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Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic retinopathy
Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic retinopathy Shivani Sinha a, Sandeep Saxena a,⁎, Senthamizh Prasad b, Abbas Ali Mahdi c, Shashi Kumar Bhasker a, Siddharth Das d, Vladimir Krasnik e, Martin Caprnda f, Radka Opatrilova g, Peter Kruzliak g,⁎⁎ a
Retina service, Department of Ophthalmology, King George's Medical University, Lucknow, India Department of Preventive and Social Medicine, King George's Medical University, Lucknow, India c Department of Biochemistry, King George's Medical University, Lucknow, India d Department of Rheumatology, King George's Medical University, Lucknow, India e Department of Ophthalmology, Faculty of Medicine, Comenius University and University Hospital, Bratislava, Slovakia f 2nd Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia g Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic b
a r t i c l e
i n f o
Article history: Received 5 September 2016 Received in revised form 1 November 2016 Accepted 16 February 2017 Available online xxxx Keywords: Proliferative diabetic retinopathy (PDR) Non-proliferative diabetic retinopathy (NPDR) Anti-myeloperoxidase (anti-MPO) antibody Retinal photoreceptor ellipsoid zone disruption Decreased visual acuity
a b s t r a c t Aim: To study the association of serum levels of anti-myeloperoxidase (MPO) antibody with retinal photoreceptor ellipsoid zone (EZ) disruption in diabetic retinopathy. Methods: Consecutive patients with type 2 DM [diabetes mellitus with no retinopathy (NODR; n = 20); non-proliferative diabetic retinopathy (NPDR; n = 18); proliferative diabetic retinopathy (PDR; n = 16)] and healthy controls (n = 20) between the ages of 40 and 65 years were included. Disruption of EZ was graded by spectral domain optical coherence tomography as no disruption of EZ and disrupted EZ. The serum levels of anti-MPO antibody was analyzed using standard protocol. Association between the variables was evaluated using multiple regression analysis. Results: A significant difference was found between the serum levels of anti-MPO antibody in various study groups (p b 0.001). A positive association was found between EZ disruption and levels of anti-MPO antibody [adjusted odd's ratio (AOR) = 1.079, CI 1.010–1.124, p = 0.04]. A significant positive correlation was found between logMAR visual acuity and grade of disruption (AOR = 1.008, CI 1.006–5.688, p = 0.04). Conclusions: An increased serum anti-MPO antibody levels is associated with retinal photoreceptor EZ disruption and decreased visual acuity in diabetic retinopathy. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is the leading cause of blindness among adults in 20 to 74 year age group. 9 Several risk factors have been implicated in the pathogenesis of DR that include poor glycemic control, hypertension, increasing age, dyslipidemia, increased serum urea, creatinine and duration of diabetes mellitus (DM). 2,8,13,17,20 Inflammation represents the inciting and final common pathway in DR. 2 In DR the inflammation is chronic in nature and the vascular injury is mediated by leukocytes. 17 Cytokine primed neutrophils have upregulation of proteinase 3 and myeloperoxidase (MPO) on their surface. This enables interaction between antineutrophilic Conflict of interest: Authors declare no conflict of interest. ⁎ Correspondence to: S. Saxena, Department of Ophthalmology, King George's Medical University, Lucknow, India. 226003. Tel.: +91 9415160528. ⁎⁎ Correspondence to: P. Kruzliak, Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho tr. 1946/1, 612 42 Brno, Czech Republic. E-mail addresses: [email protected] (S. Saxena), [email protected] (P. Kruzliak).
cytoplasmic antibody (ANCA) IgGs and their targets. 7,19 This results in robust respiratory burst with production of intracellular and extracellular reactive oxygen species (ROS), degranulation of lytic granule contents and increased adhesion of neutrophils to endothelial cells causing endothelial cell injury. 10,12,14,18,23,24 Ellipsoid zone (EZ) is the second hyperreflective region seen on OCT imaging corresponding to the ellipsoid component of photoreceptor. 25,26,29 The EZ integrity is an indicator of photoreceptor layer health and correlates well with visual acuity. 16 Our recent study highlighted the association of increased serum levels of anti-MPO antibody with increased severity of DR. 28 This study was undertaken to evaluate the association of anti-MPO antibody with retinal photoreceptor EZ disruption. 2. Material and methods The study was conducted according to the tenets of the Declaration of Helsinki after university institutional review board approval. An informed voluntary consent of the study subjects was obtained. This was a tertiary care centre based cross sectional study. Study subjects
http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011 1056-8727/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011
2
S. Sinha et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
included fifty four consecutive cases of type 2 DM and twenty healthy controls, between age group 40–60 years. On the basis of fundus photography and fluorescein angiography, cases were divided according to the ETDRS classification into three groups: patients of diabetes without retinopathy (NODR) (n = 20), non-proliferative diabetic retinopathy (NPDR) (n = 18) and proliferative diabetic retinopathy (PDR) (n = 16). Cases with ocular or systemic diseases affecting the retinal vascular pathology, cases with history of any previous ophthalmic surgical or laser interventions were excluded. Patients having systemic illness such as Alzheimer disease, connective tissue disorder, inflammatory bowel disease, end stage renal disease and with a history of myocardial infarction were also excluded. Patients on antiplatelet, anti-inflammatory and immunomodulator medications were excluded. Best corrected visual acuity (BCVA) was documented on logMAR scale. All the study subjects underwent detailed fundus evaluation using stereoscopic slit lamp biomicroscopy and indirect ophthalmoscopy. Digital fundus photography and fluorescein angiography were performed. EZ integrity was evaluated using three-dimensional spectral domain optical coherence tomography (SD-OCT) (Cirrus High Definition OCT from Carl Zeiss Meditec Inc., CA, USA) with scans passing through the fovea. Every patient underwent macular thickness analysis using the macular cube 512 × 128 feature. Two experienced observers masked to the status of DR and graded the disruption of EZ as no disruption and EZ disrupted (Fig. 1). Blood samples were collected by aseptic venepuncture from all study subjects. Hematological examination for the estimation of hemoglobin, blood sugar level, glycated hemoglobin (HbA1C), serum urea and creatinine was measured following the standard protocol on an autoanalyzer. Assay of anti-MPO antibody was carried out by using the antimyeloperoxidase ELISA (IgG) kit acquired from Euroimmun, Medizinische LaboriagnostikaA, Lubeck, Germany. Coefficient of variation (CV) for intra assay variation for a mean value of 28.4 RU/ml was 6.4% (n = 20). The CV for inter assay variation for a
mean value of 37.3 RU/ml was 13.5%. All reagents were brought to room temperature (+ 18 °C to + 25 °C) 30 min before use. Patient sample was diluted 1:101 in sample buffer. Calibrators, controls and enzyme conjugate were mixed thoroughly before use. 100 μl of the calibrators, positive and negative controls or diluted patient samples were transferred according to the pipetting protocol into the individual microplate wells. The sample was incubated at room temperature for 30 min. The wells were emptied and subsequently washed 3 times using 300 μl of working strength for each wash buffer. 100 μl of enzyme conjugate was pipetted into each of the microplate wells and incubated for 30 min at room temperature. The wells were washed. 100 μl of chromogen/substrate were pipetted into each of the microplate wells and incubated for 15 min at room temperature. 100 μl of stop solution was pipetted in the same order into each of the microplate wells and at the same speed as the chromogen/substrate solution was introduced. Photometric measurement of the color intensity was made within 30 min of adding the stop solution at a wavelength of 450 nm and a reference wavelength between 620 nm and 650 nm. The test was quantitatively analyzed using a standard curve. The upper limit of the normal range was taken as 20 relative units (RU)/ml. K.S test was used to find the normality of the data. Continuous data were summarized as mean ± SD (standard deviation) while categorical data were presented as number and percentage (%). Non-parametric (skewed) data were summarized as median and interquartile range. Continuous groups were compared by one way analysis of variance (ANOVA) and the significance of mean difference between the groups was done by LSD post hoc test after ascertaining normality by Shapiro–Wilk (W) test and homogeneity of variance by Levene's and Wald's (regression) test. Non-parametric test (Kruskal–Wallis test) were used for skewed data. Categorical groups were compared by chi-square (χ2) test. Pearson correlation analysis and linear regression was used to find the predictors of outcome variables. Independent t test was used to find association between anti-MPO titres and EZ disruption in different groups. All analyses
Fig. 1. Spectral domain optical coherence tomography showing disrupted ellipsoid zone (white arrow) on macular cube.
Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011
S. Sinha et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Table 1 Table showing median and interquartile range of biochemical parameters.
Serum urea (mg/dl) Serum creatinine (mg/dl) Hemoglobin (gm/dl) Fasting blood sugar (mg/dl) Post prandial blood sugar (mg/dl) Anti-myeloperoxidase antibody (RU/ml)
Control (n = 20)
NODR (n = 20)
NPDR (n = 18)
PDR (n = 16)
p value
23.75 0.7 14.15 85.50 124.00 17.00
24.00 0.71 14.00 99.00 136.10 16.81
28.00 0.90 13.80 108.5 179.75 19.12
28.95 1.03 12.30 114.15 189.00 31.82
b0.001 b0.001 b0.008 b0.001 b0.001 b0.001
(22.12–25.15) (0.61–0.72) (13.37–14.87) (76.7–94.75) (112.02–138.50) (12.96–19.09)
(22.12–29.60) (0.61–0.90) (13.02–14.82) (87.2–113.00) (124.20–149.75) (13.71–21.38)
were performed using SPSS software (version 22.0). The level of significance was taken as p b 0.05. 3. Results Mean age (in years) of the four groups was 51.75 ± 2.61 in controls, 51.45 ± 3.47 in NODR, 52.81 ± 2.22 in NPDR and 53.93 ± 2.23 in PDR groups. No significant difference in the age was observed among the groups (F = 1.76, p = 0.16). The χ2 test revealed similar (p N 0.05) sex proportion among all the four groups (male/female: 8/12 vs. 6/14 vs. 5/13 vs. 5/11, χ2 = 4.29, p = 0.28). Mean duration of diabetes mellitus in years was 5.65 ± 1.49 in NODR, 7.94 ± 1.78 in NPDR and 11.87 ± 2.80 in PDR groups. Duration of disease was significantly associated with increase in severity of DR (F = 46.82, p b 0.001). Mean HbA1C (%) of the four groups was 5.91 ± 0.36 in controls, 6.80 ± 0.81 in NODR, 7.21 ± 0.98 in NPDR and 7.48 ± 1.44 in PDR groups. Significant difference was found between serum levels of HbA1C among the study groups on ANOVA (F = 12.64, p b 0.001). Table 1 shows median and interquartile ranges of the biochemical parameters. On applying Kruskal–Wallis test significant difference was found among the groups. Mean logMAR BCVA was 0.32 ± 0.21 in control, 0.50 ± 0.45 in NODR, 0.96 ± 0.51 in NPDR and 1.40 ± 0.33 in PDR groups. On ANOVA, significant difference in visual acuity was found among the groups (F = 30.746., p b 0.001). Decrease in visual acuity correlated significantly with increase in serum anti-MPO antibody (F = 48.45, p b 0.001). The EZ was intact in all 20 study subjects in control and NODR groups. Disruption of EZ was present in 5 out of 18 patients with NPDR and 11 out of 16 patients with PDR. On multivariate regression analysis with EZ disruption as dependent variable and adjusting for other factors like duration of diabetes, serum urea, serum creatinine,
(26.00–33.00) (0.70–1.09) (12.17–14.20) (94.25–129.20) (139.75–240.25) (18.17–22.58)
(28.00–57.85) (0.89–1.46) (9.95–13.60) (100.72–130.65) (167.70–213.50) (25.36–39.18)
fasting blood sugar, post prandial blood sugar and HbA1C, it was observed that increased EZ disruption is associated with increased anti-MPO antibody (AOR = 1.079, CI 1.010–1.124, p = 0.04) and logMAR BCVA (AOR = 1.008, CI 1.006–5.688, p = 0.04). The box plots show relation of anti-MPO antibodies with EZ disruption depicting raised anti-MPO antibody levels in cases having EZ disruption as compared to cases with no disruption (Fig. 2). Independent t test showed that increased anti-MPO was associated with EZ disruption in both NPDR (p = 0.002) and PDR (p = 0.001) groups. 4. Discussion The present study highlights the association of increased levels of serum anti-MPO antibody with disruption of EZ in DR. A likely explanation for damage to the retinal vessel wall in DR by anti-MPO antibody can be elucidated by drawing a parallelism with action of anti-MPO antibody on glomerulus. 15 In glomerulonephritis anti-MPO antibody leads to necrosis of vessel wall resulting in damage to the glomerulus. ANCA is specific for proteins in the primary granules of neutrophils and peroxidase positive lysosomes of monocytes. Numerous in vitro studies have demonstrated that neutrophils primed with tumor necrosis factor-α (TNF-α) when incubated with ANCA IgG are stimulated to release toxic ROS and lytic granule enzymes. 6,11 This results in acute inflammation and necrosis of vessel walls leading to vasculitis. Evaluation of renal biopsy specimens from patients with glomerulonephritis demonstrates upregulation of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1) supporting their role in leukocyte recruitment. 21 The myeloperoxidase catalyzes the production of hypochlorous acid, a major strong oxidant generated by neutrophils. 31 Advanced glycation end products and ROS attacks the endothelial cells of blood vessels leading to breakdown of blood retinal barrier. 22 MPO is also a
Fig. 2. Box plot showing association of serum anti-myeloperoxidase antibody level with ellipsoid zone disruption.
Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011
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S. Sinha et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Fig. 3. Role of oxidative stress and inflammation through the myeloperoxidase enzyme in pathogenesis of EZ disruption in DR. VEGF: vascular endothelial growth factor. ICAM 1: intercellular adhesion molecule.
macrophage modulator which stimulates release of pro-inflammatory cytokine TNF in addition to ROS generated by these cells inciting a vicious cycle. Polyunsaturated fatty acids are susceptible to free radical oxidation and participate in a chain reaction that increases damage due to oxidative stress. Since photoreceptors are rich in phospholipids hence are prone to lipid peroxidation. 5 This results in increased apoptosis of the photoreceptors. In previous studies lipid peroxides levels and MPO activity have been found to be significantly elevated in the vitreous of patients with fibrovascular vitreoretinal proliferations secondary to diabetes. 4,30 Oxidative stress leads to activation of nuclear factor KB and other transcription factors leading to induction of inducible nitric oxide synthase hence increased nitric oxide (NO) production. This cascade also leads to increased ICAM-1 and vascular endothelial growth factor (VEGF) production resulting in increased leucostasis and angiogenesis. Peroxynitrite is formed by a combination of NO and superoxide. Peroxynitrates leads to induction of DNA damage and apoptosis of photoreceptors. 32 Our previous study has associated in vivo structural alterations in retina with nitrosative and oxidative stress. 27 Glycolaldehyde generated by the myeloperoxidase-H2O2-chloride system leads to Nε-carboxymethyl lysine (Nε-CML) production and thereby plays an important role in tissue damage at sites of inflammation.3 The levels of soluble ICAM-1 were found to be higher in anti-MPO antibody positive than in anti-MPO antibody-negative patients. 1 In our previous studies, in vivo structural alterations in retina have been found to be associated with increased levels of VEGF, ICAM and Nε-CML in DR. 16,20 These alterations have also been correlated with decreased visual acuity.
The present study has highlighted the complex pathway operating in the pathogenesis of EZ disruption in diabetic retinopathy. This study elucidates the integrated and intertwined role of oxidative stress and inflammation through the myeloperoxidase enzyme in pathogenesis of EZ disruption in DR (Fig. 3). The limitation of the current study is the small sample size. Study with a larger sample size will provide further insight into the pathogenesis of EZ disruption. 5. Conclusion It can be concluded that increased serum anti-MPO antibody is associated with increased severity of disruption of retinal photoreceptor EZ. References 1. Accardo-Palumbo A, Triolo G, Giardina E, Carbone MC, Ferrante A. Detection of anti-myeloperoxidase antibodies in the serum of patients with type 1 diabetes mellitus. Acta Diabetol. 1996;33:103-7. 2. Adamis AP. Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol. 2002;86:363-5. 3. Anderson MM, Requena JR, Crowley JR, Thorpe SR, Heinecke JW. The myeloperoxidase system of human phagocytes generates Nε-(carboxymethyl) lysine on proteins: A mechanism for producing advanced glycation end products at sites of inflammation. J Clin Investig. 1999;104:103-13. 4. Augustin AJ, Breipohl W, Böker T, Lutz J, Spitznas M. Increased lipid peroxide levels and myeloperoxidase activity in the vitreous of patients suffering from proliferative diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol. 1993;231: 647-50. 5. Catalá A. Lipid peroxidation of membrane phospholipids generates hydroxyl alkenals and oxidized phospholipids active in physiological and/or pathological conditions. Chem Phys Lipids. 2009;157:1-11. 6. Charles LA, Caldas ML, Falk RJ, Terrell RS, Jennette JC. Antibodies against granule proteins activate neutrophils in vitro. J Leukoc Biol. 1991;50:539-46.
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S. Sinha et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx 7. Csernok E, Ernst M, Schmitt W, Bainton DF, Gross WL. Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo. Clin Exp Immunol. 1994;95:244-50. 8. el Haddad OA, Saad MK. Prevalence and risk factors for diabetic retinopathy among Omani diabetics. Br J Ophthalmol. 1998;82:901-6. 9. Engelgau MM, Geiss LS, Saaddine JB, Boyle JP, Benjamin SM, Gregg EW, et al. The evolving diabetes burden in the United States. Ann Intern Med. 2004;140:945-50. 10. Ewert BH, Becker ME, Jennette JC, Falk RJ. Antimyeloperoxidase antibodies induce neutrophil adherence to cultured human endothelial cells. Ren Fail. 1995;17: 125-33. 11. Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA. 1990;87:4115-9. 12. Franssen CF, Huitema MG, Muller Kobold AC, Oost-Kort WW, Limburg PC, Tiebosch A, et al. In vitro neutrophil activation by antibodies to proteinase 3 and myeloperoxidase from patients with crescentic glomerulonephritis. J Am Soc Nephrol. 1999;10:1506-15. 13. Graves DT, Liu R, Alikhani M, Al-Mashat H, Trackman PC. Diabetes enhanced inflammation and apoptosis–impact on periodontal pathology. J Dent Res. 2006;85:15-21. 14. Hattar K, Bickenbach A, Csernok E, Rosseau S, Grandel U, Seeger W, et al. Wegener's granulomatosis: Antiproteinase 3 antibodies induce monocyte cytokine and prostanoid release-role of autocrine cell activation. J Leukoc Biol. 2002;71:996-1004. 15. Homeister JW, Jennette CJ, Falk RJ. Immunologic mechanisms of vasculitis. In: Alpern RJ, Caplan MJ, Moe OW, eds. The kidney. 5th ed. Elsevier Inc.; 2013. p. 2817-46. 16. Jain A, Saxena S, Khanna VK, Shukla RK, Meyer CH. Status of serum VEGF and ICAM-1 and its association with external limiting membrane and inner segment-outer segment junction disruption in type 2 diabetes mellitus. Mol Vis. 2013;19:1760-8. 17. Joussen AM, Murata T, Tsujikawa A, Kirchhof B, Bursell SE, Adamis AP. Leukocyte mediated endothelial cell injury and death in the diabetic retina. Am J Pathol. 2001;158:147-52. 18. Kettritz R, Jennette JC, Falk RJ. Crosslinking of ANCA-antigens stimulates superoxide release by human neutrophils. J Am Soc Nephrol. 1997;8:386-94. 19. Kettritz R, Schreiber A, Luft FC, Haller H. Role of mitogen-activated protein kinases in activation of human neutrophils by antineutrophil cytoplasmic antibodies. J Am Soc Nephrol. 2001;12:37-46.
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20. Mishra N, Saxena S, Shukla RK, Singh V, Meyer CH, Kruzliak P, et al. Association of serum N ε-Carboxy methyl lysine with severity of diabetic retinopathy. J Diabetes Complications. 2016;30:511-7. 21. Moon KC, Park SY, Kim HW, Hong HK, Lee HS. Expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in human crescentic glomerulonephritis. Histopathology. 2002;41:158-65. 22. Park SH, Park JW, Park SJ, Kim KY, Chung JW, Chun MH, et al. Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina. Diabetologia. 2003;46:1260-8. 23. Porges AJ, Redecha PB, Kimberly WT, Csernok E, Gross WL, Kimberly RP. Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via fc gamma RIIa. J Immunol. 1994;153:1271-80. 24. Savage CO, Pottinger BE, Gaskin G, Pusey CD, Pearson JD. Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial cells. Am J Pathol. 1992;141: 335-42. 25. Saxena S, Srivastav K, Cheung CM, Ng JY, Lai TY. Photoreceptor inner segment ellipsoid band integrity on spectral domain optical coherence tomography. Clin Ophthalmol. 2014;8:2507-22. 26. Saxena S, Srivastav K, Chod R, Akduman L. Photoreceptor inner segment ellipsoid band in diabetic retinopathy. J Ocul Biol. 2015;S:5. 27. Sharma S, Saxena S, Srivastav K, Shukla RK, Mishra N, Meyer CH, et al. Nitrosative stress is associated with severity of diabetic retinopathy and retinal structural alterations. Clin Exp Ophthalmol. 2015;43:429-36. 28. Sinha S, Saxena S, Das S, Prasad S, Bhasker SK, Mahdi AA, et al. Anti myeloperoxidase antibody is a biomarker for progression of diabetic retinopathy. J Diabetes Complications. 2016;30:700-4. 29. Staurenghi G, Sadda S, Chakravarthy U, Spaide RF, International Nomenclature for Optical Coherence Tomography (IN•OCT) Panel. Proposed lexicon for anatomic landmarks in normal posterior segment spectral-domain optical coherence tomography: the IN•OCT consensus. Ophthalmology. 2014;121:1572-8. 30. Verdejo C, Marco P, Renau-Piqueras J, Pinazo-Duran MD. Lipid peroxidation in proliferative vitreoretinopathies. Eye. 1999;13:183-8. 31. Winterbourn CC, Kettle AJ. Biomarkers of myeloperoxidase-derived hypochlorous acid. Free Radic Biol Med. 2000;29:403-9. 32. Zheng L, Kern TS. Role of nitric oxide, superoxide, peroxynitrite and PARP in diabetic retinopathy. Front Biosci. 2009;14:3974-87.
Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic retinopathy Shivani Sinha a, Sandeep Saxena a,⁎, Senthamizh Prasad b, Abbas Ali Mahdi c, Shashi Kumar Bhasker a, Siddharth Das d, Vladimir Krasnik e, Martin Caprnda f, Radka Opatrilova g, Peter Kruzliak g,⁎⁎ a
Retina service, Department of Ophthalmology, King George's Medical University, Lucknow, India Department of Preventive and Social Medicine, King George's Medical University, Lucknow, India c Department of Biochemistry, King George's Medical University, Lucknow, India d Department of Rheumatology, King George's Medical University, Lucknow, India e Department of Ophthalmology, Faculty of Medicine, Comenius University and University Hospital, Bratislava, Slovakia f 2nd Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia g Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic b
a r t i c l e
i n f o
Article history: Received 5 September 2016 Received in revised form 1 November 2016 Accepted 16 February 2017 Available online xxxx Keywords: Proliferative diabetic retinopathy (PDR) Non-proliferative diabetic retinopathy (NPDR) Anti-myeloperoxidase (anti-MPO) antibody Retinal photoreceptor ellipsoid zone disruption Decreased visual acuity
a b s t r a c t Aim: To study the association of serum levels of anti-myeloperoxidase (MPO) antibody with retinal photoreceptor ellipsoid zone (EZ) disruption in diabetic retinopathy. Methods: Consecutive patients with type 2 DM [diabetes mellitus with no retinopathy (NODR; n = 20); non-proliferative diabetic retinopathy (NPDR; n = 18); proliferative diabetic retinopathy (PDR; n = 16)] and healthy controls (n = 20) between the ages of 40 and 65 years were included. Disruption of EZ was graded by spectral domain optical coherence tomography as no disruption of EZ and disrupted EZ. The serum levels of anti-MPO antibody was analyzed using standard protocol. Association between the variables was evaluated using multiple regression analysis. Results: A significant difference was found between the serum levels of anti-MPO antibody in various study groups (p b 0.001). A positive association was found between EZ disruption and levels of anti-MPO antibody [adjusted odd's ratio (AOR) = 1.079, CI 1.010–1.124, p = 0.04]. A significant positive correlation was found between logMAR visual acuity and grade of disruption (AOR = 1.008, CI 1.006–5.688, p = 0.04). Conclusions: An increased serum anti-MPO antibody levels is associated with retinal photoreceptor EZ disruption and decreased visual acuity in diabetic retinopathy. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is the leading cause of blindness among adults in 20 to 74 year age group. 9 Several risk factors have been implicated in the pathogenesis of DR that include poor glycemic control, hypertension, increasing age, dyslipidemia, increased serum urea, creatinine and duration of diabetes mellitus (DM). 2,8,13,17,20 Inflammation represents the inciting and final common pathway in DR. 2 In DR the inflammation is chronic in nature and the vascular injury is mediated by leukocytes. 17 Cytokine primed neutrophils have upregulation of proteinase 3 and myeloperoxidase (MPO) on their surface. This enables interaction between antineutrophilic Conflict of interest: Authors declare no conflict of interest. ⁎ Correspondence to: S. Saxena, Department of Ophthalmology, King George's Medical University, Lucknow, India. 226003. Tel.: +91 9415160528. ⁎⁎ Correspondence to: P. Kruzliak, Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho tr. 1946/1, 612 42 Brno, Czech Republic. E-mail addresses: [email protected] (S. Saxena), [email protected] (P. Kruzliak).
cytoplasmic antibody (ANCA) IgGs and their targets. 7,19 This results in robust respiratory burst with production of intracellular and extracellular reactive oxygen species (ROS), degranulation of lytic granule contents and increased adhesion of neutrophils to endothelial cells causing endothelial cell injury. 10,12,14,18,23,24 Ellipsoid zone (EZ) is the second hyperreflective region seen on OCT imaging corresponding to the ellipsoid component of photoreceptor. 25,26,29 The EZ integrity is an indicator of photoreceptor layer health and correlates well with visual acuity. 16 Our recent study highlighted the association of increased serum levels of anti-MPO antibody with increased severity of DR. 28 This study was undertaken to evaluate the association of anti-MPO antibody with retinal photoreceptor EZ disruption. 2. Material and methods The study was conducted according to the tenets of the Declaration of Helsinki after university institutional review board approval. An informed voluntary consent of the study subjects was obtained. This was a tertiary care centre based cross sectional study. Study subjects
http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011 1056-8727/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011
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S. Sinha et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
included fifty four consecutive cases of type 2 DM and twenty healthy controls, between age group 40–60 years. On the basis of fundus photography and fluorescein angiography, cases were divided according to the ETDRS classification into three groups: patients of diabetes without retinopathy (NODR) (n = 20), non-proliferative diabetic retinopathy (NPDR) (n = 18) and proliferative diabetic retinopathy (PDR) (n = 16). Cases with ocular or systemic diseases affecting the retinal vascular pathology, cases with history of any previous ophthalmic surgical or laser interventions were excluded. Patients having systemic illness such as Alzheimer disease, connective tissue disorder, inflammatory bowel disease, end stage renal disease and with a history of myocardial infarction were also excluded. Patients on antiplatelet, anti-inflammatory and immunomodulator medications were excluded. Best corrected visual acuity (BCVA) was documented on logMAR scale. All the study subjects underwent detailed fundus evaluation using stereoscopic slit lamp biomicroscopy and indirect ophthalmoscopy. Digital fundus photography and fluorescein angiography were performed. EZ integrity was evaluated using three-dimensional spectral domain optical coherence tomography (SD-OCT) (Cirrus High Definition OCT from Carl Zeiss Meditec Inc., CA, USA) with scans passing through the fovea. Every patient underwent macular thickness analysis using the macular cube 512 × 128 feature. Two experienced observers masked to the status of DR and graded the disruption of EZ as no disruption and EZ disrupted (Fig. 1). Blood samples were collected by aseptic venepuncture from all study subjects. Hematological examination for the estimation of hemoglobin, blood sugar level, glycated hemoglobin (HbA1C), serum urea and creatinine was measured following the standard protocol on an autoanalyzer. Assay of anti-MPO antibody was carried out by using the antimyeloperoxidase ELISA (IgG) kit acquired from Euroimmun, Medizinische LaboriagnostikaA, Lubeck, Germany. Coefficient of variation (CV) for intra assay variation for a mean value of 28.4 RU/ml was 6.4% (n = 20). The CV for inter assay variation for a
mean value of 37.3 RU/ml was 13.5%. All reagents were brought to room temperature (+ 18 °C to + 25 °C) 30 min before use. Patient sample was diluted 1:101 in sample buffer. Calibrators, controls and enzyme conjugate were mixed thoroughly before use. 100 μl of the calibrators, positive and negative controls or diluted patient samples were transferred according to the pipetting protocol into the individual microplate wells. The sample was incubated at room temperature for 30 min. The wells were emptied and subsequently washed 3 times using 300 μl of working strength for each wash buffer. 100 μl of enzyme conjugate was pipetted into each of the microplate wells and incubated for 30 min at room temperature. The wells were washed. 100 μl of chromogen/substrate were pipetted into each of the microplate wells and incubated for 15 min at room temperature. 100 μl of stop solution was pipetted in the same order into each of the microplate wells and at the same speed as the chromogen/substrate solution was introduced. Photometric measurement of the color intensity was made within 30 min of adding the stop solution at a wavelength of 450 nm and a reference wavelength between 620 nm and 650 nm. The test was quantitatively analyzed using a standard curve. The upper limit of the normal range was taken as 20 relative units (RU)/ml. K.S test was used to find the normality of the data. Continuous data were summarized as mean ± SD (standard deviation) while categorical data were presented as number and percentage (%). Non-parametric (skewed) data were summarized as median and interquartile range. Continuous groups were compared by one way analysis of variance (ANOVA) and the significance of mean difference between the groups was done by LSD post hoc test after ascertaining normality by Shapiro–Wilk (W) test and homogeneity of variance by Levene's and Wald's (regression) test. Non-parametric test (Kruskal–Wallis test) were used for skewed data. Categorical groups were compared by chi-square (χ2) test. Pearson correlation analysis and linear regression was used to find the predictors of outcome variables. Independent t test was used to find association between anti-MPO titres and EZ disruption in different groups. All analyses
Fig. 1. Spectral domain optical coherence tomography showing disrupted ellipsoid zone (white arrow) on macular cube.
Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011
S. Sinha et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Table 1 Table showing median and interquartile range of biochemical parameters.
Serum urea (mg/dl) Serum creatinine (mg/dl) Hemoglobin (gm/dl) Fasting blood sugar (mg/dl) Post prandial blood sugar (mg/dl) Anti-myeloperoxidase antibody (RU/ml)
Control (n = 20)
NODR (n = 20)
NPDR (n = 18)
PDR (n = 16)
p value
23.75 0.7 14.15 85.50 124.00 17.00
24.00 0.71 14.00 99.00 136.10 16.81
28.00 0.90 13.80 108.5 179.75 19.12
28.95 1.03 12.30 114.15 189.00 31.82
b0.001 b0.001 b0.008 b0.001 b0.001 b0.001
(22.12–25.15) (0.61–0.72) (13.37–14.87) (76.7–94.75) (112.02–138.50) (12.96–19.09)
(22.12–29.60) (0.61–0.90) (13.02–14.82) (87.2–113.00) (124.20–149.75) (13.71–21.38)
were performed using SPSS software (version 22.0). The level of significance was taken as p b 0.05. 3. Results Mean age (in years) of the four groups was 51.75 ± 2.61 in controls, 51.45 ± 3.47 in NODR, 52.81 ± 2.22 in NPDR and 53.93 ± 2.23 in PDR groups. No significant difference in the age was observed among the groups (F = 1.76, p = 0.16). The χ2 test revealed similar (p N 0.05) sex proportion among all the four groups (male/female: 8/12 vs. 6/14 vs. 5/13 vs. 5/11, χ2 = 4.29, p = 0.28). Mean duration of diabetes mellitus in years was 5.65 ± 1.49 in NODR, 7.94 ± 1.78 in NPDR and 11.87 ± 2.80 in PDR groups. Duration of disease was significantly associated with increase in severity of DR (F = 46.82, p b 0.001). Mean HbA1C (%) of the four groups was 5.91 ± 0.36 in controls, 6.80 ± 0.81 in NODR, 7.21 ± 0.98 in NPDR and 7.48 ± 1.44 in PDR groups. Significant difference was found between serum levels of HbA1C among the study groups on ANOVA (F = 12.64, p b 0.001). Table 1 shows median and interquartile ranges of the biochemical parameters. On applying Kruskal–Wallis test significant difference was found among the groups. Mean logMAR BCVA was 0.32 ± 0.21 in control, 0.50 ± 0.45 in NODR, 0.96 ± 0.51 in NPDR and 1.40 ± 0.33 in PDR groups. On ANOVA, significant difference in visual acuity was found among the groups (F = 30.746., p b 0.001). Decrease in visual acuity correlated significantly with increase in serum anti-MPO antibody (F = 48.45, p b 0.001). The EZ was intact in all 20 study subjects in control and NODR groups. Disruption of EZ was present in 5 out of 18 patients with NPDR and 11 out of 16 patients with PDR. On multivariate regression analysis with EZ disruption as dependent variable and adjusting for other factors like duration of diabetes, serum urea, serum creatinine,
(26.00–33.00) (0.70–1.09) (12.17–14.20) (94.25–129.20) (139.75–240.25) (18.17–22.58)
(28.00–57.85) (0.89–1.46) (9.95–13.60) (100.72–130.65) (167.70–213.50) (25.36–39.18)
fasting blood sugar, post prandial blood sugar and HbA1C, it was observed that increased EZ disruption is associated with increased anti-MPO antibody (AOR = 1.079, CI 1.010–1.124, p = 0.04) and logMAR BCVA (AOR = 1.008, CI 1.006–5.688, p = 0.04). The box plots show relation of anti-MPO antibodies with EZ disruption depicting raised anti-MPO antibody levels in cases having EZ disruption as compared to cases with no disruption (Fig. 2). Independent t test showed that increased anti-MPO was associated with EZ disruption in both NPDR (p = 0.002) and PDR (p = 0.001) groups. 4. Discussion The present study highlights the association of increased levels of serum anti-MPO antibody with disruption of EZ in DR. A likely explanation for damage to the retinal vessel wall in DR by anti-MPO antibody can be elucidated by drawing a parallelism with action of anti-MPO antibody on glomerulus. 15 In glomerulonephritis anti-MPO antibody leads to necrosis of vessel wall resulting in damage to the glomerulus. ANCA is specific for proteins in the primary granules of neutrophils and peroxidase positive lysosomes of monocytes. Numerous in vitro studies have demonstrated that neutrophils primed with tumor necrosis factor-α (TNF-α) when incubated with ANCA IgG are stimulated to release toxic ROS and lytic granule enzymes. 6,11 This results in acute inflammation and necrosis of vessel walls leading to vasculitis. Evaluation of renal biopsy specimens from patients with glomerulonephritis demonstrates upregulation of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1) supporting their role in leukocyte recruitment. 21 The myeloperoxidase catalyzes the production of hypochlorous acid, a major strong oxidant generated by neutrophils. 31 Advanced glycation end products and ROS attacks the endothelial cells of blood vessels leading to breakdown of blood retinal barrier. 22 MPO is also a
Fig. 2. Box plot showing association of serum anti-myeloperoxidase antibody level with ellipsoid zone disruption.
Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011
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S. Sinha et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Fig. 3. Role of oxidative stress and inflammation through the myeloperoxidase enzyme in pathogenesis of EZ disruption in DR. VEGF: vascular endothelial growth factor. ICAM 1: intercellular adhesion molecule.
macrophage modulator which stimulates release of pro-inflammatory cytokine TNF in addition to ROS generated by these cells inciting a vicious cycle. Polyunsaturated fatty acids are susceptible to free radical oxidation and participate in a chain reaction that increases damage due to oxidative stress. Since photoreceptors are rich in phospholipids hence are prone to lipid peroxidation. 5 This results in increased apoptosis of the photoreceptors. In previous studies lipid peroxides levels and MPO activity have been found to be significantly elevated in the vitreous of patients with fibrovascular vitreoretinal proliferations secondary to diabetes. 4,30 Oxidative stress leads to activation of nuclear factor KB and other transcription factors leading to induction of inducible nitric oxide synthase hence increased nitric oxide (NO) production. This cascade also leads to increased ICAM-1 and vascular endothelial growth factor (VEGF) production resulting in increased leucostasis and angiogenesis. Peroxynitrite is formed by a combination of NO and superoxide. Peroxynitrates leads to induction of DNA damage and apoptosis of photoreceptors. 32 Our previous study has associated in vivo structural alterations in retina with nitrosative and oxidative stress. 27 Glycolaldehyde generated by the myeloperoxidase-H2O2-chloride system leads to Nε-carboxymethyl lysine (Nε-CML) production and thereby plays an important role in tissue damage at sites of inflammation.3 The levels of soluble ICAM-1 were found to be higher in anti-MPO antibody positive than in anti-MPO antibody-negative patients. 1 In our previous studies, in vivo structural alterations in retina have been found to be associated with increased levels of VEGF, ICAM and Nε-CML in DR. 16,20 These alterations have also been correlated with decreased visual acuity.
The present study has highlighted the complex pathway operating in the pathogenesis of EZ disruption in diabetic retinopathy. This study elucidates the integrated and intertwined role of oxidative stress and inflammation through the myeloperoxidase enzyme in pathogenesis of EZ disruption in DR (Fig. 3). The limitation of the current study is the small sample size. Study with a larger sample size will provide further insight into the pathogenesis of EZ disruption. 5. Conclusion It can be concluded that increased serum anti-MPO antibody is associated with increased severity of disruption of retinal photoreceptor EZ. References 1. Accardo-Palumbo A, Triolo G, Giardina E, Carbone MC, Ferrante A. Detection of anti-myeloperoxidase antibodies in the serum of patients with type 1 diabetes mellitus. Acta Diabetol. 1996;33:103-7. 2. Adamis AP. Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol. 2002;86:363-5. 3. Anderson MM, Requena JR, Crowley JR, Thorpe SR, Heinecke JW. The myeloperoxidase system of human phagocytes generates Nε-(carboxymethyl) lysine on proteins: A mechanism for producing advanced glycation end products at sites of inflammation. J Clin Investig. 1999;104:103-13. 4. Augustin AJ, Breipohl W, Böker T, Lutz J, Spitznas M. Increased lipid peroxide levels and myeloperoxidase activity in the vitreous of patients suffering from proliferative diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol. 1993;231: 647-50. 5. Catalá A. Lipid peroxidation of membrane phospholipids generates hydroxyl alkenals and oxidized phospholipids active in physiological and/or pathological conditions. Chem Phys Lipids. 2009;157:1-11. 6. Charles LA, Caldas ML, Falk RJ, Terrell RS, Jennette JC. Antibodies against granule proteins activate neutrophils in vitro. J Leukoc Biol. 1991;50:539-46.
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Please cite this article as: Sinha S, et al. Association of serum levels of anti-myeloperoxidase antibody with retinal photoreceptor ellipsoid zone disruption in diabetic reti.... Journal of Diabetes and Its Complications (2017), http://dx.doi.org/10.1016/j.jdiacomp.2017.02.011