The predictive role of markers of Inflammation and endothelial dysfunction on the course of diabetic retinopathy in type 1 diabetes
Introduction
Diabetic retinopathy (DR), a frequent microvascular complication of diabetes, is the leading cause of preventable blindness in working-age adults (Mohamed, Gillies, & Wong, 2007). Inflammation and endothelial dysfunction have been considered as contributing factors to the development of diabetic retinopathy and other microvascular complications in patients with diabetes (Adamis, 2002, Cheung et al., 2010, Goldberg, 2009, Joussen et al., 2004). Several groups have reported an association between high levels of pro-inflammatory cytokines and the development of diabetic retinopathy. Serum levels of soluble TNF receptors 1 and 2 have been reported to correlate with the severity of diabetic retinopathy in Hispanics (Kuo et al., 2012). In children with type 1 diabetes mellitus (DM), those with non-proliferative diabetic retinopathy (NPDR) had significantly higher circulating TNF levels than those without retinopathy (Myśliwska, Myśliwiec, Balcerska, & Zorena, 2007). Another study performed in type 1 diabetes mellitus also showed a significant association between serum TNF levels and proliferative diabetic retinopathy (PDR) (Gustavsson, Agardh, Bengtsson, & Agardh, 2008). This observation has been confirmed and expanded to vascular endothelial growth factor (VEGF) and IL-6 by Myśliwiec et al. Therefore, while some studies strongly suggest that inflammation markers predict the development of diabetic retinopathy, other groups have failed to find significant associations between the levels of inflammation markers and the development of diabetic nephropathy (Altinova et al., 2005, Klein et al., 2009, Nguyen et al., 2009).
Nowak et al. (2008) showed correlations between the levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble E-selectin (sE-selectin) levels and the presence of retinopathy, thus suggesting that endothelial dysfunction, assessed by increased expression of cellular adhesion molecules, is linked to the presence of diabetic retinopathy. Similar findings have been obtained by Soedamah-Muthu, Chaturvedi, and Schalkwijk (2006) who reported that sVCAM-1 and sE-selectin levels are positively associated with retinopathy thus suggesting that adhesion molecules are important in the pathogenesis of vascular complications in type 1 diabetes.
Hemostatic abnormalities have also been found to be associated with PDR. The concentrations of tissue-type plasminogen activator (t-PA) and of its fast-acting inhibitor PAI-1 were measured in the plasma collected from eight patients with type 1 diabetes and PDR, and from eight patients with type 1 diabetes and background or no retinopathy. The ratio of t-PA to PAI-1 in plasma was significantly higher in patients with proliferative retinopathy than in those without (Simpson, Booth, & Moore, 1999).
In summary, the published data, obtained mainly in studies carried out with small cohorts, reflect simple associations between the presence of retinopathy and high levels of inflammatory markers and endothelial cell dysfunction markers, although with some inconsistencies. Therefore, in order to obtain a more definitive conclusion about the importance of these biomarkers in the development of retinopathy, it was necessary to carry out this investigation in a larger cohort. The measurement of these biomarkers in plasma/serum samples collected from 1391 type 1 diabetes patients at the time of enrollment on the Diabetes Control and Complications Trial (DCCT) when the patients had either mild or moderate retinopathy or albuminuria or were completely free of complications and the fact that these patients have been carefully followed for the development/progression of complications for more than 20 years provided us not only a critical mass of patients but the ability of determining which measurements were able to predict the development/progression of retinopathy as well of recognizing biomarkers able to identify patients at high risk of prematurely developing severe retinopathy and therefore facilitate early intervention to prevent the disease.
Our study included 1391 subjects from the DCCT/EDIC cohort who had biomarkers measured in samples obtained at entry into the DCCT study as well as Early Treatment Diabetic Retinopathy Study (ETDRS) scores performed throughout the DCCT/EDIC study. This study was performed to test the hypothesis that DCCT baseline values of markers of inflammation and endothelial dysfunction would be associated with the subsequent development and progression of diabetic retinopathy. The DCCT was a randomized controlled trial of 1441 subjects who were 13–39 years of age and had type 1 diabetes for 1–15 years at study entry (DCCT Research Group, 1993). Subjects were randomized to intensive or conventional diabetes therapy. The DCCT included two cohorts, subjects in the primary prevention cohort were retinopathy-free (ETDRS = 1), had diabetes duration between 1 and 5 years, and normal albumin excretion rates (AER < 40 mg/24 hours). The subjects in the secondary intervention cohort had mild to moderate non-proliferative diabetic retinopathy (ETDRS = 2–9), had diabetes for 1–15 years, and AER ≤ 200 mg/24 hours. At DCCT Baseline (1983–1989), none of the patients had hypertension (defined as ≥ 140 (systolic) and/or ≥ 90 mmHg (diastolic) or dyslipidemia (defined as total cholesterol > 200 and/or LDL > 160 mg/dl). Subjects were followed for an average of 6.5 years before the study was terminated early, in 1993, because of its obvious positive impact on microvascular complications. In 1994, approximately 95% of the DCCT participants was enrolled into the Epidemiology of Diabetes Interventions and Complications (EDIC) study. The goal of EDIC was to assess the development of macrovascular disease in type 1 diabetes and the progression of microvascular disease (EDIC, 1999). During EDIC, subjects were under the care of their personal physicians and encouraged to practice intensive insulin therapy. Each EDIC participant underwent a standardized annual history, physical examination, resting ECG, and routine laboratory analysis that included HbA1c levels (Diabetes Control and Complications Trial (DCCT) Research Group, 1987, Epidemiology of Diabetes Interventions and Complications (EDIC), 1999). Lipid profiles and 4-hour urine collections to measure albumin excretion rates (AER) were obtained in alternate years (Diabetes Control and Complications Trial (DCCT) Research Group, 1993, Epidemiology of Diabetes Interventions and Complications (EDIC), 1999) and retinopathy assessment was performed yearly in 25% of the cohort and in the whole cohort at years 4 and 10 of EDIC.
Fasting serum samples obtained during DCCT/EDIC were sent to the DCCT/EDIC central laboratory for standard lipid analysis. Aliquots of these samples were archived for future research purposes. In 1999–2000, as part of Medical University of South Carolina Program Project Grant funded by the NIH/JDF, serum samples collected during DCCT were provided by the DCCT/EDIC Coordinating Center and NIDDK to complement the serum samples collected during EDIC. The serum samples had been stored at − 70 °C and refreezing effects were minimized by preparing aliquots of the serum when thawed for the first time and using a new frozen aliquot for each new test performed. The IRB at Medical University of South Carolina and all participating DCCT/EDIC centers approved the sample collection procedures. Written informed consent was obtained from all participants.
During DCCT, diabetic retinopathy was assessed every 6 months in all subjects. During EDIC, retinopathy was assessed in approximately one quarter of the cohort during each follow-up year and the entire cohort was assessed at EDIC years 4 and 10. Severity of retinopathy was determined using stereoscopic seven-field fundus photographs and graded according to the ETDRS protocol using methods standardized by the DCCT/EDIC group (Diabetes Control and Complications Trial (DCCT) Research Group, 1993, Early Treatment Diabetic Retinopathy Study (ETDRS) Research Group, 1991). This study used the abbreviated final version of the ETDRS scale of diabetic retinopathy severity (ETDRS Research Group, 1991), which provides a composite score on a scale of 1 to 23 for both eyes on each subject. Retinopathy severity levels were defined as follows: ETDRS score 1–3 = none–minimal retinopathy; ETDRS score 4–9 = mild–moderate non-proliferative retinopathy; ETDRS score 10–23 = severe pre-proliferative and proliferative retinopathy (SNPDR) (Lyons et al., 2004). Two pre-defined primary retinal outcomes of interest were established for this study: (1) time to significant progression of diabetic retinopathy beyond what was measured at DCCT baseline (defined as a three-step or greater increase in ETDRS from that obtained at DCCT baseline), and (2) time to develop severe non-proliferative diabetic retinopathy (SNPDR; ETDRS score ≥ 10). Patients having any scatter laser photocoagulation performed during the study were included in the groups determined to have progression of retinopathy. Measurements were collected from DCCT baseline through EDIC study year 10.
Serum levels of C-reactive protein (CRP), PAI-1 total and PAI active, sICAM-1, sVCAM-1, sE-selectin, interleukin 6 (IL-6), and of soluble tumor necrosis factor receptor 1 and 2 (sTNFR-1 and sTNFR-2) were assayed using the Signature Plus Protein Array imaging and Analysis System (Aushon BioSystems) using ArrayVision™ software for data analysis. Inter-assay coefficients of variation were respectively 2.6% for CRP, 3.4% for PAI-total, 5.9% for PAI-1 active, 3% for sICAM-1, 4% for sVCAM-1, 4% for sE-selectin, 7.5% for IL-6, 5.9% for sTNFR-1 and 2.7% for sTNFR-2. Plasma concentrations of fibrinogen were determined using a commercially available assay (Fibrinogen SPQ Test System, Diasorin Inc., Stillwater MN).
At the baseline DCCT examination, each participant completed a physical examination, medical history, electrocardiogram and laboratory testing including serum creatinine, lipid profile and hemoglobin A1c (HbA1c) (Diabetes Control and Complications Trial (DCCT) Research Group, 1987, Epidemiology of Diabetes Interventions and Complications (EDIC), 1999). Four-hour urine collections for measurement of AER and creatinine clearance were also obtained during EDIC in alternate years. Baseline covariates for the current analyses were obtained from DCCT baseline history, physical examination and laboratory data (fasting lipids and renal function). The methodology used to perform all the routine measurements used as conventional risk factors in this study has been described elsewhere (Diabetes Control and Complications Trial (DCCT) group, 1986, Diabetes Control and Complications Trial (DCCT) Research Group, 1987, Epidemiology of Diabetes Interventions and Complications (EDIC), 1999).
The concentrations of markers of inflammation and endothelial dysfunction from serum samples collected at DCCT baseline were used to determine whether increases in the levels of these biomarkers could predict elevated risk to develop or induce accelerated progression of retinopathy during 16 years of follow up in the DCCT/EDIC study. All biomarker levels were assessed for normality and transformed when necessary (i.e., CRP, IL-6, sTNF-R1, sTNF-R2 and sE-selectin required natural log transformation). Following data normalization, all biomarkers were standardized and the analysis results represent the association between a change of one standard deviation in each biomarker and the hazard ratio associated with progression of retinopathy. Composite biomarker scores were created to assess the combined impact of multiple biomarkers believed to be acting on the same pathway. Specifically, three composite scores were created by combining standardized-scores of individual biomarkers: acute phase reactants (i.e., fibrinogen and CRP); cytokines and cytokine receptors/adipokines (i.e., sTNFR 1 and 2, PAI-I active, PAI-1 total and IL-6); and thrombosis/fibrinolysis (i.e., fibrinogen, PAI-I active and PAI-1 total). Baseline demographic and clinical variables were stratified by retinopathy cohort (primary prevention; retinopathy-free) vs. secondary intervention (mild-moderate baseline retinopathy) and progression to severe retinopathy during the follow up study. A two-sample Wilcoxon rank-sum test was used to compare groups for ordinal/continuous variables while a Pearson's chi square test was used to compare categorical characteristics. Univariate comparisons of levels of markers were compared across baseline retinopathy cohorts as well as within cohort across outcomes using a two-sample Wilcoxon rank-sum test.
Due to varying time intervals between fundus photo visits for each participant, parametric hazard models for interval-censored event/survival times were used to assess the effect of the increases in biomarker levels and of clinical and demographic variables on the risk of progression of retinal disease (Odell et al., 1992, Sparling et al., 2006). Preliminary adjusted models accounted for DCCT treatment assignment, presence of baseline retinopathy, duration of diabetes, and baseline measures of AER, HbA1C, and ETDRS score. Additionally, evidence has shown a strong and consistent protective effect of HMG-CoA reductase inhibitors (‘statins’) and angiotensin-converting-enzyme inhibitors (ACE) on the risk of cardiovascular complications of diabetes as well as promise in reducing the risk of diabetic retinopathy progression (Al-Shabrawey et al., 2008, Chaturvedi, 2000). In light of this, final models were additionally adjusted for effects of ACE/angiotensin II receptor blockers (ARB) and lipid-lowering therapy [the use of these drugs increased as the DCCT/EDIC study progressed; as such, their use at any time leading up to an event or censor time (t) is entered into the model as time-varying covariate]. The final models were also adjusted for DCCT treatment group, gender, smoking, DCCT baseline retinopathy status and baseline levels of HDL-cholesterol, LDL-cholesterol, triglycerides, systolic blood pressure, and age. Interval timing was defined as the number of days since the baseline fundus for the photo sessions both before and after progression of disease as we do not have exact disease progression dates.
All statistical analyses were performed using SAS System version 9.3 (SAS Institute, Cary, NC, USA). No adjustments for multiple testing were made, as they are known to reduce statistical power and increase the probability of accepting a null hypothesis that is truly false. A type 1 error rate of 5% was used as a nominal value for statistical significance.
Section snippets
Demographics and retinopathy outcomes
In both the primary prevention (free of retinopathy at baseline) and the secondary intervention (mild to moderate retinopathy at baseline) cohorts, baseline levels of HbA1c % and the duration of diabetes were higher in those that progress to severe retinopathy (Table 1). Additionally, those who progress to severe retinopathy are less likely to be in the intensively treated study arm, and more likely to have been treated with lipid lowering drugs and ACE/ARB drugs during the study period. Of the
Discussion
In our present study, serum levels of CRP, PAI-1 total and PAI active, sICAM-1, sVCAM-1, sE-Selectin, IL-6, sTNFR-1 and sTNFR-2 were assayed. At the DCCT baseline visit, patients without retinopathy had significant lower levels of inflammatory markers (CRP and sTNFR 1 and 2) than those with mild to moderate retinopathy. Additionally, patients with mild to moderate retinopathy who progressed to SNPDR had significantly higher levels of sE-selectin at baseline. The same was true in patients
Acknowledgments
Author contributions: H.A.R. helped with data analysis and wrote and reviewed the manuscript. N.L.B. and K.J.H. analyzed data and wrote, reviewed, and edited the manuscript. P.A.C. provided the clinical data from the EDIC cohort and assisted in the writing and revision of the manuscript. R.K. supervised the performance of all of the biomarker assays performed in this study and assembled the resulting data. J.L. and P.A.C. helped with the statistical analysis, the writing, reviewing, and editing
References (38)
- et al.
Diabetic retinopathy
Lancet
(2010) - et al.
TNF-alpha is an independent serum marker for proliferative retinopathy in type 1 diabetic patients
Journal of Diabetes and its Complications
(2008) - et al.
Systemic soluble tumor necrosis factor receptors 1 and 2 are associated with severity of diabetic retinopathy in Hispanics
Ophthalmology
(2012) - et al.
Soluble vascular cell adhesion molecule-1 and soluble E-selectin are associated with micro- and macrovascular complications in type 1 diabetic patients
Journal of Diabetes and its Complications
(2006) - et al.
Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes
Diabetes
(2003) Is diabetic retinopathy an inflammatory disease?
British Journal of Ophthalmology
(2002)- et al.
Different expression pattern of serum soluble intercellular adhesion molecules-1 and neutrophilic expression of CD18 in patients with diabetic retinopathy
International Journal of Ophthalmology
(2012) - et al.
Role of NADPH oxidase and Stat3 in statin-mediated protection against diabetic retinopathy
Investigative Ophthalmology & Visual Science
(2008) - et al.
Serum inflammatory markers in diabetic retinopathy
Investigative Ophthalmology & Visual Science
(2005) - et al.
Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes
Investigative Ophthalmology & Visual Science
(2000)
Modulation of the renin–angiotensin system and retinopathy
Heart
Design and methodologic considerations for the feasibility phase
Diabetes
Feasibility of centralized measurements of glycated hemoglobin in the Diabetes Control and Complications Trial: A multicenter study
Clinical Chemistry
The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus: The Diabetes Control and Complications Trial
New England Journal of Medicine
Fundus photographic risk factors for progression of diabetic retinopathy. ETDRS report number 12
Ophthalmology
Design, implementation, and preliminary results of a long-term follow-up of the Diabetes Control and Complications Trial cohort
Diabetes Care
Coagulation and fibrinolysis parameters in type 2 diabetic patients with and without diabetic vascular complications
Medical Principles and Practice
Elevated concentrations of circulating adhesion molecules and their association with microvascular complications in insulin-dependent diabetes mellitus
Journal of Clinical Endocrinology and Metabolism
Cytokine and cytokine-like inflammation markers, endothelial dysfunction, and imbalanced coagulation in development of diabetes and its complications
Journal of Clinical Endocrinology and Metabolism
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Conflict of interest: There are no potential conflicts of interest relevant to this article.