cells. In contrast, methylglyoxal, glyoxal, and 3-deoxyglucosone, all of which are AGE precursors, had no effects on cell viability. It is well known that methylglyoxal causes oxidative damage. In the present study, however, GA failed to induce reactive oxygen species production in Schwann cells. The addition of glutathione (GSH) or N-acetyl-l-cysteine protected Schwann cells from the loss of viability induced by GA. Moreover, GA increased intracellular GSH level and γ-glutamylcysteine synthetase mRNA level. Flow cytometric analysis revealed that GA increased multidrug-resistance-associated protein 1 (MRP1) level as well. Moreover, we demonstrated that the knockdown of MRP1 with small interfering RNA (siRNA) enhanced the loss of cell viability induced by GA. Taken together, these findings suggest that MRP1, together with GSH, plays an important role in the GA-induced toxicity in Schwann cells.
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