BASIC SCIENCE

Effects of antioxidant gene therapy on the developmentof diabetic retinopathy and the metabolic memory phenomenon

Liwei Zhang & Huiming Xia & Qianqian Han &

Baihua Chen

Received: 25 June 2014 /Revised: 2 September 2014 /Accepted: 30 September 2014# Springer-Verlag Berlin Heidelberg 2014

AbstractPurpose The purpose of this study was to determine thetherapeutic effect and mechanism of AAV-MnSOD by intra-vitreal injection on diabetic retinopathy (DRP) and the meta-bolic memory phenomenon.Methods The effect of hyperglycemia and metabolic memoryon the thickness of basement membrane, ratio of pericyte areaand cross-sectional area of capillary vessels in the nerve fiberlayer and outer plexiform layer; retinal capillary cell apopto-sis; number of acellular capillaries and activities of retinalMnSOD and catalase were examined and compared withintravitreal injection of AAV-MnSOD by transmission electronmicroscopy, TUNEL assay, ELISA, and immunohistochemistry.Results Hyperglycemia increased the thickness of capillarybasement membranes in the nerve fiber layer and outer plex-iform layer, decreased the ratio of pericyte area and cross-sectional area of capillary vessels, increased numbers of acel-lular capillaries and apoptosis of retinal capillary cells, anddecreased activities of retinal MnSOD and catalase. Termina-tion of hyperglycemia cannot reverse pathological changeslisted above. Intra-vitreal injection of AAV-MnSOD dramati-cally elevated the level and activities of retinal MnSOD andcatalase, and effectively prevented the progression of DRPand the metabolic memory phenomenon.Conclusions Increasing reactive oxygen species concentra-tion and continuous decreasing of antioxidant enzyme activity

play important roles in DRP and the metabolic memory phe-nomenon. AAV-MnSOD gene therapy provides a promisingstrategy to inhibit this blinding disease.

Keywords Diabetic retinopathy .Metabolic memory .

Reactive oxygen species . Manganese superoxide dismutase

Introduction

Diabetic retinopathy (DRP) is a common and devastatingmicrovascular complication in diabetes and is the leadingcause of acquired blindness among the people of occupationalage [1]. The selective loss of pericytes and basement mem-brane hypertrophy are characteristic early pathological eventsof DRP [2].

Although hyperglycemia is the hallmark and cause ofdiabetes, a recent study by the landmark Diabetes Controland Complications Trial (DCCT) and a follow-up study bythe Epidemiology of Diabetes Interventions and Complica-tions (EDIC) have shown that conventional therapy does notprevent the development and progression of early microvas-cular complications of diabetes, even if glycemic levels weremaintained as close to the nondiabetic range as possible.These trials have suggested a ‘metabolic memory phenome-non’ of the prior glycemic exposure and have demonstratedthe importance of early glycemic control on the progression ofDRP [3–8]. Moreover, the metabolic memory phenomenonlasts longer as the history of hyperglycemia is longer [3–8].Because of the difficulty in long-term maintenance of stableand accurate glycemic levels and the risk of hypoglycemiaduring glycemic control, diabetic patients are usually diag-nosed with a long-term history of hyperglycemia. Because ofthe considerable side effects of the current therapeutic optionsfor sight-threatening, proliferative diabetic retinopathy (PDR),investigating the underlying mechanism of DRP and the

Liwei Zhang and Huiming Xia contributed equally to the work.

L. Zhang :Q. Han :B. Chen (*)Department of Ophthalmology, The Second Xiangya Hospital,Central South University, Changsha Hunan Province, People’sRepublic of Chinae-mail: chenbh2006@126.com

H. XiaDepartment of Ophthalmology, Johns Hopkins University,Baltimore, Maryland, USA

Graefes Arch Clin Exp OphthalmolDOI 10.1007/s00417-014-2827-8

metabolic memory phenomenon, and reducing its detrimentaleffect in the retina will be an extremely attractive alterna-tive other than hyperglycemic control. However, the exactmechanism responsible for this metabolic memory phe-nomenon associated with the progression of DRP remainselusive.

While various biochemical and hemodynamic pathwaysare implicated in DRP and the metabolic memory phenome-non, recent clinical studies have suggested that reactive oxy-gen species (ROS) plays a pivotal role in the development ofdiabetic complications, and is the key factor in various signal-ing pathways associated with hyperglycemia [9, 10]. Hyper-glycemia induces superoxide overproduction in the mitochon-dria of endothelial cells [9] and causes increased expression offibronectin and collagen IV. The effect persists for weeks aftertermination of hyperglycemia, which is a potential mechanismfor thickening of the basement membranes and metabolicmemory phenomenon [11]. Oxidative stress continues to in-crease while antioxidant or NADPH oxidase inhibition con-tinues to decrease for at least 1 week after re-establishment ofnormal glycemic levels [12, 13]. In animal models of DRPand the metabolic memory phenomenon, it is reported thatmitochondrial DNAwas damaged, mitochondrial fission- andfusion-related proteins were abnormally expressed, and mito-chondria transcription factors were decreased. These changesnot only reduced biosynthesis of mitochondria, but also af-fected the size, number, and function of the electron transferchain in mitochondria. In addition, due to the decreasedamount and activity of antioxidants GSH and SOD, therewas an increased level of superoxides and oxidative stresswhich caused further damage to mitochondria and exacerbat-ed DRP progression and metabolic memory phenomenondevelopment [14–24].

Kowluru et al. reported that transgenic expression of SOD2or the long-term administration of lipoic acid dramaticallyinhibited ROS and derived production, postponed retinal cap-illary cell apoptosis, reduced the amount of acellular capil-laries, and, in turn, alleviated DRP progression overall and thedevelopment of metabolic memory phenomenon [20, 25–30].However, due to the side effects of lipoic acid and thattransgenic expression of the SOD2 gene is difficult, it wouldbe more meaningful if an effective, long-lasting alternativetherapeutic strategy could be developed. Our previous studiessuggested that intra-vitreal injection of AAV-MnSOD orAAV-CAT can effectively inhibit the production of ROS,apoptosis of retinal vascular cells, the increase of acellularcapillaries, and retinal ganglion cell damage in ischemic reti-nopathy [31–33]. This suggests that antioxidant enzyme genetherapy is a potentially good candidate for the treatment ofROS-related retinal diseases including DRP and its metabolicmemory phenomenon, glaucoma, retinal ischemia/reperfu-sion, and retinopathy of prematurity. In this study, we inves-tigated the therapeutic effect and mechanism of AAV-MnSOD

on DRP and on the metabolic memory phenomenon, usingintravitreal injections in a rat DRP model .

Methods

Animals

Adult male SD rats (180–220 g) used for this study wereobtained from the Second Xiangya Hospital Laboratory Ani-mal Center, Central South University (Changsha, China). Theanimals were maintained in cages in temperature-controlledrooms featuring a 12-h light/12-h dark cycle (light period from6:00 AM to 6:00 PM). All animal studies were approved bythe Institutional Animal Care and Use Committee at CentralSouth University, and the studies were conducted in accor-dance with the principles described in the ARVO Statementfor the Use of Animals in Ophthalmic and Vision Research. Atthe end of the desired duration, animals were killed by pento-barbital overdose, one eye was fixed in 4 % paraformalde-hyde, and the other eye was used to isolate the retina under adissecting microscope by gently separating the sensory retinafrom the choroid using a micro-spatula and storing at −80 °Cfor further analyses.

Preparation for administration of MnSOD by recombinantadeno-associated virus

The recombinant AAV vector backbone pTR-UF11 was usedto accept the MnSOD cDNA. Gene expression was driven byhybrid cytomegalovirus and chicken β-actin proximal pro-moter. The resultant pTR-MnSOD plasmids were amplified,then purified and packaged as an AAV serotype 2 vector [34,35]. The resultant AAV packaged MnSOD and humanizedGFP control viruses were assayed. Each virus preparationcontained approximately 1012 genome copies/ml (AAV-MnSOD was a gift from Dr. Alfred S. Lewin, Department ofOphthalmology, University of Florida, USA). One microliterof AAV-MnSOD was injected into one eye of each animal inthe vitreous cavity 5 months after the induction of diabetes.

Establishment of diabetic retinopathy and metabolic memorymodel in rats

Diabetes was induced in rats using an injection ofstreptozotocin (50 mg/kg body weight), and blood glucosewas measured 3 days later. Animals were randomly assignedto one of four groups: good glycemic control (glycated hemo-globin<7 %) for 12 months (control group, CG), high glyce-mic control (glycated hemoglobin>11%) for 12months (highglycemic group, HG), high glycemic control (glycated hemo-globin>11 %) for 6 months followed by good glycemiccontrol for 6 additional months (metabolic memory group,

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MM), or high glycemic control for 6 months followed bygood glycemic control for 6 months, with intravitreal injectionof AAV-MnSOD (1 μl) at 5 months (treatment group, TG) toallow sufficient time for expression of the AAV-deliveredtransgene (MnSOD). Rats maintained in high glycemic con-trol received 1-2 U insulin, four to five times per week toallow for slow weight gain while maintaining hyperglycemiaand preventing ketosis. Those maintained at a normal glyce-mic level received insulin twice daily (7–8 U total). Allanimals were housed in metabolic cages and received a pow-dered diet. Their bodyweights were measured twice per week,blood glucose once a week (Johnson Lifescan Corporation,USA), and glycated hemoglobin (GHb) every 2–3 monthsusing a Alc ELISA kit from Shanghai Yi Li Biotechnology.Their 24-h urine samples were collected and tested for gly-cosuria and ketonuria (Johnson Lifescan Corporation, USA)once a week.

Transmission electron microscopy

The enucleated eyes were dissected along the equators andimmediately stored in 2.5 % glutaraldehyde solution with100 mM phosphate buffer (pH 7.4) for 24 h. The retinas werepostfixed with 2 % buffered osmium tetroxide and dehydratedwith graded ethanol solutions. The samples were embedded in812 resin (Electron Microscope Science, Hitachi, Japan), andultrathin transverse sections (500 nm, Sweden ultramicrotomeLKB-III) of selected areas near the retinal microvasculaturewere prepared. The sections were stained with uranyl acetateand lead citrate and the retinal ganglion cell layer and photo-receptor cell layer were viewed by transmission electron mi-croscopy (TEM). At least eight random images were recordedat 20,000 magnification from each independent preparation.Quantification was done by a IBAS-2000 image analyzer(model EM 900; Carl Zeiss Meditec, Oberkochen, Germany).Basement membrane area (BMA), basement membrane pe-rimeter (BMP), basement membrane length (BML), and base-ment membrane thickness (BMT) were calculated as follows:BML BMP/2, BMT BMA/BML. Trans-sectional areas ofretinal capillaries (TA) and pericyte areas (PA) were examinedand the PA/TA ratio was calculated.

Preparation of retinal vasculature

Retinal vasculature was prepared from freshly isolated eyesthat had been fixed with 4 % paraformaldehyde for 1 day.Retinas were isolated, washed in PBS overnight, and incubat-ed with 2.5 % trypsin (AMERSCO corporation, USA) at37 °C for 3 h with occasional gentle shaking. Nonvascularcells were gently brushed away from the vasculature, and theisolated vasculature was laid out onto slides and used forTUNEL assay and examination of acellular capillaries.

Retinal vasculature TUNEL assay

The TUNEL reaction (In Situ Cell Death Detection kit;Roche, Mannheim, Germany) was performed to detect retinalcell death in the isolated vasculature. In each assay, a positivecontrol was set up by treatment with DNase (50 U/100 μL) for

10 min to fragment DNA. The retinal vasculature (isolatedby the trypsin digest method) was washed extensively in PBSand then permeabilized with 1 % Triton X-100 in PBS. TheTUNEL reaction was performed in a humidified atmosphereat 37 °C for 1 h. The number of TUNEL-positive nuclei indifferent groups was counted throughout the entire retinalvasculature.

Quantitation of degenerated (acellular) capillaries

After counting TUNEL-positive cells, the coverslips weregently soaked away from the slides. Sections were thenstained with periodic acid Schiff-hematoxylin. Acellular cap-illaries were identified as small vessel tubes with no nucleianywhere along their lengths and are reported per squaremillimeter of retinal area. They were quantitated by our pre-vious method [31].

Detection of the retinal MnSOD and catalase activity

The enzyme activity of MnSOD was measured in 5–10 μg ofretinal protein using a kit fromCayman Chemical (AnnArbor,MI, USA). The method uses tetrazolium salt to quantify O2

−

generated by xanthine oxidase and hypoxanthine. The stan-dard curve was generated using a quality-controlled SODstandard. MnSOD activity was determined by performingthe assay in the presence of potassium cyanide to inhibit Cu-ZnSOD, thus measuring the residual MnSOD activity [32].

Catalase activity was assessed as previously described [33].Individual retinas was homogenized in 50 mM phosphatebuffer, pH 7.4. Then, 200 ml of the homogenate was mixedwith 25 ml of 20 % Triton X-100; the mixture was incubatedfor 5 min at 4 °C and centrifuged at 10,000× g for 1 min. Thesupernatant (50 ml) was diluted with 900 ml of 50 mMphosphate buffer, and the reaction was started by adding50 ml of 200 mM H2O2 in 50 mM phosphate buffer. Werecorded the absorbance at 240 nm at 37 °C every 10 s for100 s. The enzymatic activity was calculated using the extinc-tion coefficient of H2O2 at 240 nm, 0.0394 mM−1, cm−1, andthe results were expressed as nmol H2O2/min/mg of protein.

Statistical analysis

All results are expressed as mean and SD. Statistical analysiswas carried out using commercially available software (SPSS

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17.0 for Windows). Data were analyzed by the nonparametricKruskal-Wallis test followed by the Mann–Whitney U test,except for the results of immunohistochemistry and TUNELassay on retinal sections. Those tests were analyzed byANOVA (immunohistochemistry) and by nonparametric signtest (TUNEL). Differences were considered statistically sig-nificant at P≤0.05.

Results

Development of hyperglycemia in rats

Rats in the high glycemic group (HG) had significantly higherglycated hemoglobin (GHb) values and lower body weightsthan did the age-matched control rats (CG) (blood glucose28.62±4.2/mol/l vs. 8.6±2.4 mmol/l, GHb 14.5 %±2.3 % vs.6.5 %±0.8 %, body weight 301±44 g vs. 486±53 g, 24-hurine sample 104±28 ml vs. 13±3, respectively; P<0.05). Inthe metabolic memory group (MG), during the 6-month highglycemic period, GHb (13.6±2.1 %), body weight (298±45 g), blood glucose (26.4±3.6 mmol/L), and 24-h urinesample (91±24 ml) were similar to those in the HG. However,during the following 6 months of the good glycemic period,GHb (6.8±1.2 %), body weight (438±33), blood glucose(10.6±2.2 mmol/L), and 24-h urine sample (16±5ml) becamesimilar to those of the CG. Intravitreal injection of AAV-MnSOD did not influence the severity of hyperglycemiaduring either the poor control period or the reversal period.

Changes of retinal capillary microstructure in diabetic rats

As shown in the control group (CG), capillary vessels in theretinal nerve fiber layer and outer plexiform layer are com-posed of healthy endothelial cells, pericytes, and basementmembrane, with a regular lumen (Fig. 1a and b). The base-ment membranes of capillary vessels in the retinal nerve fiberlayers and outer plexiform layers were significantly increasedin high blood glucose rats (HG). The thicknesses increased bytwofold as compared to the control group. The retinal vascularlumen was significantly narrower or even occluded. The PA/TA ratio was decreased by approximately 16-fold as com-pared to the control group (Fig. 1c and d, Table 2). Afternormal glycemic control, the basement membrane thickening,apoptosis of pericytes, and PA/TA ratio decreases continued(Fig. 1e and f). These changes are statistically significant(P<0.01) as compared to the control group, but not signifi-cantly different from the high glucose group (P>0.05)(Tables 1 and 2). Intravitreal injection of AAV-MnSODrobustly alleviated the damage to retinal vascular lumenduring metabolic memory (Fig. 1g and h) and significantlyinhibited the decrease of the PA/TA ratio as compared tothe high glucose group (P<0.01) (Tables 1 and 2).

Apoptosis of retinal capillary cells in diabetic rats

In DRP, loss of retinal microvascular endothelial cells andpericytes leads to acellular capillaries. Retinal microvascula-ture in the control group was regular in the pattern and size ofthe lumen with rare apoptotic cells. On the other hand, in thehigh glucose group, the retina showed an irregular pattern anduneven lumen size with an increased number of apoptoticcells (26.25±2.872/mm2 retina) as compared to the controlgroup (7.50±1.732/mm2 retina). The number of apoptoticretinal capillary cells was 3.5-fold compared to the controlgroup. After 6 months of exposure to high glucose, the num-ber of apoptotic retinal capillary cells kept increasing evenafter reversal of hyperglycemia. There is no significant differ-ence in numbers of apoptotic retinal capillary cells betweenthe high glucose level group (26.25±2.872/mm2 retina) andmetabolic memory group (29.25±2.872/mm2 retina); both aresignificantly higher than the control group (7.50±1.732/mm2

retina) (P<0.01 when compared the to control group; P>0.05when compared the to high glucose group). Intravitreal injec-tion of AAV-MnSOD can effectively inhibit the apoptosis ofretinal capillary cells by more than 50 % (11.25±0.957/mm2

retina) (P<0.01 as compared the to metabolic memory group)(Fig. 2a and e).

Retinal acellular capillaries in diabetic rats

Retinal microvasculature in the control group was regular inthe pattern and size of the lumen with rare apoptotic cells. Onthe contrary, in the high glucose group, the retinas showed anirregular pattern and reduced lumen sizes, decreased numbersof endothelial cells and pericytes, and increased acellularcapillaries (6.250±0.500/mm2 retina), as compared to thecontrol group (1.500±0.577/mm2 retina). After maintainingat a high glucose level for 6 months, reinstallation of normalglucose levels could not stop the steady increase in retinalacellular capillaries (6.750±0.957/mm2 retina) (P<0.01 ascompared to control group and P>0.05 as compared to highglucose group). Treatment with the MnSOD gene led to adecrease in the number of acellular capillaries (3.500±0.577/mm2 retina) that was proportional to the decrease in apoptoticnuclei within the retinal vessels (P<0.01 as compared tometabolic memory group, Fig. 3a and d). Quantitative analysisof acellular capillaries/mm2 retina showed a significant thera-peutic effect by MnSOD gene delivery, but not by goodglycemic control (Fig. 3e).

Activity of MnSOD and catalase in the retina

As compared to the control group, after high glucose exposurefor 12 months, there was a 50 % decrease and 37 % decreasein MnSOD and catalase activity, respectively. Six months ofgood glycemic control after 6 months of poor glycemic

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Fig. 1 MnSOD gene deliveryreduced basement membranethickness. Examination ofbasement membrane thickness byTEM showed thickening ofbasement membrane of retinalcapillary vessels in the nerve fiberlayer (c) and outer plexiform layer(d) in the high glycemia groupand in the metabolic memorygroup (e and f) as compared to thecontrol group (a and b). MnSODgene delivery effectively inhibitedthe thickening of basementmembrane in both the nerve fiberlayer (g) and outer plexiformlayer (h). Scale Bar=2 μm

Table 2 Ratio of pericyte area and cross-section area of capillaryvessel(%)

Group Nerve fiber layer Outer plexiform layer

Control Group 0.460±0.043 0.488±0.125

High Glycemia Group 0.026±0.010* 0.024±0.006*

Metabolic Memory Group 0.021±0.005* 0.017±0.007*

MnSOD Treatment Group 0.244±0.046Δ 0.364±0.022Δ#

*P<0.01 compared to the CG;Δ P<0.01 compared to the MG; #P>0.05compared to the CG

Table 1 Basement membrane of retinal vessels (nM)

Group Nerve fiber layer Outer plexiform layer

Control Group 40.23±8.90 37.70±10.91

High Glycemia Group 120.93±15.98* 109.56±20.63*

Metabolic Memory Group 112.11±11.58* 93.02±14.05*

MnSOD Treatment Group 60.49±13.24Δ# 59.52±13.41Δ#

*P<0.05 compared to the CG;Δ P<0.05 compared to the MG; #P>0.05compared to the CG

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control did not reverse the inhibition of MnSOD and catalaseactivity; the enzyme activities in the HG and MG groups weresimilar. Intravitreal injection of AAV-MnSOD significantlyrestored MnSOD and catalase activities in the retina(P<0.01 as compared the to high glucose group and P<0.01as compared to the metabolic memory group, Fig. 4a and b).

Discussion

Studies on diabetic patients and animal models suggest anincrease on oxidative stress and superoxide level, and inhibi-tion of CAT, GSH, and MnSOD activities during DRP, which,in turn, affects biogenesis and the electron transport chain inmitochondria. The pathological changes are irreversible evenafter hyperglycemia termination [6, 7, 15, 16, 20, 24, 25,

36–38]. It is reported that overexpression of MnSOD caneffectively quench high glucose-induced oxidative stress andnitrative stress in transfected endothelial cells and hemizygoustransgenic mice. It inhibited diabetes-induced oxidative dam-age to mitochondria, apoptosis of retinal endothelium, andDRP progression. It also prevented DRP progression afternormal glucose control [25–27]. Here we have reported, forthe first time, that elevating the level of MnSOD and catalaseactivity in retinal tissue by intravitreal injection of AAV-MnSOD can effectively treat STZ-induced DRP pathology,including thickening of the retinal vascular basement mem-brane, apoptosis of retinal capillary cells, and increase ofacellular capillaries. AAV-mediated MnSOD expression alsoprevents the development of the metabolic memory phenom-enon when the blood glucose returns to normal levels.

DRP is a slowly progressing and time-dependent disease; ittakes many decades in humans and about 1 year in rodents to

Fig. 2 MnSOD gene deliveryinhibited retinal capillaryapoptosis in retinal vasculature ofdiabetic rats by trypsin digestion.(a) Retinal vasculature in the CG.(b) Retinal vasculature in the HG.(C) Retinal vasculature in theMG. (d) Retinal vasculature in theTG. (e) Number of apoptotic cellsin all groups. Apoptotic cells(arrows) in the retinal vasculature.(×200). (n=6 in each group;*P<0.01 compared with the CG;Δ P<0.01 compared with theMG and HG)

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develop [3, 17, 22, 39]. While histopathological characteris-tics of DRP are not observed until 10–12months of diabetes inrats, retinal capillary cell apoptosis can be seen around 6–8months after induction of diabetes [22, 39]. Pathogenesis ofDRP includes selective loss of pericytes, thickening of the

retinal vascular basement membrane, blood-retinal barrierbreakdown, blockage of retinal capillaries, angiogenesis, fi-broblast proliferation, and finally detachment of the retina.The capillary basement membrane is essential for maintainingcapillary walls and controlling permeability. Thickening of the

Fig. 3 MnSOD gene deliveryinhibited capillary degenerationand reduced acellular capillariesin retinal vasculature of diabeticrats. (a) Retinal vasculatures inthe CG. (b) Retinal vasculature inthe HG. (c) Retinal vasculature intheMG. (d) Retinal vasculature inthe TG. (e) Number of acellularcapillaries in all groups. Acellularcapillaries (arrows) in the retinalvasculature (×200) (n=6 in eachgroup; *P<0.01 compared withthe CG; Δ P<0.01 comparedwith the MG and HG)

Fig. 4 MnSOD gene delivery ledto increased activities of retinalMnSOD and catalase. (a)MnSOD activity. (b) Catalaseactivity. Retinas were dissectedand proteins were prepared forELISA (n=6 in each group;*P<0.01 compared with the CG;Δ P<0.01 compared with theMG and HG)

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basement membrane will cause apoptosis of pericytes andendothelial cells, causing further changes on the permeabilityof retinal capillaries. Increased retinal capillary leakage willblock capillaries and cause the development of acellular cap-illaries and areas of retinal nonperfusion. Resulting patholog-ical angiogenesis in turn promotes DRP progression andeventually leads to irreversible vision loss. [39, 40].

Because thickening of the capillary basement membrane isclosely associated with the seriousness of DRP, capillary base-ment membrane thickness can be used as a criterion to evaluatethe severity of DRP. Our results revealed that high glucoselevels can induce thickening of the capillary basement mem-brane in the nerve fiber layer and outer plexiform layer, as wellas a decreased ratio of pericyte area and cross-sectional area ofcapillary vessels in a diabetic rat retina. We also observed thatthe above events continue for at least 6 months even whenblood glucose levels returned to normal. This suggests thatthickening of the capillary basement membrane can also beused as a criterion for evaluation of the severity of themetabolicmemory phenomenon in DRP. Intravitreal injection of AAV-MnSOD effectively reduced the retinal capillary basementmembrane thickness, alleviating the detrimental effects of themetabolic memory phenomenon on the retinal capillary base-ment membrane. This suggests that an increase of superoxidesand decrease of MnSOD activities are among the main reasonsfor DRP and the metabolic memory phenomenon.

Besides thickening of the retinal capillary basement mem-brane, the selective loss of retinal pericytes is another impor-tant pathological event at early stages of DRP. The loss ofretinal pericytes will further cause thickening of the basementmembrane, changing of capillary permeability, formation ofacellular capillaries, and finally pathological angiogenesis[41]. Pericytes play an important role in maintaining retinalcapillary integrity. Loss of pericytes will lead to angiogenesis,formation of thrombi, damage to endothelial cells, and causeall the clinical symptoms of DRP [42–44]. As a result, theextent of pericyte apoptosis can also be used as a criterion forthe severity of DRP. In vitro studies suggested that high bloodglucose could increase ROS in retinal capillary endothelialcells and lead to the release of cytochrome C into cytoplasm,translocation of Bax into mitochondria, decreased expressionof connexin [43], change of mitochondrial morphology, dam-aged mitochondrial DNA, decreased expression of functionalproteins in the respiration chain, reduced mitochondrial mem-brane potential, and finally the apoptosis of endothelial cells[45–48]. Antioxidant heme oxygenase-1 or PEDF exert aprotective effect on retinal capillary endothelial cells culturedunder high glucose levels in vitro [49, 50]. High blood glucoseor fluctuation of blood glucose levels can also induce theincrease of oxidative stress, decrease GSH, and cause mor-phological and metabolic changes of mitochondria in retinalcapillaries [51, 52]. Activation of capase-3 will lead to theapoptosis of pericytes [51]. Antioxidants including PEDF,

NAC, and peroxiredoxins inhibit pericyte apoptosis throughdecreased production of ROS, reduced DNA damage, andinhibited activation of capase-3 [51, 53, 54]. In vivo studiesalso revealed the increase of oxidative stress and nitrativestress during DRP, apoptosis of capillary cells, increasednumber of acellular capillaries, decreased activity of MnSOD,and compromised mitochondria function. Those events con-tinue even when the blood glucose level is adjusted to normallevels suggesting the occurrence of the metabolic memoryphenomenon [17, 19–21, 23–30, 55–59]. In our study, wefound that after maintaining a high glucose level for12 months, retinal capillaries display distortive and irregularshapes with smaller lumens. There was an increase in theapoptosis of retinal capillaries and number of acellular capil-laries as compared to the control group. After strict control ofblood glucose levels with insulin, retinal pathological changescontinued, which was consistent with other reports whichshowed that islet transplantation cannot stop retinal capillaryblockage [60] and reversal of hyperglycemia cannot alleviateDRP after maintaining high glucose levels for 2.5 years in thediabetic dog [61]. This was also true in a study carried out withgalactosemic rats and dogs. After feeding with galactose for aperiod of time, discontinuing feeding of galactose did not slowthe progression of retinal pathogenesis [62]. However, ourstudy suggested that intravitreal injection of AAV-MnSODcould effectively inhibit the apoptosis of retinal capillariesand reduce acellular capillaries after reversal of hyperglyce-mia and this result was consistent with the study using thetransgenic overexpression of MnSOD in rats [25]. Our resultsindicate the intravitreal injection has long-lasting therapeuticeffects on the progression of diabetic capillary pathogenesisand the occurrence of the metabolic memory phenomenon.

Superoxide is a critical factor linking high blood glucose withdiabetic vascular complications [9]. The retina is particularlysusceptible to oxidative stress because of its high consumptionof oxygen, high proportion of polyunsaturated fatty acids, andexposure to visible light [9]. Oxidative stress is increased in theretina in diabetes, and increased oxidative stress has been shownto contribute to the pathogenesis of DRP [10]. Accumulatingevidence suggests that during the development of retinopathy,there is an obvious increase in oxidative stress. After 6months ofhigh blood glucose exposure, superoxide levels remain high inretinal tissue even after switching back to normal blood glucoselevels. Furthermore, due to the reduction of antioxidant enzyme(MnSOD, GSH, CAT) activity, mitochondrial DNA damagecontinued, and electron transport was interrupted, which in turnincreased the production of superoxide, forming a vicious feed-back loop, and finally led to retinal capillary disease [27, 30, 54,55]. MnSOD is the only antioxidant enzyme in mitochondriaand the first barrier against superoxide ions. In mitochondria,MnSOD catalyzes the transformation of superoxide to hydrogenperoxide and diatomic oxygen [52], and it protects the mito-chondrial membranes from damage. In rodent animal models, a

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decrease of MnSOD activity damaged mitochondrial functionand antedated diabetic retinopathy [30]. Overexpression ofMnSOD in transgenic rats can prevent a decrease in GSH levelsand increase antioxidant capability in the retina. Retinal endo-thelial MnSOD overexpression will ameliorate increased ROSproduction and VEGF expression in diabetic mice [63] suggest-ing an indispensable role byMnSOD inDRP [25]. The results ofour study showed that there is an obvious decrease of retinalMnSOD activity even when blood glucose was restored back tonormal levels. The compromised ability to clear ROS superox-ides continues to increase and facilitate the progression of retinalcapillary lesions, which was consistent with the results byKowluru et al. [24]. ReducedMnSOD activity may be attributedto the nonenzymatic glycosylation or epigenetic modification inthe activity center of MnSOD [37, 38]. Meanwhile, the compro-mised ROS clearance activity may also be attributed to thereduced activity of catalase in the retina. Intravitreal injectionof AAV-MnSOD can conspicuously increase the activity ofMnSOD and catalase in the retina and inhibit progression ofDRP after reversal of hyperglycemia. This suggests that AAV-MnSOD gene therapy is a promising way to treat DRP and themetabolic memory phenomenon.

In conclusion, we have shown that after a long period ofhyperglycemia, the diabetic rat retina developed thickening ofbasement membrane and had a decreased ratio of pericyte areaover cross-sectional capillary area, which did not end even afterreversal of hyperglycemia. We characterized this “metabolicmemory” phenomenon and its pathological effect through mi-crostructure examination. Overexpression of MnSOD via intra-vitreal AAV-MnSOD injection effectively elevatedMnSOD andcatalase activities, inhibited thickening of the basement mem-brane and reduced the apoptosis of retinal capillary cells andproduction of acellular capillaries. Our results indicate that intra-vitreal injection of AAV-MnSOD is a promising strategy for thetreatment of DRP and related metabolic memory phenomenon.The treatment effect lasts for at least 6 months. Further studiesneed to be carried out to determine the therapeutic frame andrelated underlying mechanisms.

The project was sponsored by grants from the National Natural ScienceFoundation of China (30973260). The authors would like to thank AlfredS. Lewin for the gift of AAV-MnSOD.

Conflict of interest The authors declare that they have no conflict ofinterest.

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