2. materials and methods - hindawi publishing corporationmediators of inammation retinal...

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 986217 7 pageshttpdxdoiorg1011552013986217

Clinical StudyGene Expression of IGF1 IGF1R and IGFBP3 inEpiretinal Membranes of Patients with ProliferativeDiabetic Retinopathy Preliminary Study

Dorota Romaniuk1 Malgorzata W Kimsa2 Barbara Strzalka-Mrozik2

Magdalena C Kimsa2 Adam Kabiesz1 Wanda Romaniuk1 and Urszula Mazurek2

1 Clinical Department of Ophthalmology Medical University of Silesia Ceglana Street 35 40-952 Katowice Poland2Department of Molecular Biology Medical University of Silesia Narcyzow Street 1 41-200 Sosnowiec Poland

Correspondence should be addressed to Dorota Romaniuk d romaniukhotmailcom

Received 20 August 2013 Accepted 4 November 2013

Academic Editor Dorota Raczynska

Copyright copy 2013 Dorota Romaniuk et al 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

The molecular mechanism formation of secondary epiretinal membranes (ERMs) after proliferative diabetic retinopathy (PDR) orprimary idiopathic ERMs is still poorly understood Therefore the present study focused on the assessment of IGF1 IGF1R andIGFBP3 mRNA levels in ERMs and PBMCs from patients with PDR The examined group comprised 6 patients with secondaryERMs after PDR and the control group consisted of 11 patients with idiopathic ERMs Quantification of IGF1 IGF1R and IGFBP3mRNAs was performed by real-time QRT-PCR technique In ERMs IGF1 and IGF1R mRNA levels were significantly higher inpatients with diabetes compared to control subjects In PBMCs there were no statistically significant differences of IGF1 IGF1Rand IGFBP3 expression between diabetic and nondiabetic patients In conclusion our study indicated IGF1 and IGF1R differentialexpression in ERMs but not in PBMCs of diabetic and nondiabetic patients suggesting that these factors can be involved in thepathogenesis or progression of proliferative vitreoretinal disorders This trial is registered with NCT00841334

1 Introduction

Proliferative diabetic retinopathy (PDR) is a common andspecific microvascular complication of diabetes mellitusInflammation angiogenesis and fibrosis are primary pro-cesses involved in the pathogenesis of PDR [1] MoreoverSnead et al [2] suggested that hypoxia and neovascularcytokine production may play an important role in epiretinalmembrane (ERM) formation of PDR patients

Epiretinal membrane is a layer of pathologic tissue whichgrows on the internal surface of the retina [3 4] ERMscan be idiopathic (iERM) or secondary to some patho-logic conditions including proliferative diabetic retinopathyproliferative vitreoretinopathy (PVR) macular pucker highmyopia uveitis and other retinal degenerative diseases [5]Depending on duration and severity of ERMs the symptomsfelt by patient can differ frommild to severemetamorphopsia

decrease in visual acuity caused by retinal distortion andproblems such as vitreoretinal tractions or even tractionalretinal detachment [6] It is known that ERMs in PDRare mainly composed of new pathological vessels On thecontrary the idiopathic ERMs consist of a nonangiogenicfibroglial tissue [7] However the molecular mechanismformation of both the primary and secondary ERMs indiabetic and nondiabetic patients is still poorly understood

Several cytokines including insulin-like growth factor(IGF) are involved in the progression of ERMs [8ndash10] TheIGF family is comprised of ligands IGF1 IGF2 insulinsix binding proteins (IGFBP1 through -6) and cell surfacereceptors including IGF1R IGF2R and insulin receptors [11]Many recent studies suggested that IGF1 and IGF2 may beinvolved in the pathogenesis of PDR however the exact rolehas not been well understood yet [12 13] Recent reportssuggest that increases in IGF1 activity may contribute to

2 Mediators of Inflammation

retinal neovascularization characteristic for PDR [14 15]IGFs are also involved in stimulation of epiretinal membranecontraction [9] Additionally IGF system is known to beassociated with inflammation process [16 17]

Numerous attempts have been made to identify variousproteins in serum or mRNA in peripheral blood mononu-clear cells (PBMCs) which could be easily accessed and actas markers of intratissue processes in various diseases [13 18]However local concentrations of different growth factorsin the retina may be more important than systemic levelsin the pathogenesis of diabetic retinopathy There are onlyfew published data regarding differences between mRNAlevels of IGF in the eye tissues of diabetic and nondiabeticpatients [19ndash22] Therefore the present study focused on theassessment of mRNA levels of IGF1 IGF1R and IGFBP3 bothin ERMs and PBMCs frompatients with proliferative diabeticretinopathy

2 Materials and Methods

21 Subjects The blood samples from 17 patients and sur-gically removed ERMs from 17 eyes of these patients wereobtained and categorized into diabetic and nondiabetic onesThe examined group comprised 6 patients (3 females and3 males mean age 729 years range 66ndash80 years) withsecondary ERMs after PDR and the control group consistedof 11 patients (8 females and 3males mean age 697 range 61ndash79 years)with iERMs (Table 1) All the patientswere treated inthe Department of Ophthalmology University Hospital No5 Medical University of Silesia Katowice Poland

The period between the diagnosis of the condition andthe actual surgery for the condition was between 2 and 16months Some patients presented and had surgery withintwo month When patients were asymptomatic or had goodvisual acuity ERMs were diagnosed clinically that did notrequire immediate surgery The average duration in whichthe membranes were present in the eye before removal was64 months (range 3ndash10 months) for PDR and 86 months(range 2ndash16 months) for iERM All subjects underwenta complete ophthalmic examination best corrected visualacuity (BCVA) tonometry slit lamp examination of anteriorand posterior segment ultrasonography of the eye andoptical coherence tomography (OCT) of the central retinaThe criteria for inclusion into the molecular analysis were asfollows patients of Caucasian race both males and femalesaged ge55 years suffering from idiopathic ERMs or secondaryERMs in PDR Presence of ERM was proven in OCTexamination All patients were qualified to surgical treatmentof ERMmdashunilateral pars plana vitrectomy (PPV)

The study was approved by the Bioethics Committee ofthe Medical University in Katowice (KNW0022KB18212)in accordance with the Declaration of Helsinki regardingmedical research involving human subjects All patients wereinformed about the research and signed an informed consentform

22 Tissues Samples of all ERMs were collected duringpars plana vitrectomy using intraocular microforceps and

Table 1 Selected clinical features of patients with secondary ERMsafter proliferative diabetic retinopathy and the control group withidiopathic ERMs

Characteristic Secondary ERMsgroup (119899 = 6)

Idiopathic ERMsgroup (119899 = 11)

GenderFemale 3 8Male 3 3

Age (years) 729 (66ndash80)lowast 697 (61ndash79)Eye

Right 2 5Left 4 6

Mean time intervalbetween ERM diagnosisand surgery (months)

64 (3ndash10) 86 (2ndash16)

Visual acuitydagger 03 (001ndash05) 02 (002ndash04)lowastValues of clinical parameters are expressed as means (minimumndashmaximum)daggerSnellen chart

were stored for 48 h at minus70∘C until RNA extraction Venousblood samples were collected into EDTA-containing tubesand peripheral mononuclear cells were isolated from 5mLspecimens derived from each patients by using Ficoll-Conraydensity gradient centrifugation for 30min at 1500 rpm atroom temperature immediately after blood collection (spe-cific gravity 1077 Immunobiological Co Gunma Japan)

23 Ribonucleic Acid Extraction from Tissue Specimens TotalRNA was extracted from ERMs and PBMCs using a TRIzolreagent (Invitrogen Carlsbad CA USA) RNA extracts weretreated with DNase I (MBI Fermentas Vilnius Lithuania)according to the manufacturerrsquos instructions The quality ofextracts was checked electrophoretically using 08 agarosegel stained with ethidium bromideThe results were analyzedand recorded using the 1D Bas-Sys gel documentation system(Biotech-Fisher Perth Australia) Total RNA concentrationwas determined by spectrophotometric measurement in 5120583Lcapillary tubes using the Gene Quant II RNADNA Calcula-tor (Pharmacia Biotech Cambridge UK)

24 Quantitative Real-Time Polymerase Chain Reaction AssayDetection of the expression of IGF1 IGF1R IGFBP3 andglyceraldehyde-3-phosphate (GAPDH) mRNAs was carriedout using a QRT-PCR technique with SYBR Green chem-istry (SYBR Green Quantitect RT-PCR Kit Qiagen) andan Opticon DNA Engine Continuous Fluorescence detector(MJ Research) as described previously [23] All sampleswere tested in triplicate GAPDH for each sample was mea-sured to exclude possible RT-PCR inhibitorsOligonucleotideprimers specific for IGF1 IGF1R and IGFBP3 were designedon the basis of reference sequences (GenBank accession noNM 0006183 NM 0008753 andNM 0005984 resp) usingPrimer ExpressTM Version 20 software (PE Applied Biosys-tems Foster City CA USA) Detection of GAPDH mRNAswas performed as described previously [23] (Table 2)

Mediators of Inflammation 3

Table 2 Characteristics of primers used for real-time QRT-PCR

Gene Sequence of primers Length of amplicon (bp)lowast Tm (∘C)dagger

IGF1 Forward 51015840-TGCTTCCGGAGCTGTGATC-31015840 221 776Reverse 51015840-GATCCTGCGGTGGCATGTCACTCTTCACT-31015840

IGF1R Forward 51015840-ACGCCAATAAGTTCGTCCACAGAGACCT-31015840 187 774Reverse 51015840-GAAGACTCCATCCTTGAGGGACTCAG-31015840

IGFBP3 Forward 51015840-GCTACAGCATGCAGAGCAAGT-31015840 102 824Reverse 51015840-CAGCTGCTGGTCATGTCCTT-31015840

GAPDH Forward 51015840-GAAGGTGAAGGTCGGAGTC-31015840 226 800Reverse 51015840-GAAGATGGTGATGGGATTC-31015840

lowastbp base pairsdaggerTm melting temperature

The thermal profile for one-step RT-PCR was as followsreverse transcription at 50∘C for 30min denaturation at95∘C for 15min and 40 cycles consisting of the followingtemperatures and time intervals 94∘C for 15 s 60∘C forGAPDH 611∘C for IGF1 564∘C for IGF1R and 542∘C forIGFBP3 for 30 s and 72∘C for 30 s Each run was completedusing melting curve analysis to confirm the specificity ofamplification and the absence of primer dimers RT-PCRproducts were separated on 6 polyacrylamide gels andvisualized with silver salts

25 Quantification of Expression of Target Genes To quantifythe results obtained with RT-PCR for IGF1 IGF1R IGFBP3and GAPDH a standard curve method was used [23 24]Commercially available standards of 120573-actin cDNA (TaqManDNA Template Reagent Kit PE Applied Biosystems FosterCA) were used at five different concentrations (06 12 3060 and 120 ng120583L) to simultaneously detect the expressionprofile of each investigated gene For standards the calcu-lation of copy number values was based on the followingrelationship 1 ng of DNA = 333 genome equivalents (PEApplied Biosystems) Amplification plots for each dilutionof a commercially available standard template were used todetermine the Ct values A standard curve was generated byplotting the Ct values against the log of the known amountof 120573-actin cDNA copy numbers Correlation coefficients forstandard curves ranged from 0988 to 0995 indicating a highdegree of confidence for measurement of the copy number ofmolecules in each sample

26 Statistical Analyses Statistical analyses were performedusing Statistica 90 software (StatSoft Tulsa OK USA) andthe level of significance was set at 119875 lt 005 Values wereexpressed as median with the 25th and 75th quartiles andminimum and maximum The Mann-Whitney 119880-test wasapplied to assess differences in the expression level of IGF1IGF1R and IGFBP3 Correlations were evaluated using theSpearman rank correlation test

3 Results

31 Specificity of the Real-Time Reverse Transcription Poly-merase Chain Reaction Assay RT-PCR specificity for the

target genes was confirmed experimentally on the basis ofamplimers melting temperatures For each RT-PCR producta single peak at expected temperatures was observed IGF1776∘C IGF1R 774∘C IGFBP3 824∘C and GAPDH 800∘C(data not shown) Gel electrophoresis also revealed thepresence of a single product of predicted length (data notshown)

32 Differences in IGF1 IGF1R and IGFBP3 Expression Levelbetween Diabetic and Nondiabetic Patients IGF1 and IGF1RmRNAs were detected in all tested ERMs and PBMCsobtained from the control and study groups HoweverIGFBP3 was only detected in PBMCs of all patients In theERMs themRNA level of IGF1was significantly higher in thepatientswith diabetes (median= 271mRNAcopies120583g of totalRNA) compared to the control iERMs of nondiabetic group(median = 85mRNA copies120583g of total RNA) In the caseof IGF1R the expression was significantly greater in ERMsafter PDR (median = 1341mRNA copies120583g of total RNA)compared to the idiopathic ERMs (median = 938 mRNAcopies120583g of total RNA) (119875 = 00224 Mann-Whitney 119880-test)(Figure 1)

In PBMCs there were no significantly different expres-sions of IGF1 IGF1R and IGFBP3 between diabetic(median = 163mRNA copies120583g of total RNA 298mRNAcopies120583g of total RNA 1211mRNA copies120583g of totalRNA resp) and nondiabetic patients (median = 144mRNAcopies120583g of total RNA 308mRNA copies120583g of total RNA1209mRNA copies120583g of total RNA resp) (IGF1 119875 = 06353IGF1R 119875 = 07250 IGFBP3 119875 = 09599 Mann-Whitney119880-test) (Figure 2)

33 Correlations between IGF1 IGF1R and IGFBP3ExpressionLevel and Time Interval between Diagnosis and SurgeryThere was no correlation between the time interval betweendiagnosis of the condition and the actual surgery and theexpression of IGF1 IGF1R and IGFBP3 both in the ERMsand PBMCs of the diabetic samples Corresponding to theresults obtained for diabetic patients in iERMs and PBMCsfrom the nondiabetic patients no correlations between theexpression of IGF1 IGF1R and IGFBP3 genes and timeinterval between diagnosis of the condition and the actualsurgery were detected (Table 3)


Table 3 The correlations between relative expression of IGF1 IGF1R and IGFBP3 and time interval between diagnosis and surgery in theERMs and PBMCs of diabetic and nondiabetic patients

Diabetic patientsERMs IGF1 IGF1R PBMCs IGF1 IGF1R IGFBP3Time interval betweendiagnosis and surgery 119903 = 0316 119903 = minus0316

Time interval betweendiagnosis and surgery 119903 = 0462 119903 = 0103 119903 = minus0462

Nondiabetic patientsiERMs IGF1 IGF1R PBMCs IGF1 IGF1R IGFBP3Time interval betweendiagnosis and surgery 119903 = 0065 119903 = 0497


Statistical significance lowast119875 lt 005 spearman rank correlation test

Median Min-max

IGF1 IGF1R

0

200

400

600

800

1000

1200

1400

1600

PDRiERM

mRN

A co

pies

120583g

of to

tal R

NA

lowast

lowast

25ndash75

Figure 1 The mRNA levels of IGF1 and IGF1R in epiretinalmembranes of patients with proliferative diabetic retinopathy (PDR)and in the control subjects with idiopathic ERMs (iERM) (Box andwhisker plots present medians plusmn quartiles and extreme values ofcopy numbers per 1 120583g of total RNA statistical significance lowast119875 lt005 Mann-Whitney 119880-test)

4 Discussion

IGF1IGF1RIGFBPs complex can participate in the phys-iological and pathological events that occur in the retinaPrevious research has revealed IGF1 IGF1R and IGFBPsexpression in microvascular endothelial cells pericytes andretinal epithelial cells (RPE) in vitro [25ndash27] Thereforeit is suggested that these growth factors can also causeprogression of ERMs especially during proliferative diabeticretinopathy [11] On the other hand the level of IGFs in theeye tissues may result in part from damage to the blood-retinal barrier Furthermore the association between levelof IGFs in serumPBMCs and the development of PDR isstill not fully explained [12] Therefore determination ofdifferences between mRNA levels of IGFs in ERMs and

IGF1 IGF1R IGFBP3

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

mRN

A co

pies

120583g

of to

tal R

NA

Median Min-max

PDRiERM

25ndash75

Figure 2 The mRNA levels of IGF1 IGF1R and IGFBP3 in periph-eral blood mononuclear cells of patients with proliferative diabeticretinopathy (PDR) and in the control subjects with idiopathic ERMs(iERM) (Box and whisker plots present medians plusmn quartiles andextreme values of copy numbers per 1120583g of total RNA statisticalsignificance lowast119875 lt 005 Mann-Whitney 119880-test)

PBMCs in diabetic compared to nondiabetic patients usingreal-time QRT-PCR seems to be important In the presentstudy IGF1 IGF1R and IGFBP3 mRNA levels were detectedin PBMC samples but in ERMs only IGF1 and IGF1RmRNAswere determined It can be explained that IGFBP3 did notcause proliferation of retinal cells [28] Moreover Spoerriet al [29] showed that exogenous administration of IGFBP3can induce growth inhibition and apoptosis of retinal cellsOn the other hand it was reported that IGFBP3 is neu-roprotective antiapoptotic anti-inflammatory after retinalinjury [30 31] and may prevent the progression of PDR [32]Previous research showed that also IGFs are not involved inproliferation of these cells but in stimulation of epiretinalmembrane contraction [9] In contrast Spraul et al [12] andTakagi et al [25] indicate that IGF1 and IGF2 can influence


RPE cell migration and proliferation In turn Ruberte et al[33] determined that neovascularization was consistent withincreased vascular endothelial growth factor level induced byIGF1 in retinal cells

Statistically significant higher mRNA levels of IGF1 andIGF1R in the ERMs of patients with PDR compared to thecontrol subjects with iERMs in our results confirmed thatthe IGFs may play a key role in development or progressionof PDR Similar results were demonstrated by other authorswho found significantly higher protein level of IGF1 in PDRthan in control patient but in human vitreous samples [34ndash36] whereas Gerhardinger et al [37] reported a lower mRNAlevels of IGF1 in diabetic retinas obtained postmortem fromhumandonors and they did not observe a changed expressionof IGF1R

Interestingly our results for the quantitative analysis ofIGF1 IGF1R and IGFBP3 in PBMCs revealed no statisticallysignificant differences of all three genes between diabetic andnondiabetic patients which might suggest that the processesoccurring in the eye tissues are not affected by systemicexpression levels of these cytokines during diabetes Corre-sponding to our results Payne et al [13] and Spranger et al[36] found that diabetic subjects had similar IGF1 level com-pared to nondiabetic subjects but in serum samples Similarlyother authors showed no correlation between serum IGF1levels and the development or progression of PDR [13 38]Onthe other hand several studies contradict these findings forexample Dills et al [39] andMerimee et al [40] have revealedhigher serum IGF1 levels in diabetic patients MoreoverChen et al [14] suggested that serum IGF1 level may be acontributing factor in diabetic retinopathy However thesedifferences may be a result of the assay method used tomeasure IGF1 levels Additionally transport mechanisms forIGFs and IGFBPs between the blood and the eye are not verywell known and may depend on the blood-retinal barrierwhose disruption is an important event in pathogenesisof diabetic retinopathy and other ocular disorders [12]Haurigotrsquos studies demonstrated that intraocular IGF1 butnot systemic IGF1 is sufficient to trigger blood-retinal barrierbreakdown [15] It may suggest that treatment strategiesshould be designed to counteract local than systemic IGF1effects [15] In contrast Spranger et al [36] indicated thatbreakdown of the blood-retinal barrier and serum levels ofIGF mainly determined intravitreal IGF levels rather thanlocal production The disruption of the blood-retinal barriercan cause also the infiltration of immunologically competentcells into PDR membranes therefore the inflammatoryreactions may play a role in the development of the epiretinalmembrane in PDR [41] It is also known that IGF1 hasanti-inflammatory effects and it can be negatively correlatedwith inflammatory markers [16] No correlation between thevitreous and serum levels of various growth factors in patientswith PDR and nonproliferative diabetic retinopathy was alsoobserved [42ndash44] The lack of correlations between serumand intraocular tissue levels can suggest that serumPBMClevels of various growth factors may not be a useful predictorof the diabetic retinopathy severity [42 43] Moreover ourresearch did not show any correlation between IGF1 IGF1R

and IGFBP3 mRNA levels and the time interval betweendiagnosis of the condition and the surgery

In conclusion our study indicated IGF1 and IGF1R dif-ferential expression in ERMs but not in PBMCs of diabeticand nondiabetic patients suggesting that these factors can beinvolved in the pathogenesis or progression of proliferativevitreoretinal disorders Unfortunatelymajor limitation of ourstudy is a relatively small number of samples However analy-sis of ocular samples is often difficult because of the technicalobstacles associated with extracting sufficient quantities ofthe samples Therefore there is a need to study a largerpopulation and carry out further analysis for example onprotein levels in order to clarify the contribution of theseintravitreal growth factors in the development of diabetic andnondiabetic ERMs Moreover this will help to identify bettertreatment strategies for proliferative diabetic retinopathy

Conflict of Interests

The authors declare that there is no conflict of interests

References

[1] A M Abu El-Asrar M Imtiaz Nawaz D Kangave M M Sid-diquei and K Geboes ldquoOsteopontin and other regulators ofangiogenesis and fibrogenesis in the vitreous from patients withproliferative vitreoretinal disordersrdquo Mediators of Inflamma-tion vol 2012 Article ID 493043 8 pages 2012

[2] D R J Snead S James and M P Snead ldquoPathological changesin the vitreoretinal junction 1 epiretinal membrane formationrdquoEye vol 22 no 10 pp 1310ndash1317 2008

[3] W E Smiddy A M Maguire W R Green et al ldquoIdiopathicepiretinal membranes ultrastructural characteristics and clini-copathologic correlation 1989rdquoRetina vol 25 no 5 pp 811ndash8212005

[4] A T Yazici N Alagoz H U Celik et al ldquoIdiopathic andsecondary epiretinal membranes do they differ in terms ofmorphology An optical coherence tomography-based studyrdquoRetina vol 31 no 4 pp 779ndash784 2011

[5] X Zhang G Barile S Chang et al ldquoApoptosis and cell prolif-eration in proliferative retinal disorders PCNA Ki-67 caspase-3 and PARP expressionrdquo Current Eye Research vol 30 no 5pp 395ndash403 2005

[6] S Y L Oberstein J Byun D Herrera E A Chapin S KFisher and G P Lewis ldquoCell proliferation in human epiretinalmembranes characterization of cell types and correlation withdisease condition and durationrdquo Molecular Vision vol 17 pp1794ndash1805 2011

[7] Y Yamaji S Yoshida K Ishikawa et al ldquoTEM7 (PLXDC1) inneovascular endothelial cells of fibrovascular membranes frompatients with proliferative diabetic retinopathyrdquo InvestigativeOphthalmology and Visual Science vol 49 no 7 pp 3151ndash31572008

[8] C Harada Y Mitamura and T Harada ldquoThe role of cytokinesand trophic factors in epiretinal membranes involvement ofsignal transduction in glial cellsrdquo Progress in Retinal and EyeResearch vol 25 no 2 pp 149ndash164 2006

[9] A Bringmann and PWiedemann ldquoInvolvement of Muller glialcells in epiretinal membrane formationrdquo Graefersquos Archive forClinical and Experimental Ophthalmology vol 247 no 7 pp865ndash883 2009


[10] L Iannetti M Accorinti R Malagola et al ldquoRole of theintravitreal growth factors in the pathogenesis of idiopathicepiretinal membranerdquo Investigative Ophthalmology and VisualScience vol 52 no 8 pp 5786ndash5789 2011

[11] R Simo E Carrasco M Garcıa-Ramırez and C HernandezldquoAngiogenic and antiangiogenic factors in proliferative diabeticretinopathyrdquo Current Diabetes Reviews vol 2 no 1 pp 71ndash982006

[12] C W Spraul C Kaven J Amann G K Lang and G E LangldquoEffect of insulin-like growth factors 1 and 2 and glucose on themigration and proliferation of bovine retinal pigment epithelialcells in vitrordquo Ophthalmic Research vol 32 no 5 pp 244ndash2482000

[13] J F Payne V Tangpricha J Cleveland M J Lynn R Ray andS K Srivastava ldquoSerum insulin-like growth factor-I in diabeticretinopathyrdquoMolecular Vision vol 17 pp 2318ndash2324 2011

[14] H-S Chen T-E Wu L-C Hsiao and S-H Lin ldquoInteractionbetween glycaemic control and serum insulin-like growthfactor 1 on the risk of retinopathy in type 2 diabetesrdquo EuropeanJournal of Clinical Investigation vol 42 no 4 pp 447ndash454 2012

[15] V Haurigot P Villacampa A Ribera et al ldquoIncreased intraoc-ular insulin-like growth factor-I triggers blood-retinal barrierbreakdownrdquo The Journal of Biological Chemistry vol 284 no34 pp 22961ndash22969 2009

[16] M J Lee D H Shin K I Ko et al ldquoAssociation betweenthe ratio of insulin-like growth factor-I to insulin-like growthfactor binding protein-3 and inflammation in incident auto-mated peritoneal dialysis patientsrdquo Growth Hormone and IGFResearch vol 23 no 5 pp 170ndash174 2013

[17] A Kluge R Zimmermann D Weihrauch et al ldquoCoordi-nate expression of the insulin-like growth factor system aftermicroembolisation in porcine heartrdquo Cardiovascular Researchvol 33 no 2 pp 324ndash331 1997

[18] B Strzalka-Mrozik A Nowak J Gola M Kowalczyk MKapral and U Mazurek ldquoFactors associated with changes inendothelin-1 gene expression in patients with diabetic retinopa-thy in type 2 diabetes mellitusrdquo Molecular Vision vol 16 pp1272ndash1279 2010

[19] C Guidry and J L King ldquoIsolation and characterization of vit-reous insulin-like growth factor binding proteinsrdquo InvestigativeOphthalmology and Visual Science vol 52 no 1 pp 303ndash3092011

[20] C P Burren J L Berka S R Edmondson G A Werther andJ A Batch ldquoLocalization of mRNAs for insulin-like growthfactor-I (IGF-I) IGF-I receptor and IGF binding proteins in rateyerdquo Investigative Ophthalmology and Visual Science vol 37 no7 pp 1459ndash1468 1996

[21] Y C Wu B R Buckner M Zhu H D Cavanagh and D MRobertson ldquoElevated IGFBP3 levels in diabetic tears a negativeregulator of IGF-1 signaling in the corneal epitheliumrdquo TheOcular Surface vol 10 no 2 pp 100ndash107 2012

[22] N Sengupta A Afzal S Caballero et al ldquoParacrinemodulationof CXCR4 by IGF-1 and VEGF implications for choroidalneovascularizationrdquo Investigative Ophthalmology and VisualScience vol 51 no 5 pp 2697ndash2704 2010

[23] P Berezowski B Strzalka-Mrozik M Forminska-Kapusciket al ldquoPosttraumatic temporal TGF-120573mRNA expression in lensepithelial cells of paediatric patientsrdquo Folia Biologica vol 58 pp24ndash29 2012

[24] M Kapral J Wawszczyk M Jurzak A Hollek and L WeglarzldquoThe effect of inositol hexaphosphate on the expression of

selected metalloproteinases and their tissue inhibitors in IL-1120573-stimulated colon cancer cellsrdquo International Journal of Colorec-tal Disease vol 27 no 11 pp 1419ndash1428 2012

[25] H Takagi N Yoshimura H Tanihara and Y Honda ldquoInsulin-like growth factor-related genes receptors and binding proteinsin cultured human retinal pigment epithelial cellsrdquo InvestigativeOphthalmology and Visual Science vol 35 no 3 pp 916ndash9231994

[26] P Moriarty M Boulton A Dickson and D McLeod ldquoProduc-tion of IGF-I and IGF binding proteins by retinal cells in vitrordquoBritish Journal of Ophthalmology vol 78 no 8 pp 638ndash6421994

[27] P E Spoerri E A Ellis R W Tarnuzzer and M B GrantldquoInsulin-like growth factor receptor and binding proteins inhuman retinal endothelial cell cultures of diabetic and non-diabetic originrdquo Growth Hormone and IGF Research vol 8 no2 pp 125ndash132 1998

[28] J L King and C Guidry ldquoVitreous IGFBP-3 effects on Mullercell proliferation and tractional force generationrdquo InvestigativeOphthalmology amp Visual Science vol 53 no 1 pp 93ndash99 2012

[29] P E Spoerri S Caballero S H Wilson L C Shaw and M BGrant ldquoExpression of IGFBP-3 by human retinal endothelialcell cultures IGFBP-3 involvement in growth inhibition andapoptosisrdquo Investigative Ophthalmology and Visual Science vol44 no 1 pp 365ndash369 2003

[30] J L Kielczewski P Hu L C Shaw et al ldquoNovel protective prop-erties of IGFBP-3 result in enhanced pericyte ensheathmentreduced microglial activation increased microglial apoptosisand neuronal protection after ischemic retinal injuryrdquo TheAmerican Journal of Pathology vol 178 no 4 pp 1517ndash15282011

[31] Q Zhang C Soderland and J J Steinle ldquoRegulation of retinalendothelial cell apoptosis through activation of the IGFBP-3receptorrdquo Apoptosis vol 18 no 3 pp 361ndash368 2013

[32] F M Regan R M Williams A McDonald et al ldquoTreatmentwith recombinant human insulin-like growth factor (rhIGF)-IrhIGF binding protein-3 complex improves metabolic controlin subjects with severe insulin resistancerdquo Journal of ClinicalEndocrinology and Metabolism vol 95 no 5 pp 2113ndash21222010

[33] J Ruberte E Ayuso M Navarro et al ldquoIncreased ocular levelsof IGF-1 in transgenic mice lead to diabetes-like eye diseaserdquoJournal of Clinical Investigation vol 113 no 8 pp 1149ndash11572004

[34] R Burgos C Mateo A Canton C Hernandez J Mesa and RSimo ldquoVitreous levels of IGF-I IGF binding protein 1 and IGFbinding protein 3 in proliferative diabetic retinopathy a case-control studyrdquo Diabetes Care vol 23 no 1 pp 80ndash83 2000

[35] R Meyer-Schwickerath A Pfeiffer W F Blum et al ldquoVitreouslevels of the insulin-like growth factors I and II and theinsulin-like growth factor binding proteins 2 and 3 increasein neovascular eye disease Studies in nondiabetic and diabeticsubjectsrdquo Journal of Clinical Investigation vol 92 no 6 pp2620ndash2625 1993

[36] J Spranger J Buhnen V Jansen et al ldquoSystemic levels con-tribute significantly to increased intraocular IGF-I IGF-II andIFG-BP3 in proliferative diabetic retinopathyrdquo Hormone andMetabolic Research vol 32 no 5 pp 196ndash200 2000

[37] C Gerhardinger K D McClure G Romeo F Podesta and MLorenzi ldquoIGF-I mRNA and signaling in the diabetic retinardquoDiabetes vol 50 no 1 pp 175ndash183 2001


[38] Q Wang D G Dills R Klein B E K Klein and S E MossldquoDoes insulin-like growth factor I predict incidence and pro-gression of diabetic retinopathyrdquo Diabetes vol 44 no 2 pp161ndash164 1995

[39] D G Dills S E Moss R Klein and B E K Klein ldquoAssociationof elevated IGF-I levels with increased retinopathy in late-onsetdiabetesrdquo Diabetes vol 40 no 12 pp 1725ndash1730 1991

[40] T J Merimee J Zapf and E R Froesch ldquoInsulin-like growthfactors Studies in diabetics with and without retinopathyrdquoTheNew England Journal of Medicine vol 309 no 9 pp 527ndash5301983

[41] S Kase W Saito S Ohno and S Ishida ldquoProliferative diabeticretinopathy with lymphocyte-rich epiretinal membrane associ-ated with poor visual prognosisrdquo Investigative Ophthalmologyand Visual Science vol 50 no 12 pp 5909ndash5912 2009

[42] A Praidou I Klangas E Papakonstantinou et al ldquoVitreousand serum levels of platelet-derived growth factor and theircorrelation in patients with proliferative diabetic retinopathyrdquoCurrent Eye Research vol 34 no 2 pp 152ndash161 2009

[43] A Praidou E Papakonstantinou S Androudi N GeorgiadisG Karakiulakis and S Dimitrakos ldquoVitreous and serumlevels of vascular endothelial growth factor and platelet-derivedgrowth factor and their correlation in patients with non-proliferative diabetic retinopathy and clinically significantmac-ula oedemardquo Acta Ophthalmologica vol 89 no 3 pp 248ndash2542011

[44] A Canton R Burgos C Hernandez et al ldquoHepatocyte growthfactor in vitreous and serum from patients with proliferativediabetic retinopathyrdquo British Journal of Ophthalmology vol 84no 7 pp 732ndash735 2000

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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


retinal neovascularization characteristic for PDR [14 15]IGFs are also involved in stimulation of epiretinal membranecontraction [9] Additionally IGF system is known to beassociated with inflammation process [16 17]

Numerous attempts have been made to identify variousproteins in serum or mRNA in peripheral blood mononu-clear cells (PBMCs) which could be easily accessed and actas markers of intratissue processes in various diseases [13 18]However local concentrations of different growth factorsin the retina may be more important than systemic levelsin the pathogenesis of diabetic retinopathy There are onlyfew published data regarding differences between mRNAlevels of IGF in the eye tissues of diabetic and nondiabeticpatients [19ndash22] Therefore the present study focused on theassessment of mRNA levels of IGF1 IGF1R and IGFBP3 bothin ERMs and PBMCs frompatients with proliferative diabeticretinopathy

2 Materials and Methods

21 Subjects The blood samples from 17 patients and sur-gically removed ERMs from 17 eyes of these patients wereobtained and categorized into diabetic and nondiabetic onesThe examined group comprised 6 patients (3 females and3 males mean age 729 years range 66ndash80 years) withsecondary ERMs after PDR and the control group consistedof 11 patients (8 females and 3males mean age 697 range 61ndash79 years)with iERMs (Table 1) All the patientswere treated inthe Department of Ophthalmology University Hospital No5 Medical University of Silesia Katowice Poland

The period between the diagnosis of the condition andthe actual surgery for the condition was between 2 and 16months Some patients presented and had surgery withintwo month When patients were asymptomatic or had goodvisual acuity ERMs were diagnosed clinically that did notrequire immediate surgery The average duration in whichthe membranes were present in the eye before removal was64 months (range 3ndash10 months) for PDR and 86 months(range 2ndash16 months) for iERM All subjects underwenta complete ophthalmic examination best corrected visualacuity (BCVA) tonometry slit lamp examination of anteriorand posterior segment ultrasonography of the eye andoptical coherence tomography (OCT) of the central retinaThe criteria for inclusion into the molecular analysis were asfollows patients of Caucasian race both males and femalesaged ge55 years suffering from idiopathic ERMs or secondaryERMs in PDR Presence of ERM was proven in OCTexamination All patients were qualified to surgical treatmentof ERMmdashunilateral pars plana vitrectomy (PPV)

The study was approved by the Bioethics Committee ofthe Medical University in Katowice (KNW0022KB18212)in accordance with the Declaration of Helsinki regardingmedical research involving human subjects All patients wereinformed about the research and signed an informed consentform

22 Tissues Samples of all ERMs were collected duringpars plana vitrectomy using intraocular microforceps and

Table 1 Selected clinical features of patients with secondary ERMsafter proliferative diabetic retinopathy and the control group withidiopathic ERMs

Characteristic Secondary ERMsgroup (119899 = 6)

Idiopathic ERMsgroup (119899 = 11)

GenderFemale 3 8Male 3 3

Age (years) 729 (66ndash80)lowast 697 (61ndash79)Eye

Right 2 5Left 4 6

Mean time intervalbetween ERM diagnosisand surgery (months)

64 (3ndash10) 86 (2ndash16)

Visual acuitydagger 03 (001ndash05) 02 (002ndash04)lowastValues of clinical parameters are expressed as means (minimumndashmaximum)daggerSnellen chart

were stored for 48 h at minus70∘C until RNA extraction Venousblood samples were collected into EDTA-containing tubesand peripheral mononuclear cells were isolated from 5mLspecimens derived from each patients by using Ficoll-Conraydensity gradient centrifugation for 30min at 1500 rpm atroom temperature immediately after blood collection (spe-cific gravity 1077 Immunobiological Co Gunma Japan)

23 Ribonucleic Acid Extraction from Tissue Specimens TotalRNA was extracted from ERMs and PBMCs using a TRIzolreagent (Invitrogen Carlsbad CA USA) RNA extracts weretreated with DNase I (MBI Fermentas Vilnius Lithuania)according to the manufacturerrsquos instructions The quality ofextracts was checked electrophoretically using 08 agarosegel stained with ethidium bromideThe results were analyzedand recorded using the 1D Bas-Sys gel documentation system(Biotech-Fisher Perth Australia) Total RNA concentrationwas determined by spectrophotometric measurement in 5120583Lcapillary tubes using the Gene Quant II RNADNA Calcula-tor (Pharmacia Biotech Cambridge UK)

24 Quantitative Real-Time Polymerase Chain Reaction AssayDetection of the expression of IGF1 IGF1R IGFBP3 andglyceraldehyde-3-phosphate (GAPDH) mRNAs was carriedout using a QRT-PCR technique with SYBR Green chem-istry (SYBR Green Quantitect RT-PCR Kit Qiagen) andan Opticon DNA Engine Continuous Fluorescence detector(MJ Research) as described previously [23] All sampleswere tested in triplicate GAPDH for each sample was mea-sured to exclude possible RT-PCR inhibitorsOligonucleotideprimers specific for IGF1 IGF1R and IGFBP3 were designedon the basis of reference sequences (GenBank accession noNM 0006183 NM 0008753 andNM 0005984 resp) usingPrimer ExpressTM Version 20 software (PE Applied Biosys-tems Foster City CA USA) Detection of GAPDH mRNAswas performed as described previously [23] (Table 2)












3 Results













Median Min-max

IGF1 IGF1R

0

200

400

600

800

1000

1200

1400

1600

PDRiERM

mRN

A co

pies

120583g

of to

tal R

NA

lowast

lowast

25ndash75


4 Discussion


IGF1 IGF1R IGFBP3

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

mRN

A co

pies

120583g

of to

tal R

NA

Median Min-max

PDRiERM

25ndash75











References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results













Median Min-max

IGF1 IGF1R

0

200

400

600

800

1000

1200

1400

1600

PDRiERM

mRN

A co

pies

120583g

of to

tal R

NA

lowast

lowast

25ndash75


4 Discussion


IGF1 IGF1R IGFBP3

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

mRN

A co

pies

120583g

of to

tal R

NA

Median Min-max

PDRiERM

25ndash75











References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Median Min-max

IGF1 IGF1R

0

200

400

600

800

1000

1200

1400

1600

PDRiERM

mRN

A co

pies

120583g

of to

tal R

NA

lowast

lowast

25ndash75


4 Discussion


IGF1 IGF1R IGFBP3

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

mRN

A co

pies

120583g

of to

tal R

NA

Median Min-max

PDRiERM

25ndash75











References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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