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BJO Online First, published on April 15, 2015 as 10.1136/bjophthalmol-2014-306341 Clinical science

Six-month changes in cytokine levels after intravitreal bevacizumab injection for diabetic macular oedema and macular oedema due to central retinal vein occlusion Jing Wen,1,2,3 Yanrong Jiang,1,2,3 Xiaoxue Zheng,1,2,3 Ying Zhou1,2,3 1

Department of Ophthalmology, Peking University People’s Hospital, Beijing, China 2 Key Laboratory of Vision Loss and Restoration (Peking University), Ministry of Education, Beijing, China 3 Beijing Key Laboratory of Diagnosis and Therapy of Retinal and Choroid Diseases, Beijing, China Correspondence to Dr Yanrong Jiang, Department of Ophthalmology, Peking University People’s Hospital, 11 Xizhimen South Street, Xicheng District, Beijing 100044, China; [email protected] Received 4 November 2014 Revised 1 March 2015 Accepted 12 March 2015

ABSTRACT Background/aims This study evaluated the impact of intravitreal injection of bevacizumab (IVB) on the microenvironment of the eyes of diabetic macular oedema (DMO) and macular oedema due to central retinal vein occlusion (CRVO-MO) patients. Methods This study comprised 136 patients, including 51 patients in the DMO group, 70 in the CRVO-MO group and 15 in the control group, who were followed for 6 months after IVB. Angiogenic cytokines, inflammatory cytokines and growth factors concentrations in the aqueous humour were measured before and after IVB using suspension array technology. We compared the levels of cytokines among DMO patients, CRVO-MO patients and control patients. We compared the levels of cytokines among groups according to the interval between the first and second injections of bevacizumab and according to the number of injections received during the 6-month follow-up period. Results Significantly higher concentrations of vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), interleukin 6 (IL-6), serum amyloid A (SAA) and monocyte chemoattractant protein-1 (MCP-1) were found in the aqueous humour of DMO and CRVO-MO patients compared with cataract patients. One month after IVB, the intraocular concentrations of VEGF were significantly decreased in the eyes of DMO ( p=0.045) and CRVO-MO ( p=0.002) patients compared with baseline. No other cytokine was significantly altered by bevacizumab therapy. Conclusions Angiogenic, inflammatory and growth factors are involved in the development of DMO and CRVO-MO. In addition to VEGF, IVB did not cause significant differences in other inflammatory cytokines and growth factors in DMO and CRVO-MO patients.

INTRODUCTION

To cite: Wen J, Jiang Y, Zheng X, et al. Br J Ophthalmol Published Online First: [ please include Day Month Year] doi:10.1136/bjophthalmol2014-306341

Macular oedema (MO) is a retinal complication of diabetic retinopathy (DR) and central retinal vein occlusion (CRVO) that is the predominant contributor to vision loss.1 The breakdown of the blood–retina barrier and the consequent vascular leakage and thickening of the retina are the primary events involved in disease pathogenesis. VEGF is a potent angiogenic factor associated with retinal neovascularisation and the increased vascular permeability leading to MO. Numerous other chemical mediators and cytokines have been identified in the ocular fluid of patients with DR and CRVO, such as transforming

growth factor β (TGF-β),2 hepatocyte growth factor (HGF),3 4 basic fibroblast growth factor (bFGF),5 monocyte chemoattractant protein-1 (MCP-1),6 serum amyloid A (SAA)7 and interleukin 6 (IL-6),6 enhancing our understanding of the ocular biochemical milieu of this condition. Furthermore, SAA and IL-6 have been shown to associate with central macular thickness (CMT) in CRVO patients.5 Several studies have demonstrated the utility of the intravitreal injection of anti-VEGF agents such as bevacizumab for the management of diabetic macular oedema (DMO) and CRVO-MO. However, the effects of this treatment differ for each disease, and the results have varied between studies.8–11 In addition, several studies have shown that the angio-fibrotic switch has been observed in diabetic fibrovascular proliferative membranes after bevacizumab treatment12 and that intravitreal injection of bevacizumab (IVB) increases the intraocular levels of inflammatory cytokines in patients with proliferative diabetic retinopathy (PDR).2 13 The aetiology of these changes after IVB remains unclear. Therefore, in this study, we measured and compared the concentrations of angiogenic factors, inflammatory cytokines and growth factors in the aqueous humour from the eyes of DMO and CRVO-MO patients before and after IVB. We investigated how anti-VEGF treatment affected intraocular cytokine levels in DR and CRVO patients based on a 6-month observation.

MATERIALS AND METHODS The samples were collected after receiving approval from the Institutional Review Board of the People’s Hospital affiliated with Peking University and in accordance with the Declaration of Helsinki. Written informed consent for all examinations and procedures was obtained from each participant.

Study subjects We included patients who were treated with IVB from May 2012 to August 2013. The inclusion criteria for MO secondary to DR or CRVO were as follows: (1) a decrease in visual acuity, (2) diffuse MO based on fundus fluorescein angiography (FFA), (3) a CMT greater than 250 μm based on optical coherence tomography (OCT) and (4) a follow-up visit at least 6 months after IVB. The exclusion criteria were as follows: (1) vitreous haemorrhaging, tractional retinal detachment (TRD) or glaucoma before IVB; (2) previous ocular surgery;

Wen J, et al. Br J Ophthalmol 2015;0:1–7. doi:10.1136/bjophthalmol-2014-306341

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Clinical science Table 1 Demographic characteristics of the patients in the DMO, CRVO-MO and control groups DMO (n=51) Age, years Gender (female), % With hypertension, % Duration of symptoms, months

CRVO-MO (n=70)

57.9±11.76 57.91±14.8 45.1 48.6 43.1 55.4 12.0 (0.75–24) 9.0 (0.5–48)

Control (n=15)

p Value

58.9±4.66 66.7 40.0 NA

0.962* 0.348† 0.297† 0.074‡

The data are presented as the mean±SD, number (%) or median (range). *ANOVA. †Fisher’s exact test. ‡Mann–Whitney U test. CRVO-MO, macular oedema due to central retinal vein occlusion; DMO, diabetic macular oedema; NA, not applicable.

(3) intravitreal injection of corticosteroids or bevacizumab or pan-retinal photocoagulation within 6 months prior to the study and (4) MO caused by any retinal condition other than DR or CRVO. All patients received an intravitreal injection of 1.25 mg of bevacizumab per 0.05 mL of saline solution (Avastin; Genentech, San Francisco, California, USA). Reference samples were obtained from patients undergoing cataract surgery. Ophthalmic examinations were performed before and after IVB, including a best corrected visual acuity (BCVA) test using manifest refraction and the logarithm of the minimum angle of resolution (logMAR) visual acuity chart, non-contact tonometry, slit-lamp examination, fundus examination and FFA, which was performed using a fundus camera (TRC-50EX; Tokyo Optical, Tokyo, Japan). CMT was defined as the value of a 1 mm central area based on OCT (Zeiss-Humphrey, Dublin, California, USA). The patients were categorised into four groups according to the interval between the first and second injections: the baseline group, patients from whom the aqueous humour was collected before the first injection; the Month 1 group, patients who received a second treatment at 1 month; the Month 3 group, patients who received a second treatment at 3 months and the Month 6 group, patients who received a second treatment at 6 months. For the last three groups, we collected aqueous humour samples just before the second IVB.

The patients were also separated into three groups according to the number of injections received during the 6-month follow-up period. In the 1-injection group, patients received one injection during the study period. The sample was taken before the second injection, which was 6 months after the first injection. In the 2-injections group, patients received two injections during the study period. The sample was taken before the third injection, which was at least 1 month after the second injection. In the 3-injections group, patients received three injections during the study period. The sample was taken before the fourth injection, which was at least 1 month after the third injection.

Sample collection All injections and cataract surgeries were performed by the same surgeon (YJ) at the People’s Hospital affiliated with Peking University. Undiluted aqueous humour samples were collected during intravitreal injection or cataract surgery. Approximately 200 μl of aqueous humour samples were obtained via anterior chamber paracentesis. These samples were stored in a sterilised plastic tube (2 mL; Corning, Troy, Michigan, USA) and were immediately frozen at −80°C until analysis. The samples were assayed within 6 months of collection.

Measurement of cytokines Procarta Cytokine Assay Kits (Panomics, Fremont, California, USA) were used to measure the concentrations of TGF-β, HGF, bFGF, MCP-1, SAA, IL-6 and VEGF in the aqueous humour samples. These assays employed suspension array technology (xMAP; Luminex, Austin, Texas, USA) using multi-analyte profiling beads to detect and quantify multiple protein targets simultaneously, the detailed process of which was reported in a similar study.14

Statistical analysis All statistical analyses were performed using SPSS V.21.0 for Windows (SPSS, Chicago, Illinois, USA). The levels of TGF-β, HGF, bFGF, MCP-1, SAA, IL-6 and VEGF in the aqueous humour were expressed as the means±SD. A one-sample Kolmogorov–Smirnov test was performed to examine whether the samples were normally distributed. Differences in the clinical characteristics between the DMO and CRVO-MO groups at baseline and after IVB were estimated using the Mann–Whitney U test. When appropriate, the Mann–Whitney U test and the t test were used to compare the levels of cytokines between the

Table 2 Clinical characteristics of the patients in the DMO and CRVO-MO groups

BCVA (logMAR) IOP (mm Hg) Outer CMT (μm) Complete CMT (μm) Vitreoretinal condition, no. Vitreous haemorrhage Rubeosis iridis Fibroneovascular membranes TRD

DMO before IVB (n=11)

DMO after IVB (n=40)

p Value

CRVO-MO before IVB (n=20)

CRVO-MO after IVB (n=50)

p Value

0.79±0.32 16.45±2.73 435.09±149.83 551.64±173.81

0.67±0.45 14.91±4.07 387.23±181.41 505.50±170.37

0.415 0.147 0.252 0.372

1.26±0.47 15.93±3.94 534.44±248.46 732.84±364.51

0.97±0.53 15.18±3.45 470.86±248.34 607.39±251.58

0.027* 0.495 0.251 0.193

0 0 0 0

0 0 0 0

– – – –

0 0 0 0

0 0 0 0

– – – –

The data are presented as the mean±SD or the numbers. Mann–Whitney U test. *Indicates p

Six-month changes in cytokine levels after intravitreal bevacizumab injection for diabetic macular oedema and macular oedema due to central retinal vein occlusion.

This study evaluated the impact of intravitreal injection of bevacizumab (IVB) on the microenvironment of the eyes of diabetic macular oedema (DMO) an...
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