RetractionRetracted: Hyperglycemia Induces Toll-Like Receptor-2 and -4Expression and Activity in Human Microvascular RetinalEndothelial Cells: Implications for Diabetic Retinopathy

Journal of Diabetes Research

Received 24 September 2020; Accepted 24 September 2020; Published 31 October 2020

Copyright © 2020 Journal of Diabetes Research. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Journal of Diabetes Research has retracted the article titled“Hyperglycemia Induces Toll-Like Receptor-2 and -4Expression and Activity in Human Microvascular RetinalEndothelial Cells: Implications for Diabetic Retinopathy”[1] due to concerns identified with Figures 1 and 2.

Following the publication of an erratum to correct animage duplication in Figure 2 [2], concerns have been identi-fied in the original publication, and in the revised figureprovided by the authors. Our concerns are as follows:

Figure 1(a)

(i) GADPH bands C and 15 are the same as bands 25and M (horizontally flipped).

(ii) In the TLR4 bands, there is an undeclared gel splicebetween the C and 15 bands

Figure 1(b)

(i) There is an undeclared gel splice between the 25 andM lanes of TLR4 which is not consistent with theloading control

Figure 2(c)

(i) There is an undeclared gel splice between the C and15 lanes of TRIF and IRF3

(ii) There is an undeclared gel splice between the 25 andM lanes of TRIF and IRF3

(iii) These gel splices are not consistent with the loadingcontrol

With the agreement with the Chief Editor, this article istherefore being retracted due to the above concerns.

References

[1] U. Rajamani and I. Jialal, “Hyperglycemia Induces Toll-LikeReceptor-2 and -4 Expression and Activity in Human Micro-vascular Retinal Endothelial Cells: Implications for DiabeticRetinopathy,” Journal of Diabetes Research, vol. 2014, ArticleID 790902, 15 pages, 2014.

[2] U. Rajamani and I. Jialal, “Erratum to “Hyperglycemia InducesToll-Like Receptor-2 and -4 Expression and Activity in HumanMicrovascular Retinal Endothelial Cells: Implications for Dia-betic Retinopathy”,” Journal of Diabetes Research, vol. 2016,Article ID 8976945, 2 pages, 2016.

HindawiJournal of Diabetes ResearchVolume 2020, Article ID 5071954, 1 pagehttps://doi.org/10.1155/2020/5071954

https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://doi.org/10.1155/2020/5071954

RETRACTEDResearch ArticleHyperglycemia Induces Toll-Like Receptor-2 and -4 Expressionand Activity in Human Microvascular Retinal Endothelial Cells:

Implications for Diabetic Retinopathy

Uthra Rajamani1 and Ishwarlal Jialal1,2

1Laboratory for Atherosclerosis and Metabolic Research, Division of Endocrinology, Diabetes and Metabolism,Department of Pathology, University of California Davis Medical Center, Research Building 1, Room 3000,4635 Second Avenue, Sacramento, CA 95817, USA2Veterans Affairs Medical Center, Mather, CA 95655, USA

Correspondence should be addressed to Ishwarlal Jialal; ijialal@ucdavis.edu

Received 31 October 2014; Accepted 10 December 2014; Published 31 December 2014

Academic Editor: Subrata Chakrabarti

Copyright © 2014 U. Rajamani and I. Jialal. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Diabetic retinopathy (DR) causes visual impairment in working age adults and hyperglycemia-mediated inflammation is central inDR. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, scanty data is available ontheir role in DR. Hence, in this study, we examined TLR2 and TLR4 mRNA and protein expression and activity in hyperglycemichuman retinal endothelial cells (HMVRECs). HMVRECs were treated with hyperglycemia (HG) or euglycemia and mRNA andprotein levels of TLR-2, TLR-4, MyD88, IRF3, and TRIF as well as NF-𝜅B p65 activation were measured. IL-8, IL-1𝛽, TNF-𝛼 andMCP-1, ICAM-1, and VCAM-1 as well as monocyte adhesion to HMVRECs were also assayed. HG (25mM) significantly inducedTLR2 andTLR4mRNAandprotein inHMVRECs. It also increased bothMyD88 andnon-MyD88pathways, nuclear factor-𝜅B (NF-𝜅B), biomediators, andmonocyte adhesion.This inflammationwas attenuated by TLR-4 or TLR-2 inhibition, and dual inhibition bya TLR inhibitory peptide aswell as TLR2 and 4 siRNA.Additionally, antioxidant treatment reducedTLR-2 andTLR4 expression anddownstream inflammatory markers. Collectively, our novel data suggest that hyperglycemia induces TLR-2 and TLR-4 activationand downstream signaling mediating increased inflammation possibly via reactive oxygen species (ROS) and could contribute toDR.

1. Introduction

Diabetes is a growing global epidemic affecting nearly 36million people in USA alone and nearly 350 million world-wide. Diabetic retinopathy (DR) is the leading cause ofvision impairment and blindness among working adultsand afflicts around 30 percent of patients [1, 2]. Diabetesis a proinflammatory state characterized by elevated lev-els of C-reactive protein (CRP), inflammatory cytokines,chemokines, adhesionmolecules, monocyte activity, and adi-pose tissue dysregulation [3–6]. Hyperglycemia in diabetescontributes to microvascular complications and reduction ofglycemia reduces progression of microvascular disease suchas retinopathy [1, 2]. Mechanisms that have been advanced to

explain how hyperglycemia can induce DR include the polyolpathway, activation of protein kinase-C (PKC), increasedoxidative stress, advanced glycation end product (AGE)formation, and increased inflammation [2, 7–9]. Also bothincreased oxidative stress and AGE receptor engagementcan result in increased inflammation. Several studies havereported a role for inflammation in the development of DR[2, 7–10]. Inflammation results in an increase in NF-𝜅Bactivity, cytokines, chemokines, adhesion molecules, leuko-cyte adhesion, and leukostasis [2, 9, 10]. However the exactmechanism behind hyperglycemia-mediated inflammationleading to microvascular complications is unclear.

Toll-like receptors (TLRs) are pathogen-associated mo-lecular pattern receptors and play a role in innate immune

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2014, Article ID 790902, 15 pageshttp://dx.doi.org/10.1155/2014/790902

http://dx.doi.org/10.1155/2014/790902

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response [11, 12]. Their activation triggers a signaling cascadewhich results in the production of cytokines/chemokines,and so forth, thereby initiating an inflammatory response [11,12]. Among the various TLRs, our group has shown increasedTLR-2 andTLR-4 in type 1 and type 2 diabetes [13, 14]. Severalother groups have also reported increased TLR expressionand/or activity in muscle, B cells, and adipose tissue indiabetic patients [15, 16]. TLR-2 and TLR-4 have been shownto play an important role in atherosclerosis [16–18] and to beupregulated in patients with diabetic microvascular disease[19]. Furthermore genetic deficiency of TLR-2 and TLR-4 hasresulted in an amelioration of diabetic nephropathy [20, 21].However the role of TLRs in the pathogenesis of DR hasnot been explored. To this end, we determined the effect ofhyperglycemia onTLR-2 andTLR-4 activity and on leukocyteadhesion in human microvascular retinal endothelial cells(HMVRECs) to gain insights for a role of TLRs in DR.

2. Methods

2.1. Human Microvascular Retinal Endothelial Cell Culture.Human microvascular retinal endothelial cells (HMVREC)were obtained from Cell Systems (Kirkland, WA, USA). Thecells were isolated by elutriation and their endothelial naturewas confirmed by positive immunofluorescence with vWF,CD31, and Dil-Ac-LDL uptake. There was no evidence ofMuller cell (absence of CRALBP), pericyte as determinedby absence of staining for 𝛼-smooth muscle actin, and theabsence of glial cell contamination was determined by CD68staining.Theyweremaintained in endotoxin-free endothelialmicrovascular growth medium (Lonza, Walkersville, MD)supplemented with FBS and other growth factors at 37∘Cwith 5% CO

2in attachment factor treated culture vessels.

The cells, when confluent, were subsequently treated with5.5mM, 15mM, and 25mM glucose for 24 hours. Duringthe course of HG treatment, the cells were maintained inbasal endothelial growth media to avoid activation of TLRsby serum and other supplements. 19.5mMmannitol/5.5mMglucose was employed as an osmotic control.

2.2. Treatment. We employed the specific TLR-4 inhibitor,TAK 242 (0.5 𝜇M and 1 𝜇M) (Calbiochem, MA, USA), TLR-2 neutralizing antibody (100 nM and 200 nM) (Abcam, MA,USA), andTLR-2 andTLR-4 inhibitory peptide (TIP) (25𝜇Mand 50 𝜇M) (Imgenex, CA, USA) to test the effect of TLRinhibition on HG-mediated effects. Additionally, siRNA forTLR-2 and TLR-4 were employed. Antioxidants apocynin(Sigma,MO,USA) andN-acetyl cysteine (NAC) (Sigma,MO,USA) were employed to study the role of oxidative stress inTLR activation.

Subsequently, the cell supernatants and cell lysates werecollected for assays and western blots. Additionally, nuclearextracts were separately collected for NF-𝜅B p65 assay.

2.3. siRNA Transfection. Prevalidated TLR-2 and TLR-4small interfering RNA (siRNA) were obtained from SantaCruz Biotechnology (Dallas, TX). HMVRECs were treatedwith 0.75𝜇g siRNA for 18–24 hours in transfection media.

The cells were replaced in complete media and allowed tosettle for 24 hours. Subsequently cells were serum starved for2 hours and HG treatments given as explained above.

2.4. Western Blot. Cell lysates (50 𝜇g protein) were resolved,transferred, and probed with TLR-4, TLR-2, myeloid differ-entiation factor 88 (MyD88), TRIF, and IRF3 antibodies asdescribed previously [22]. Strippedmembranes were used for𝛽-actin as a loading control. Representative blots from fourdifferent experiments were used for analysis and in figures arepresentative blot is shown.

2.5. PCR. RNA from treated HMVRECs was extracted usingtrizol (Invitrogen, Carlsbad, CA). RT-PCR was performedusing primers specific for TLR-2 and TLR-4 (InvivoGen,San Diego, CA), with glyceraldehyde-3-phosphate dehydro-genase (GAPDH) as control (R&D Systems, Minneapolis,MN). Band intensities were determined using ImageJ. TLR-2 and TLR-4 mRNA was expressed as a ratio of GAPDH asdescribed previously [13, 22].

2.6. ELISA. Interleukin- (IL-) 1𝛽, IL-8, tumor necrosis factor-(TNF-) 𝛼, monocyte chemoattractant protein- (MCP-) 1,vascular cell adhesion molecule- (VCAM-) 1, and intracel-lular adhesion molecule- (ICAM-) 1 were measured in thesupernatants of HMVRECs by ELISA (Life Technologies,Grand Island, NY), as reported previously [23] and NF-kBp65wasmeasured in nuclear extracts (ActiveMotif, Carlsbad,CA) [22].

2.7. Endothelial Cell Adhesion Assay. HMVREC monolayerswere cultured and treated as above. THP-1 cells were loadedwith fluorescent CFDA-SE (carboxyfluorescein diacetate-succinimidyl ester) dye as described previously [24] for30 minutes at 37∘C. The labeled THP-1 cells were addedto confluent HMVREC cells and incubated for 90min.Thereafter the cells were gently washed using endothelialgrowth media and the number of bound cells were assayedby fluorescence excitation (485 nm) and emission (535 nm).TNF-𝛼 (10 ng/mL) served as the positive control.

2.8. ROS Measurement. Fluorescence microscopy was usedto measure cellular ROS levels. Briefly, HMVRECs in culturewerewashedwithwarmPBS and treatedwith 25𝜇MH

2DCF-

DA dye for 45 minutes at 37∘C. Subsequently the cells werewashedwith PBS twice and immediately viewed using a FITCgreen filter (Ex 485/Em 535). ROS production in the cells wasquantified bymeasuring themean fluorescence intensity of atleast 10 cells per image.

2.9. Statistical Analysis. Results from the experimental stud-ies were reported as mean ± SD. Differences were analyzedby ANOVA with Newman-Keuls post hoc analyses. A prob-ability value of 𝑃 < 0.05 was considered significant. Allstatistical analyses were performed using GraphPad PrismSoftware (San Diego, CA).

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3. Results

3.1. High Glucose Increases TLR-4 and TLR-2 Expression.HMVRECs were exposed to 5.5mM (normal glucose),15mM, and 25mM (HG) for 24 hours andmRNA expressionand protein levels of TLR-2 and TLR-4 were determined.We observed that both 15mM and 25mM glucose treat-ments significantly increased TLR-4 (Figure 1(a)) and TLR-2(Figure 1(c)) mRNA expression compared to 5.5mM glucose(𝑃 < 0.01, 𝑛 = 5). We did not observe any mRNA changesin TLR-2 or TLR-4 expression with the osmotic controlof 19.5mM mannitol/5.5mM glucose compared to 5.5mMglucose, suggesting that this increase in TLR-2 and TLR-4expression is not an osmotic effect.

TLR-2 and TLR-4 protein levels in the normal and HGexperiments were quantitated by western blots. High glucose(15mM and 25mM) increased TLR-2 and TLR-4 proteinlevels significantly (𝑃 < 0.05, 𝑛 = 5) compared to nor-mal glucose and mannitol consistent with our mRNA data(Figures 1(b) and 1(d)). It was also noted that 25mM glucosetreatment resulted in a greater increase in expression andreceptor protein abundance compared to 15mM glucose.

3.2. TLR Downstream-Signaling via MyD88 and Non-MyD88Pathways. TLR-4 is known to signal via both MyD88 andnon-MyD88 pathways while TLR-2 signals through theMyD88 pathway only [11, 12, 16]. Hence we measured sig-naling mediators of both MyD88 and non-MyD88 pathwayssince we showed that high glucose upregulates both TLR-2 and TLR-4. HG significantly induced MyD88, TRIF, andIRF3 suggesting that both MyD88 dependent and indepen-dent pathways are activated (Figure 2). Furthermore, highglucose (15mM and 25mM) also increased NF-𝜅B activityas evident from an increase in nuclear p65 compared to thenormoglycemic control (𝑃 < 0.001, 𝑛 = 5). With increasesin TLR-2 and TLR-4 protein and NF-𝜅B activity, we alsoquantitated secreted inflammatory biomediators.

3.3. HighGlucose Increases Circulating Biomediators of Inflam-mation. Secreted inflammatory biomediators IL-1𝛽, IL-8,TNF-𝛼, and MCP-1 were also increased significantly in15mM and 25mM treatment with 25mM treatment showingsignificantly higher induction compared to 15mM (Figure 3).

3.4. High Glucose Increases Monocyte Adhesion. HG treat-ment resulted in increased secretion of the cell adhesionmolecules (CAMs), ICAM-1, and VCAM-1. Endothelial celladhesion assay measuring monocyte adhesion to HMVRECsalso showed that there was increased monocyte adhesionwith high glucose (Figure 4).

3.5. TLR-4/2 Inhibition Attenuates High Glucose-MediatedInflammation. Since we have shown an increase in bothTLR-2 and TLR-4 with high glucose, we next tested therelative contributions of these receptors to the increasedinflammation including monocyte adhesion. The high glu-cose mediated increase in inflammatory biomediators suchas IL-8, TNF-𝛼, and MCP-1 were significantly attenuated

on treatment with TAK 242, a small molecule specificinhibitor of TLR-4 adaptor protein-protein interaction andsignaling [25]. It binds to TLR-4’s intracellular domain at thecysteine-474 [26]. We employed 0.5𝜇M and 1.0 𝜇M for TLR-4 inhibition and both concentrations (0.5 𝜇M and 1.0 𝜇M)attenuated TLR-4 signaling with 1.0𝜇Mshowingmore potentattenuation compared to 0.5 𝜇M (Figure 5(a)). Similarly bothconcentrations of TAK 242 decreased high glucose-mediatedincreases NF-𝜅B p65 activity, ICAM-1, and VCAM-1 andmonocyte adhesion to HMVRECs (Figure 5(b)).

We next employedTIRAP inhibitory peptide (TIP)whichis a TIRAP (toll-interleukin 1 receptor (TIR) domain adaptorprotein) decoy protein that blocks both TLR-2 andTLR-4 sig-naling by interfering with signaling complex assembly [27].Similar to TAK 242 treatment, TIP also significantly inhibitedhigh glucose induced biomediator secretion (IL-8, TNF-𝛼,and MCP-1) with more inhibition seen with 50𝜇M con-centration (Figure 6(a)). Also TIP treatment decreased highglucose induced nuclear p65 activity, ICAM-1, and VCAM-1and monocyte adhesion to HMVRECs (Figure 6(b)).

We also employed a TLR-2 neutralizing antibody toinhibit TLR-2 signaling since there is no available smallmolecular inhibitor. Compared to high glucose, the TLR-2 neutralizing antibody significantly attenuated biomediatorsecretion (IL-8, TNF-𝛼, and MCP-1) compared to high glu-cose at both concentrations (100 nM and 200 nM) with moreinhibition seen with 200 nM concentration (Figure 7(a)).Nuclear p65, ICAM-1, and VCAM-1 levels as well as mono-cyte adhesion were significantly decreased by TLR-2 neutral-izing antibody compared to high glucose (Figures 7(a) and7(b)). We also employed an irrelevant control antibody andobserved that it did not result in any significant inhibition inany of the biomediators measured.

Small interfering RNA (siRNA) against TLR-2 and TLR-4resulted in at least 50%knockdownof the respective receptorsas determined bywestern blotting for receptor levels (data notshown). In agreement with the above data, siRNA for bothTLR-2 andTLR-4 also showed decreases inNF-𝜅B p65 as wellas in secreted inflammatory biomediators such as TNF-𝛼 andIL-8 (Figure 8). In addition, increased THP-1 adhesion due toHGwas also significantly reduced by siRNA treatment whichfurther confirms our previous inhibitor studies.

3.6. High Glucose Mediated ROS Activates TLR-2 and TLR-4.Previously it has been shown that ROS is important in theinduction of TLRs in monocytes [22]. HG treatment showedsignificant increases (approximately 2.5fold) in ROS levels asshown by H

2DCFDA staining while both apocynin and NAC

significantly lowered ROS levels (Figure 9). These changes inROS were paralleled by an increase in both TLR-2 and TLR-4 mRNA expression and protein levels that were reducedwith the inhibitors (Figure 9). There was almost a 1.6-foldincrease in TLR-4 protein levels and a 2-fold increase in TLR-2 protein levels which were reduced to near normal levels bythe antioxidants apocynin and NAC. HG-mediated increasesin NF-𝜅B p65 levels were also attenuated significantly byboth antioxidants (Figure 9). However it was noted that bothapocynin and NAC showed similar decreases in ROS levelsindicating that HG induces superoxide predominantly and

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Figure 1: High glucose (HG) induces toll-like receptor-4 (TLR-4) and TLR-2: confluent HMVRECs were serum-starved for 2 hours andincubated for 24 hours with 5.5, 15, and 25mM glucose or mannitol (M) 19.5mM/5.5mM glucose for 24 hours. Thereafter the cells wereharvested for mRNA in trizol or protease inhibitor buffer for cell lysates. (a) High glucose induces TLR-4 mRNA expression in humanmicrovascular endothelial cells (HMVRECs). Bar graphs representing hyperglycemia-induced TLR-4 mRNA expression normalized toGAPDH. ∗𝑃 < 0.001 versus control and †𝑃 < 0.01 versus 15mM; (b) HG induces TLR-4 protein levels in HMVRECs. Bar graph representingTLR-4 protein levels normalized to 𝛽-actin. ∗𝑃 < 0.01 versus control, ∗∗𝑃 < 0.001 versus control, and †𝑃 < 0.001 versus 15mM; (c)high glucose induces TLR-2 mRNA expression in HMVRECs. Bar graphs representing hyperglycemia-induced TLR-2 mRNA expressionnormalized to GAPDH. ∗𝑃 < 0.001 versus control and †𝑃 < 0.001 versus 15mM; (d) HG induces TLR-2 protein levels in HMVRECs. Bargraph representing TLR-2 protein levels normalized to 𝛽-actin. ∗𝑃 < 0.05 versus control, ∗∗𝑃 < 0.01 versus control, and †𝑃 < 0.05 versus15mM.

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Figure 2: High glucose induces both MyD88 and Non-MyD88 pathways: HMVRECs were incubated and treated with 5.5, 15, and 25mMglucose andmannitol as described inMethods and legend of Figure 1 and protein lysates were harvested. (a)Western blots showing increasedMyD88 protein levels normalized against 𝛽-actin. ∗𝑃 < 0.01 versus control and ∗∗𝑃 < 0.001 versus control. †𝑃 < 0.05 versus 15mM, (b)increased nuclear p65 levels withHG treatment. ∗𝑃 < 0.001 versus control. †𝑃 < 0.05 versus 15mM, (c) representative blots showing increasedTRIF and IRF3 protein levels with HG. Blots are normalized against 𝛽-actin. TRIF: ∗𝑃 < 0.05 versus control; ∗∗𝑃 < 0.001 versus control;IRF3 ∗𝑃 < 0.01 versus control, ∗∗𝑃 < 0.001 versus control, and †𝑃 < 0.05 versus 15mM.

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Figure 3: High glucose induces increased inflammatory biomediator release: HMVRECs were incubated and treated with 5.5, 15, and 25mMglucose and mannitol as described in Methods and legend of Figure 1. Thereafter cell supernatants were collected for ELISA. (a) IL-8, (b)IL-1𝛽, (c) TNF-𝛼, and (d) MCP-1 levels are increased under hyperglycemic conditions. ∗𝑃 < 0.001 versus controls and †𝑃 < 0.001 versus15mM.

that the TLR-2 and TLR-4 related effects observed could bemainly due to superoxide.

4. Discussion

Diabetic retinopathy (DR) is a chronic low-grade inflamma-tory disease of the retinal microvasculature [28]. Chronicinflammatory features such as increased microvascular per-meability, leukostasis [29, 30], cytokine, chemokine, adhe-sion molecule expression [31], and neovascularization [32]are seen in DR. Several studies have implicated increasedinflammation in DR [9, 10, 33, 34].

The primary aim of the present study was to determinethe role of the pathogen recognition receptors, toll-likereceptors (TLRs) specifically TLR-2 and TLR-4 in DR byinvestigating TLR activity in a pivotal cell in the pathogenesisof DR, that is, retinal ECs [2, 9, 28]. In this study we observethat both TLR-2 and TLR-4 were significantly increasedin HMVREC by HG compared to normoglycemia. Thisobservation is in agreement with previous reports from ourgroup and others demonstrating increased expression andactivity of TLR-2 and TLR-4 in monocytes, muscle, andadipose tissue of diabetic patients as we reviewed previ-ously [16]. However it is the first report on TLR demon-stration on microvascular endothelium that is increasedwith hyperglycemia. TLR-2/4 mediated MyD88 pathway wasupregulated as shown by increased MyD88 protein levels

and nuclear p65. Additionally, TLR-4-mediated Non-MyD88pathway was also activated as indicated by the increasedprotein levels of TRIF and IRF3. These findings are in linewith previous studies implicating hyperglycemia in renalmicrovascular complications. Kaur et al. reported increasedTLR-4 expression and activity under hyperglycemic in renalmesangial cells incriminating TLR-4 in contributing to dia-betic nephropathy [35]. Lin et al. showed increased TLR-4expression under hyperglycemic conditions in human prox-imal tubular epithelial cells pointing towards a role for TLR-4 in tubulointerstitial inflammation in diabetic nephropathy[21]. We now extend these findings to a crucial cell in DR,theHMVRECs by showing increased TLR2 and 4 activity andbiomediator release.

In this study we also show high glucose-mediatedincreases in inflammatory cytokines/chemokines such as IL-8, IL-1𝛽, MCP-1, and TNF-𝛼. It seems plausible that TLR-4/2promotes inflammation, abnormal endothelial cell functionin the retina, and retinal endothelial cell damage in DR. Pre-vious studies suggest that oxidative stress appears to mediatethe upregulation of TLRs under hyperglycemic conditions[22, 36]. Dasu et al. demonstrated in human monocytesthat high glucose activated TLR2 and TLR4 expression,induction of MyD88, and non-MyD88-mediated signalingand NF-𝜅B activity. This effect appeared to be mediated viaPKC activation of NADPH oxidase [22]. Another group alsoshowed that knockout of the P47phox subunit of NADPH

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Figure 4: High glucose induced cell adhesion: HMVRECs were incubated and treated with 5.5, 15, and 25mM glucose and mannitol asdescribed in Methods and legend of Figure 1. Thereafter cell supernatants were collected for ELISA. Adhesion assay is described in Methodssection. (a) Increased ICAM-1 levels with HG. ∗𝑃 < 0.01 versus controls and ∗∗𝑃 < 0.001 versus controls, (b) increased sVCAM-1 levels withHG. ∗𝑃 < 0.001 versus controls; (c) cell adhesion assay showing increased cell adhesion with HG. ∗𝑃 < 0.001 versus control.

oxidase prevented upregulation of both TLR2 and TLR4 indiet-induced obesity [36].

Kowluru and Abbas showed elevated superoxide levels inretina of diabetic rats as well as retinal cells incubated in HG[37].They also showed that antioxidant defense is diminishedin diabetic retinae [38, 39]. Also Safi et al. using identicalHMVRECs showed that high glucose induced both increasein biomarkers of oxidative stress and apoptosis [40]. HoweverBusik et al. showed that HG did not stimulate endogenousROS in retinal endothelial cells [41]. Furthermore, Busiket al. attributed HG-mediated retinal endothelial injury tocytokines released by other cells such as Muller cells and notdirectly to HG. However we have confirmed in our cultureof HMVRECs the absence of other cells types such as Mullercells, pericytes (𝛼-smoothmuscle actin), ormicroglia (CD68)using immunofluorescence staining (data not shown) andhence can attribute the effects observed in HMVRECs. Inour report on HMVRECs we show that high glucose inducesROS and both TLR2 and TLR4. Furthermore antioxidants,apocynin, and NAC reduced both ROS and TLR2 and TLR4with a reduction in downstream signaling.

We further probed effects of HG by measuring adhesionmolecules alongside performing monocyte adhesion assayas a measure of leukostasis, another important feature ofDR. Several previous studies incriminate CAMs and celladhesion in DR. Miyamoto et al. convincingly showed a role

for increased ICAM-1 in leukostasis within retinal vessels ofSTZ-induced diabetes by demonstrating that a monoclonalantibody to ICAM-1 decreases both retinal leukostasis andvascular leakage [42]. Joussen et al. [34] showed that dia-betic mice deficient in ICAM-1 or its ligand, CD18, wereafforded protection from early features of DR includingleukostasis, pericyte loss, and increased permeability. Melethet al. reported that diabetic patients exhibit elevated levels ofserum inflammatory markers including adhesion moleculescompared to nondiabetic controls. They showed increasedlevels of RANTES, MCP-1, ICAM-1, VCAM-1, and VEGFadditionally reporting that the levels of these cytokines weredirectly proportional to the severity of retinopathy [33]. Inthe current study we report that, in HMVRECs, there is anincrease in adhesion molecules such as ICAM-1 and VCAM-1 and increased monocyte adhesion under hyperglycemicconditions, which is in agreement with previously publishedliterature providing further validation for the model studiedin this report. In contrast to our findings Vagaja et al. [43]showed that in hyperglycemic mice the classic ligand forTLR4, lipopolysaccharide, significantly reduced leukostasisbut exacerbated two key features of DR, namely, injury tothe endothelial cells and thinning of the retina. It needsto be pointed out that the TLR4 pathway (TLR-4 proteinand downstream signaling) was not explored in this study.Moreover LPS treatment was only for 24 hours explaining

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Figure 6: (a) TLR-2/4 inhibition by TIP attenuates downstream inflammatory biomediators: HMVRECs were serum starved for 2 hoursand treated with 25 and 50 𝜇M TIP for 2 hours. Thereafter 5.5 and 25mM glucose treatments were performed as described in Figure 3.(I) NF-𝜅B, (II) TNF-𝛼, (III) IL-8, and (IV) MCP-1 levels are significantly decreased upon TLR-2/4 inhibition compared to HG treatment.#𝑃 < 0.001 versus control, ∗𝑃 < 0.001 versus 25mM, and †𝑃 < 0.01 versus 25 𝜇M TIP. (b) Cell adhesion molecules ICAM-1 and VCAM-1attenuation upon TIP treatment. HMVRECswere serum starved for 2 hours and treated with 25 and 50𝜇MTIP for 2 hours.Thereafter 5.5 and25mM glucose treatments were performed as described in Figure 4. Adhesionmolecules (I) ICAM-1 and (II) VCAM-1 levels are significantlydecreased upon TIP treatment. #𝑃 < 0.001 versus control and ∗𝑃 < 0.001 versus 25mM. (III) Adhesion assay showing decreased cell adhesionwith TLR-2/4 inhibition. #𝑃 < 0.001 versus control and ∗𝑃 < 0.01 versus 25mM.

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𝑃 < 0.001 versus 25mM; Sc: scrambled sense RNA.

their paradoxical finding with respect to leukostasis, sincestudy duration needs to be several months in order toappreciate the pathology of DR [34].

Interestingly, the use of TAK 242, a specific small mo-lecule inhibitor to TLR4, significantly attenuated the above-mentioned increases in biomediators. TAK-242 (resatorvid)is a specific inhibitor of TLR-4 signaling that inhibits theproduction of TLR-4-triggered inflammatory mediators bybinding to the intracellular domain of TLR4 [25, 26]. Thissynthetic inhibitor blocks TLR-4’s intracellular domain TIR(toll/IL-1 receptor) [25, 26]. It impairs TLR-4’s ability to asso-ciate with adaptor molecules, namely, TIRAP (MyD88 path-way) and TRAM (TIR-domain-containing adapter-inducinginterferon-𝛽-related adaptor molecule) (non-MyD88 path-way), blocking subsequent signal transduction. In this studywe report TAK-242-mediated attenuation of inflammatorybiomediators such as IL-8, IL-1𝛽, TNF-𝛼, andMCP-1 andNF-𝜅B activity in HG treated HMVRECs. These results clearlydemonstrate that with high glucose the increases in p65NF-𝜅B and inflammatory biomediators could be the result,in part, of an increase in TLR-4 signaling. Several studieshave suggested a role for TLR-4 in HG mediated increasedinflammation. Devaraj et al. showed in a STZ-diabetic TLR-4 knockout mouse model that there were reduced levelsof MyD88, TRIF, and IRF3 and decreased NF-𝜅B activitycompared to wild type diabetic mice [44]. Additionally they

also showed reduced biomediator release (IL-1𝛽, IL-6, IL-8, MCP-1, Interferon-𝛽, and TNF-𝛼) in diabetic TLR-4KOcompared to diabetic wild type. Using a TLR-4mutantmousethat impairs signal transduction, the investigators showedthat TLR-4 signaling is involved in retinal damage andinflammation triggered by ischemic injury in a nondiabeticmodel [45]. Our data with TAK 242 further confirms therole of TLR-4 in inflammation and extends it to the diabeticretinal microcirculatory milieu.

Furthermore, TAK 242 treatment attenuates ICAM-1and VCAM-1 levels in HG treated HMVRECs and showsa decrease in monocyte adhesion. Our findings in thisstudy support previous reports that TLR-4 mediated ICAM-1 expression is attenuated with TLR-4 inhibition thus con-firming TLR-4s’ role in cell adhesion also in a retinalmicrovascular environment. Hence our study not only fur-ther strengthens the role of TLR-4 inHGmediated inflamma-tion including leukocyte adhesion but also for the first timesuggests a possible role in diabetic retinopathy.

We also employed a dual inhibitor, to both TLR-2 andTLR-4 to further elucidate the contributions of TLR-4 andTLR-2, using TIRAP inhibitory peptide (TIP), which isknown to be an inhibitor of both TLR-2 and TLR-4 receptors.The TIRAP inhibitory peptide used in our study is a TR6with a peptide sequence PGFLRDPWCKYQML. Coutureet al. demonstrated that TIRAP inhibitory peptide TR6

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2

DCFDA staining. #𝑃 < 0.001 versuscontrol and ∗𝑃 < 0.001 versus 25mM; (b) TLR-2 and TLR-4 protein levels measured using western blot. #𝑃 < 0.01 versus control and∗

𝑃 < 0.01 versus 25mM; (c) TLR-2 and TLR-4 mRNA expression measured by PCR. #𝑃 < 0.01 versus control and #𝑃 < 0.05 versus 25mM.(d) NF-𝜅B p65 measurement by ELISA, #𝑃 < 0.001 versus control and ∗𝑃 < 0.001 versus 25mM.

significantly inhibits MyD88 recruitment in both TLR-2 andTLR-4 receptors. Additionally MyD88-independent pathwaywas also inhibited byTIP and the authors suggest that adaptorprotein recruitment might occur sequentially at differentcellular locations and controlled by TLR-4 trafficking [27].The addition of TIP attenuated inflammatory cytokine secre-tion as measured by IL-8, TNF-𝛼, and MCP-1 levels showeddecreased NF-𝜅B p65 activation and decreased levels ofICAM-1 and VCAM-1 accompanied by decreased monocyteadhesion compared to HG treatment. Although TIP inhibitsboth TLR-2 and TLR-4, the level of attenuation of inflamma-tory cytokines or adhesion molecules was not significantlydifferent from that of TAK242. A possible explanation for this

finding could be that althoughTLR-2 andTLR-4 expression isincreased by high glucose, both inhibitors work by inhibitingthe same downstream crucial signal transduction pathway,for example, NF-𝜅B. Tang et al. examined the role of deletionof MyD88 only from bone marrow derived cells on retinalpathology. They showed that MyD88 deletion in leukocytesinhibited diabetes-induced leukostasis, ICAM-1 expression,and retinal superoxide production. They also reported thatdeletion of TLR-2 and TLR-4 in leukocytes caused partialattenuation suggesting that both TLR2/4 and IL-1𝛽 signalingpathway play a role in diabetes-induced leukostasis andICAM-1 expression and superoxide generation in retina [46].Furthermore they showed that an agonist of TLR2 but not

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TLR4 induced both IL-8 and IL-6 in retina. Whilst theyfocused on the effect of leukocyte perturbations on retinalpathology their data support a role especially for TLR2 inDR.

TLR-2 inhibition using a TLR-2 neutralizing antibodyonce again attenuated inflammatory cytokine secretion asmeasured by IL-8, TNF-𝛼, and MCP-1 levels, decreasednuclear p65, and decreased levels of ICAM-1 and VCAM-1accompanied by decreased monocyte adhesion compared toHG treatment. TLR-2 has been implicated by some groups indiabetes-mediated microvascular complications. Devaraj etal. showed the involvement of TLR-2 in diabetic nephropa-thy. They utilized TLR-2 knockout animals and showedthat absence of TLR-2 decreased proinflammatory state ofdiabetes alongside attenuation in inflammatory cytokines,albuminuria, podocyte effacement, and M1 macrophages inthe kidney [20]. TLR2 signaling in intrinsic kidney cells hasbeen shown to be required for the full development of inflam-mation, kidney damage, and fibrosis in diabetic nephropathy[47]. Collectively, these studies suggest a role for TLR-2 inHG mediated inflammation and support our current novelfindings that TLR-2 inhibition attenuates inflammation andleukostasis in HMVRECs.

In an attempt to deduce the mechanism behind HGmediated activation of TLR-2 and TLR-4 in HMVRECs, wefound that high glucose-mediated increase in ROS levelswas associated with an increase in both TLR-2 and TLR-4 mRNA and protein expression as well as nuclear p65activation. Several studies have shown the involvement ofNADPH oxidase (Nox) in HG mediated ROS productionin endothelial cells. Taye et al. showed increased expressionof Nox subunits p47 (phox) and Nox 2 under high glucosein human umbilical artery endothelial cells [48]. The linkbetween HG, Nox, and TLRs in monocytes was previouslydemonstrated by our group [22]. In the current studywe showa link between HG and Nox using apocynin, an NADPHoxidase inhibitor, which attenuated ROS levels significantlyproving that the ROS produced is by Nox predominantly.In addition, we make the novel finding in HMVRECs thatapocynin treatment showed decreases in TLR-2 and TLR-4 mRNA and protein levels. We have previously shownin monocytes that HG mediated superoxide release is viaPKC-𝛼 activation [22, 49]. Thallas-Bonke et al. showed indiabetic renal disease that PKC-𝛼 is a keymechanism for Noxactivation [50]. Taken together these studies point towardsNox activation and subsequent superoxide in our settingbeing PKC-𝛼mediated.

We also employed another antioxidant N-acetyl cysteine(NAC) which serves as a prodrug to L-cysteine that actsas a precursor for glutathione. NAC, similar to apocynin,decreased ROS levels as well as mRNA and protein levelsof both TLR-2 and TLR-4. It was however noted that NACtreatment brought about a similar degree of change in TLR-2 and TLR-4 levels as apocynin. This goes on to furtherprove that, in our experimental setting, superoxide was thepredominant ROS produced due to HGwhich activates TLR-2 and TLR-4 in the absence of TLR ligands such as bacterialendotoxins.

However we would also like to point out that althoughcare was taken to avoid endotoxin contamination in the

media the presence of small amounts of endotoxins in thecellular environment is possible given the fact that the cellswere cultured in the presence of serum and other cellularsupplements which could be sources of endotoxins. Also itneeds to be pointed out that Morigi et al. have previouslydocumented an increase in leukocyte adhesion and NF-kBactivity with hyperglycemia in the absence of other agonists[51].

5. Conclusion

The current study sheds light on an area which has notbeen explored thus far, microvascular endothelial TLRs inDR. Our study clearly points towards a role for TLR-4-and TLR-2-induced inflammation in the genesis of DR. Inaddition to showing upregulation and increased activity ofboth TLR-2 and TLR-4 withHG, we also show using differentinhibitors that we can attenuate the TLR-mediated increasedinflammation. Additionally, we show that this TLR-2 andTLR-4 activation is ROS mediated, specifically Nox derivedsuperoxide-mediated and that antioxidant treatment helpedin abrogation of TLR activation. We make the novel observa-tion that both TLR-2 and TLR-4 are induced by high glucosein microvascular retinal endothelial cells and could possiblycontribute to DR by inducing increased inflammation. Thisreport could prove an impetus for future studies testing therole of both TLR-4 and TLR-2 in vivo in DR by using diabeticwild type and knockout mice or small molecule inhibitors.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

References

[1] D. A. Antonetti, A. J. Barber, S. K. Bronson et al., “Diabeticretinopathy: seeing beyond glucose-inducedmicrovascular dis-ease,” Diabetes, vol. 55, no. 9, pp. 2401–2411, 2006.

[2] N. Cheung, P. Mitchell, and T. Y. Wong, “Diabetic retinopathy,”The Lancet, vol. 376, no. 9735, pp. 124–136, 2010.

[3] M. T. Schram, N. Chaturvedi, C. Schalkwijk et al., “Vascularrisk factors andmarkers of endothelial function as determinantsof inflammatory markers in type 1 diabetes: the EURODIABprospective complications study,” Diabetes Care, vol. 26, no. 7,pp. 2165–2173, 2003.

[4] N. Shanmugam,M.A. Reddy,M.Guha, andR.Natarajan, “Highglucose-induced expression of proinflammatory cytokine andchemokine genes in monocytic cells,” Diabetes, vol. 52, no. 5,pp. 1256–1264, 2003.

[5] S. Devaraj, N. Glaser, S. Griffen, J. Wang-Polagruto, E.Miguelino, and I. Jialal, “Increased monocytic activity andbiomarkers of inflammation in patients with type 1 diabetes,”Diabetes, vol. 55, no. 3, pp. 774–779, 2006.

[6] S. Devaraj, M. R. Dasu, and I. Jialal, “Diabetes is a proin-flammatory state: a translational perspective,” Expert Review ofEndocrinology & Metabolism, vol. 5, no. 1, pp. 19–28, 2010.

[7] M. V. van Hecke, J. M. Dekker, G. Nijpels et al., “Inflammationand endothelial dysfunction are associated with retinopathy:

RETRACTED

14 Journal of Diabetes Research

the Hoorn Study,” Diabetologia, vol. 48, no. 7, pp. 1300–1306,2005.

[8] B. E. K. Klein, M. D. Knudtson, M. Y. Tsai, and R. Klein, “Therelation of markers of inflammation and endothelial dysfunc-tion to the prevalence and progression of diabetic retinopa-thy: wisconsin epidemiologic study of diabetic retinopathy,”Archives of Ophthalmology, vol. 127, no. 9, pp. 1175–1182, 2009.

[9] J. Tang and T. S. Kern, “Inflammation in diabetic retinopathy,”Progress in Retinal and Eye Research, vol. 30, no. 5, pp. 343–358,2011.

[10] J. M. Tarr, K. Kaul, M. Chopra, E. M. Kohner, and R. Chibber,“Pathophysiology of diabetic retinopathy,” ISRN Ophthalmol-ogy, vol. 2013, Article ID 343560, 13 pages, 2013.

[11] S. Akira and K. Takeda, “Toll-like receptor signalling,” NatureReviews Immunology, vol. 4, no. 7, pp. 499–511, 2004.

[12] B. Beutler, “Inferences, questions and possibilities in Toll-likereceptor signalling,” Nature, vol. 430, no. 6996, pp. 257–263,2004.

[13] S. Devaraj, M. R. Dasu, J. Rockwood, W. Winter, S. C. Griffen,and I. Jialal, “Increased toll-like receptor (TLR) 2 and TLR4expression in monocytes from patients with type 1 diabetes:further evidence of a proinflammatory state,” The Journal ofClinical Endocrinology&Metabolism, vol. 93, no. 2, pp. 578–583,2008.

[14] M. R. Dasu, S. Devaraj, S. Park, and I. Jialal, “Increased Toll-Like Receptor (TLR) activation and TLR ligands in recentlydiagnosed type 2 diabetic subjects,” Diabetes Care, vol. 33, no.4, pp. 861–868, 2010.

[15] S. J. Creely, P. G. McTernan, C. M. Kusminski et al.,“Lipopolysaccharide activates an innate immune systemresponse in human adipose tissue in obesity and type 2diabetes,” American Journal of Physiology—Endocrinology andMetabolism, vol. 292, no. 3, pp. E740–E747, 2007.

[16] I. Jialal and H. Kaur, “The role of toll-like receptors in diabetes-induced inflammation: implications for vascular complica-tions,” Current Diabetes Reports, vol. 12, no. 2, pp. 172–179, 2012.

[17] K. S. Michelsen, M. H. Wong, P. K. Shah et al., “Lack of toll-like receptor 4 or myeloid differentiation factor 88 reducesatherosclerosis and alters plaque phenotype in mice deficientin apolipoprotein E,” Proceedings of the National Academy ofSciences of the United States of America, vol. 101, no. 29, pp.10679–10684, 2004.

[18] A. E. Mullick, P. S. Tobias, and L. K. Curtiss, “Modulation ofatherosclerosis in mice by Toll-like receptor 2,” The Journal ofClinical Investigation, vol. 115, no. 11, pp. 3149–3156, 2005.

[19] S.Devaraj, I. Jialal, J.-M.Yun, andA. Bremer, “Demonstration ofincreased toll-like receptor 2 and toll-like receptor 4 expressioninmonocytes of type 1 diabetesmellitus patients withmicrovas-cular complications,” Metabolism: Clinical and Experimental,vol. 60, no. 2, pp. 256–259, 2011.

[20] S. Devaraj, P. Tobias, B. S. Kasinath, R. Ramsamooj, A. Afify,and I. Jialal, “Knockout of toll-like receptor-2 attenuates boththe proinflammatory state of diabetes and incipient diabeticnephropathy,” Arteriosclerosis, Thrombosis, and Vascular Biol-ogy, vol. 31, no. 8, pp. 1796–1804, 2011.

[21] M. Lin,W. H. Yiu, H. J. Wu et al., “Toll-like receptor 4 promotestubular inflammation in diabetic nephropathy,” Journal of theAmerican Society of Nephrology, vol. 23, no. 1, pp. 86–102, 2012.

[22] M. R. Dasu, S. Devaraj, L. Zhao, D. H. Hwang, and I. Jialal,“High glucose induces toll-like receptor expression in humanmonocytes: mechanism of activation,” Diabetes, vol. 57, no. 11,pp. 3090–3098, 2008.

[23] I. Jialal, B. A. Huet, H. Kaur, A. Chien, and S. Devaraj,“Increased toll-like receptor activity in patients with metabolicsyndrome,” Diabetes Care, vol. 35, no. 4, pp. 900–904, 2012.

[24] S. K. Venugopal, S. Devaraj, I. Yuhanna, P. Shaul, and I.Jialal, “Demonstration that C-reactive protein decreases eNOSexpression and bioactivity in human aortic endothelial cells,”Circulation, vol. 106, no. 12, pp. 1439–1441, 2002.

[25] N. Matsunaga, N. Tsuchimori, T. Matsumoto, and M. Ii, “TAK-242 (resatorvid), a small-molecule inhibitor of Toll-like receptor(TLR) 4 signaling, binds selectively to TLR4 and interfereswith interactions between TLR4 and its adaptor molecules,”Molecular Pharmacology, vol. 79, no. 1, pp. 34–41, 2011.

[26] K. Takashima, N. Matsunaga, M. Yoshimatsu et al., “Analysisof binding site for the novel small-molecule TLR4 signaltransduction inhibitor TAK-242 and its therapeutic effect onmouse sepsis model,” British Journal of Pharmacology, vol. 157,no. 7, pp. 1250–1262, 2009.

[27] L. A. Couture, W. Piao, L. W. Ru, S. N. Vogel, and V. Y.Toshchakov, “Targeting toll-like receptor (TLR) signaling bytoll/interleukin-1 receptor (TIR) domain-containing adapterprotein/MyD88 adapter-like (TIRAP/Mal)-derived decoy pep-tides,” Journal of Biological Chemistry, vol. 287, no. 29, pp. 24641–24648, 2012.

[28] T. S. Kern, “Contributions of inflammatory processes to thedevelopment of the early stages of diabetic retinopathy,” Exper-imental Diabetes Research, vol. 2007, Article ID 95103, 14 pages,2007.

[29] M. K. Mathews, C. Merges, D. S. McLeod, and G. A. Lutty,“Vascular endothelial growth factor and vascular permeabilitychanges in human diabetic retinopathy,” Investigative Ophthal-mology and Visual Science, vol. 38, no. 13, pp. 2729–2741, 1997.

[30] T. Hikichi, N. Fujio, J. Akiba, Y. Azuma, M. Takahashi, and A.Yoshida, “Association between the short-term natural historyof diabetic macular edema and the vitreomacular relationshipin type II diabetes mellitus,” Ophthalmology, vol. 104, no. 3, pp.473–478, 1997.

[31] H. Canataroglu, I. Varinli, A. A. Ozcan, A. Canataroglu, F.Doran, and S. Varinli, “Interleukin (IL)-6, interleukin (IL)-8levels and cellular composition of the vitreous humor in prolif-erative diabetic retinopathy, proliferative vitreoretinopathy, andtraumatic proliferative vitreoretinopathy,” Ocular Immunologyand Inflammation, vol. 13, no. 5, pp. 375–381, 2005.

[32] S. Schroder, W. Palinski, and G. W. Schmid-Schonbein, “Acti-vatedmonocytes and granulocytes, capillary nonperfusion, andneovascularization in diabetic retinopathy,”American Journal ofPathology, vol. 139, no. 1, pp. 81–100, 1991.

[33] A. D.Meleth, E. Agrón, C.-C. Chan et al., “Serum inflammatorymarkers in diabetic retinopathy,” Investigative Ophthalmologyand Visual Science, vol. 46, no. 11, pp. 4295–4301, 2005.

[34] A. M. Joussen, V. Poulaki, M. L. Le et al., “A central role forinflammation in the pathogenesis of diabetic retinopathy,” TheFASEB Journal, vol. 18, no. 12, pp. 1450–1452, 2004.

[35] H. Kaur, A. Chien, and I. Jialal, “Hyperglycemia induces tolllike receptor 4 expression and activity inmousemesangial cells:Relevance to diabetic nephropathy,” The American Journal ofPhysiology—Renal Physiology, vol. 303, no. 8, pp. F1145–F1150,2012.

[36] J.-X. Chen and A. Stinnett, “Critical role of the NADPH oxidasesubunit p47phox on vascular TLR expression and neointimallesion formation in high-fat diet-induced obesity,” LaboratoryInvestigation, vol. 88, no. 12, pp. 1316–1328, 2008.

RETRACTED

Journal of Diabetes Research 15

[37] R. A. Kowluru and S. N. Abbas, “Diabetes-induced mitochon-drial dysfunction in the retina,” InvestigativeOphthalmology andVisual Science, vol. 44, no. 12, pp. 5327–5334, 2003.

[38] R. A. Kowluru, J. Tang, and T. S. Kern, “Abnormalities ofretinal metabolism in diabetes and experimental galactosemia.VII. Effect of long-term administration of antioxidants on thedevelopment of retinopathy,” Diabetes, vol. 50, no. 8, pp. 1938–1942, 2001.

[39] T. S. Kern, R. A. Kowluru, and R. L. Engerman, “Abnormalitiesof retinal metabolism in diabetes or galactosemia: ATPases andglutathione,” Investigative Ophthalmology & Visual Science, vol.35, no. 7, pp. 2962–2967, 1994.

[40] S. Z. Safi, R. Qvist, G. O. Yan, and I. S. Ismail, “Differentialexpression and role of hyperglycemia induced oxidative stressin epigenetic regulation of 𝛽1, 𝛽2 and 𝛽3-adrenergic receptorsin retinal endothelial cells,” Acta Veterinaria Scandinavica, vol.7, article 29, 2014.

[41] J. V. Busik, S. Mohr, and M. B. Grant, “Hyperglycemia-inducedreactive oxygen species toxicity to endothelial cells is dependenton paracrine mediators,” Diabetes, vol. 57, no. 7, pp. 1952–1965,2008.

[42] K. Miyamoto, S. Khosrof, S.-E. Bursell et al., “Preventionof leukostasis and vascular leakage in streptozotocin-induceddiabetic retinopathy via intercellular adhesion molecule-1 inhi-bition,” Proceedings of the National Academy of Sciences of theUnited States of America, vol. 96, no. 19, pp. 10836–10841, 1999.

[43] N. N. Vagaja, N. Binz, S. McLenachan, E. P. Rakoczy, and P. G.McMenamin, “Influence of endotoxin-mediated retinal inflam-mation on phenotype of diabetic retinopathy in Ins2Akitamice,” British Journal of Ophthalmology, vol. 97, no. 10, pp. 1343–1350, 2013.

[44] S. Devaraj, P. Tobias, and I. Jialal, “Knockout of toll-likereceptor-4 attenuates the pro-inflammatory state of diabetes,”Cytokine, vol. 55, no. 3, pp. 441–445, 2011.

[45] G. Dvoriantchikova, D. J. Barakat, E. Hernandez, V. I.Shestopalov, and D. Ivanov, “Toll-like receptor 4 contributes toretinal ischemia/reperfusion injury,” Molecular Vision, vol. 16,pp. 1907–1912, 2010.

[46] J. Tang, C. Allen Lee, Y. Du et al., “MyD88-dependent pathwaysin leukocytes affect the retina in diabetes,” PLoS ONE, vol. 8, no.7, Article ID e68871, 2013.

[47] J. Ma, H. Wu, C. Y. Zhao, U. Panchapakesan, C. Pollock, andS. J. Chadban, “Requirement for TLR2 in the developmentof albuminuria, inflammation and fibrosis in experimentaldiabetic nephropathy,” International Journal of Clinical andExperimental Pathology, vol. 7, no. 2, pp. 481–495, 2014.

[48] A. Taye, A. H. Saad, A. H. Kumar, and H. Morawietz, “Effect ofapocynin on NADPH oxidase-mediated oxidative stress-LOX-1-eNOS pathway in human endothelial cells exposed to highglucose,” European Journal of Pharmacology, vol. 627, no. 1–3,pp. 42–48, 2010.

[49] M. R.Dasu, S. Devaraj, and I. Jialal, “High glucose induces IL-1𝛽expression in human monocytes: mechanistic insights,” Ameri-can Journal of Physiology—Endocrinology and Metabolism, vol.293, no. 1, pp. E337–E346, 2007.

[50] V. Thallas-Bonke, S. R. Thorpe, M. T. Coughlan et al., “Inhi-bition of NADPH oxidase prevents advanced glycation endproduct-mediated damage in diabetic nephropathy through aprotein kinase C-𝛼-dependent pathway,”Diabetes, vol. 57, no. 2,pp. 460–469, 2008.

[51] M. Morigi, S. Angioletti, B. Imberti et al., “Leukocyte-endothelial interaction is augmented by high glucose concen-trations and hyperglycemia in a NF-kB-dependent fashion,”Journal of Clinical Investigation, vol. 101, no. 9, pp. 1905–1915,1998.