Journal of Diabetes and Its Complications 29 (2015) 245–249
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
Neutrophil–Lymphocyte ratio is associated with arterial stiffness in diabetic retinopathy in type 2 diabetes Rui-tao Wang a, Ji-rong Zhang a, Ying Li a, b, Tiemin Liu c, Kai-jiang Yu d,⁎ a
Department of Geriatrics, the Second Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China International Physical Examination and Healthy Center, the Second Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China Division of Hypothalamic Research, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, 75390, USA d Department of Intensive Care Unit, the Third Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China b c
a r t i c l e
i n f o
Article history: Received 17 August 2014 received in revised form 3 November 2014 accepted 12 November 2014 Available online 20 November 2014 Keywords: Type 2 diabetes mellitus Diabetic retinopathy Neutrophil–Lymphocyte ratio Arterial stiffness Brachial-ankle pulse wave velocity
a b s t r a c t Aims: Diabetic retinopathy (DR) is the most common complication of type 2 diabetes mellitus (T2DM). Inflammation plays a considerable role in the pathogenesis of T2DM and DR. Emerging evidence revealed that the neutrophil–lymphocyte ratio (NLR) may be a useful marker of cardiovascular disease. The brachial-ankle pulse wave velocity (baPWV) is an indicator for early atherosclerotic changes. Therefore, this study aimed to investigate the association of NLR with baPWV in patients with DR. Methods: In this cross-sectional study, we investigated the relationship between NLR and baPWV in 402 participants. Participants were divided into the following three groups: 133 control subjects without T2DM; 138 diabetic subjects without DR; and 131 patients with DR. Results: NLR and baPWV were elevated both in T2DM and in DR. Moreover, compared to T2DM, NLR and baPWV were higher in DR. There was a positive correlation between NLR and baPWV in patients with T2DM and DR after adjusting confounding factors. Multiple linear regression analysis further revealed that NLR was an independent and significant determinant for elevated baPWV (for T2DM, β = 0.170; p = 0.041; for DR, β = 0.188; p = 0.022, respectively). Conclusions: The findings showed that NLR and baPWV are elevated both in T2DM and in DR. In addition, NLR is independently associated with baPWV. Early detection of abnormal NLR levels may be helpful for the search of subclinical atherosclerosis in patients with T2DM and DR. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and is the major cause of acquired blindness in working-age adults. Currently, there is increasing evidence that inflammatory processes play a considerable role in the pathogenesis of DR (Zhou, Wang, and Xia, 2012). Adhesion of leukocytes to the retinal vasculature is a contributing factor in the vascular pathology of DR. The neutrophil–lymphocyte ratio (NLR), an inexpensive inflammatory marker, has emerged as a useful index to predict cardiovascular risk. Moreover, NLR predicts presence, severity and extent of coronary artery disease, myocardial infarction in high risk patients for coronary artery disease, and peripheral arterial disease prognostication (Arbel et al., 2012; Bhat et al., 2013; Duffy et al., 2006; Horne et al., 2005; Nunez et al.,
Conflict of interest: The authors have not declared any conflicts of interest. ⁎ Corresponding author at: Department of Intensive Care Unit, the Third Affiliated Hospital, Harbin Medical University, NO.150 Haping ST, Nangang District, Harbin, 150081, China. Tel./fax: +86 451 86298036. E-mail address: [email protected] (K. Yu). http://dx.doi.org/10.1016/j.jdiacomp.2014.11.006 1056-8727/© 2015 Elsevier Inc. All rights reserved.
2008). More recently, some reports revealed that NLR is associated with glycated hemoglobin, different grades of glucose intolerance and insulin resistance, and the severity of DR (Sefil et al., 2014; Shiny et al., 2014; Woo, Ahn, Ahn, Park, and Lee, 2011). Elevated arterial stiffness, an indicator of subclinical atherosclerosis, is an independent predictor of cardiovascular morbidity and mortality in type 2 diabetes mellitus (T2DM) (Laurent et al., 2006). Pulse wave velocity (PWV) is a simple, noninvasive, and highly reproducible measure of central arterial stiffness and is widely used as an index of arterial stiffness. Brachial-ankle PWV (baPWV) measurement is closely correlated with aortic PWV (Yamashina et al., 2002). Previous reports revealed that increased baPWV is associated with microalbuminuria, diabetic neuropathy, and DR (Ogawa et al., 2008; Yokoyama, Aoki, Imahori, and Kuramitsu, 2004, Yokoyama, Yokota, Tada, and Kanno, 2007). Therefore, baPWV is a useful marker for evaluation of complications in patients with T2DM (Aso et al., 2003). Chronic hyperglycemia causes the progression of arterial stiffness and DR (Tanaka et al., 2013). Furthermore, increased NLR is associated with elevated hemoglobin A1c (HbA1c) levels in T2DM (Sefil et al., 2014). However, little research has been conducted to investigate the
246
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249
relationship between NLR and baPWV in DR. Therefore, the aim of the study was to evaluate the changes of NLR and baPWV in patients with T2DM and DR. 2. Patients and methods 2.1. Study population We studied 402 subjects who visited the International Physical Examination and Healthy Center, Harbin, China, from January 2011 through December 2012. Of the 402 participants enrolled, 179 (44.5%) were male, and 223 (55.5%) were female. The mean ages were 61.4 ± 6.9 and 62.2 ± 6.7 years old, respectively. Participants were divided into the following three groups: control subjects without T2DM (control group); diabetic subjects without DR (T2DM group); and DR group. We obtained informed consent from all subjects. The study protocol was approved by the Ethics Committee of the Second Hospital of Harbin Medical University, China. 2.2. Clinical examination All participants underwent a standardized clinical examination and interview using a detailed questionnaire to obtain information, including medical history, current smoking status and medication use. Anthropometric measurements were taken according to standardized procedures. Blood pressure was measured in millimeters of mercury (mmHg) using an aneuroid sphygmomanometer and was taken as the mean of 2 consecutive measurements after at least 5 minutes of sitting. Body mass index (BMI) was calculated as weight divided by height squared (kg/m 2). 2.3. Biochemical analyses After a 10 hour of fasting, a venous blood sample was obtained from each subject. Laboratory tests were evaluated, which consisted of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), and fasting plasma glucose (FPG). The assays were conducted at the Laboratory of Analytical Biochemistry at the Second Hospital of Harbin Medical University, Harbin, using a biochemical analyzer (Modular Analytics, Roche, Mannheim, Germany). The HbA1c level was measured using high-performance liquid chromatography by an HbA1c analyzer (Variant™ II; Bio-Rad, Hercules, CA, USA). Complete blood count was performed in an automated hematology analyzer (Sysmex XE-2100, Kobe, Japan), including a white blood cell (WBC) differential. The NLR was calculated from the differential count by dividing the absolute neutrophil count by the absolute lymphocyte count. All samples were processed within 1 hour after blood collection. 2.4. Measurement of baPWV All subjects fasted overnight and were asked to refrain from caffeine intake and smoking within 4 hours prior to study visit. BaPWV was measured using an automatic device (model MB3000, M&B Electronic Instruments, Beijing, China). The baPWV was automatically calculated from the distance between two arterial recording sites divided by the transit time according to the formula (L/PTT). L was the difference between the length from the heart to ankle and the length from the heart to brachium. PTT was the pulse transit time between the brachial and tibial arterial waveforms. The mean value of right and left baPWV was obtained for analysis. All measurements were conducted by a single examiner who was blinded to the clinical data. The reproducibility and validity of the baPWV measurement using this method has been previously demonstrated (Li, Cao, Zhang, Li, and Wang, 2014, Li, Meng, Meng, Yu, and Wang, 2011).
2.5. Diagnosis and exclusion criteria T2DM was defined as fasting serum glucose was ≥7.0 mmol/L or nonfasting serum glucose was ≥11.1 mmol/L or as taking prescription medications. For the controls or the patients with impaired fasting glucose, T2DM was diagnosed if the 2-hr post-glucose level after a 75-g oral glucose tolerance test ≥11.1 mmol/L. For the patients without symptomatic hyperglycemia, if a single laboratory test result is in the diabetes range, a repeat confirmatory test was performed on another day. The duration of diabetes was the period between the age at diagnosis and the age at the baseline examination. Hypertension was diagnosed if systolic blood pressure ≥ 140 mmHg and/or diastolic pressure ≥ 90 mmHg, or as antihypertensive treatment. The modification of diet in renal disease (MDRD) equation was used to estimate glomerular filtration rate (eGFR). MDRD equation was: eGFR = 186.3 × (SCr)−1.154 × (age)−0.203(×0.742 if female). Coronary artery disease was defined as the occurrence of a nonfatal myocardial infarction, a percutaneous coronary angioplasty, or other forms of acute or chronic ischemic heart disease. Retinopathy was examined by fundoscopy according to the Airlie House classification (1991). Exclusion criteria for this study included tumor, hematological disorders, active inflammation, chronic inflammatory diseases, chronic liver and kidney diseases, type 1 diabetes, coronary artery disease, stroke, and ankle brachial index less than 0.9 to rule out peripheral arterial occlusion. 2.6. Statistical analyses All data were expressed as means ± SD or median (IQR) or percentage. The chi-square statistical test was utilized for dichotomous variables, while one-way ANOVA test or Kruskal–Wallis H test was used for continuous variables. Post hoc analyses using two-tailed Tukey's HSD were conducted to compare the differences for normally distributed data between the groups. Mann–Whitney U test was used to compare the differences for non-parametric data between the groups. Correlations between NLR and baPWV were tested by partial correlation. Multiple linear regression analysis was used to identify significant determinants of baPWV. Skewed variables such as TG, HDL, and FPG had a remarkably good fit to normal distribution after log transformation, and they were expressed as median values (25–75th percentile). Exact P values b 0.05, were considered statistically significant. Data analysis was performed with SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA). 3. Results The clinical and biochemical characteristics of the subjects are shown in Table 1. There was a significant difference in mean age, BMI, SBP, DBP, FPG, TC, TG, HDL, LDL, neutrophils, lymphocytes, eGFR, hemoglobin A1c, T2DM duration, and the percentage of hypertension and current use of angiotensin-converting enzyme inhibitor/angiotensin II receptor antagonist (ACEI/ARB), and calcium channel blocker (CCB) drugs in three groups. In addition, current use of insulin had a higher prevalence in DR group. However, WBC count and the percentage of sex, current smokers, drinking, and statin use showed no difference. The means of NLR in the three groups are shown in Fig. 1. NLR levels were elevated both in patients with T2DM and in patients with DR compared with control subjects (for T2DM vs. control, p b 0.001, post hoc Tukey test; for DR vs. control, p b 0.001, post hoc Tukey test). Moreover, NLR levels in patients with DR were higher compared to those in patients with T2DM (p b 0.001, post hoc Tukey test). NLR values in control, T2DM and DR group were 1.5 ± 0.7, 2.1 ± 1.3 and 3.7 ± 1.4, respectively (p b 0.001). The partial correlations between NLR and baPWV are summarized in Table 2. No statistically significant correlation was observed among control subjects. After adjustment for age, sex, BMI, drinking,
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249
247
Table 1 Clinical and biochemical characteristics of subjects. Variables
Control
T2DM
DR
p value
N Age (years) Male sex (%) BMI (kg/m2) Smoker (%) Drinking (%) SBP (mmHg) DBP (mmHg) FPG (mmol/L) TC (mmol/L) TG (mmol/L) HDL (mmol/L) LDL (mmol/L) WBC (×109/L) Neutrophils (×109/L) Lymphocytes (×109/L) BaPWV (cm/s) eGFR (mL/min/1.73 m2) Hemoglobin A1c (%) T2DM duration (years) Hypertension (%) ACEIs/ARBs (%) CCBs (%) Statins (%) Insulin (%) Sulfonylureas (%) Metformin (%) Acarbose (%) Insulin secretagogues (%)
133 58.7 ± 5.9 45.9 24.5 ± 3.4 29.3 24.8 132.3 ± 13.6 79.1 ± 6.4 5.18 (4.81–5.48) 4.71 ± 0.93 2.31 (1.76–2.85) 1.53 (1.38–1.70) 2.42 ± 0.70 5.8 ± 1.8 5.11 ± 1.03 3.84 ± 0.83 1576.7 ± 114.4 76.1 ± 15.7 5.4 ± 0.7 – 18.8 11.3 12.8 22.6 – – – – –
138 60.3 ± 6.0 47.1 25.2 ± 2.9 23.9 23.9 136.7 ± 11.9 80.8 ± 5.3 6.47 (6.03–6.91) 5.25 ± 0.90 3.12 (2.57–4.00) 1.27 (1.10–1.43) 3.03 ± 0.64 6.1 ± 1.6 5.71 ± 1.02 3.33 ± 0.98 1669.1 ± 115.6 73.3 ± 16.4 6.9 ± 1.1 2.7 ± 1.7 27.5 15.9 16.7 21.7 16.7 37.7 28.3 19.6 13.0
131 66.6 ± 5.8 40.5 25.8 ± 2.7 22.9 28.2 143.5 ± 11.4 84.2 ± 5.8 7.93 (7.63–8.41) 5.67 ± 0.77 3.29 (2.68–4.10) 1.15 (1.02–1.32) 4.22 ± 0.71 6.2 ± 1.5 6.85 ± 0.96 2.08 ± 0.76 1838.2 ± 129.7 71.1 ± 15.8 7.7 ± 0.7 8.6 ± 1.7 57.3 31.3 34.4 27.5 41.2 16.0 19.1 35.1 22.1
b0.001 0.511 0.002 0.433 0.278 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.108 b0.001 b0.001 b0.001 0.038 b0.001 b0.001 b0.001 b0.001 b0.001 0.494 b0.001⁎ b0.001⁎ 0.077⁎ 0.004⁎ 0.050⁎
Data are expressed as means (SD) or median (inter-quartile range) or percentage. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; WBC, white blood cell; Neutrophils, neutrophils absolute count; TC, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; FPG, fasting plasma glucose; eGFR, estimated glomerular filtration rate; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor antagonist; CCB, calcium channel blocker. p value was calculated by one-way ANOVA test or Kruskal–Wallis H or chi-square test. ⁎ p value was obtained by comparison of diabetic subjects and the subjects with DR using independent samples t test.
smoking status, SBP, DBP, FPG, TC, TG, HDL, LDL, eGFR, hemoglobin A1c, statin, CCB, ACEIs/ARBs, T2DM duration, and hypertension, NLR statistically correlated with baPWV both in patients with T2DM and in patients with DR (for T2DM, r = 0.222, p = 0.015; for DR, r = 0.233, p = 0.013; respectively). To determine independent predictors of baPWV, multiple regression analysis is performed using the stepwise procedure (Table 3). Factors showing a value p b 0.10 in univariate analysis were selected to enter in the model. The analysis showed that for control subjects, age, DBP, and TC were significant determinants for increased baPWV. However, for patients with T2DM, the independent predictors of baPWV were age, BMI, and NLR (for NLR, β = 0.170; p = 0.041). For patients with DR, the independent predictors of baPWV were SBP, NLR and T2DM duration (for NLR, β = 0.188; p = 0.022).
4. Discussion In this study, we found that NLR and baPWV elevated both in T2DM and in DR. Moreover, there was a positive correlation between NLR and baPWV in diabetic patients after adjusting confounding factors. Multiple regression analysis further revealed that NLR was an independent and significant determinant for increased baPWV both in T2DM and in DR. Chronic inflammation may constitute the pathophysiological basis for our present findings. Inflammatory response likely contributes to T2DM occurrence by causing insulin resistance, and is in turn intensified in the presence of hyperglycemia to promote long-term complications of diabetes (Lontchi-Yimagou, Sobngwi, Matsha, and Kengne, 2013). Insulin resistance has been attributed to adipose tissue activation associated with an increased release of inflammatory cytokines such as TNF-a, IL-6, C-reactive protein (CRP) and decreasing
Table 2 Partial correlation coefficient (r) for NLR in relation to baPWV levels. BaPWV (cm/s)
For control subjects NLR For patients with T2DM NLR For patients with DR NLR Fig. 1. Boxplot showing the NLR levels in control, T2DM, and DR groups. The top, bottom, and middle lines in each box correspond to the 75th (top quartile), 25th (bottom quartile), and 50th percentiles (median), respectively.
r
p value
0.077
0.407
0.222
0.015
0.233
0.013
Adjusted for age, sex, BMI, drinking, smoking status, SBP, DBP, FPG, TC, TG, HDL, LDL, eGFR, hemoglobin A1c, statin, CCB, ACEIs/ARBs, T2DM duration, and hypertension. Variables such as TG, HDL, and FPG were logarithmically transformed before statistical analysis. Abbreviations: see to Table 1.
248
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249
Table 3 Stepwise multivariate linear regression analysis with baPWV (cm/s) as the dependent variable. Variables For control subjects Age (years) TC (mmol/L) DBP (mmHg) For patients with T2DM Age (years) BMI (kg/m2) NLR For patients with DR SBP (mmHg) T2DM duration (years) NLR
β
SE
Partial correlation
p value
0.221 0.175 0.167
1.618 10.230 1.477
0.227 0.181 0.176
0.009 0.038 0.045
0.218 0.177 0.170
1.563 3.209 7.255
0.226 0.183 0.175
0.008 0.033 0.041
0.284 0.216 0.188
0.929 6.309 7.533
0.296 0.228 0.201
0.001 0.009 0.022
β, standardized regression coefficients. SE, standard error. NLR, neutrophil/lymphocyte ratio. The p value for entry was set at 0.05, and the p value for removal was set at 0.10. For control subjects, adjusted R2 = 0.080, p = 0.030. For patients with T2DM, adjusted R2 = 0.051, p = 0.019. For patients with DR, adjusted R2 = 0.158, p = 0.022. TG, HDL, and FPG were log-transformed before statistical analysis. Abbreviations: see to Table 1.
and arterial stiffness may exert beneficial effects on the prognosis of diabetic patients. There are several potential limitations of our study. Firstly, because of the cross-sectional design of our study, we had no direct evidence for a cause–effect relationship. The association between NLR and baPWV in T2DM requires further investigation by the prospective studies. Secondly, although we took into account most of the known risk factors of atherosclerosis progression in association with NLR and baPWV in T2DM, we cannot definitely exclude potential confounding factors that may possibly affect our models. Thirdly, blood pressure was measured only on one occasion for arterial hypertension diagnosis. In conclusion, our study showed that NLR and arterial stiffening are elevated both in NDR and in DR compared with those in control subjects. Moreover, NLR is independently associated with baPWV even after adjusting other cardiovascular risk factors. Early detection of abnormal NLR may be helpful for the search of undetected subclinical atherosclerosis in patients with DR.
Acknowledgments production of IL-10, an anti-inflammatory cytokine (Cruz et al., 2013). Moreover, there is increasing evidence that inflammatory process has a considerable role in the pathogenesis of DR with multiple studies showing an association of various systemic as well as local (vitreous and aqueous fluid) inflammatory factors and the progression of DR (Kastelan, Tomic, Gverovic, Salopek, and Ljubic, 2013). Increased retinal neutrophil count damages retinal vascular endothelial cells and blood-retinal barrier through a Fas-Fas ligand-dependent pathway (Harris, Skalak, and Hatchell, 1994; Joussen et al., 2003; McLeod, Lefer, Merges, and Lutty, 1995). Recent reports confirmed that NLR is increased in T2DM and DR (Sefil et al., 2014; Shiny et al., 2014; Woo et al., 2011). In addition, elevated NLR was independently related with the severity of DR (Woo et al., 2011). The processes of development and progression of DR and diabetesaccelerated atherosclerosis are tightly linked (Ellis et al., 2013). The accumulation of advanced glycation end products due to chronic glycemic exposure might be the common mechanism of arterial stiffness and microangiopathy in T2DM (Airaksinen et al., 1993; Singh, Barden, Mori, and Beilin, 2001). Recent studies reported that increased baPWV is associated with microalbuminuria, urinary albumin excretion, neuropathy, and retinopathy in T2DM (Ogawa et al., 2008; Shin et al., 2013; Sjoblom, Nystrom, Lanne, Engvall, and Ostgren, 2014; Yokoyama et al., 2004, 2007). In addition, PWV was positively correlated with the stage of DR (Ogawa et al., 2008; Tanaka et al., 2013). Therefore, it is considered that baPWV is a useful indicator for evaluation of complications in type 2 diabetic patients (Aso et al., 2003). In this study, we observed an association between NLR and baPWV in diabetic patients. Consistent with the result, some reports have documented the association between arterial stiffening and inflammatory markers, such as IL-6 and CRP (Nakhai-Pour, Grobbee, Bots, Muller, and van der Schouw, 2007; Nishida et al., 2007; Oh et al., 2012; Wang et al., 2013). Furthermore, it has been proposed that neutrophils may be one of the key factors contributing to microvasculature changes and inflammation when neutrophils adhere to the endothelial cell wall (Sala and Folco, 2001). Endothelial dysfunction and increased expression of proinflammatory cytokines could increase vascular inflammation, smooth muscle cell proliferation and subsequently elevated arterial stiffness (Pasceri, Willerson, and Yeh, 2000). Our findings are of great practical implications because, contrary to other inflammatory markers, NLR is inexpensive, easy to assess, and is part of the routine laboratory assessment. Early detection of NLR may be helpful for the search of undetected subclinical atherosclerosis in patients with T2DM and DR. Moreover, early evaluation of baPWV may be necessary for monitoring of disease progression in T2DM. Identification of a treatment that could improve both inflammation
This study was supported by the Science Foundation of Health and Family Planning Commission of Heilongjiang Province (No.2007.295).
References Airaksinen, K. E., Salmela, P. I., Linnaluoto, M. K., Ikaheimo, M. J., Ahola, K., & Ryhanen, L. J. (1993). Diminished arterial elasticity in diabetes: Association with fluorescent advanced glycosylation end products in collagen. Cardiovascular Research, 27(6), 942–945. Grading diabetic retinopathy from stereoscopic color fundus photographs–an extension of the modified Airlie House classification. ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research Group. Ophthalmology, 98(5 Suppl). (1991)., 786–806. Arbel, Y., Finkelstein, A., Halkin, A., Birati, E. Y., Revivo, M., Zuzut, M., et al. (2012). Neutrophil/lymphocyte ratio is related to the severity of coronary artery disease and clinical outcome in patients undergoing angiography. Atherosclerosis, 225(2), 456–460. Aso, K., Miyata, M., Kubo, T., Hashiguchi, H., Fukudome, M., Fukushige, E., et al. (2003). Brachial-ankle pulse wave velocity is useful for evaluation of complications in type 2 diabetic patients. Hypertension research: official journal of the Japanese Society of Hypertension, 26(10), 807–813. Bhat, T., Teli, S., Rijal, J., Bhat, H., Raza, M., Khoueiry, G., et al. (2013). Neutrophil to lymphocyte ratio and cardiovascular diseases: A review. Expert Review of Cardiovascular Therapy, 11(1), 55–59. Cruz, N. G., Sousa, L. P., Sousa, M. O., Pietrani, N. T., Fernandes, A. P., & Gomes, K. B. (2013). The linkage between inflammation and Type 2 diabetes mellitus. Diabetes Research and Clinical Practice, 99(2), 85–92. Duffy, B. K., Gurm, H. S., Rajagopal, V., Gupta, R., Ellis, S. G., & Bhatt, D. L. (2006). Usefulness of an elevated neutrophil to lymphocyte ratio in predicting long-term mortality after percutaneous coronary intervention. The American Journal of Cardiology, 97(7), 993–996. Ellis, T. P., Choudhury, R. H., Kaul, K., Chopra, M., Kohner, E. M., Tarr, J. M., et al. (2013). Diabetic retinopathy and atherosclerosis: Is there a link. Current Diabetes Reviews, 9 (2), 146–160. Harris, A. G., Skalak, T. C., & Hatchell, D. L. (1994). Leukocyte-capillary plugging and network resistance are increased in skeletal muscle of rats with streptozotocininduced hyperglycemia. International journal of microcirculation, clinical and experimental/sponsored by the European Society for Microcirculation, 14(3), 159–166. Horne, B. D., Anderson, J. L., John, J. M., Weaver, A., Bair, T. L., Jensen, K. R., et al. (2005). Which white blood cell subtypes predict increased cardiovascular risk. Journal of the American College of Cardiology, 45(10), 1638–1643. Joussen, A. M., Poulaki, V., Mitsiades, N., Cai, W. Y., Suzuma, I., Pak, J., et al. (2003). Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 17(1), 76–78. Kastelan, S., Tomic, M., Gverovic, A. A., Salopek, R. J., & Ljubic, S. (2013). Inflammation and pharmacological treatment in diabetic retinopathy. Mediators of Inflammation, 2013, 213130. Laurent, S., Cockcroft, J., Van Bortel, L., Boutouyrie, P., Giannattasio, C., Hayoz, D., et al. (2006). Expert consensus document on arterial stiffness: Methodological issues and clinical applications. European Heart Journal, 27(21), 2588–2605. Li, R. Y., Cao, Z. G., Zhang, J. R., Li, Y., & Wang, R. T. (2014). Decreased serum bilirubin is associated with silent cerebral infarction. Arteriosclerosis, Thrombosis, and Vascular Biology, 34(4), 946–951. Li, Y., Meng, S. Y., Meng, C. C., Yu, W. G., & Wang, R. T. (2013). Decreased serum bilirubin is associated with arterial stiffness in men. Nutrition, metabolism, and cardiovascular diseases: NMCD, 23(4), 375–381.
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249 Lontchi-Yimagou, E., Sobngwi, E., Matsha, T. E., & Kengne, A. P. (2013). Diabetes mellitus and inflammation. Current Diabetes Reports, 13(3), 435–444. McLeod, D. S., Lefer, D. J., Merges, C., & Lutty, G. A. (1995). Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid. The American Journal of Pathology, 147(3), 642–653. Nakhai-Pour, H. R., Grobbee, D. E., Bots, M. L., Muller, M., & van der Schouw, Y. T. (2007). C-reactive protein and aortic stiffness and wave reflection in middle-aged and elderly men from the community. Journal of Human Hypertension, 21(12), 949–955. Nishida, M., Moriyama, T., Ishii, K., Takashima, S., Yoshizaki, K., Sugita, Y., et al. (2007). Effects of IL-6, adiponectin, CRP and metabolic syndrome on subclinical atherosclerosis. Clinica chimica acta; international journal of clinical chemistry, 384 (1–2), 99–104. Nunez, J., Nunez, E., Bodi, V., Sanchis, J., Minana, G., Mainar, L., et al. (2008). Usefulness of the neutrophil to lymphocyte ratio in predicting long-term mortality in ST segment elevation myocardial infarction. The American Journal of Cardiology, 101 (6), 747–752. Ogawa, O., Hiraoka, K., Watanabe, T., Kinoshita, J., Kawasumi, M., Yoshii, H., et al. (2008). Diabetic retinopathy is associated with pulse wave velocity, not with the augmentation index of pulse waveform. Cardiovascular Diabetology, 7, 11. Oh, E. G., Kim, S. H., Bang, S. Y., Hyun, S. S., Im, J. A., Lee, J. E., et al. (2012). Highsensitivity C-reactive protein is independently associated with arterial stiffness in women with metabolic syndrome. The Journal of Cardiovascular Nursing, 27(1), 61–67. Pasceri, V., Willerson, J. T., & Yeh, E. T. (2000). Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation, 102(18), 2165–2168. Sala, A., & Folco, G. (2001). Neutrophils, endothelial cells, and cysteinyl leukotrienes: A new approach to neutrophil-dependent inflammation. Biochemical and Biophysical Research Communications, 283(5), 1003–1006. Sefil, F., Ulutas, K. T., Dokuyucu, R., Sumbul, A. T., Yengil, E., Yagiz, A. E., et al. (2014). Investigation of neutrophil lymphocyte ratio and blood glucose regulation in patients with type 2 diabetes mellitus. The Journal of International Medical Research, 42(2), 581–588. Shin, D. I., Seung, K. B., Yoon, H. E., Hwang, B. H., Seo, S. M., Shin, S. J., et al. (2013). Microalbuminuria is independently associated with arterial stiffness and vascular
249
inflammation but not with carotid intima-media thickness in patients with newly diagnosed type 2 diabetes or essential hypertension. Journal of Korean Medical Science, 28(2), 252–260. Shiny, A., Bibin, Y. S., Shanthirani, C. S., Regin, B. S., Anjana, R. M., Balasubramanyam, M., et al. (2014). Association of neutrophil-lymphocyte ratio with glucose intolerance: An indicator of systemic inflammation in patients with type 2 diabetes. Diabetes Technology & Therapeutics, 16(8), 524–530. Singh, R., Barden, A., Mori, T., & Beilin, L. (2001). Advanced glycation end-products: A review. Diabetologia, 44(2), 129–146. Sjoblom, P., Nystrom, F. H., Lanne, T., Engvall, J., & Ostgren, C. J. (2014). Microalbuminuria, but not reduced eGFR, is associated with cardiovascular subclinical organ damage in type 2 diabetes. Diabetes & Metabolism, 40(1), 49–55. Tanaka, K., Kawai, T., Saisho, Y., Meguro, S., Harada, K., Satoh, Y., et al. (2013). Relationship between stage of diabetic retinopathy and pulse wave velocity in Japanese patients with type 2 diabetes. Journal of Diabetes Research, 2013, 193514. Wang, X., Du, Y., Fan, L., Ye, P., Yuan, Y., Lu, X., et al. (2013). Relationships between HDL-C, hs-CRP, with central arterial stiffness in apparently healthy people undergoing a general health examination. PLoS one, 8(12), e81778. Woo, S. J., Ahn, S. J., Ahn, J., Park, K. H., & Lee, K. (2011). Elevated systemic neutrophil count in diabetic retinopathy and diabetes: A hospital-based cross-sectional study of 30,793 Korean subjects. Investigative Ophthalmology & Visual Science, 52(10), 7697–7703. Yamashina, A., Tomiyama, H., Takeda, K., Tsuda, H., Arai, T., Hirose, K., et al. (2002). Validity, reproducibility, and clinical significance of noninvasive brachial-ankle pulse wave velocity measurement. Hypertension research: official journal of the Japanese Society of Hypertension, 25(3), 359–364. Yokoyama, H., Aoki, T., Imahori, M., & Kuramitsu, M. (2004). Subclinical atherosclerosis is increased in type 2 diabetic patients with microalbuminuria evaluated by intimamedia thickness and pulse wave velocity. Kidney International, 66(1), 448–454. Yokoyama, H., Yokota, Y., Tada, J., & Kanno, S. (2007). Diabetic neuropathy is closely associated with arterial stiffening and thickness in type 2 diabetes. Diabetic medicine: a journal of the British Diabetic Association, 24(12), 1329–1335. Zhou, J., Wang, S., & Xia, X. (2012). Role of intravitreal inflammatory cytokines and angiogenic factors in proliferative diabetic retinopathy. Current Eye Research, 37(5), 416–420.
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
Neutrophil–Lymphocyte ratio is associated with arterial stiffness in diabetic retinopathy in type 2 diabetes Rui-tao Wang a, Ji-rong Zhang a, Ying Li a, b, Tiemin Liu c, Kai-jiang Yu d,⁎ a
Department of Geriatrics, the Second Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China International Physical Examination and Healthy Center, the Second Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China Division of Hypothalamic Research, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, 75390, USA d Department of Intensive Care Unit, the Third Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang, China b c
a r t i c l e
i n f o
Article history: Received 17 August 2014 received in revised form 3 November 2014 accepted 12 November 2014 Available online 20 November 2014 Keywords: Type 2 diabetes mellitus Diabetic retinopathy Neutrophil–Lymphocyte ratio Arterial stiffness Brachial-ankle pulse wave velocity
a b s t r a c t Aims: Diabetic retinopathy (DR) is the most common complication of type 2 diabetes mellitus (T2DM). Inflammation plays a considerable role in the pathogenesis of T2DM and DR. Emerging evidence revealed that the neutrophil–lymphocyte ratio (NLR) may be a useful marker of cardiovascular disease. The brachial-ankle pulse wave velocity (baPWV) is an indicator for early atherosclerotic changes. Therefore, this study aimed to investigate the association of NLR with baPWV in patients with DR. Methods: In this cross-sectional study, we investigated the relationship between NLR and baPWV in 402 participants. Participants were divided into the following three groups: 133 control subjects without T2DM; 138 diabetic subjects without DR; and 131 patients with DR. Results: NLR and baPWV were elevated both in T2DM and in DR. Moreover, compared to T2DM, NLR and baPWV were higher in DR. There was a positive correlation between NLR and baPWV in patients with T2DM and DR after adjusting confounding factors. Multiple linear regression analysis further revealed that NLR was an independent and significant determinant for elevated baPWV (for T2DM, β = 0.170; p = 0.041; for DR, β = 0.188; p = 0.022, respectively). Conclusions: The findings showed that NLR and baPWV are elevated both in T2DM and in DR. In addition, NLR is independently associated with baPWV. Early detection of abnormal NLR levels may be helpful for the search of subclinical atherosclerosis in patients with T2DM and DR. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and is the major cause of acquired blindness in working-age adults. Currently, there is increasing evidence that inflammatory processes play a considerable role in the pathogenesis of DR (Zhou, Wang, and Xia, 2012). Adhesion of leukocytes to the retinal vasculature is a contributing factor in the vascular pathology of DR. The neutrophil–lymphocyte ratio (NLR), an inexpensive inflammatory marker, has emerged as a useful index to predict cardiovascular risk. Moreover, NLR predicts presence, severity and extent of coronary artery disease, myocardial infarction in high risk patients for coronary artery disease, and peripheral arterial disease prognostication (Arbel et al., 2012; Bhat et al., 2013; Duffy et al., 2006; Horne et al., 2005; Nunez et al.,
Conflict of interest: The authors have not declared any conflicts of interest. ⁎ Corresponding author at: Department of Intensive Care Unit, the Third Affiliated Hospital, Harbin Medical University, NO.150 Haping ST, Nangang District, Harbin, 150081, China. Tel./fax: +86 451 86298036. E-mail address: [email protected] (K. Yu). http://dx.doi.org/10.1016/j.jdiacomp.2014.11.006 1056-8727/© 2015 Elsevier Inc. All rights reserved.
2008). More recently, some reports revealed that NLR is associated with glycated hemoglobin, different grades of glucose intolerance and insulin resistance, and the severity of DR (Sefil et al., 2014; Shiny et al., 2014; Woo, Ahn, Ahn, Park, and Lee, 2011). Elevated arterial stiffness, an indicator of subclinical atherosclerosis, is an independent predictor of cardiovascular morbidity and mortality in type 2 diabetes mellitus (T2DM) (Laurent et al., 2006). Pulse wave velocity (PWV) is a simple, noninvasive, and highly reproducible measure of central arterial stiffness and is widely used as an index of arterial stiffness. Brachial-ankle PWV (baPWV) measurement is closely correlated with aortic PWV (Yamashina et al., 2002). Previous reports revealed that increased baPWV is associated with microalbuminuria, diabetic neuropathy, and DR (Ogawa et al., 2008; Yokoyama, Aoki, Imahori, and Kuramitsu, 2004, Yokoyama, Yokota, Tada, and Kanno, 2007). Therefore, baPWV is a useful marker for evaluation of complications in patients with T2DM (Aso et al., 2003). Chronic hyperglycemia causes the progression of arterial stiffness and DR (Tanaka et al., 2013). Furthermore, increased NLR is associated with elevated hemoglobin A1c (HbA1c) levels in T2DM (Sefil et al., 2014). However, little research has been conducted to investigate the
246
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249
relationship between NLR and baPWV in DR. Therefore, the aim of the study was to evaluate the changes of NLR and baPWV in patients with T2DM and DR. 2. Patients and methods 2.1. Study population We studied 402 subjects who visited the International Physical Examination and Healthy Center, Harbin, China, from January 2011 through December 2012. Of the 402 participants enrolled, 179 (44.5%) were male, and 223 (55.5%) were female. The mean ages were 61.4 ± 6.9 and 62.2 ± 6.7 years old, respectively. Participants were divided into the following three groups: control subjects without T2DM (control group); diabetic subjects without DR (T2DM group); and DR group. We obtained informed consent from all subjects. The study protocol was approved by the Ethics Committee of the Second Hospital of Harbin Medical University, China. 2.2. Clinical examination All participants underwent a standardized clinical examination and interview using a detailed questionnaire to obtain information, including medical history, current smoking status and medication use. Anthropometric measurements were taken according to standardized procedures. Blood pressure was measured in millimeters of mercury (mmHg) using an aneuroid sphygmomanometer and was taken as the mean of 2 consecutive measurements after at least 5 minutes of sitting. Body mass index (BMI) was calculated as weight divided by height squared (kg/m 2). 2.3. Biochemical analyses After a 10 hour of fasting, a venous blood sample was obtained from each subject. Laboratory tests were evaluated, which consisted of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL), and fasting plasma glucose (FPG). The assays were conducted at the Laboratory of Analytical Biochemistry at the Second Hospital of Harbin Medical University, Harbin, using a biochemical analyzer (Modular Analytics, Roche, Mannheim, Germany). The HbA1c level was measured using high-performance liquid chromatography by an HbA1c analyzer (Variant™ II; Bio-Rad, Hercules, CA, USA). Complete blood count was performed in an automated hematology analyzer (Sysmex XE-2100, Kobe, Japan), including a white blood cell (WBC) differential. The NLR was calculated from the differential count by dividing the absolute neutrophil count by the absolute lymphocyte count. All samples were processed within 1 hour after blood collection. 2.4. Measurement of baPWV All subjects fasted overnight and were asked to refrain from caffeine intake and smoking within 4 hours prior to study visit. BaPWV was measured using an automatic device (model MB3000, M&B Electronic Instruments, Beijing, China). The baPWV was automatically calculated from the distance between two arterial recording sites divided by the transit time according to the formula (L/PTT). L was the difference between the length from the heart to ankle and the length from the heart to brachium. PTT was the pulse transit time between the brachial and tibial arterial waveforms. The mean value of right and left baPWV was obtained for analysis. All measurements were conducted by a single examiner who was blinded to the clinical data. The reproducibility and validity of the baPWV measurement using this method has been previously demonstrated (Li, Cao, Zhang, Li, and Wang, 2014, Li, Meng, Meng, Yu, and Wang, 2011).
2.5. Diagnosis and exclusion criteria T2DM was defined as fasting serum glucose was ≥7.0 mmol/L or nonfasting serum glucose was ≥11.1 mmol/L or as taking prescription medications. For the controls or the patients with impaired fasting glucose, T2DM was diagnosed if the 2-hr post-glucose level after a 75-g oral glucose tolerance test ≥11.1 mmol/L. For the patients without symptomatic hyperglycemia, if a single laboratory test result is in the diabetes range, a repeat confirmatory test was performed on another day. The duration of diabetes was the period between the age at diagnosis and the age at the baseline examination. Hypertension was diagnosed if systolic blood pressure ≥ 140 mmHg and/or diastolic pressure ≥ 90 mmHg, or as antihypertensive treatment. The modification of diet in renal disease (MDRD) equation was used to estimate glomerular filtration rate (eGFR). MDRD equation was: eGFR = 186.3 × (SCr)−1.154 × (age)−0.203(×0.742 if female). Coronary artery disease was defined as the occurrence of a nonfatal myocardial infarction, a percutaneous coronary angioplasty, or other forms of acute or chronic ischemic heart disease. Retinopathy was examined by fundoscopy according to the Airlie House classification (1991). Exclusion criteria for this study included tumor, hematological disorders, active inflammation, chronic inflammatory diseases, chronic liver and kidney diseases, type 1 diabetes, coronary artery disease, stroke, and ankle brachial index less than 0.9 to rule out peripheral arterial occlusion. 2.6. Statistical analyses All data were expressed as means ± SD or median (IQR) or percentage. The chi-square statistical test was utilized for dichotomous variables, while one-way ANOVA test or Kruskal–Wallis H test was used for continuous variables. Post hoc analyses using two-tailed Tukey's HSD were conducted to compare the differences for normally distributed data between the groups. Mann–Whitney U test was used to compare the differences for non-parametric data between the groups. Correlations between NLR and baPWV were tested by partial correlation. Multiple linear regression analysis was used to identify significant determinants of baPWV. Skewed variables such as TG, HDL, and FPG had a remarkably good fit to normal distribution after log transformation, and they were expressed as median values (25–75th percentile). Exact P values b 0.05, were considered statistically significant. Data analysis was performed with SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA). 3. Results The clinical and biochemical characteristics of the subjects are shown in Table 1. There was a significant difference in mean age, BMI, SBP, DBP, FPG, TC, TG, HDL, LDL, neutrophils, lymphocytes, eGFR, hemoglobin A1c, T2DM duration, and the percentage of hypertension and current use of angiotensin-converting enzyme inhibitor/angiotensin II receptor antagonist (ACEI/ARB), and calcium channel blocker (CCB) drugs in three groups. In addition, current use of insulin had a higher prevalence in DR group. However, WBC count and the percentage of sex, current smokers, drinking, and statin use showed no difference. The means of NLR in the three groups are shown in Fig. 1. NLR levels were elevated both in patients with T2DM and in patients with DR compared with control subjects (for T2DM vs. control, p b 0.001, post hoc Tukey test; for DR vs. control, p b 0.001, post hoc Tukey test). Moreover, NLR levels in patients with DR were higher compared to those in patients with T2DM (p b 0.001, post hoc Tukey test). NLR values in control, T2DM and DR group were 1.5 ± 0.7, 2.1 ± 1.3 and 3.7 ± 1.4, respectively (p b 0.001). The partial correlations between NLR and baPWV are summarized in Table 2. No statistically significant correlation was observed among control subjects. After adjustment for age, sex, BMI, drinking,
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249
247
Table 1 Clinical and biochemical characteristics of subjects. Variables
Control
T2DM
DR
p value
N Age (years) Male sex (%) BMI (kg/m2) Smoker (%) Drinking (%) SBP (mmHg) DBP (mmHg) FPG (mmol/L) TC (mmol/L) TG (mmol/L) HDL (mmol/L) LDL (mmol/L) WBC (×109/L) Neutrophils (×109/L) Lymphocytes (×109/L) BaPWV (cm/s) eGFR (mL/min/1.73 m2) Hemoglobin A1c (%) T2DM duration (years) Hypertension (%) ACEIs/ARBs (%) CCBs (%) Statins (%) Insulin (%) Sulfonylureas (%) Metformin (%) Acarbose (%) Insulin secretagogues (%)
133 58.7 ± 5.9 45.9 24.5 ± 3.4 29.3 24.8 132.3 ± 13.6 79.1 ± 6.4 5.18 (4.81–5.48) 4.71 ± 0.93 2.31 (1.76–2.85) 1.53 (1.38–1.70) 2.42 ± 0.70 5.8 ± 1.8 5.11 ± 1.03 3.84 ± 0.83 1576.7 ± 114.4 76.1 ± 15.7 5.4 ± 0.7 – 18.8 11.3 12.8 22.6 – – – – –
138 60.3 ± 6.0 47.1 25.2 ± 2.9 23.9 23.9 136.7 ± 11.9 80.8 ± 5.3 6.47 (6.03–6.91) 5.25 ± 0.90 3.12 (2.57–4.00) 1.27 (1.10–1.43) 3.03 ± 0.64 6.1 ± 1.6 5.71 ± 1.02 3.33 ± 0.98 1669.1 ± 115.6 73.3 ± 16.4 6.9 ± 1.1 2.7 ± 1.7 27.5 15.9 16.7 21.7 16.7 37.7 28.3 19.6 13.0
131 66.6 ± 5.8 40.5 25.8 ± 2.7 22.9 28.2 143.5 ± 11.4 84.2 ± 5.8 7.93 (7.63–8.41) 5.67 ± 0.77 3.29 (2.68–4.10) 1.15 (1.02–1.32) 4.22 ± 0.71 6.2 ± 1.5 6.85 ± 0.96 2.08 ± 0.76 1838.2 ± 129.7 71.1 ± 15.8 7.7 ± 0.7 8.6 ± 1.7 57.3 31.3 34.4 27.5 41.2 16.0 19.1 35.1 22.1
b0.001 0.511 0.002 0.433 0.278 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.108 b0.001 b0.001 b0.001 0.038 b0.001 b0.001 b0.001 b0.001 b0.001 0.494 b0.001⁎ b0.001⁎ 0.077⁎ 0.004⁎ 0.050⁎
Data are expressed as means (SD) or median (inter-quartile range) or percentage. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; WBC, white blood cell; Neutrophils, neutrophils absolute count; TC, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; FPG, fasting plasma glucose; eGFR, estimated glomerular filtration rate; ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor antagonist; CCB, calcium channel blocker. p value was calculated by one-way ANOVA test or Kruskal–Wallis H or chi-square test. ⁎ p value was obtained by comparison of diabetic subjects and the subjects with DR using independent samples t test.
smoking status, SBP, DBP, FPG, TC, TG, HDL, LDL, eGFR, hemoglobin A1c, statin, CCB, ACEIs/ARBs, T2DM duration, and hypertension, NLR statistically correlated with baPWV both in patients with T2DM and in patients with DR (for T2DM, r = 0.222, p = 0.015; for DR, r = 0.233, p = 0.013; respectively). To determine independent predictors of baPWV, multiple regression analysis is performed using the stepwise procedure (Table 3). Factors showing a value p b 0.10 in univariate analysis were selected to enter in the model. The analysis showed that for control subjects, age, DBP, and TC were significant determinants for increased baPWV. However, for patients with T2DM, the independent predictors of baPWV were age, BMI, and NLR (for NLR, β = 0.170; p = 0.041). For patients with DR, the independent predictors of baPWV were SBP, NLR and T2DM duration (for NLR, β = 0.188; p = 0.022).
4. Discussion In this study, we found that NLR and baPWV elevated both in T2DM and in DR. Moreover, there was a positive correlation between NLR and baPWV in diabetic patients after adjusting confounding factors. Multiple regression analysis further revealed that NLR was an independent and significant determinant for increased baPWV both in T2DM and in DR. Chronic inflammation may constitute the pathophysiological basis for our present findings. Inflammatory response likely contributes to T2DM occurrence by causing insulin resistance, and is in turn intensified in the presence of hyperglycemia to promote long-term complications of diabetes (Lontchi-Yimagou, Sobngwi, Matsha, and Kengne, 2013). Insulin resistance has been attributed to adipose tissue activation associated with an increased release of inflammatory cytokines such as TNF-a, IL-6, C-reactive protein (CRP) and decreasing
Table 2 Partial correlation coefficient (r) for NLR in relation to baPWV levels. BaPWV (cm/s)
For control subjects NLR For patients with T2DM NLR For patients with DR NLR Fig. 1. Boxplot showing the NLR levels in control, T2DM, and DR groups. The top, bottom, and middle lines in each box correspond to the 75th (top quartile), 25th (bottom quartile), and 50th percentiles (median), respectively.
r
p value
0.077
0.407
0.222
0.015
0.233
0.013
Adjusted for age, sex, BMI, drinking, smoking status, SBP, DBP, FPG, TC, TG, HDL, LDL, eGFR, hemoglobin A1c, statin, CCB, ACEIs/ARBs, T2DM duration, and hypertension. Variables such as TG, HDL, and FPG were logarithmically transformed before statistical analysis. Abbreviations: see to Table 1.
248
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249
Table 3 Stepwise multivariate linear regression analysis with baPWV (cm/s) as the dependent variable. Variables For control subjects Age (years) TC (mmol/L) DBP (mmHg) For patients with T2DM Age (years) BMI (kg/m2) NLR For patients with DR SBP (mmHg) T2DM duration (years) NLR
β
SE
Partial correlation
p value
0.221 0.175 0.167
1.618 10.230 1.477
0.227 0.181 0.176
0.009 0.038 0.045
0.218 0.177 0.170
1.563 3.209 7.255
0.226 0.183 0.175
0.008 0.033 0.041
0.284 0.216 0.188
0.929 6.309 7.533
0.296 0.228 0.201
0.001 0.009 0.022
β, standardized regression coefficients. SE, standard error. NLR, neutrophil/lymphocyte ratio. The p value for entry was set at 0.05, and the p value for removal was set at 0.10. For control subjects, adjusted R2 = 0.080, p = 0.030. For patients with T2DM, adjusted R2 = 0.051, p = 0.019. For patients with DR, adjusted R2 = 0.158, p = 0.022. TG, HDL, and FPG were log-transformed before statistical analysis. Abbreviations: see to Table 1.
and arterial stiffness may exert beneficial effects on the prognosis of diabetic patients. There are several potential limitations of our study. Firstly, because of the cross-sectional design of our study, we had no direct evidence for a cause–effect relationship. The association between NLR and baPWV in T2DM requires further investigation by the prospective studies. Secondly, although we took into account most of the known risk factors of atherosclerosis progression in association with NLR and baPWV in T2DM, we cannot definitely exclude potential confounding factors that may possibly affect our models. Thirdly, blood pressure was measured only on one occasion for arterial hypertension diagnosis. In conclusion, our study showed that NLR and arterial stiffening are elevated both in NDR and in DR compared with those in control subjects. Moreover, NLR is independently associated with baPWV even after adjusting other cardiovascular risk factors. Early detection of abnormal NLR may be helpful for the search of undetected subclinical atherosclerosis in patients with DR.
Acknowledgments production of IL-10, an anti-inflammatory cytokine (Cruz et al., 2013). Moreover, there is increasing evidence that inflammatory process has a considerable role in the pathogenesis of DR with multiple studies showing an association of various systemic as well as local (vitreous and aqueous fluid) inflammatory factors and the progression of DR (Kastelan, Tomic, Gverovic, Salopek, and Ljubic, 2013). Increased retinal neutrophil count damages retinal vascular endothelial cells and blood-retinal barrier through a Fas-Fas ligand-dependent pathway (Harris, Skalak, and Hatchell, 1994; Joussen et al., 2003; McLeod, Lefer, Merges, and Lutty, 1995). Recent reports confirmed that NLR is increased in T2DM and DR (Sefil et al., 2014; Shiny et al., 2014; Woo et al., 2011). In addition, elevated NLR was independently related with the severity of DR (Woo et al., 2011). The processes of development and progression of DR and diabetesaccelerated atherosclerosis are tightly linked (Ellis et al., 2013). The accumulation of advanced glycation end products due to chronic glycemic exposure might be the common mechanism of arterial stiffness and microangiopathy in T2DM (Airaksinen et al., 1993; Singh, Barden, Mori, and Beilin, 2001). Recent studies reported that increased baPWV is associated with microalbuminuria, urinary albumin excretion, neuropathy, and retinopathy in T2DM (Ogawa et al., 2008; Shin et al., 2013; Sjoblom, Nystrom, Lanne, Engvall, and Ostgren, 2014; Yokoyama et al., 2004, 2007). In addition, PWV was positively correlated with the stage of DR (Ogawa et al., 2008; Tanaka et al., 2013). Therefore, it is considered that baPWV is a useful indicator for evaluation of complications in type 2 diabetic patients (Aso et al., 2003). In this study, we observed an association between NLR and baPWV in diabetic patients. Consistent with the result, some reports have documented the association between arterial stiffening and inflammatory markers, such as IL-6 and CRP (Nakhai-Pour, Grobbee, Bots, Muller, and van der Schouw, 2007; Nishida et al., 2007; Oh et al., 2012; Wang et al., 2013). Furthermore, it has been proposed that neutrophils may be one of the key factors contributing to microvasculature changes and inflammation when neutrophils adhere to the endothelial cell wall (Sala and Folco, 2001). Endothelial dysfunction and increased expression of proinflammatory cytokines could increase vascular inflammation, smooth muscle cell proliferation and subsequently elevated arterial stiffness (Pasceri, Willerson, and Yeh, 2000). Our findings are of great practical implications because, contrary to other inflammatory markers, NLR is inexpensive, easy to assess, and is part of the routine laboratory assessment. Early detection of NLR may be helpful for the search of undetected subclinical atherosclerosis in patients with T2DM and DR. Moreover, early evaluation of baPWV may be necessary for monitoring of disease progression in T2DM. Identification of a treatment that could improve both inflammation
This study was supported by the Science Foundation of Health and Family Planning Commission of Heilongjiang Province (No.2007.295).
References Airaksinen, K. E., Salmela, P. I., Linnaluoto, M. K., Ikaheimo, M. J., Ahola, K., & Ryhanen, L. J. (1993). Diminished arterial elasticity in diabetes: Association with fluorescent advanced glycosylation end products in collagen. Cardiovascular Research, 27(6), 942–945. Grading diabetic retinopathy from stereoscopic color fundus photographs–an extension of the modified Airlie House classification. ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research Group. Ophthalmology, 98(5 Suppl). (1991)., 786–806. Arbel, Y., Finkelstein, A., Halkin, A., Birati, E. Y., Revivo, M., Zuzut, M., et al. (2012). Neutrophil/lymphocyte ratio is related to the severity of coronary artery disease and clinical outcome in patients undergoing angiography. Atherosclerosis, 225(2), 456–460. Aso, K., Miyata, M., Kubo, T., Hashiguchi, H., Fukudome, M., Fukushige, E., et al. (2003). Brachial-ankle pulse wave velocity is useful for evaluation of complications in type 2 diabetic patients. Hypertension research: official journal of the Japanese Society of Hypertension, 26(10), 807–813. Bhat, T., Teli, S., Rijal, J., Bhat, H., Raza, M., Khoueiry, G., et al. (2013). Neutrophil to lymphocyte ratio and cardiovascular diseases: A review. Expert Review of Cardiovascular Therapy, 11(1), 55–59. Cruz, N. G., Sousa, L. P., Sousa, M. O., Pietrani, N. T., Fernandes, A. P., & Gomes, K. B. (2013). The linkage between inflammation and Type 2 diabetes mellitus. Diabetes Research and Clinical Practice, 99(2), 85–92. Duffy, B. K., Gurm, H. S., Rajagopal, V., Gupta, R., Ellis, S. G., & Bhatt, D. L. (2006). Usefulness of an elevated neutrophil to lymphocyte ratio in predicting long-term mortality after percutaneous coronary intervention. The American Journal of Cardiology, 97(7), 993–996. Ellis, T. P., Choudhury, R. H., Kaul, K., Chopra, M., Kohner, E. M., Tarr, J. M., et al. (2013). Diabetic retinopathy and atherosclerosis: Is there a link. Current Diabetes Reviews, 9 (2), 146–160. Harris, A. G., Skalak, T. C., & Hatchell, D. L. (1994). Leukocyte-capillary plugging and network resistance are increased in skeletal muscle of rats with streptozotocininduced hyperglycemia. International journal of microcirculation, clinical and experimental/sponsored by the European Society for Microcirculation, 14(3), 159–166. Horne, B. D., Anderson, J. L., John, J. M., Weaver, A., Bair, T. L., Jensen, K. R., et al. (2005). Which white blood cell subtypes predict increased cardiovascular risk. Journal of the American College of Cardiology, 45(10), 1638–1643. Joussen, A. M., Poulaki, V., Mitsiades, N., Cai, W. Y., Suzuma, I., Pak, J., et al. (2003). Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 17(1), 76–78. Kastelan, S., Tomic, M., Gverovic, A. A., Salopek, R. J., & Ljubic, S. (2013). Inflammation and pharmacological treatment in diabetic retinopathy. Mediators of Inflammation, 2013, 213130. Laurent, S., Cockcroft, J., Van Bortel, L., Boutouyrie, P., Giannattasio, C., Hayoz, D., et al. (2006). Expert consensus document on arterial stiffness: Methodological issues and clinical applications. European Heart Journal, 27(21), 2588–2605. Li, R. Y., Cao, Z. G., Zhang, J. R., Li, Y., & Wang, R. T. (2014). Decreased serum bilirubin is associated with silent cerebral infarction. Arteriosclerosis, Thrombosis, and Vascular Biology, 34(4), 946–951. Li, Y., Meng, S. Y., Meng, C. C., Yu, W. G., & Wang, R. T. (2013). Decreased serum bilirubin is associated with arterial stiffness in men. Nutrition, metabolism, and cardiovascular diseases: NMCD, 23(4), 375–381.
R. Wang et al. / Journal of Diabetes and Its Complications 29 (2015) 245–249 Lontchi-Yimagou, E., Sobngwi, E., Matsha, T. E., & Kengne, A. P. (2013). Diabetes mellitus and inflammation. Current Diabetes Reports, 13(3), 435–444. McLeod, D. S., Lefer, D. J., Merges, C., & Lutty, G. A. (1995). Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid. The American Journal of Pathology, 147(3), 642–653. Nakhai-Pour, H. R., Grobbee, D. E., Bots, M. L., Muller, M., & van der Schouw, Y. T. (2007). C-reactive protein and aortic stiffness and wave reflection in middle-aged and elderly men from the community. Journal of Human Hypertension, 21(12), 949–955. Nishida, M., Moriyama, T., Ishii, K., Takashima, S., Yoshizaki, K., Sugita, Y., et al. (2007). Effects of IL-6, adiponectin, CRP and metabolic syndrome on subclinical atherosclerosis. Clinica chimica acta; international journal of clinical chemistry, 384 (1–2), 99–104. Nunez, J., Nunez, E., Bodi, V., Sanchis, J., Minana, G., Mainar, L., et al. (2008). Usefulness of the neutrophil to lymphocyte ratio in predicting long-term mortality in ST segment elevation myocardial infarction. The American Journal of Cardiology, 101 (6), 747–752. Ogawa, O., Hiraoka, K., Watanabe, T., Kinoshita, J., Kawasumi, M., Yoshii, H., et al. (2008). Diabetic retinopathy is associated with pulse wave velocity, not with the augmentation index of pulse waveform. Cardiovascular Diabetology, 7, 11. Oh, E. G., Kim, S. H., Bang, S. Y., Hyun, S. S., Im, J. A., Lee, J. E., et al. (2012). Highsensitivity C-reactive protein is independently associated with arterial stiffness in women with metabolic syndrome. The Journal of Cardiovascular Nursing, 27(1), 61–67. Pasceri, V., Willerson, J. T., & Yeh, E. T. (2000). Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation, 102(18), 2165–2168. Sala, A., & Folco, G. (2001). Neutrophils, endothelial cells, and cysteinyl leukotrienes: A new approach to neutrophil-dependent inflammation. Biochemical and Biophysical Research Communications, 283(5), 1003–1006. Sefil, F., Ulutas, K. T., Dokuyucu, R., Sumbul, A. T., Yengil, E., Yagiz, A. E., et al. (2014). Investigation of neutrophil lymphocyte ratio and blood glucose regulation in patients with type 2 diabetes mellitus. The Journal of International Medical Research, 42(2), 581–588. Shin, D. I., Seung, K. B., Yoon, H. E., Hwang, B. H., Seo, S. M., Shin, S. J., et al. (2013). Microalbuminuria is independently associated with arterial stiffness and vascular
249
inflammation but not with carotid intima-media thickness in patients with newly diagnosed type 2 diabetes or essential hypertension. Journal of Korean Medical Science, 28(2), 252–260. Shiny, A., Bibin, Y. S., Shanthirani, C. S., Regin, B. S., Anjana, R. M., Balasubramanyam, M., et al. (2014). Association of neutrophil-lymphocyte ratio with glucose intolerance: An indicator of systemic inflammation in patients with type 2 diabetes. Diabetes Technology & Therapeutics, 16(8), 524–530. Singh, R., Barden, A., Mori, T., & Beilin, L. (2001). Advanced glycation end-products: A review. Diabetologia, 44(2), 129–146. Sjoblom, P., Nystrom, F. H., Lanne, T., Engvall, J., & Ostgren, C. J. (2014). Microalbuminuria, but not reduced eGFR, is associated with cardiovascular subclinical organ damage in type 2 diabetes. Diabetes & Metabolism, 40(1), 49–55. Tanaka, K., Kawai, T., Saisho, Y., Meguro, S., Harada, K., Satoh, Y., et al. (2013). Relationship between stage of diabetic retinopathy and pulse wave velocity in Japanese patients with type 2 diabetes. Journal of Diabetes Research, 2013, 193514. Wang, X., Du, Y., Fan, L., Ye, P., Yuan, Y., Lu, X., et al. (2013). Relationships between HDL-C, hs-CRP, with central arterial stiffness in apparently healthy people undergoing a general health examination. PLoS one, 8(12), e81778. Woo, S. J., Ahn, S. J., Ahn, J., Park, K. H., & Lee, K. (2011). Elevated systemic neutrophil count in diabetic retinopathy and diabetes: A hospital-based cross-sectional study of 30,793 Korean subjects. Investigative Ophthalmology & Visual Science, 52(10), 7697–7703. Yamashina, A., Tomiyama, H., Takeda, K., Tsuda, H., Arai, T., Hirose, K., et al. (2002). Validity, reproducibility, and clinical significance of noninvasive brachial-ankle pulse wave velocity measurement. Hypertension research: official journal of the Japanese Society of Hypertension, 25(3), 359–364. Yokoyama, H., Aoki, T., Imahori, M., & Kuramitsu, M. (2004). Subclinical atherosclerosis is increased in type 2 diabetic patients with microalbuminuria evaluated by intimamedia thickness and pulse wave velocity. Kidney International, 66(1), 448–454. Yokoyama, H., Yokota, Y., Tada, J., & Kanno, S. (2007). Diabetic neuropathy is closely associated with arterial stiffening and thickness in type 2 diabetes. Diabetic medicine: a journal of the British Diabetic Association, 24(12), 1329–1335. Zhou, J., Wang, S., & Xia, X. (2012). Role of intravitreal inflammatory cytokines and angiogenic factors in proliferative diabetic retinopathy. Current Eye Research, 37(5), 416–420.
