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Superoxide Anions and Endothelial Cell Proliferation inNormoglycemia and Hyperglycemia
Michela Zanetti, Ralph M. Zwacka, John F. Engelhardt, Zvonimir S. Katusic, Timothy O’Brien
Abstract—Oxygen free radicals are believed to play a key role in cellular proliferation, and increased concentrations ofthese molecules have been implicated in the pathogenesis of endothelial dysfunction in diabetes mellitus. Our aim wasto study the role of superoxide anions in endothelial cell proliferation under conditions of normoglycemia andhyperglycemia. Human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs)exposed to adenoviral vectors encoding CuZnSOD (AdCuZnSOD),b-galactosidase (Adbgal), or diluent (control) werecultured in normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 28 mmol/L) medium. Cell proliferation wascompared by use of [3H]thymidine incorporation and cell count in transduced and control cells in the setting of NG andHG. Transgene expression was detected in transduced cells by X-gal staining and by Western analysis and SOD activityassay in AdCuZnSOD-transduced cells. Superoxide production was significantly (P,0.05) decreased in AdCuZnSOD-transduced cells cultured in both NG and HG medium. In NG, AdCuZnSOD-transduced endothelial cells had decreasedproliferation compared with control cells. After 48 hours in HG, superoxide levels were increased and DNA synthesiswas decreased (P,0.05) in control and Adbgal-transduced but were not affected in AdCuZnSOD-transduced cells. Inaddition, after 7 days in HG, cell counts were reduced (P,0.05) in control (7362.5%) and Adbgal-transduced(7563.4%) but not in AdCuZnSOD-transduced cells (8963.4%). These results suggest that either a deficiency or anexcess of superoxide anions inhibits endothelial cell proliferation, and the inhibitory effect of increased superoxide dueto hyperglycemia can be reversed by CuZnSOD overexpression.(Arterioscler Thromb Vasc Biol. 2001;21:195-200.)
A lthough oxygen-derived free radicals have been impli-cated in causing cell damage and cell death,1 it has
become clear in recent years that they can also play aphysiological role in the intracellular signaling pathways. Inparticular, the importance of superoxide anion as a mediatorinvolved in the regulation of cell growth in vascular smoothmuscle cells (VSMCs) has been outlined by several re-ports.2–5At variance with VSMCs, sporadic data are availableon redox-regulated changes in endothelial cell proliferation,because antioxidants and agonists that upregulate superoxideproduction have been shown to produce few or no effects onendothelial cell growth.3,4 Therefore, it has been hypothesizedthat endothelial cells are less susceptible to growth regulationby redox state, although some data contradict this view.6
It is well established that diabetes mellitus is associatedwith increased oxidative stress,7,8 which is believed to play akey role in the pathogenesis of diabetic vascular dysfunction.9
Endothelial cells from both microvessels and macrovesselscultured in high glucose show delayed replication,10–14 ab-normal cell cycling,15 and increased apoptosis,16 along withan increased expression and activity of endogenous antioxi-dant enzymes.14 It has been hypothesized that the upregula-tion of antioxidant enzymes by glucose is insufficient to
reverse the deleterious effects of the increased oxidativestress characteristic of this condition. Glucose-induced endo-thelial cell toxicity may be reversed by exposure of cells toantioxidants17 or their precursors,18 whereas increased apo-ptosis is prevented by the administration of exogenousSOD.19 Taken together, these data indicate that adequate freeradical scavenging is imperative for normal endothelial func-tion and survival in diabetes mellitus.
Therapeutic approaches designed to deliver genes encod-ing antioxidant enzymes to intracellular sites at sustainedlevels and in a durable manner may have advantages overdelivery of the protein in diabetic vascular disease. Disad-vantages of delivery of recombinant antioxidant proteinsinclude the short half-life of these substances in the blood-stream along with an inability to detoxify intracellular reac-tive oxygen species. These limitations could possibly beovercome by use of gene therapy to create an endogenoussource of enzyme to provide sustained antioxidant protection.
Superoxide dismutase (SOD) is responsible for scavengingO2
z 2 in eukaryotic cells. Three isoforms of the enzyme exist,which differ in their subcellular localization as well as in thecofactors required for catalytic activity. The cytosolic isoformrequires copper and zinc (CuZn), whereas the mitochondrial
Received May 11, 2000; revision accepted November 21, 2000.From the Endocrine Research Unit, Mayo Clinic and Foundation, Rochester, Minn (M.Z., Z.S.K., T.O.), and the Departments of Anatomy and Cell
isoform requires manganese.20 An immunodistinct tetramericextracellular SOD is also CuZn-dependent.21 Gene transferstrategies may allow the effects of various isoforms to becompared and may provide insights into the cellular source ofexcess free radical generation associated with hyperglycemia.
MethodsCell CultureHuman aortic endothelial cells (HAECs) and human umbilical veinendothelial cells (HUVECs) were obtained from Clonetics. Theywere cultured in modified MCDB-131 medium (EBM, Clonetics)supplemented with 2% FBS, gentamycin (50mg/mL), bovine brainextract (12mg/mL), hydrocortisone (1mg/mL), and human epider-mal growth factor (10 ng/mL). HAECs were first grown to conflu-ence at 37°C in a humidified atmosphere containing 5% CO2.Cultures between the third and the seventh subpassages were usedfor the experiments.
Construction, Propagation, and Purification ofAdenoviral VectorsA recombinant adenovirus containing the cDNA encoding the humanCuZnSOD gene driven by a cytomegalovirus promoter (AdCuZn-SOD) was generated as previously described.22 It was propagated,isolated, and quantified by standard techniques.22,23
AdCMVLacZ (Adbgal), used in all experiments as a control, wasa kind gift from Dr James M. Wilson (University of Pennsylvania,Philadelphia). Viral stocks were stored at270°C.
Gene Transfer to Endothelial CellsHAECs and HUVECs were plated at the optimal density for eachexperiment and cultured overnight in regular medium. For all theexperiments, cells were transduced with adenoviral vectors 24 hoursafter plating. Cells were incubated with various multiplicities ofinfection (MOIs) (25 or 50) of AdCuZnSOD or Adbgal in PBS/0.5%albumin for 1 hour at 37°C. Additional cells (control) were exposedto diluent alone. The viral solution was then removed and replacedwith regular medium.
Assessment of Transgene ExpressionTransgene expression was demonstrated by X-gal staining ofAdbgal-transduced cells, Western blot analysis of CuZnSOD pro-tein, and SOD activity assay.
Detection ofb-GalactosidaseFor X-gal staining, HAECs were transduced with increasing MOIs(0, 25, 50) of Adbgal as described above. After gene transfer,incubation was continued in normal glucose or high glucose mediumfor an additional 48 hours to allow transgene expression. Thereafter,cells were washed with PBS and fixed for 5 minutes in 4%paraformaldehyde, 0.4% glutaraldehyde in PBS. One milliliter of asolution containing 500mg/mL 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) (Boehringer Mannheim Corp) was addedto each experimental well and then incubated for 2 hours at 37°C.Each well was examined under a light microscope, and the efficiencyof gene transfer to the endothelial monolayer was visually assessed.
Western Blot Analysis for Human CuZnSOD ProteinFor Western blot analysis of human CuZnSOD protein, HAECs wereplated at a density of 23106 in 100-mm plates, transduced the nextday with adenoviral vectors at MOI 50, and incubated for 48 hoursin regular medium. Then soluble proteins were extracted by lysingand sonication of the pellets. After centrifugation, the supernatantwas collected and total protein concentration determined by thebicinchoninic acid assay. Prestained protein markers (Bio-Rad) and15 mg of protein were loaded on 4% stacking/15% separatingSDS-PAGE. The resolved proteins were transferred to 0.2-mmnitrocellulose membrane on a semidry electrophoretic transfer sys-tem (Bio-Rad) for Western blot analysis. Blots were blocked with5% nonfat dry milk in PBS buffer/0.1% Tween 20 overnight at 4°C.The membrane was then incubated with a sheep anti-human CuZn-
SOD (1:100, Biodesign) in blocking buffer overnight at 4°C. Theblots were next incubated with peroxidase-conjugated anti-sheepsecondary antibody (1:2500, Biodesign) for 1 hour at room temper-ature. Specific CuZnSOD protein was detected by enhanced chemi-luminescence (ECL, Amersham Life Science).
Determination of SOD ActivityHAECs were plated, transduced the next day with AdCuZnSOD orAdbgal at MOI 50, and incubated for 48 hours in medium containing28 mmol/L glucose. Then cells were scraped with a rubber police-man and sonicated in 13PBS/0.1% Triton X-100 (pH 7.4) on icewith two 30-second bursts. SOD activity was measured by thereduction of cytochromec, as described.24 Briefly, xanthine/xanthineoxidase was used to generate O2
z 2, which was detected by thereduction of cytochromec. Spectrophotometric measurement of therate of reduction of cytochromec in the presence of increasingamounts of SOD protein was performed. Total SOD activity wasdetermined from the amount of inhibition of cytochromec reduction.
Measurement of Superoxide ProductionAfter transduction with adenoviral vectors encoding Adbgal orAdCuZnSOD, cells were grown for 48 hours in 5.5 or 28 mmol/Lglucose medium. Secretion of superoxide by the endothelial cellswas determined by SOD-inhibitable reduction of cytochromec.25
Cells were incubated for 1 hour in phenol red–free medium in thepresence of 20mmol/L cytochromec. O2
z 2 release was calculatedfrom the difference of absorbance at 550 nm; a molar extinction of21 000 was used.
Assessment of Cell ProliferationCell proliferation was determined by [3H]thymidine incorporationand cell count. For both experiments, HAECs were plated atsubconfluence and transduced with 50 MOI of AdCuZnSOD orAdbgal. To assess [3H]thymidine incorporation, cells were firstrendered quiescent for 24 hours with medium supplemented with0.1% FBS and then stimulated for 44 hours with regular mediumcontaining either 5.5 or 28 mmol/L glucose. [3H]thymidine incorpo-ration was determined by addition of 1mCi of 3H-labeled thymidine(Amersham Life Science) for 4 hours at 37°C. Then cells werewashed, DNA was extracted with 0.5N NaOH, and radioactivity wascounted by scintillation spectroscopy.
Cell count was performed in both HAECs and HUVECs. For thisexperiment, after gene transfer, cells were incubated for 7 days inregular medium containing 5.5 or 28 mmol/L glucose, which waschanged every 48 hours.
Cells were counted in a Coulter Counter (model ZM, CoulterElectronics Ltd, ) on day 7.
Statistical AnalysesDifferences between mean values of multiple groups were analyzedby 1-way ANOVA with Fisher analysis. Values ofP#0.05 wereconsidered to be statistically significant.
ResultsAssessment of Transgene Expression inEndothelial Cells
Assessment ofb-Galactosidase Expression Under Normaland High-Glucose ConditionsAs assessed by X-gal staining in Adbgal-transduced HAECscultured under normal-glucose conditions, gene transfer re-sulted in efficient transgene expression (Figure 1). In con-trast, there was no evidence of X-gal staining in control cells(Figure 1).
To assess whether incubation in high-glucose conditionsmight affect efficiency of transduction, transgene expressionwas determined after incubation in normal- or high-glucosemedium for 48 hours. X-gal–positive cells were 47.262% inmedium containing 5.5 mmol/L glucose and 51.862.6% inmedium containing 28 mmol/L glucose (P50.17).
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Western Blot Analysis of Recombinant CuZnSODWestern blot analysis of CuZnSOD protein showed a single16-kDa band corresponding to human CuZnSOD in lysatesprepared from AdCuZnSOD-transduced HAECs (Figure 2).
Determination of SOD ActivityCell lysates were next examined for antioxidant enzymeactivities. After transduction with AdCuZnSOD and incuba-tion in medium containing 28 mmol/L glucose for 48 hours,total SOD activity was significantly(P,0.0005) increased inAdCuZnSOD-transduced HAECs (435.86101.4 U/mg pro-tein) compared with control (68.368.6 U/mg protein) andAdbgal-transduced (57.669.4 U/mg protein) cells.
Measurement of Superoxide ProductionIn medium containing 5.5 mmol/L glucose, O2
z 2 productionwas significantly (P,0.007) decreased in AdCuZnSOD-transduced HAECs (1.660.2 nmolz h21 z well21) comparedwith control and Adbgal-transduced cells (2.560.13 and2.460.2 nmolz h21 z well21, respectively) (Figure 3A).
After 48 hours in medium containing 28 mmol/L glucose,control and Adbgal-transduced HAECs released in the me-dium 4.060.1 and 4.260.1 nmol O2
z 2 per hour per well,respectively (Figure 3B). Transduction with AdCuZnSODsignificantly (P,0.03) reduced the amount of O2
z 2 measuredin the culture medium (3.260.2 nmolz h21 z well21) (Figure3B).
Effect of Adenovirus-Mediated Gene Transfer onEndothelial Cell ProliferationUnder normal-glucose conditions, transduction with Adbgalresulted in a significant inhibition of DNA synthesis (Adbgal,
1347693.5 and control, 18756132.9 dpm/well,P,0.05)(Figure 4A) and cell count (Adbgal, 68.666.43103 andcontrol, 81.369.43103 cells/well, P,0.05) (Figure 4B).Thus, adenoviral vector per se had an inhibitory effect onendothelial cell proliferation in this cell line. To determinewhether the effect of adenovirus on cell proliferation is cellline–specific, additional experiments were performed onanother endothelial cell line (HUVECs). Transduction withAdbgal did not significantly affect cell count in this endo-thelial cell line (Adbgal, 50.065.03103 and control,57.162.43103 cells/well).
Figure 1. X-Gal staining of HAECs transduced with adenoviral solutions containing increasing concentrations of Adbgal. A, 0 MOI (novirus); B, 25 MOI; C, 50 MOI.
Figure 2. Effects of CuZnSOD gene transfer on expression ofCuZnSOD protein. HAECs were incubated with 50 MOI ofAdCuZnSOD or Adbgal in regular medium, and incubation wascontinued for 48 hours more to allow expression. Medium wasthen removed, and cells were harvested and sonicated. HumanCuZnSOD protein in cell lysate was detected by Western blotanalysis. Cell lysates (15 mg protein) were loaded. CuZnSODprotein expression (lane 3) was markedly increased inAdCuZnSOD-transduced cells compared with control (lane 1)and Adbgal-transduced (lane 2) cells.
Figure 3. Effects of CuZnSOD gene transfer on O2z 2 produc-
tion. HAECs were transduced with 50 MOI of AdCuZnSOD orAdbgal and exposed to 5.5 or 28 mmol/L glucose medium.After 48 hours, cells were washed and incubated in 1 mLserum-free, phenol red–free medium containing 20 mmol/L cyto-chrome c for 1 hour. O2
z 2 levels were calculated from amountof cytochrome c reduction at 550 nm. A, O2
z 2 production in5.5 mmol/L glucose; B, O2
z 2 production in 28 mmol/L glucose.Each bar represents mean6SEM of 2 values from 3 separateexperiments. *P,0.03 vs control and Adbgal.
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Effect of CuZnSOD Overexpression on EndothelialCell Proliferation in Normal GlucoseTo assess the effect of superoxide anion on endothelial cellproliferation under normoglycemic conditions, adenovirus-mediated gene transfer of CuZnSOD to HAECs was per-formed. In normal glucose, [3H]thymidine incorporation wasinhibited in AdCuZnSOD-transduced (875657.7 dpm/well)compared with Adbgal-transduced (1347693.5 dpm/well,P,0.05) HAECs (Figure 4A). This finding was confirmed bycell count (49.166.53103 cells/well in the AdCuZnSODversus 68.666.43103 cells/well in the Adbgal group,P,0.05, Figure 4B).
Transduction with AdCuZnSOD also resulted in a signif-icant inhibition of cell proliferation in HUVECs(30.062.43103 cells/well versus 57.162.43103 in controland 50.0653103 in Adbgal,P,0.05), confirming the role ofsuperoxide anions in endothelial cell proliferation underconditions of normal glucose.
Effects of High Glucose Concentrations onCell ProliferationHigh glucose resulted in decreased proliferation in bothHAECs and HUVECs. After 48 hours’ exposure to highglucose, [3H]thymidine incorporation was 86.963.6% and82.162.9% of each control in normal glucose in control andAdbgal-transduced HAECs (Figure 5A), consistent withresults previously reported.11 After 7 days in 28 mmol/Lglucose, cell counts in both control and Adbgal-transducedHAECs were significantly (P,0.05) less than the corre-sponding group grown in physiological glucose (7362.5%and 7563.4%, respectively) (Figure 5B). Similar cell countwas obtained with HUVECs (control, 81.565.2%; Adbgal,
63.667.6%, P,0.05 versus each control group in5.5 mmol/L glucose).
Effects of CuZnSOD Gene Transfer on CellProliferation in High GlucoseThe effect of CuZnSOD overexpression on endothelial cellproliferation in the setting of high glucose was next deter-mined. In contrast to control and Adbgal-transduced cells,DNA synthesis as assessed by [3H]thymidine incorporationwas not decreased in AdCuZnSOD-transduced HAECs ex-posed to high concentrations of glucose (Figure 5A). Further-more, cell counts were not significantly reduced inAdCuZnSOD-transduced HAECs (Figure 5B) grown in highglucose. Similar results were showed with HUVECs(AdCuZnSOD-transduced cells, 91.364% of the controlgroup in 5.5 mmol/L glucose).
DiscussionSuperoxide anions play a crucial role in cell proliferation. Inthe present study, we show that overexpression of CuZnSODin endothelial cells cultured in normal glucose decreases the
Figure 4. Effect of CuZnSOD gene transfer on HAEC prolifera-tion in normal glucose. HAECs were transduced with 50 MOI ofAdbgal or AdCuZnSOD as described before and then incubatedin regular medium. Cell proliferation was assessed by [3H]thymi-dine incorporation 72 hours after gene transfer (A) and by cellcount 7 days after transduction (B). Results are expressed asmean6SEM of 3 different experiments, each of them performedin triplicate. *P,0.05 vs Adbgal and AdCuZnSOD; #P,0.05 vsAdCuZnSOD.
Figure 5. Effects of CuZnSOD gene transfer on cell proliferationin HAECs cultured in high glucose. HAECs were transducedwith 50 MOI of either AdCuZnSOD or Adbgal. Control cells wereexposed to diluent alone. Immediately after gene transfer, cellswere made quiescent for 24 hours and then stimulated for 44hours with regular medium containing either 5.5 or 20 mmol/Lglucose. [3H]thymidine was added for final 4 hours of incubationtime. For cell count, HAECs were transduced as describedbefore, and then cultured in 5.5 or 28 mmol/L glucose for 7days. A, [3H]thymidine in HAECs; B, cell count. Results areexpressed as % of each control in 5.5 mmol/L glucose,assumed as 100% from 3 different experiments, performed intriplicate; *P,0.05 vs each respective group in 5.5 mmol/Lglucose.
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production of superoxide anion and inhibits cell proliferation.Furthermore, culturing cells in high glucose increases theproduction of superoxide anion and also inhibits cell prolif-eration. Finally, we demonstrate that the effect of highglucose on cell proliferation can be prevented by CuZnSODoverexpression. These results suggest that regulation ofsuperoxide anions plays a crucial role in the modulation ofendothelial cell proliferation and that perturbations inducedby pathological states such as hyperglycemia may be over-come by gene transfer of antioxidant enzymes.
Extensive data from the literature support the role ofsuperoxide anion as a growth-promoting factor in VSMCs.2–5
The data on the effect of superoxide on endothelial cellproliferation are sporadic and somewhat contradictory, how-ever, because exposure to antioxidants does not affect endo-thelial cell growth,4 whereas superoxide anion appears to beinvolved in the regulation of basal, nonstimulated endothelialcell proliferation.6
To assess the effect of SOD overexpression on cellproliferation in normal- and high-glucose conditions, we useda strategy of adenovirus-mediated gene transfer. It should benoted that adenovirus-mediated gene transfer per se inhibitedproliferation of the HAEC line used in these experiments.When cells were cultured in normal glucose, thymidineincorporation and cell counts were decreased in the Adbgalgroup compared with control cells, suggesting a toxic effectof the vector. Adenovirus-mediated inhibition of cell prolif-eration has previously been described for a number of celllines26 but never to our knowledge for a vascular cell line.When experiments were repeated in HUVECs, however, cellcounts were not significantly decreased in Adbgal-transducedcells, suggesting that the effect may be cell line–specific. Thisconcept is in keeping with previous results from our groupusing porcine coronary artery SMCs and HUVECs in whicha toxic effect of an adenoviral vector encodingb-galactosidase was not observed.23,27Thus, the sensitivity toadenovirus-mediated toxicity may vary depending on the cellline under consideration. We chose to continue our studies inhigh glucose with HAECs because of their strategic locationin the arterial bed and because this cell line has somedistinctive characteristics that are not shared byHUVECs.28,29
When cultured in normal-glucose medium, cells expressingCuZnSOD had decreased superoxide production and agonist-induced proliferation compared with Adbgal-expressing cellsand cells exposed to diluent alone. Thus, in both endothelialcell lines, overexpression of CuZnSOD was associated withdecreased cell proliferation in normal glucose concentrations.Our results are similar to those observed when fibroblasts aretreated with the antioxidant N-acetylcysteine, which results ininhibition of cell proliferation,30 and suggest that superoxideanion plays a critical role in cell proliferation. Although themechanism of this effect is unclear, a role for superoxide inthe Ras signaling pathway has been suggested.30 These resultssuggest that scavenging superoxide anions by SOD overex-pression in normal-glucose conditions inhibits endothelialcell proliferation and that a critical concentration of superox-ide anions appears to be necessary for cell proliferation.
In the present study, we evaluated the effect of highglucose on endothelial cell proliferation. In vitro, high glu-cose has been demonstrated to delay endothelial cell replica-
tion,15 cause cell death,10 and trigger apoptosis via increasedsuperoxide production.16,19 The latter effect is prevented byantioxidants. Ho et al31 recently showed that reactive oxygenspecies induced by high glucose mediate apoptosis in endo-thelial cells via JNK activation, which triggers caspase 3,whereas Pieper et al32 demonstrated that incubation of endo-thelial cells in high glucose results in the activation of nuclearfactor-kB. Thus, increased apoptosis may represent one of thepossible mechanisms by which increased concentrations ofsuperoxide anion found in association with high glucoseaffect cell viability.
For the reasons outlined above, the data concerning theeffect of high glucose concentrations on HAEC cell prolifer-ation are expressed in relation to cell counts in normalglucose for each experimental condition. In keeping withprevious reports on glucose-mediated cytotoxicity,10–14 theseresults clearly demonstrate that proliferation of control andAdbgal-transduced endothelial cells is inhibited by highglucose concentrations via increased generation of superox-ide radicals. In the present study, culture of endothelial cellsin high glucose resulted in increased superoxide generation,which was reversed by CuZnSOD overexpression. This effectwas associated with prevention of glucose-mediated decreaseof cell proliferation, thus suggesting that increased superox-ide generation was responsible for the inhibition of endothe-lial cell proliferation.
It was demonstrated previously that delayed endothelialcell replication due to oxidative stress in the setting of highglucose may be reversed by administration of SOD pro-tein.17,19However, this approach is limited by the fact that theexogenously administered SOD remains in an extracellularlocation, whereas superoxide radical is generated inside thecell. In contrast, overexpression of CuZnSOD via genetransfer has previously been shown to result in the correctcytoplasmic location of the transgene product.33 In addition,this approach may result in a longer duration of proteinexpression.
In conclusion, these data suggest that either a deficiency oran excess of superoxide levels inhibits endothelial cell pro-liferation. Furthermore, the inhibitory effect of increasedsuperoxide anion levels on endothelial cell proliferationobserved in diabetes mellitus may be reversed by overexpres-sion of superoxide dismutase.
AcknowledgmentsThis work was supported in part by National Heart, Lung, and BloodInstitute grant HL-58080 (T.O.B.), funds from the Bruce and RuthRappaport Program in Vascular Biology, and the Mayo Foundation.T.O.B. holds a Juvenile Diabetes Foundation Career DevelopmentAward. The authors are grateful to Dr R.J. Singh for helping to setup the SOD activity assay and to S. Stephan for technical assistance.
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