Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling
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A long-term ‘memory’ of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding.
Materials and methods
HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus α-lipoic acid in the last week.
In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-β, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADP-ribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant α-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H2DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with α-lipoic acid added to the last week of normalised glucose.
These results provide proof-of-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation.
KeywordsAntioxidants Diabetic complications Endothelial cells Endothelial dysfunction Hyperglycaemia Memory Oxidative stress Retinal cells Retinopathy
Bcl-2-associated X protein
B cell lymphoma protein-2
protein kinase C
reactive oxygen species
uncoupling protein 2
The hallmark of diabetes is hyperglycaemia, which, evidence suggests, plays a major role in the pathogenesis of the disease’s complications, both micro and macrovascular in nature .
Dysfunction of the endothelium is an integral cause of development of diabetic vascular disease , with many studies confirming that high glucose in vitro can directly affect endothelial cell function . Recently, an excess of reactive oxygen species (ROS) generated in the endothelium and in target organs such as the retina, kidney and heart in response to hyperglycaemia has been shown to form a causative link between high glucose and diabetic complications [4, 5]. In particular, endothelial dysfunction in diabetes has been found to be mediated through an excess of cellular ROS  involving respiratory chain uncoupling , protein kinase C (PKC) activation , NAD(P)H oxidase activation , formation of single strand DNA breaks and activation of poly(ADP-ribose) (PAR) polymerase (PARP) , basement membrane thickening  and activation of pro-apoptotic proteins .
Almost 20 years ago, the laboratory of Lorenzi showed that there was a persistence or ‘memory’ of the induced expression of basement membrane mRNAs (fibronectin, collagen IV) long after high glucose levels were normalised in endothelial cells in culture [10, 11] and in diabetic rats , suggesting the possibility of a long-lasting deleterious effect of hyperglycaemia on these cells, independent of the actual level of glucose. These data are consistent with findings that retinopathy progression can persist in dogs even after the normalisation of glycaemic control .
The aim of this study was to duplicate the previous findings in endothelial cells, retinal cells and in the retina of diabetic animals, and to evaluate the possible role of the persistence of ROS production after glucose normalisation as a possible explanation for the phenomenon termed here ‘memory.’
Materials and methods
‘Memory’ experiments in cells
HUVECs were grown in MCDB-131 medium (GIBCo Life Technologies, Grand Island, NY, USA) with 15% cosmic calf serum (HyClone, Logan, UT), epidermal growth factor (10 ng/ml; Peprotech, Rocky Hill, NJ, USA) and penicillin/streptomycin. ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) were grown in DMEM with 10% cosmic calf serum (HyClone) and penicillin/streptomycin. As previously described , cells were incubated in 5 mmol/l glucose, with 25 mmol/l mannitol (Sigma Chemical, St Louis, MO, USA) for osmolarity normalisation, or in 30 mmol/l glucose for 3 weeks or up to 1 week in normal glucose (5 mmol/l) after being exposed for 2 weeks to continuous high glucose (30 mmol/l). Fresh medium was added to cells every other day and 24 h before the end of the experiment.
Effects of ROS inhibition on the ‘memory’ phenomenon in HUVECs
In the last week of normal glucose in HUVECs, glucose was given with or without agents capable of inhibiting ROS production as follows: 62.5 μmol/l α-lipoic acid (ALA), an antioxidant working also at the mitochondria level ; 10 μmol/l apocynin ; 10 μmol/l oxypurinol ; 1 μmol/l PJ34 ; and 25 to 100 plaque-forming units per cell of an uncoupling protein 2 (UCP2) overexpressing adenovirus, used to specifically inhibit mitochondrial ROS production . Inhibitors were replaced every other day and 24 h before the end of the experiment. Inhibitors were purchased from EMD Bioscience (San Diego, CA, USA), except oxypurinol and ALA, which were from Sigma Chemical and the UCP2 adenovirus, which was a kind gift of M. H. Zou (Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma, OK, USA). Cells were grown at 25 to 30% confluence in 25 or 75 cm2 flasks until 100% confluent.
Rat streptozotocin-induced hyperglycaemia mode
All animal studies were conducted in accordance with guidelines approved by the Association for Assessment and Accreditation of Laboratory Animal Care International using a protocol approved by the Institutional Animal Care and Use Committee at the University of Oklahoma. To make 6- to 8-week-old female Brown Norway rats (Charles River Laboratories, Wilmington, MA, USA) hyperglycaemic, a single intraperitoneal injection of 50 mg/kg streptozotocin (Sigma Chemical) in 10 mmol/l citrate buffer, pH 4.5, was given as previously described . Similarly to cell experiments, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus ALA during the last week. Rats receiving insulin were implanted with a continuous-release insulin implant (LinShin, Scarborough, ONT, Canada)  under the skin of the upper abdomen. Blood glucose levels were in the following ranges: 5.8–8.0 mmol/l in non-diabetic rats, 17–22.8 mmol/l in diabetic rats and 4.8–5.6 mmol/l in the last week of treatment in insulin-treated animals. ALA (racemic mixture; Sigma Chemical) was prepared in 0.9% sodium chloride, pH 7.4, snap-frozen, stored at −80°C and aliquots thawed daily just before injection . Animals receiving ALA were injected intraperitoneally with 75 mg/kg ALA each day, while all other animals received daily injections of 0.9% sodium chloride, pH 7.4. Blood glucose of animals receiving insulin and ALA during the last week was 16.0–22.2 mmol/l during the first 2 weeks and 6.0–8.8 mmol/l in the last week.
Western blot analysis
Whole-cell lysates were made using M-PER lysis buffer (Pierce Chemical, Rockford, IL, USA) for endothelial cells with 300 mmol/l sodium chloride and containing protease inhibitor cocktail (EMD Bioscience) and phosphatase inhibitors (Sigma Chemical). Equal amounts of protein lysates (30 or 50 μg; determined by Pierce micro BCA protein assay) were separated using 8–12% SDS-PAGE gels, transferred to 0.2 μmol/l nitrocellulose (Schleicher and Schuell, Keene, NH, USA), blocked in SuperBlock (Pierce Chemical), incubated with primary antibodies, either overnight at 4°C, or for 2 h at room temperature, washed three times in Tris-buffered saline to which 0.25% Tween-20 secondary antibodies (Pierce Chemical) was added, washed again with 0.25% Tween-20, to which SuperSignal Dura chemiluminescence substrate (Pierce Chemical) was added and subjected to digital imaging using a charge-coupled device camera (ImageStation 4000; Kodak, New Haven, CT, USA). Densitometry of bands was measured using Image J 1.30 analysis software (http://rsb.info.nih.gov/nih-image/). In all cases, confirmation of equal loading was performed by Memcode staining (Pierce Chemical) and by probing blots against vinculin . Primary antibodies and dilutions used were as follows: 3-nitrotyrosine (3-NY) (1:100; Upstate Biotech, Lake Placid, NY, USA); B cell lymphoma protein-2 (BCL-2)-associated X protein (Bax) (1:1,000; Cell Signaling Technology, Danvers, MA, USA); fibronectin human (H-300, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA); manganese superoxide dismutase (1:650; Abcam, Cambridge, MA, USA); p47phox human (1:500; Upstate Biotech); PAR (1:400; Alexis Biochemicals, San Diego, CA, USA); phospho PKC-α/βII (1:1,000; Cell Signaling); PKC-βII (1:200; Santa Cruz).
CM-H2DCFDA ROS fluorescence assay
HUVECs were exposed for 3 weeks to 5 or 30 mmol/l glucose or for 2 weeks to 30 mmol/l glucose, followed by 1 week in 5 mmol/l glucose with or without inhibitors as described above. The day preceding the end of the experiment, HUVECs were plated at a density of 7,000 cells (70% confluence) on to white 96-well microplates with clear bottoms (3610; Costar, Cambridge, MA, USA) in 5 mmol/l with or without inhibitors. At the end of the experiment, medium was removed from the cells and 2 μg/ml of the cell-permeable CM-H2DCFDA probe (Molecular Probes-Invitrogen, Eugene, OR, USA) in Hank’s Balanced Salt Solution was added to each well of the plates and incubated for 15 min at 37°C as previously described . Fluorescence intensity was then measured using a plate reader (BMG Labtech, Durham, NC, USA) using 488 nm excitation and 530 nm emission filters.
Data were analysed using one-way ANOVA to compare the means of all the groups. The Neuman−Keuls multiple comparisons procedure was used to determine which pairs of means were different. Differences were considered significant at p < 0.05.
Persistence of high glucose stress markers after glucose normalisation in endothelial and retinal cells
The role of ROS in the ‘memory’ of high glucose stress in endothelial cells
‘Memory’ of hyperglycaemic stress in the retina of diabetic animals
The role of ROS in ‘memory’ of high glucose stress in diabetic retina
The next experiment was to determine whether the persistence of hyperglycaemic stress could be interrupted in the rat diabetic model in vivo, using the mitochondrial antioxidant nutritional supplement, ALA, which has previously been shown to interrupt target organ damage when given chronically to diabetic animals . The addition of ALA to the last week of normalised glucose resulted in significant decreases in most of the hyperglycaemic stress markers in the retina of diabetic animals (Fig. 5).
In this study we were able to confirm that a persistence or cellular ‘memory’ of high glucose stress exists after glucose levels are normalised in endothelial cells. However, for the first time, we have demonstrated that persisting overproduction of ROS may explain this phenomenon. Also, for the first time, we have found that the inhibition of mitochondrial ROS production with ALA  or UCP2 transfection  can interrupt aspects of this ‘memory’ phenomenon. Moreover, inhibiting extra-mitochondrial ROS production or PARP activity also reduced several, but not all, of the persistently induced stress markers.
There is convincing evidence for the notion that a persisting increase of ROS generation provides the mechanistic basis underlying the hyperglycaemic ‘memory’ of high glucose stress marker induction. The fact that the general antioxidant ALA, which works at the mitochondrial level , was capable of disrupting the ‘memory’ for all of the high glucose stress markers in isolated endothelial cells and rat retinas as well as the induction of free ROS in endothelial cells, supports this hypothesis. Moreover, the differential effects of inhibiting mitochondrial ROS compared with extra-mitochondrial ROS in endothelial cells suggest that the persistence of the ‘memory’ phenomenon is related to a mitochondrial effect of hyperglycaemia. This is in keeping with previous findings, which have demonstrated that in conditions of high glucose mitochondrial overproduction of ROS accounts for the activation of all the other pathways involved in damaging endothelial cells, including those involved in amplifying the production of ROS, such as PKC and NAD(P)H excess [33, 34, 35].
Our data confirm the existence of a ‘memory’ of hyperglycaemia-induced damage in retinal cells in culture and in the retina of diabetic rats, suggesting that this phenomenon is linked to persistence of the oxidative stress. This hypothesis is consistent with a previous finding that the return to good glycaemic control in rats after 6 months of very poor control was ineffective in decreasing 3-NY levels or other markers of oxidative stress in the retina of these diabetic animals . Moreover, the effect of ALA in our study supports the finding of Lin et al., showing protective effects of ALA in experimental diabetic retinopathy through the inhibition of oxidative and nitrosative stress .
The 1 week duration of the ‘memory’ of high glucose stress is too long to be explained by simple signalling mechanisms or the half-lives of messenger RNA or protein. Thus there must be something propagating this ‘memory’ phenomenon. Based on the excess of free ROS observed after glucose normalisation and the ability of different mechanisms to inhibit ROS and interrupt aspects of the persistence of high glucose stress, an excess of intracellular ROS in target cells is a likely candidate. In addition, although the particular regulatory mechanisms of the high glucose stress markers assessed in this paper differ, all of these markers are directly or indirectly induced by ROS [34, 37, 38, 39, 40, 41, 42], again implicating an excess of cellular ROS in the ‘memory’ phenomenon. Interestingly, ALA and in particular overexpression of UCP2 were able to reduce the ‘memory’ effect for all the studied parameters, suggesting that persistent mitochondrial generation of ROS plays a key role in producing this phenomenon. A purely speculative explanation is the possibility that mitochondrial proteins are glycated during the 2 week exposure to high glucose. These premises are important because a recent study, for the first time, has described a direct relationship between the formation of intracellular AGE on mitochondrial respiratory chain proteins, the decline in mitochondrial function and excess formation of reactive species [43, 44]. Thus, AGE-modified mitochondrial respiratory chain proteins are prone to produce more ROS, independently of the level of glucose [43, 44], and could therefore also be propagating the ‘memory.’ Another possible alternative explanation is persistent activation of AGE receptors (RAGE) . Further studies will be needed to explore these hypotheses.
In our study, only the inhibition of mitochondrial production of ROS seemed to completely abolish the ‘memory’ effect of hyperglycaemia, while the inhibition of cytoplasmic free radicals only partially interrupted this effect This is not surprising because mitochondrial generation of free radicals is considered to be only the first step of ROS generation, which can proceed through several other intracellular sources [46, 47, 48].
It is also intriguing that several markers not only failed to normalise or remain at the level reached during high glucose exposure, but in fact increased further after glucose normalisation. However, because expression of these markers has been shown to be regulated by free radical generation, the persistence of ROS production in the cells may also account for this phenomenon.
In summary, our results provide a proof-of-principle of cellular ‘memory’ of induction of six high glucose stress markers of basement membrane thickening, oxidative stress, cell death, cell signalling and DNA damage, as well as of free ROS in endothelial and retinal pigment epithelial cells and in the retina of diabetic rats long after glucose normalisation. Furthermore, antioxidants and inhibitors of mitochondrial ROS and to a lesser extent extra-mitochondrial ROS production were shown to interrupt aspects of this ‘memory’ phenomenon. These data support and extend the hypothesis that an excess of ROS is the causal link between hyperglycaemia and complications of diabetes . Although the length of the experiment was very short, our data still show that 1 week of tight glucose control is not sufficient to normalise the damage induced by hyperglycaemia. Our data therefore confirm the need for early tight glycaemic control to avoid diabetic complications. At the same time, they also further strengthen the use of antioxidant agents, together with glucose normalisation, to mitigate the progression of these complications.
This study was funded by a NIH/NCRR Center for Biomedical Research Excellence program project, ‘Mentoring Vision Research in Oklahoma’ (1P20RR017703-01), to M. A. Ihnat (promising junior investigator). It was also supported by the Hungarian National Scientific Research Fund (grants D-45933, T-049621, AT-049488) and Hungarian Health Science Council (grants 248/2003, 249/2003) to C. Szabó.
Duality of interest
The authors all state that they have no conflict of interest with respect to publication of this work.