Main text and abstract. Journal name Diabetes. Running title A population-based cohort study. Denmark. Australia. University of Singapore, Singapore

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1 Main text and abstract Journal name Diabetes Manuscript title Retinal Vessel Calibers Predict Long-term Microvascular Complications in Type 1 Diabetes Mellitus: The Danish Cohort of Pediatric Diabetes 1987 (DCPD1987) Running title A population-based cohort study Authors Rebecca Broe, 1,2,3 Malin L Rasmussen, 1,2 Ulrik Frydkjaer-Olsen, 1,2 Birthe S Olsen, 4,5 Henrik B Mortensen, 4,5 Lauren Hodgson, 6 Tien Y Wong, 6,7 Tunde Peto, 8,9 Jakob Grauslund 1,2 1 Department of Ophthalmology, Odense University Hospital, Odense, Denmark 2 The Clinical Research Institute, University of Southern Denmark, Odense, Denmark 3 OPEN Odense Patient data Explorative Network, Odense University Hospital, Odense, Denmark 4 Department of Pediatrics E, Herlev Hospital, Herlev, Denmark 5 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 6 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, University of Melbourne, Melbourne, VIC, Australia 7 Singapore Eye Research Institute, Singapore National Eye Center, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 8 The NIHR Biomedical Research Centre, Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom 9 The Clinical Research Institute, University of Southern Denmark, Odense, Denmark Corresponding author Rebecca Broe Department of Ophthalmology, Odense University Hospital Sdr. Boulevard 29 DK-5000 Odense C, Denmark Phone: rebecca.broe@rsyd.dk Abstract word count 200 Main text word count 3712 Abstract Number of tables and figure 4 tables and 1 figure 1 Diabetes Publish Ahead of Print, published online June 9, 2014

2 Diabetic neuropathy, nephropathy and retinopathy cause significant morbidity in patients with type 1 diabetes, even though improvements in treatment modalities delay the appearance and reduce the severity of these complications. To prevent or further delay the onset, it is necessary to better understand common underlying pathogenesis and to discover pre-clinical biomarkers of these complications. Retinal vessel calibers have been associated with the presence of microvascular complications but their long-term predictive value has only been sparsely investigated. We examined retinal vessel calibers as 16-year predictors of diabetic nephropathy, neuropathy and proliferative retinopathy in a young population-based Danish cohort with type 1 diabetes. We used semi-automated computer software to analyze vessel diameters on baseline retinal photos. Calibers of all vessels coursing through a zone disc diameter from the disc margin were measured and summarized as the central artery and vein equivalents. In multiple regression analyzes we found both wider venular diameters and smaller arteriolar diameters to be predictive of the 16-year development of nephropathy, neuropathy and proliferative retinopathy. Early retinal vessel caliber changes are seemingly early markers of microvascular processes, precede the development of microvascular complications and a potential non-invasive predictive test on future risk of diabetic retinopathy, neuropathy and nephropathy. Introduction 2

3 Diabetic microvascular complications, namely diabetic peripheral neuropathy (DPN), diabetic nephropathy (DN) and diabetic retinopathy (DR), are common in type 1 diabetes (1) despite advances in metabolic care. Identifying new predictors of microvascular disease could be helpful for early individual risk-stratification, which may provide better opportunity for timely implementation of effective interventions. For this purpose, the retinal vasculature provides a unique opportunity to assess vascular health directly and non-invasively in vivo. Studies have investigated how retinal vessel calibers are associated with microvascular complications in both type 1 and type 2 diabetes (2-14). The evidence to date suggest that wider retinal venular diameters are associated with presence of both DN and severe levels of DR in several cross-sectional studies (3; 8-10), and furthermore with incident DN (4; 5) and progression to severe DR(13; 14) in prospective studies. No relations between retinal venular diameters and DPN have been shown thus far (2; 4; 6; 7). Concurrently, narrower arteriolar diameters have been linked to severe DR (3; 8) and DN (2; 3) in cross-sectional reports but for DPN the results have been inconclusive (2; 6; 7). Two prospective studies proposed that in type 1 diabetes, wider arteriolar diameters are associated with increased risk of incident DR (11; 12) while other groups showed no longitudinal correlation between arteriolar diameters and diabetic microvascular complications (4; 5; 13). As most previous studies on retinal vascular calibers and diabetic complications are cross-sectional or short-term follow-up studies, there is very limited information on the predictive value of these measurements. To define the link between retinal vascular calibers and diabetic microvascular complications prospective studies with long follow-up periods are needed. 3

4 The aim of this study was to investigate the predictive value of retinal vascular calibers on microvascular complications in a 16-year prospective study of a young population-based Danish cohort with type 1 diabetes mellitus. Methods Study population The participants in this study were identified from a nationwide population-based pediatric cohort of Danish children with type 1 diabetes who were initially studied in (n=720); the Danish Cohort of Pediatric Diabetes 1987 (DCPD1987). Detailed characteristics of this cohort have been reported elsewhere (15-22). Of this cohort, 339 were included in a baseline examination in 1995 and thus eligible for 16-year follow-up in Of these 15 (4.4%) were excluded for follow-up due to missing baseline retinal photographs, 13 (3.8%) had died and 63 (18.6%) were unreachable because they had emigrated or could not be contacted due to research protection. Of the remaining 248 patients, 185 (74.6%) participated at follow-up in 2011 and 63 (25.4%) declined to participate. The study was performed in accordance with the criteria of the Helsinki II Declaration and was approved by the local scientific ethics committee. All patients gave a written informed consent at both baseline and follow-up examinations. Baseline examinations and measurements of retinal vascular caliber In 1995, information on sex, age, diabetes duration, HbA 1c, blood pressure, BMI, vibration perception threshold (VPT), determination of mean albumin excretion rate (AER) and presence of DR was collected. The methods used have been described elsewhere (16), however; baseline microand macroalbuminuria was defined as mean AER µg/min and > 200µg/min, in at least two timed overnight urine collections. Color retinal photographs were taken after pupillary dilation by 4

5 certified operators using 40-60º retinal cameras in accordance with the European Diabetes Study Group (EURODIAB) recommendations (23). The film slides were later digitalized with DigitDia 5000 Filmscanner (Reflecta, Rottenburg, Germany). Two photographs were taken of each eye, a macular-temporal field and a disc-nasal field. The latter was used for analyzes of vessel diameters. A high inter-eye correlation has been shown previously and consequently we only used right eye for assessment of vessel calibers (24). If the photo of the right eye was not of sufficient quality for grading, left eye was used. Retinal vascular calibers were assessed by the same certified and validated grader with a computer-based program following a previously validated protocol (24; 25). Diameters of all vessels coursing completely through a zone disc diameter from the disc margin were measured with image analysis software (IVAN, Department of Ophthalmology Visual Science, University of Wisconsin, Madison, WI, USA). The calibers of the central retinal artery and vein were estimated using the Big-6 formula ; a method that combines diameter measurements of the six largest arterioles and the six largest venules into the central artery and vein equivalents (CRAE and CRVE) (26). In other words, the diameter of the six largest arterioles in the specified zone is used to calculate the diameter of the central retinal artery as it enters from the optic disc. CRAE is therefore an estimated central retinal artery diameter based on measurements of its branches. The method for calculating CRVE is similar. For assessment of baseline DR, all photos were graded by the same certified grader by a modified ETDRS protocol, allowing for non-standard photography (15; 27). Outcome measures in 2011 The examinations took place between 1 January and 1 November The participants underwent a clinical examination where VPT was measured in triplicate on the apex of the right first toe with a 5

6 hand-held biothesiometer (Bio-Medical Instrument Co, Newbury, Ohio, USA). The mean of the last two measurements was calculated and a mean VPT>25Volt was considered abnormal. Participants with abnormal mean VPT were classified as having DPN. Spot urine samples were collected and analyzed centrally to determine urine albumin-creatinine ratio (ACR). Participants with ongoing infections or who were menstruating were asked to hand in urine samples at a later time. Laboratory measurement of albumin and creatinine were performed on an Abbott Architect analyzer by immunoturbidimetric and enzymatic assays, respectively. A spot urine ACR was then calculated and microalbuminuria was defined as ACR=30-299mg/g and macroalbuminuria as ACR 300mg/g. Participants with macroalbuminuria at follow-up, or who had had a renal transplant or had received dialysis were classified as having DN. We used the Danish Patient Registry to identify those who had dialysis/renal transplant among the patients who declined to participate. Participants underwent an ophthalmological examination including retinal photography where seven 45º fields were taken of each eye in accordance with the Early Treatment of Diabetic Retinopathy Study (ETDRS) standards (28). Non-stereoscopic, digital color retinal photographs were performed in mydriasis (Tropicamide 1% and epinephrine 10%) by the same trained and certified operator using 3D OCT-2000 Spectral Domain OCT, Topcon, Tokyo, Japan. Images were captured at a resolution of 4.288x2.848 pixels. The retinal photographs were graded by the same certified grader by a modified ETDRS scale (15; 27). Participants were classified as having proliferative diabetic retinopathy (PDR) if they had ETDRS level 61 or above on the eye included in the vessel analyzes. We used the Danish Patient Registry to identify those who had had panretinal photocoagulation among the patients who declined to participate to get an estimate of the incidence rate of PDR in this group. 6

7 Statistical methods Categorical data are presented as percentages, whereas continuous data are presented as means ± standard deviations (SD). We used the Mann-Whitney test for differences between two groups, Cuzick s test for trend for several groups, and the Spearman rank correlation to test for associations between two continuous variables. Multiple logistic regression analyzes with backwards selection were performed to estimate Odds Ratios (OR) for incident DPN, DN and PDR. All baseline variables (sex, age, diabetes duration, HbA1c, blood pressure, BMI, VPT, mean AER and level of retinopathy) were included in each of these models. All participants with VPT<25Volt at baseline were considered at risk of incident DPN and all participants without PDR at baseline were at risk of incident PDR. Patients with baseline mean AER>200µg/min (overt DN) were excluded from analyzes on renal function at follow-up. The 95% CI are given for estimates of ORs and were considered statistically significant when they did not cross 1.0. Findings with a p-value<0.05 were considered statistically significant. For analyzes on cutoff points for CRAE and CRVE, we generated a series of dichotomous variables for each 10µm increase in diameter; i.e. over/under CRAE=140µm, over/under CRAE=150µm etc. We ran multiple logistic regressions for every new CRAE and CRVE variable, for each endpoint, and plotted receiver operating characteristics curves for each regression model to determine the area under the curve (AUC). We furthermore tested the equality of the highest vs. the lowest AUC for each outcome with respect to both CRAE and CRVE cutoffs to determine if there was a significant difference in the predictive value of the models. If a significant difference was found between the lowest vs. highest AUC we tested the second lowest AUC against the highest - if this difference was 7

8 significant we proceeded on to the third lowest vs. highest etc. to establish the span of the investigated threshold. For the cutoffs producing the highest AUCs the ORs were used to calculate an estimate of the risk increase (as a percentage) of a given outcome for participants below vs. above this threshold. All statistical calculations were performed with STATA 11.1 (StataCorp LP, College Station, Texas, USA). Results Characteristics of the study population All 185 participants had at least one gradable baseline retinal photo in terms of vessel analyzes and thus none were excluded for this reason. The mean age and diabetes duration at baseline of the participants were 21.0 and 13.5 years, respectively, and 49.7% were males. A comparison was done between the participants and the patients who were available for follow-up but declined to participate (Table 1). The patients who declined had significantly higher HbA1c values at baseline compared to the participants but the groups did not differ in any other respect. For the 185 participants the range of CRAE and CRVE were µm and µm, respectively, whereas they were µm and µm, respectively, for the 63 who declined. The distribution of CRAE and CRVE in these two groups is shown in Figure 1. Relationship between retinal vascular calibers and clinical baseline characteristics In Table 2 it is demonstrated how various baseline characteristics of the participants were associated with CRAE and CRVE when looked at separately. CRVE was significantly related to level of retinopathy, diabetes duration, VPT, and systolic blood pressure, when analyzed one by one. 8

9 Furthermore, there was a borderline association with diastolic blood pressure. CRAE was significantly related to systolic and diastolic blood pressure, respectively. Associations of retinal vascular calibers and incident DPN DPN was evaluated in 162 patients at follow-up, who had had VPT<25Volt at baseline in Of these, 10.5% (n=17) had developed DPN during 16 years. As shown in Table 3, multiple logistic regressions found both CRAE (OR 2.96 per 10µm decrease, 95%CI ) and CRVE (OR 1.52 per 10µm increase, 95%CI ) to be significantly associated with the 16-year incidence of DPN. Other baseline risk factors associated with DPN were increasing age (OR 1.62 per 1 year increase, 95% CI ), male sex (OR 3.62 vs. female sex, 95% CI ), increasing HbA 1c (OR 3.53 per 1% increase, 95% CI ), increasing diastolic blood pressure (OR 2.77 per 10 mmhg increase, 95% CI ) and increasing VPT (OR 1.62 per 1 Volt increase, 95% CI ). Associations of retinal vascular calibers and DN A total of 166 participants without macroalbuminuria at baseline in 1995 handed in urine samples at follow-up. They were included in analyzes of vessel diameters and incident DN. Of these, 13 patients (7.8%) had developed macroalbuminuria, had had a kidney transplant (n=2) and/or had received dialysis (n=4) at follow-up. Among the 63 patients who declined to participate at followup, one had had a kidney transplant while none had received dialysis. Both CRAE (OR 2.63 per 10µm decrease, 95%CI ) and CRVE (OR 1.76 per 10µm increase, 95%CI ) were significantly associated with the 16-year incidence of DN in the multiple logistic regression model (Table 3). Of the other risk factors we adjusted for in the model, only increasing diastolic blood pressure (OR 4.06 per 10 mmhg increase, 95% CI ) and 9

10 increasing levels of HbA 1c (OR 2.18 per 1% increase, 95% CI ) were associated with nephropathy. Associations of retinal vascular calibers and incident PDR Of the 185 participants, one had PDR at baseline in 1995 and was excluded from this analysis. Of the 184 patients at risk, 27.2% (n=50) progressed to PDR during the 16 year period on the eye chosen for vessel analyzes. Among the 63 patients who declined participation in 2011, 26 (41.3%) had had pan-retinal photocoagulation performed in at least one eye. As shown in Table 3, multiple logistic regressions found both CRAE (OR 1.56 per 10µm decrease, 95%CI ) and CRVE (OR 1.36 per 10µm increase, 95%CI ) to be significantly associated with the 16-year incidence of PDR. Other baseline risk factors associated with PDR were HbA 1c (OR 2.44 per 1% increase, 95% CI ), diastolic blood pressure (OR 1.95 per 10 mmhg increase, 95% CI ) and baseline retinopathy at ETDRS level 35 (OR 6.15 vs. level 10, 95% CI ). Baseline retinopathy at levels 20 and 43 vs. level 10 was not significantly associated with incident PDR. Cutoff points for CRAE and CRVE as predictors of microvascular complications We used the following dichotomous variables for CRAE: over/under 140, 150, 160, 170 and 180µm, and for CRVE: over/under 210, 220, 230, 240, 250, 260, 270, and 280µm. In Table 4 the calculated AUCs for each multiple regression is shown. For CRAE the highest AUC was produced with the cutoff 170µm with respect to incident DPN and DN, and with 180µm for incident PDR. For each outcome, no significant difference was found when comparing the highest AUC with the lowest (p=0.10, 0.10, and 0.18, respectively). Participants with CRAE below 170µm had an increased risk of incident DPN and DN of 803% and 10

11 1090%, respectively, as compared to participants above this threshold (DPN: OR 9.03, 95%CI ; DN: OR 11.90, 95%CI ). Participants below CRAE 180µm had an 436% increased risk of incident PDR compared to participants above (OR 5.36, 95%CI ). For CRVE the highest AUC was seen with the cutoff 260µm with respect to incident DPN and with 250 µm for DN and PDR. No significant difference was found when comparing the highest AUC with the lowest for all three outcomes (p=0.25, 0.25, and 0.13, respectively). Participants with CRVE above 260µm had an increased risk of incident DPN of 592% compared to participants below this threshold (OR 6.92, 95%CI ). Participants above CRVE 250µm had an 341% and 224% increased risk of incident DN and PDR, respectively, compared to participants below (DN: OR 4.41, 95%CI ; PDR: OR 3.24, 95%CI ). Discussion In this prospective study of young patients with type 1 diabetes, changes in retinal vascular caliber (wider venular caliber and narrow arteriolar caliber) were consistently associated with the 16-year incidence of the major diabetic microvascular complications, namely DPN, DN and PDR. Most previous studies have examined these complications separately and no previous studies have investigated the relation between retinal vessel calibers and DPN prospectively; with cross-sectional reports inconclusive (2; 4; 6; 7). Our results show a strong association between DPN and both narrow arteriolar and wide venular diameters, despite the relatively small number of cases. A possible link between retinal vessel diameters and renal disease was illustrated earlier in the Wisconsin Epidemiologic Study of Diabetic Retinopathy where larger retinal venular caliber was independently associated with the 10-year incidence of DN in type 2 diabetes (4) and the 16-year 11

12 incidence in type 1 diabetes (5). Nevertheless, they found no associations concerning arteriolar caliber. Our data support the concept that retinal venular dilation is related to the development of DN in people with type 1 diabetes. Moreover, we observed a relation between narrower arteriolar calibers and the 16-year incidence of nephropathy which has not been previously demonstrated longitudinally, but a similar association was presented in cross sectional reports (2; 3). Hypertension is known to accelerate the progression of renal disease in patients with diabetes (29). In addition to the well-known association between narrow arteriolar calibers and coexisting hypertension (30-32), a meta-analysis and data pooling study showed that narrow retinal arteriolar calibers appear to precede clinical hypertension (33). Therefore, a plausible explanation for the link between narrower arteriolar caliber and incident DN seen in this population, compared to previously studied cohorts, is a higher prevalence of hypertension subsequently to the baseline examination. Large venular calibers (3; 8-10) and narrow arteriolar calibers (3; 8) were formerly associated with the presence of severe DR in cross sectional reports. We showed the same relation for the 16-year progression to PDR. Large venular calibers have previously been related to progression to severe DR in some studies with shorter follow-up periods (13; 14). In contrast to our data, two prospective studies found larger arteriolar caliber associated with increased risk of incident DR (11; 12) but another group could not show this correlation (13). However, these studies focused on the shortterm incidence of DR and general progression of the disease and not incident PDR. Relations between hypertension and DR have been demonstrated earlier (34). As with our findings on retinal arteriolar narrowing and incident DN, we speculate that a high prevalence of hypertension subsequent to baseline examinations partly explains the association between narrower arteriolar caliber and PDR seen in our cohort. 12

13 The underlying pathophysiological mechanisms of the venular dilation observed in this study are not fully known. One hypothesis is retinal hypoxia (35; 36). Another possible mechanism is inflammation as larger retinal venules were linked to higher levels of inflammatory biomarkers (37; 38). Additionally, studies on vasodilatory response to flicker-light stimulation, a process reflecting the endothelial function of retinal vessels, propose that larger retinal venular and arteriolar caliber indicate endothelial dysfunction(39; 40). Our findings on arteriolar calibers do not comply with this, though. It is possible, that mechanisms stronger than endothelial dysfunction have superior impact on the arterioles in our cohort, such as hypertensive mechanisms. In the pursuit for cutoff values, participants with CRAE values below µm seemed to be at higher risk of microvasculopathy than those above. However, this was only an observed trend as we could not show a significant difference between the highest AUCs and even the lowest ones. Thus it cannot be concluded that these thresholds are the best in predicting microvascular disease. Similarly we observed the highest AUCs for CRVE cutoffs in an area of µm but found no significant difference when comparing to the lowest AUCs. We speculate that the relatively low number of participants in the study might be the reason why these observed trends are not significant. Correspondingly, the number of participants is a plausible explanation for the relatively high risk increase calculated for each outcome for patients below/above a given threshold compared to the opposite group. The logistic regression with CRAE and CRVE categorized by a chosen threshold produced rather large CIs and interpretation of these numbers should therefore be done with caution. Future studies are clearly needed to establish the cutoffs and the risk estimations. 13

14 The strengths of this study include the population-based cohort, the long follow-up time, and use of quantitative methods for assessment of vascular calibers by standardized protocols. None of the participants had ungradable retinal photos and all were graded by the same, certified grader. Most participants had little or no vascular disease at baseline which provides a better estimate of retinal vessel calibers predictive value as progression to end stage of any complication is a major increase. A limitation to this study is the relatively small number of patients, however; our results were statistically significant irrespective of this. Intermediate measurements on the cohort would provide valuable information on the timeline in development of complications but such data are not available, unfortunately. Nephropathy at baseline and follow-up was assessed with different methods; at baseline mean AER was used and at follow-up ACR. Classification issues have previously been shown when comparing these two methods (41); however, only smaller differences were observed for classification of macroalbuminuria as we used for defining DN in this study and we therefore speculate that this is a minor issue. Assessing DR from two fundus photos at baseline compared to seven at follow-up make underestimation of DR at baseline a possibility. Nevertheless, it has previously been shown that the two EURODIAB fields are acceptable for grading DR compared to the seven ETDRS fields (23). In conclusion, our study supports that changes in retinal vascular calibers are preclinical biomarkers of early diabetic neuropathy, nephropathy and retinopathy in persons with type 1 diabetes. This could possibly be indications of a common pathogenic pathway for diabetic microvascular 14

15 complications and thus retinal imaging could provide a non-invasive instrument to identify patients at risk of microvasculopathy (42). Author contributions RB, JG, BSO and HBO contributed to the study concept and design. RB, MLR, UF, LH, BSO and HBM contributed to the acquisition of data. Data analyses were performed by RB, who also wrote the initial draft of the paper. MLR, BSO, HBM, LH, TYW, TP and JG were involved in the interpretation of data and revised the paper critically for intellectual content. RB is the guarantor of this work and, as such, had full access to all the data in the study and takes full responsibility for the integrity of the data, the accuracy of the data analysis, and for the decision to submit for publication. All authors approved the final version of the paper. Acknowledgements This work was supported by Fight for Sight, Denmark, Synoptik Foundation, Medivit Aps, Gangsted Foundation, Foundation of Karen Svankjaer Yde, Lykfeldts Grant, The A.P. Moeller Foundation for the Advancement of Medical Science, The Region of Southern Denmark and The University of Southern Denmark. No potential conflicts of interest relevant to this article were reported. 15

16 References 1. Pambianco G, Costacou T, Ellis D, Becker DJ, Klein R, Orchard TJ: The 30-year natural history of type 1 diabetes complications: the Pittsburgh Epidemiology of Diabetes Complications Study experience. Diabetes 2006;55: Grauslund J, Hodgson L, Kawasaki R, Green A, Sjolie AK, Wong TY: Retinal vessel calibre and micro- and macrovascular complications in type 1 diabetes. Diabetologia 2009;52: Klein R, Klein BE, Moss SE, Wong TY, Hubbard L, Cruickshanks KJ, Palta M: Retinal vascular abnormalities in persons with type 1 diabetes: the Wisconsin Epidemiologic Study of Diabetic Retinopathy: XVIII. Ophthalmology 2003;110: Klein R, Klein BE, Moss SE, Wong TY: Retinal vessel caliber and microvascular and macrovascular disease in type 2 diabetes: XXI: the Wisconsin Epidemiologic Study of Diabetic Retinopathy. Ophthalmology 2007;114: Wong TY, Shankar A, Klein R, Klein BE: Retinal vessel diameters and the incidence of gross proteinuria and renal insufficiency in people with type 1 diabetes. Diabetes 2004;53: Ding J, Cheung CY, Ikram MK, Zheng YF, Cheng CY, Lamoureux EL, Tai ES, Subramaniam T, Wong TY: Early retinal arteriolar changes and peripheral neuropathy in diabetes. Diabetes care 2012;35: Sabanayagam C, Tai ES, Lee J, Lim SC, Wong TY: Retinal vessel caliber and peripheral neuropathy in diabetic participants. Microcirculation (New York, NY : 1994) 2010;17: Klein R, Klein BE, Moss SE, Wong TY, Sharrett AR: Retinal vascular caliber in persons with type 2 diabetes: the Wisconsin Epidemiological Study of Diabetic Retinopathy: XX. Ophthalmology 2006;113: Cheung CY, Lamoureux E, Ikram MK, Sasongko MB, Ding J, Zheng Y, Mitchell P, Wang JJ, Wong TY: Retinal vascular geometry in Asian persons with diabetes and retinopathy. Journal of diabetes science and technology 2012;6: Tsai AS, Wong TY, Lavanya R, Zhang R, Hamzah H, Tai ES, Cheung CY: Differential association of retinal arteriolar and venular caliber with diabetes and retinopathy. Diabetes research and clinical practice 2011;94: Cheung N, Rogers SL, Donaghue KC, Jenkins AJ, Tikellis G, Wong TY: Retinal arteriolar dilation predicts retinopathy in adolescents with type 1 diabetes. Diabetes care 2008;31: Alibrahim E, Donaghue KC, Rogers S, Hing S, Jenkins AJ, Chan A, Wong TY: Retinal vascular caliber and risk of retinopathy in young patients with type 1 diabetes. Ophthalmology 2006;113: Klein R, Klein BE, Moss SE, Wong TY, Hubbard L, Cruickshanks KJ, Palta M: The relation of retinal vessel caliber to the incidence and progression of diabetic retinopathy: XIX: the Wisconsin Epidemiologic Study of Diabetic Retinopathy. Archives of ophthalmology 2004;122: Roy MS, Klein R, Janal MN: Retinal venular diameter as an early indicator of progression to proliferative diabetic retinopathy with and without high-risk characteristics in African Americans with type 1 diabetes mellitus. Archives of ophthalmology 2011;129: Broe R, Rasmussen ML, Frydkjaer-Olsen U, Olsen BS, Mortensen HB, Peto T, Grauslund J: The 16-year incidence, progression and regression of diabetic retinopathy in a young populationbased Danish cohort with type 1 diabetes mellitus: The Danish cohort of pediatric diabetes 1987 (DCPD1987). Acta diabetologica 2013; 16. Olsen BS, Johannesen J, Sjolie AK, Borch-Johnsen K, Hougaard P, Thorsteinsson B, Pramming S, Marinelli K, Mortensen HB: Metabolic control and prevalence of microvascular complications in young Danish patients with Type 1 diabetes mellitus. Danish Study Group of Diabetes in Childhood. Diabetic medicine : a journal of the British Diabetic Association 1999;16:

17 17. Olsen BS, Sjolie A, Hougaard P, Johannesen J, Borch-Johnsen K, Marinelli K, Thorsteinsson B, Pramming S, Mortensen HB: A 6-year nationwide cohort study of glycaemic control in young people with type 1 diabetes. Risk markers for the development of retinopathy, nephropathy and neuropathy. Danish Study Group of Diabetes in Childhood. Journal of diabetes and its complications 2000;14: Olsen BS, Sjolie AK, Hougaard P, Johannesen J, Marinelli K, Jacobsen BB, Mortensen HB: The significance of the prepubertal diabetes duration for the development of retinopathy and nephropathy in patients with type 1 diabetes. Journal of diabetes and its complications 2004;18: Mortensen HB, Hartling SG, Petersen KE: A nation-wide cross-sectional study of glycosylated haemoglobin in Danish children with type 1 diabetes. Diabetic medicine : a journal of the British Diabetic Association 1988;5: Mortensen HB, Hougaard P, Ibsen KK, Parving HH: Relationship between blood pressure and urinary albumin excretion rate in young Danish type 1 diabetic patients: comparison to non-diabetic children. Danish Study Group of Diabetes in Childhood. Diabetic medicine : a journal of the British Diabetic Association 1994;11: Mortensen HB, Marinelli K, Norgaard K, Main K, Kastrup KW, Ibsen KK, Villumsen J, Parving HH: A nation-wide cross-sectional study of urinary albumin excretion rate, arterial blood pressure and blood glucose control in Danish children with type 1 diabetes mellitus. Danish Study Group of Diabetes in Childhood. Diabetic medicine : a journal of the British Diabetic Association 1990;7: Mortensen HB, Villumsen J, Volund A, Petersen KE, Nerup J: Relationship between insulin injection regimen and metabolic control in young Danish type 1 diabetic patients. The Danish Study Group of Diabetes in Childhood. Diabetic medicine : a journal of the British Diabetic Association 1992;9: Aldington SJ, Kohner EM, Meuer S, Klein R, Sjolie AK: Methodology for retinal photography and assessment of diabetic retinopathy: the EURODIAB IDDM complications study. Diabetologia 1995;38: Wong TY, Knudtson MD, Klein R, Klein BE, Meuer SM, Hubbard LD: Computer-assisted measurement of retinal vessel diameters in the Beaver Dam Eye Study: methodology, correlation between eyes, and effect of refractive errors. Ophthalmology 2004;111: Hubbard LD, Brothers RJ, King WN, Clegg LX, Klein R, Cooper LS, Sharrett AR, Davis MD, Cai J: Methods for evaluation of retinal microvascular abnormalities associated with hypertension/sclerosis in the Atherosclerosis Risk in Communities Study. Ophthalmology 1999;106: Knudtson MD, Lee KE, Hubbard LD, Wong TY, Klein R, Klein BE: Revised formulas for summarizing retinal vessel diameters. Current eye research 2003;27: Anonymous: 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 1991;98: Anonymous: Early Treatment Diabetic Retinopathy Study (ETDRS): Manual of Operations. U.S. Department of Commerce, National Technical Information Service, Giunti S, Barit D, Cooper ME: Mechanisms of diabetic nephropathy: role of hypertension. Hypertension 2006;48: Cheung CY, Ikram MK, Sabanayagam C, Wong TY: Retinal microvasculature as a model to study the manifestations of hypertension. Hypertension 2012;60: Wong TY, Mitchell P: Hypertensive retinopathy. The New England journal of medicine 2004;351:

18 32. Wong TY, Mitchell P: The eye in hypertension. Lancet 2007;369: Chew SK, Xie J, Wang JJ: Retinal arteriolar diameter and the prevalence and incidence of hypertension: a systematic review and meta-analysis of their association. Current hypertension reports 2012;14: Aiello LP, Cahill MT, Wong JS: Systemic considerations in the management of diabetic retinopathy. American journal of ophthalmology 2001;132: de Jong FJ, Vernooij MW, Ikram MK, Ikram MA, Hofman A, Krestin GP, van der Lugt A, de Jong PT, Breteler MM: Arteriolar oxygen saturation, cerebral blood flow, and retinal vessel diameters. The Rotterdam Study. Ophthalmology 2008;115: Stefansson E, Landers MB, 3rd, Wolbarsht ML: Oxygenation and vasodilatation in relation to diabetic and other proliferative retinopathies. Ophthalmic surgery 1983;14: Klein R, Klein BE, Knudtson MD, Wong TY, Tsai MY: Are inflammatory factors related to retinal vessel caliber? The Beaver Dam Eye Study. Archives of ophthalmology 2006;124: Wong TY, Islam FM, Klein R, Klein BE, Cotch MF, Castro C, Sharrett AR, Shahar E: Retinal vascular caliber, cardiovascular risk factors, and inflammation: the multi-ethnic study of atherosclerosis (MESA). Investigative ophthalmology & visual science 2006;47: Nguyen TT, Kawasaki R, Wang JJ, Kreis AJ, Shaw J, Vilser W, Wong TY: Flicker lightinduced retinal vasodilation in diabetes and diabetic retinopathy. Diabetes care 2009;32: Nguyen TT, Kawasaki R, Kreis AJ, Wang JJ, Shaw J, Vilser W, Wong TY: Correlation of lightflicker-induced retinal vasodilation and retinal vascular caliber measurements in diabetes. Investigative ophthalmology & visual science 2009;50: Younes N, Cleary PA, Steffes MW, de Boer IH, Molitch ME, Rutledge BN, Lachin JM, Dahms W: Comparison of urinary albumin-creatinine ratio and albumin excretion rate in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications study. Clinical journal of the American Society of Nephrology : CJASN 2010;5: Cheung N, Wong TY: Predicting risk of diabetic retinopathy from retinal vessel analysis: personalized medicine in transition. Archives of ophthalmology 2012;130:

19 Table 1 Baseline characteristics (1995) of participants and non-participants (declined) in the 16-year follow-up examination in 2011 Baseline characteristic 1995 Participants (N=185) Non-participants (N=63) n Mean ± SD n Mean ± SD p* CRAE (µm) ± ± CRVE (µm) ± ± Age (years) ± ± Duration of diabetes (years) ± ± HbA1c (%) ± ± 1.4 <0.01 HbA1c (mmol/mol) ± ± 16 <0.01 Systolic BP (mmhg) ± ± Diastolic BP (mmhg) ± ± Body mass index (kg/m 2 ) ± ± VPT (Volt) ± ± n % n % p** Sex (male) Albuminuria None (meanaer<20µg/min Micro (meanaer= µg/min) Macro (meanaer>200µg/min) Retinopathy None Non-proliferative Proliferative

20 CRAE = Central retinal arteriolar equivalent; CRVE = Central retinal venular equivalent; VPT = Vibration perception threshold Continuous data are presented as means with standard deviations (SD); categorical as percentages. *Comparison with participants using Mann-Whitney tests ** Comparison with participants using Chi-squared tests 20

21 Table 2 Baseline characteristics in 185 participants in relation to CRAE and CRVE Retinal arteriolar caliber (CRAE in µm) Retinal venular caliber (CRVE in µm) Clinical characteristic n Mean ± SD p* Mean ± SD p* Sex Male ± ±23.7 Female ± ±24.9 Albuminuria None ± ±24.2 Micro ± ±28.7 Macro ± ±33.4 Retinopathy 0.57 <0.02 ETDRS ± ±23.7 ETDRS ± ±24.4 ETDRS ± ±24.6 ETDRS ± ±20.5 ETDRS Retinal arteriolar caliber (CRAE per 1 µm) Retinal venular caliber (CRVE per 1 µm) rho p** rho p** Age (per 1 year) Duration of diabetes (per 1 year) <0.01 HbA1c (per 1%) HbA1c (per 1 mmol/mol) Systolic blood pressure (per 1 mmhg) < <0.05 Diastolic blood pressure (per 1 mmhg) < BMI (per 1 kg/m 2 ) VPT (per 1 Volt) <

22 CRAE = Central retinal arteriolar equivalent; CRVE = Central retinal venular equivalent; VPT = Vibration perception threshold Data are presented as means with standard deviations (SD) when comparing groups. *For comparison between two groups Mann-Whitney tests was used and for comparison between three groups Cuzick tests was used. ** To test for correlations between continuous variables the Spearman rank correlation test was used. 22

23 Table 3 Associations between Central Retinal Arteriolar Equivalent (CRAE), Central Retinal Venular Equivalent (CRVE) and 16-year incidence of microvascular disease among 185 young patients with type 1 diabetes from the DCPD1987 study. Peripheral neuropathy Nephropathy Proliferative DR Change OR (95% CI) p* OR (95% CI) p* OR (95% CI) p* CRAE -10 µm 2.96 ( ) < ( ) < ( ) <0.03 CRVE 10 µm 1.52 ( ) < ( ) < ( ) <0.02 OR = odds ratio; CI = confidence interval; DR = diabetic retinopathy *Calculated using multiple logistic regression models. 23

24 Table 4 Area under receiving operator characteristics curves (AUCs) for multiple logistic regression models on incident peripheral neuropathy, nephropathy and proliferative retinopathy in 185 young patients with type 1 diabetes from the DCPD1987 study. Cutoff values Peripheral neuropathy Nephropathy Proliferative retinopathy AUC p* AUC p* AUC p* CRAE (µm) ** ** CRVE (µm) ** *Comparison between highest and lowest AUC **The variables CRAE 140 and 180 as well as CRVE 280 were omitted due to empty groups in the regression model with nephropathy as outcome. 24

25 Figure 1 The distribution of the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE) in the 185 participants at follow-up and in the 63 people who declined participation. Top left, CRAE in the 185 participants at follow-up in Top right, CRAE in the 63 who declined to participate at follow-up. Bottom left, CRVE in the 185 participants. Bottom right, CRVE in the 63 who declined participation. 25

26 Diabetes Page 26 of x94mm (600 x 600 DPI)