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Arterial Stiffness and Endothelial Dysfunction in Children with Type 1 Diabetes

HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Materials and methods
 Arterial stiffness findings
 Endothelial dysfunction findin...
 Conclusions
 References
 Biographical sketch
 

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1 ARTERIAL STIFFNESS AND ENDOTHELIAL DYSFUNCTION IN CHILDREN WITH TYPE 1 DIABETES By MICHAEL JAMES HALLER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FUFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 By Michael James Haller

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3 To the children who face the challenges of diabetes every day.

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4 ACKNOWLEDGMENTS I thank the members of my supervisor y committee for their mentoring, the many participants in my research st udies, and the Childrens Miracle Network (CMN) and the Diabetes Action Research and Education Foundation (DAR E) for financial support. Additional funding was provided by NIH grants 42288-05 and 3925006 and GCRC grant MO1-RR00082. I thank the Florida Camp for Children and Youth with Diabetes (FCCYD) and Benton Pediatrics for allowing patient recruitment, and Kelvin Lee an d Jennifer Stein for performing radial tonometry and Endo-PAT. I thank my parents for providing unwavering support of my intellectual curiosity and academic interests. I thank my wife for filling my life with joy and meaning every day.

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5 TABLE OF CONTENTS ACKNOWLEGEMENTS LIST OF TABLES...7 LIST OF FIGURES.8 ABSTRACT. CHAPTER 1 INTRODUCTION.........11 2 MATERIALS AND METHODS.......13 Arterial Stiffness Studies... Subjects .....13 Study Protocol Measurement of Arterial S tiffness by Radial Tonometry......15 Measurement of Lipids, HbA1c, Glucose Measurement of Cytokines.... Measurement of Autoantibodies....16 Statistical Considerations ..17 Endothelial Dysfunction Studies ...18 Subjects..18 Study Protocol Measurement of Endothelial Function... Statistical Considerations...20 3 ARTERIAL STI FFNESS FINDINGS...22 Augmentation Index (AI)... Lipids. Blood Pressure...22 Length of Diabetes and Control.22 Cytokines Exercise.. Family History... Gender....23 Discussion.. 4 ENDOTHELIAL DYSFUNCTION FINDINGS...30

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6 Endo-PAT Score....30 Discussion.... 5CONCLUSIONS... APPENDIX LIST OF REFERENCES... BIOGRAPHICAL SKETCH.39

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7 LIST OF TABLES Table Page 3-1 Matched T1D Subjects and Controls in AI Study.....29 3-2 Spearman Correlations in T1D Subjects and Controls in AI Study..29 4-1 Laboratory and Endo-PAT characteristics of cont rols and T1D subjects......33

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8 LIST OF FIGURES Figure Page 2-1 Radial artery tonometry.....20 2-2 Radial artery and corre sponding aortic pulse waves.....21 2-3 Schematic of Endo-PAT in use.......21 4-1 Box plot of Endo-PAT score in controls and subjec ts with Type 1 Diabetes.33

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ARTERIAL STIFFNESS AND ENDOTHELIAL DYSFUNCTION IN CHILDREN WITH TYPE 1 DIABETES By Michael James Haller December 2006 Chair: Marian C. Limacher Major Department: Medical Sciences--Clinical Investigation To determine if children with type 1 diab etes mellitus (T1D) have increased arterial stiffness and endothelial dysfunc tion, we performed two separate case-control studies using radial artery tonometry and augmen tation index to estimate arterial stiffness and peripheral artery tonometry (PAT) and Endo-PAT score to estimate endothelial func tion. In our arterial stiffness study we studied children aged 10-18 years, 98 with T1D and 57 healthy controls matched for age, sex, race, and BMI, generating 43 matched pa irs. Radial artery tonometry was performed under basal resting conditions, immediately befo re a fasting blood sample was collected for analysis of fasting lip ids, HbA1c, glucose, and cytokine s on all children. In our endothelial dysfunction studies, 44 children with T1D (age 14.6 2.7 years; duration of diabetes 6.01 4 years; range of diabetes durati on 1-16 years; HbA1c 8.34% 1.2) and 20 control children (age 14.1 1.5 years) underwent endothe lial function testing after an overnight fast using the EndoPAT finger tip device. Each child had height weight, BMI, blood pr essure, fasting lipid profile, and glucose determin ations. All children with T1D underwent a second Endo-PAT study 4 weeks after their initial st udy in order to determine the in tra-patient variability of the technique.

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10 We determined that children with diabet es had a significantly higher augmentation index corrected to a hear t rate of 75 (AI75) than their matched controls. Mean AI75 in T1D subjects was 1.11.15 versus -3.32.36 in controls. Th e case-control difference was 5.20.02 (p=0.0031). Children with T1D had endothelial dysfunction as evidenced by lower mean EndoPAT scores (1.63 vs 1.95, p=0.01) when compared to control children. Th e mean intra-patient standard deviation of Endo-PAT score in the children with T1D was 0.261. Children with T1D had higher High Densisty Lipopr otein (HDL) cholesterol (p = 0.0001), mean systolic BP (p=0.02), and mean total cholestero l (p=0.03) than control childre n. No significant differences in age, body mass index (BMI), diastolic blood pressure, Low Density Lipoprotein (LDL) cholesterol, or triglycerides we re observed between the children with T1D and control children. Using radial artery tonome try derived measures of arte rial stiffness and Endo-PAT derived measures of endothelial function, we dete rmined that children with T1D have increased arterial stiffness and endothelial dysfunction compar ed to matched controls. Such early arterial abnormalities likely contribute to accelerated prog ression to cardiovascular disease. Future studies will be able to use th ese noninvasive techniques to assess the impact of specific interventions on arterial health in children with T1D. Radial artery tonometry and Endo-PAT are promising noninvasive techniques that can be used to assess arte rial stiffness and endothelial dysfunction in children with T1D. Non-invasive measures like ra dial artery tonometry and EndoPAT may provide additional risk stratification data needed to justify more aggressive primary prevention of CVD in children with T1D.

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11 CHAPTER 1 INTRODUCTION Type 1 diabetes (T1D) is a well established risk factor fo r the development of premature cardiovascular disease (CVD)(1). Despite advances in medical practice over the last 25 years, the incidence of early CVD in the T1D populati on remains disproportionately high (2-5). The Diabetes Control and Complications Tria l (DCCT) and its long itudinal follow up, the Epidemiology of Diabetes Interventions and Co mplications Study (EDIC) have demonstrated that the risk of T1D-related mi crovascular and macrovascular co mplications is related to longterm glycemic control (6; 7). Unfortunately, even with intensive insulin th erapy, the majority of children with T1D are unable to maintain near-normal glycemia. Childre n in the intensive arm of the DCCT were only able to achieve an average HbA1c of 8.1% (8). However, glycemic control is only one of several important risk factors in defining CVD risk. Mini mizing the long term risks for CVD in patients with T1D may require early and aggressive mana gement of other important CVD risk factors such as blood pressure and lipids (9; 10). Ma ny adult studies have de monstrated that the incidence of cardiovascula r events can be lowered through reduc tion of plasma cholesterol levels and optimal management of hypertension. Unfortuna tely, the majority of patients who are being treated aggressively have already mani fested cardiovascular complications. Because children rarely expe rience cardiovascular events, surrogate markers of CVD are needed to provide the additional risk stratificatio n needed to justify and monitor the effects of more aggressive therapy (11). Brachial artery reactivity is a technique that measures the endothelium-dependent dilation of the brachial artery in resp onse to reactive hyperemia. In patients with endothelial dysfunctio n, the ability of the artery to dilate is impaired. Endothelial dysfunction, as measured by decreased brachial react ivity, and arterial stiffness, as measured by

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12 pulse wave analyses, have both been shown to be independent predicto rs of cardiovascular events (12). Impaired brachial reactivity has been demonstrated in adults and in a small group of children with T1D (13; 14). Unfort unately, standard brachial reac tivity studies are difficult to perform, require expensive equipment and involve subjective analysis of th e results. Several new methods for measuring endothelial function and arterial stiffness are now av ailable and may be more applicable for everyday clinical use. To date, studies of arterial stiffness and endothelial function in ch ildren with T1D using these newer techniques have not been perfor med. Because radial tonometry and PAT can be performed in nearly any clinic setting, are easy and affordable to perform, and because they provide the user with an instant analysis of the patients arte rial stiffness or endothelial function, tonometry and PAT have a potential advantage over standard brachial reactivity and carotid IMT as clinically useful tools. To test the utility of radial tonom etry and PAT, we studied whether children with T1D had increased arterial stiffn ess and endothelial dysfunction when compared to healthy controls.

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13 CHAPTER 2 MATERIALS AND METHODS Arterial Stiffness Studies Subjects We evaluated arterial stiffness in 98 ch ildren with type 1 diabetes and 57 control volunteers using radial ar tery tonometry. Children were re cruited from the Florida Diabetes Camp, the University of Florida diabetes and primary care clinics, a nd general pediatrics practices in the area. Children with T1D were recruited by letters sent to the parents of all children registered for diabetes camp offering free lipid and HbA1c analyses in return for participation. Controls were recruited from gene ral pediatrics practices by letters sent to the parents of clinic patients offering free analyses of lipids, glucose, and HbA1c in return for participation. While children with T1D would likely have been routin ely tested for lipids, glucose, and HbA1c, healthy controls would not. Thus, excluding diabetes, there may have been differences in background cardiovascular risk be tween cases and controls as controls whose physician or family perceived them as being at in creased risk may have been more inclined to participate. Inclusion criteria fo r both children with diabetes a nd controls were age between 10 and 18 years, no known cardiovascular disease, and no history of using antihypertensive or lipid lowering medications. Children with diabetes were included only if they had been diagnosed for at least one year. Children were classified as having T1D based on a history of acute onset of polyuria, polydipsia, polyphagia, weight loss, and ketosis. When the hist ory was not clear, islet cell, glutamic acid decarboxylase, or insulin auto antibody status was used to confirm T1D. There were no children with diabetes included that did not have either a well do cumented history or at least one positive diabetes-related autoantibody. From the total group, 43 matched pairs were

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14 identified who met all inclusion criteria. The gr oups were matched for age (yrs), sex, race, and BMI (kg/m2). The study was approved by the Institutional Revi ew Board of the University of Florida. Subjects parents provided written consent before their child was enrolled in the study and the subjects provided assent. Subject s parents completed a brief ques tionnaire that included age, race, medications, family history, and level of ex ercise. Family history was specified in the questionnaire as pertaining to only 1st and 2nd degree relatives of the child, and definitions of hypercholesterolemia, hypertension, and early heart disease were provided. Level of exercise was graded on a 1 to 4 scale (1 = no exercise, 2 = minimal exercise, 3 = m oderate exercise, and 4 = extreme exercise). The most recent HbA1c, duration of diagnosis, and history of recent illnesses was obtained from the medical record. Study Protocol Radial artery tonometry was performed a nd blood samples were obtained between 6 am and 10 am on the same day, with the child supine and relaxed. Study subjec ts were required to fast after midnight and to abstain from ca ffeine for 24 hours before the study. Height was measured on a wall mounted, calibrated stadiomete r (Genentech, San Fran cisco, California) and weight was read from a calibrated digital scale. After a 5 minute rest in the supine position, the subjects had their blood pressure measured w ith a digital oscillometr ic device (Omron model HEM-739, Omron Healthcare Inc., Vernon Hills, Illinois). Radial tonometry was then performed. Following the completion of the ra dial tonometry, a ve nous blood sample was obtained for glucose, HbA1c, total choleste rol, LDL, HDL, triglycerides, hsCRP, IL-1 and IL6. Subjects with T1D had their morning insuli n injection postponed until all studies were completed.

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15 Measurement of Arterial Stiffness by Radial Tonometry Augmentation index (AI) and augmentation i ndex corrected to a h eart rate of 75 (AI75) were measured using the SphygmoCor Vx versio n 7.01 (AtCor Medical, Sydney, Australia). In brief, a high-fidelity micromanometer with a fr equency response of > 2kHz (Millar Instruments, Houston, Texas) was placed on the right radial artery and gentle pressure was applied until a consistent waveform was produced. After 10-20 sequential waveforms had been acquired, the integrated software was used to generate an averaged peri pheral and corre sponding central waveform that was used for the determination of the AI and AI75 (Figure 2-1). The algorithm used to convert the radial pulse wave to an aortic wave form was derived from invasive arterial pressure and flow da ta obtained by cardiac catheterization and has been validated in several adult studies (15-17). Validation studies ar e underway to confirm that the same algorithm can be applied in children. A quality index is displa yed and represents the reproducibility of the waveform. A value greater than 70 is consid ered to demonstrate excellent waveform consistency. For this study, only meas urements with a quality index above 80 were accepted. Two acceptable measurements were obtained on each subject. AI is defined as the difference between the first and second peaks of the central arterial waveform, expressed as a percentage of the pulse pressure and measures th e contribution that the wa ve reflection makes to the arterial pressure waveform. The amplitude a nd timing of the reflected wave depends largely on the stiffness of the small and large arteries. T hus, AI provides a measure of systemic arterial stiffness. AI75 allows for a true comparison of the a ugmentation of central pressure between study subjects by discounting differe nces related to heart rate va riation (18). An elevated or positive AI suggests stiffer arteries than a low or negative AI (Figure 2-2).

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16 Measurement of Lipids, HbA1c, Glucose Serum was collected from study participants using standard venipunc ture techniques and Vacutainer serum separator tubes (BD Biosciences San Diego, CA, USA). After collection, samples to be analyzed for lipid, HbA1c, a nd glucose were immediat ely refrigerated and transported to the Shands Hospital laboratory at the University of Florida. Samples were analyzed in the clinical laborat ory using standard technique. Measurement of Cytokines Serum for cytokine and autoantibody analys is was separated into serial aliquots and frozen at -80 C within 1 hour of the blood draw. All serum analyses were conducted following a single freeze-thaw cycle. Cytokine measurem ents from serum were performed using a commercially available multiplexed kit (Beadlyte Human Multi-Cytokine Detection System 3, Upstate, Lake Placid, NY) and the Luminex100 LabMAPTM System. Quantitative evaluation of the serum cytokines IL-1 and IL-6 was performed. Serum samples were subjected to a 1:2 dilution in serum diluent provided by the manuf acturer in order to reduce the effects of interfering heterophile species (19; 20). High sensitivity CRP (Alpco, Windham NH, USA) levels were measured by standard sandwich ELISA techniques according to manufacturers instructions. Serum analyte concentrations were calculated using 4-parameter analysis utilizing SoftMax Pro Software, Ver. 2.2.1 (Molecula r Devices Corp., Sunnyvale, CA, USA). Measurement of Autoantibodies Autoantibodies against two T1D-associated autoantigens were tested from serum obtained from all study particip ants including thos e against GAD65 and IA-2. Assays were performed as previously described (20). The inve stigators are regular participants in workshops and proficiency tests sponsored by the Immunolo gy of Diabetes Society and CDC to validate

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17 assay performance. At the most recent effo rt (DASP 2003), our performance for GADA assay indicated 80% sensitivit y/95% specificity for type 1 diabet es, while our IA-2A assay provided 64% sensitivity/100% specificity. Statistical Considerations As described above, the study wa s planned as a matched pair design. However, if matching factors are treated as c ovariates all conclusions qualitatively remained the same when analysis was performed on all entr ants, independent of match avai lability. When analyzing the matched pairs, case-control comparisons were as sessed with one sample paired t-tests for the following dependent variables: AI and AI75 (primary) and total cholesterol, HDL, LDL, triglycerides, blood pressure, HbA1c, and glucos e (secondary). All p-values were two-sided. The original study was planned for a sample size of 100 matched pairs. We were unable to recruit the anticipated numbers of matched controls within the planned time-frame due to strict matching criteria and unwillingness of control su bjects to participate in a blood draw, However, the matched variations in the variables AI and AI75 were smaller than originally anticipated, and a retrospective power calculation, using ma tched standard deviations of 11.6 and 11.0 respectively, demonstrates that the actual sample size of 43 matched pairs yields sensitivity to differences in the paired means of 5.6 and 5.3 re spectively at p=0.025 two-si ded and 80% power. As a secondary objective, separate anal yses for associations with AI and AI75 were conducted within controls (n=57) and T1D cases (n=98). Due to the potential for outliers in hsCRP and the qualitative nature of some of the va riables, Spearmans corr elation was utilized to examine the relationship between AI, AI75 and total cholesterol, LDL, HDL, triglycerides, blood pressure, HbA1c, glucose, hsCRP, IL-1 IL6, family history, and exercise regimen.

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18 Endothelial Dysfunction Studies Subjects Forty-four children (22 Male, 22 Female) with T1D for at least 1 year were recruited from the University of Florida pediatric endocrinology clinic fo r this study. Twenty control children (12 Male, 8 Female) we re recruited from the Mayo Clinic, Rochester, MN. Control subjects were non-smokers and community base d, and did not have any co-existing medical conditions or any family history of premature car diovascular disease or hyperlipidemia. All T1D and control patients who participated in this study were Caucasian. Study Protocol Following an overnight fast and using iden tical protocols, endot helial function was assessed in all children using the Endo-PAT device (Itamar Medical Ltd, Caesarea, Israel). Height, weight, and blood pressure were recorded before Endo-PAT testing. Fasting blood work was performed immediately following the Endo-PAT assessment. Following Endo-PAT testing, blood was obtained for lipid profile and glucose determinati ons in all subjects and also for HbA1c determination in children with T1D. Laboratory analysis of the blood samples was performed at the separate sites using identical la boratory platforms. Four weeks after their initial test, T1D subjects had repeat Endo-PAT testing to determine intra-patient variability. Measurement of Endothelial Function Endo-PAT is a non-invasive device that comb ines the traditional flow mediated dilatation technique with pneumatic finger-tip pr obes to measure arterial pulse wave amplitude and provide an objective measure of endothelial function. The Endo-PAT device is an operator independent device that allows affordable and objective measurements of endothelial function and eliminates the need for an ultrasound techni cian or interpretation of the ultrasound signal.

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19 The Endo-PAT system uses the identical arterial occlusive procedure used to induce reactive hyperemia in standard brachial re activity measurements of endothelial function. However, instead of measuring brachial artery diameter, Endo-PAT uses finger-tip plethysmography probes to measure the changes in pulse wave amplitude observed before and after the period of reactive hyperemia. An Endo-PAT score is then calculated to provide a measure of endothelial func tion. In adults, the Endo-PAT score has shown excellent correlation with measures of coronary and peripheral endot helial dysfunction (21; 22). Correlations of EndoPAT score with invasive measures of endothelial function have not been performed in children. Only one previous study has used the Endo-PAT device to demonstrate endothelial dysfunction in children with T1D(11). This stud y was designed to confirm the usefulness of Endo-PAT as a surrogate measure of CVD risk in children with T1D. We expanded the scope of the initial trial by studying a larger group of children with T1D and healthy controls and measuring the reproducibility of Endo-PAT with serial testing. We hypothesized that children with T1D would have endothelial dysfunction (decr eased Endo-PAT score) when compared to healthy controls and that Endo-PAT would have acceptable intra-patient variability. To perform Endo-PAT, the patient sits in a reclining ch air with the hands at heart level and propped in a comfortable position such that the fingers are hanging freely. Fingertip probes are placed on both index fingers and pulse wave amplitudes are recorded for the duration of the study. After 5 minutes of baseline measurement, arterial flow to the non-dominant arm is occluded for 5 minutes using a blood pressure cu ff inflated to 40mmHg a bove systolic pressure. After the 5 minute occlusion, the cu ff is rapidly deflated to allo w for reactive or flow-mediated hyperemia. Pulse wave amplitudes are recorded for at least 5 minutes after the cuff is deflated. The Endo-PAT system compares the ratio of arterial pr essure in the two fingers before and after

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20 the occlusion to calculate the Endo-PAT score. The Endo-PAT score is calculated as the ratio of the average pulse wave amplitude measur ed over 60 seconds starting 1 minute after cuff deflation divided by the average pulse wave ampl itude measured at baseline. This ratio is normalized to the concurrent signal from the cont ra-lateral finger to correct for changes in systemic vascular tone (Figure 2-3). Statistical Considerations The primary endpoint for this study was the Endo-PAT score. HbA1c, LDL, HDL, total cholesterol, triglyceride s, glucose, systolic and diastolic bl ood pressure, and BMI were analyzed as secondary endpoints. The two groups of patien ts (TID and control) were compared using a two-sided two sample t-test. Intra-pa tient standard deviations for Endo-PAT were estimated from the repeated measures and averaged over the pati ents within each subgroup. With a sample of 44 TID patients and 20 controls, a two sided, two-sample t-test had 80% power at P=0.05 to detect a difference of 0.78 sta ndard deviations between the tw o groups. Assuming a standard deviation of 0.4, this corresponds to sensitivity to an Endo-PAT score difference of 0.32 units. Figure 2-1. Radial artery tonometry. A high fidelity tonometer is pl aced on the radial artery until a consistent waveform is generated by the attached software program.

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21 Figure 2-2. Radial artery and corresponding aortic pulse waves. Augmentation index increases with age as arteries stiffen. Figure 2-3. Schematic of Endo-PAT in use. This figure demonstrates the set up and use of the Endo-PAT. The probes are placed on the index fingers of both arms. A reactive hyperemia procedure is performed by occludi ng the brachial artery of one arm for 5 minutes. Data are automatic ally analyzed by the software package and the EndoPAT score is provided. (With permission from Itamar Medical, Ltd.)

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22 CHAPTER 3 ATERIAL STIFFNESS FINDINGS Augmentation Index (AI) Laboratory and tonometry characteristics of the matched diabetes and control groups are shown in Table 3-1. Spearman correlations of AI and AI75 are shown in Table 3-2. T1D was associated with increased arterial stiffness as evidenced by the higher AI75. The mean AI75 in T1D subjects was 1.89.75 whereas the AI75 for controls was -3.32 10.36. Using a paired difference two-sided t-test, the AI75 demonstrated a case-control difference of 5.2.0 (p=0.003). There was no significant differen ce in the uncorrected AI (p=0.37). Lipids Total cholesterol, LDL, and HDL analyses did not reveal any significant case-control differences. Triglyceride levels, however, were significantly higher in the control population with a case-control difference of -25.0.6 (p=0.012). Even when lipid values among only the T1D subjects were analyzed, no significant associ ations were noted between total cholesterol, LDL, HDL, or triglyceri des and either AI or AI75 (Table 2-2). Blood Pressure Systolic and diastolic blood pressure analysis revealed significantly higher values in the controls, with differences of -5.2.6 (p =0.025) and -3.67.9 (p=0.019), respectively. However, no significant associations were noted between systolic or diastolic blood pressure and AI or AI75 (Table 2-2). Length of Diabetes and Control Duration of diabetes, HbA1c, and blood glucose did not demonstrate a significant association with AI or AI75 (Table 2-2). Due to the lack of substantive variation in ages, the

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23 study was not adequately powered to dete rmine if the difference in AI or AI75 between controls and T1D subjects became apparent at a specific age. Cytokines There were no significant a ssociations between AI or AI75 and IL-1, IL-6, or hsCRP (Table 2-2). Exercise Level of exercise was graded on a 1 to 4 s cale (1 = no exercise, 2 = minimal exercise, 3 = moderate exercise, and 4 = extreme exercise). Di fferences in the reported exercise level between T1D cases (2.89) and controls (2.79) were not sta tistically significant. Reported exercise level among T1D cases failed to demonstrate a significant correlation with AI or AI75. Reported exercise level among controls al one did, however, reveal a signifi cant association with both AI (r = -0.44 p<.001) and AI75 (r = -0.33 p=0.015)(Table 2-2). Family History Among T1D cases, family history failed to demonstrate a significant correlation with either AI or AI75. Among controls significant associations were seen between AI and family history of hypercholesterolemi a (r = 0.29, p = 0.030 for AI) and ear ly heart disease (r = 0.38, p = 0.004 for AI, r = 0.42, p = 0.001 for AI75), but not family history of hypertension (Table 2-2). Gender There were 26 male matched pairs and 17 fema le matched pairs for a total of 43 matched pairs in the case-control an alysis. The case-control difference among paired females demonstrated a significant difference in mean AI75. Case-control difference of mean AI75 among paired males did not reveal any significant di fferences. Although the case-control difference between AI75 in matched females reached signifi cance and the difference between matched

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24 males did not, there was no signifi cant difference between males and females. Hence, the overall conclusion should take precedence over the gender-specific conclusion. Discussion This study confirms that children as young as 10 years of age with T1D have increased arterial stiffness when compar ed to matched controls. Althou gh several recent studies present similar findings (14; 23-25), this is the first study to use radial artery tonometry and augmentation index to demonstrate increased arte rial stiffness in children with T1D. Arterial stiffness as a result of endothelial dysfunction is an early sign of cardiovascular disease occuring more often and at an earlier age in patients with T1D compared to those without diabetes. Endothelial dysfunction in T1D is pr imarily due to increased production of advanced glycation end products (AGEs) causing decreased production and action of nitric oxide and a concomitant decrease in arterial compliance ( 26). The resultant endothelial dysfunction allows for premature integration of lipid laden macr ophages in arterial wa lls. In addition, the hyperglycemic environment results in qualitative changes in LDL particle size, oxidation, and glycation that have been implicated in early increases in carotid ar tery IMT and endothelial dysfunction (27; 28). Despite the fact that most T1D patients in reasonable metabolic control have normal cholesterol profiles, decreasing these modified LDLs may be a plausible intervention to reduce cardiovasc ular disease in T1D. Decreas ing LDL values in relatively young T1D patients (average age 34) with norma l initial LDL values resulted in improved endothelial function after just 6 weeks of statin therapy (29). Because management of hyperglycemia is the cornerstone of therapy in T1D, optimal glucose control has been suggested as the primar y target to minimize the risk of macrovascular disease. However, even the DCCTs intensiv e therapy group (average HbA1c of 7.2%), while

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25 showing improved carotid IMT when compared to the conventional treatment group (average HbA1c 9.1%), had significantly increased carotid IMT compared to controls (30; 31). This difference correlated significantly with blood pr essure and cholesterol levels (32). Optimal management of all co-morbidities is needed to minimize risk. Nevertheless, many clinicians believe that di abetes related cardiova scular risk is not sufficiently affected by metabolic derangements in childhood to pursue optimal management of lipids and hypertension in children with T1D. Data from the Pittsburgh Epidemiology of Complications Study, a 10 year follow up of patie nts who developed T1D before the age of 17, showed that high blood pressure and increased LDL were independent risk factors for microvascular disease, macrovascular disease, and mortality (3). Aut opsy studies of over 3000 children found that children have ev idence of aortic fatty streaks as early as 3 years of age and raised fibrous plaques as early as 8 years of age (33; 34).T he Muscatine Heart Study and the Bogalusa Heart Study have demonstrated that ca rdiovascular risk fact ors apparent in childhood predict future coronary artery disease (35; 36). Radial artery tonometry is an easily lear ned, affordable, noninvasive, reproducible and accurate technique that can be used to monito r arterial stiffness and, therefore, future cardiovascular risk in both high-risk children and adults (13; 37-39). Ra dial artery tonometry has been shown to have excellent intra-observer and intra-patient reproducib ility and to provide a reliable assessment of endothelial dysfunction (40; 41). In adults with T1D, augmentation index is increased prematurely (38). This is the first study to evaluate radial artery tonometry in children with T1D. Although we were unable to show any si gnificant correlations between AI in children with T1D and established cardiovascular risk fact ors, it is possible that the T1D children with the most elevated

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26 AIs might have demonstrated correlations if th e study had been adequately powered to evaluate these relationships. Nevertheless, we did demonstrate a clear difference in AI75 between controls and subjects with T1D. While the exact mechanis ms remain unclear, the observed difference in AI75 documents that there are iden tifiable abnormalities in vessel sti ffness in children with T1D. Using a combination of flow-mediated dilata tion and carotid IMT, Jrvisalo, et al. and Singh et al., demonstrated endothe lial dysfunction in children w ith T1D (14; 23). Both studies revealed correlations with endothelial dysfunction and LDL cholesterol. The two studies disagreed, however, when describing the relation ship between increased carotid IMT and T1D. While the disparate findings may be explained by di fferences in technique, it is possible that the differences are related to the age and pubertal st atus of the patient populations. The average age of the T1D children in the two studies was 11 and 15 years, respectively. Although endothelial dysfunction is present in young ch ildren with T1D, perhaps the correlations with endothelial function and other cardiovascular risk f actors do not become apparent after puberty. In our study, the average age of the T1D ch ildren was 12.9 years. The differences in age and pubertal status, in addition to the different methods used to measure endothelial function, could explain differences in between various stud ies. Unfortunately, the age distribution of our study population was too narrow to determine at wh at age the difference between controls and children with T1D becomes apparent. Studies wi th broader age distribu tions are needed to determine the effects of age and puberty. Adult and pediatric studie s of endothelial dysfunction in diabetes patients have demonstrated significant associations with high LDL, low HDL, hypertension, glycemic control, duration of disease, folic acid status, exercise regimen, and gender (13; 23; 24; 42). Although we did demonstrate that T1D was associated with incr eased arterial stiffness, this study did not find

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27 any significant associations with lipids, blood pressure, glycemic control, cytokines, family history, or gender in the childre n with diabetes. The inabilit y to demonstrate a correlation between established cardiovascular risk factors and increased AI75 in children with diabetes is most likely related to the small sample size, na rrow age range and lack of longitudinal follow-up. Nonetheless, the risk factors measured are well established as being rele vant to the development of atherosclerosis. In children, however, it may be necessary to measure variables that may be more directly related to arterial stiffness such as AGEs, qualitative lipids, nitrite, and superoxide dismutase. In control children, we found a significant a ssociation between elev ated AI and family history of hypercholesterolemia. This may ha ve represented a volunteer bias as the control families who volunteered for the study were more likely to have a positive family history of heart disease. This could explain why the control children had signifi cantly higher triglycerides and blood pressures than the T1D subjects. A bias to wards increased cardiovascul ar risk in controls would have decreased the difference in AI75, between controls and children with diabetes. Nonetheless, T1D children demonstrated greater arte rial stiffness than controls. That higher exercise levels were associated with decreased arte rial stiffness in the controls but not in the T1D subjects suggests that the metabo lic derangements of diabetes obscu re some of the advantages of exercise. Despite the novel findings provi ded by this study, there are se veral important limitations. The Sphygmocor Vx software used to analyze the wave reflections has not been fully validated in children. The transfer function used to calcula te the aortic pressure wave was validated using directly measured aortic and radial pressure waves in adults. While ag e and AI are linearly associated in adults, such that the same transf er function could be used at all ages, no childhood

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28 data are yet available to confirm the transf er functions validit y. Although case-control comparisons eliminated the need for known age adjusted normal values, the argument could be made that the transfer function may not accurately determine aortic pressure waves in children. In a recent study of adults with type 2 diabetes, Hope et al. suggested th at generalized transfer functions may not be valid at all in patients with diabetes (43). While their concerns about the applicability of transfer functions to specific populations is valid, they also found no significant differences between directly m easured and transfer function de rived AI in their diabetes population. The strict matching criteria that were employed resulted in a lower than expected total number of matches. With a la rger study, associations between AI and lipids, blood pressure, and cytokines might have been uncovered. Another weakness of this study was the omission of Tanner staging which may have re sulted in significant pubertal di fferences between age-matched pairs. Pubertal status may be an important de terminant in deciphering when children with T1D develop increased arterial stiffness and should be included in all future studies. Finally, the failure to include albuminuria as a study variable is a recognized limitation. Several other studies have demonstrated the importance of microalbum inuria in predicting atherosclerosis in T1D patients (44). Despite these limitations, the study does provi de additional evidence that children with T1D have endothelial dysfuncti on. Further studies are needed to confirm the relationship between arterial stiffness and other modifiable ca rdiovascular risk factors. Future interventional studies should be developed to provide longitudinal comparisons of optimal blood pressure, lipid, and glucose control with e ndothelial function in T1D children.

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29 Table 3-1. Matched T1D Subject s and Controls in AI Study. (n=43) Means, standard deviations, paired differences of the means, and p values are shown. T1D Controls Mean SD Mean SD Paired Difference SD P value AI 1.11 10.15 -0.47 9.791.59 11.61 0.37 AI75 1.88 10.75 -3.31 10.365.20 11.02 0.0031 HbA1c 8.41 1.29 5.2 0.253.17 1.23 <0.0001 T Cholesterol 151.19 29.46 152.66 36.1-5.12 35.72 0.35 Triglycerides 61.05 26.32 89.36 54.72-25.0 62.60 0.012 HDL 56.67 8.2 53.09 12.264.05 15.43 0.093 LDL 85.18 34.1 86.02 25.25-4.09 32.31 0.41 Glucose 159.67 68.78 85.22 9.5875.89 70.18 <0.001 SBP 109.93 13.6 115.02 10.31-5.09 14.51 0.025 DBP 67.45 8.8 71.18 7.96-3.73 9.80 0.015 Table 3-2. Spearman Correlations in T1D S ubjects and Controls in AI Study. Spearman values and p-values are shown for AI a nd AI75 in all T1D (n=98) subjects and Controls (n=57). T1DM (n=98) Controls (n=57) AI (p value) AI75 (p value) AI (p value) AI75 (p value) HbA1c 0.076 (0.46) 0.089 (0.39) 0.060 (0.66) 0.094 (0.49) T Cholesterol 0.055 (0.60) 0.056 (0.59) 0.150 (0.27) 0.198 (0.14) Triglycerides 0.096 (0.35) 0.061 (0.55) 0.018 (0.89) 0.046 (0.73) HDL -0.027 (0.80) 0.086 (0.41) 0.192 (0.15) 0.126 (0.35) LDL 0.062 (0.54) -0.013 (0.89) 0.071 (0.16) 0.076 (0.58) Glucose 0.164 (0.11) 0.132 (0.20) 0.004 (0.97) -0.0004 (0.997) SBP -0.132 (0.20) -0.144 (0.15) -0.289 (0.03) -0.169 (0.21) DBP 0.094 (0.36) 0.094 (0.35) -0.059 (0.66) 0.058 (0.67) IL-1 -0.190 (0.06) -0.079 (0.44) 0.210 (0.12) 0.010 (0.46) IL-6 -0.150 (0.14) -0.126 (0.22) -0.152 (0.26) -0.074 (0.59) hsCRP -0.005 (0.96) 0.004 (0.96) 0.059 (0.66) 0.133 (0.33) FH of HTN 0.120 (0.24) 0.149 (0.14) -0.114 (0.44) -0.066 (0.63) FH of Early CVD -0.031 (0.44) -0.078 (0.45) 0.345 (0.0086) 0.381 (0.0034) FH of Cholesterol -0.132 (0.20) -0.101 (0.33) 0.303 (0.02) 0.218 (0.10) Exercise 0.027 (0.79) 0.032 (0.76) -0.346 (0.0083) -0.449 (0.0005) Years of Diabetes 0.041 (0.44) 0.080 (0.44) N/A N/A

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30 CHAPTER 4 ENDOTHELIAL DYSFUNCTION FINDINGS Endo-PAT Score The summary statistics for the comparisons are presented in Table 4-1. The mean intrapatient standard deviation of Endo-PAT in the T1D patients was 0.261. Children with T1D (n=44) had endothelial dysfunction as evidenced by lower mean Endo-PAT scores (1.63 vs 1.95, p=0.01) when compared to control childre n (n=20). However, the range of Endo-PAT scores was wide. Children with T1D had a range of Endo-PAT scores of 1.01 to 3.01 while control children had a range of 1.5 to 2.5 (Fi gure 4-1). Children with T1D had higher mean systolic BP (p=0.02), mean total cholesterol (p=0.03), and mean HDL (p=0.0001) than control children. It was not clear why there were such large differen ces in blood pressure or HDL between the two groups. No signifi cant differences in age, BMI, diastolic blood pressure, LDL, or triglycerides were observed between th e children with T1D a nd control children. Discussion This study confirms that Endo-PAT can reliably evaluate endothelial function in children with T1D. Significant differences in Endo-PAT score, consistent with endothelial dysfunction, were observed between children with T1D and controls. Several characteristics of our study population warrant furt her comment. The two study populations compared were of similar age and gende r, racially homogeneous, and tested using an identical protocol and technique despite the fact that they derived from different locations in North America (Florida and Minnesota). Our T1D population had higher systolic BP, HDL, and total cholesterol. In addition to the hyperglyce mia associated with T1D, these important CVD risk factors could explain some of the difference in Endo-PAT score between the two groups. We must accept, therefore, that the difference in endothelial dysfunction seen in the two groups

PAGE 31

31 may be partially explained by selection bias a nd not by CVD risk associated solely with the disease state of T1D. In a ddition, our study was not designed to determine the relative importance of glycemic control, blood pressure, cholesterol, BMI, family history, or any other CVD risk factors on Endo-PAT score. Additional studies will be needed to furt her investigate the relative importance of other traditional CVD risk factors on Endo-PAT score. Finally, EndoPAT has not yet been used to eval uate the risk of future cardi ovascular events in adult or pediatric populations. Despite the limitations of this pa rticular study, we be lieve that Endo-PAT has several potential advantages over tr aditional reactive hy peremia measurements. Specifically, EndoPAT testing is affordable, reproducible, and operato r independent. Therefor e, the procedure is not subject to the subjective inte rpretations of blood vessel diam eter associated with brachial artery ultrasound imaging methods. While larger studies are need ed to confirm the association between low Endo-PAT score and traditional measures of endothelial dysfunction, Endo-PAT may be useful as both a research and clinical tool in assessing e ndothelial function in populations at high risk for developing premature CVD. Recent Epidemiology of Diabetes Interven tions and Complicaitons (EDIC) study data demonstrate that intensive diabetes management significantly decreases cardiovascular events amongst patients with T1D (7). Unfortunately, th e overall risk of CVD in T1D patients remains disproportionately high. Because T1D is still asso ciated with a 2-3 fold increased risk of premature CVD, further efforts are needed to accu rately identify and trea t those patients at high risk for CVD. CVD is a process rooted in early childhood. A multitude of studies now demonstrate that risk factors pr esent in childhood pred ict cardiovascular ev ent rates in adulthood (33; 35). Furthermore, autopsy studies have show n that permanent plaques associated with CVD

PAGE 32

32 appear in the arteries of children as young as 8 years of age (36). Still, outside of striving for tight glycemic control in children with T1D, many pediatric endocrino logists struggle with which measures to adopt to appropriately identify and manage comorbidities associated with CVD risk (45). The average HbA1c in our T1D study populat ion (8.34%) approached the HbA1c of the adolescents in the intensive ther apy arm of the DCCT and yet e ndothelial dysfunction was easily identified in our T1D study population. Thus, until technological advances provide for marked improvements in glycemic control fo r all children with T1D, we are likely to continue to observe average HbA1c levels well above those that elimin ate risk of complicati ons even in relatively compliant patients. Similarly, while slightly hi gher than those seen in the control population, the average LDL cholesterol (LDL) in our T1D population (8 7.6 mg/dl) and systolic blood pressure (BP) (109 mmHg) easily met American Diabetes Associ ation and American Hear t Association goals. Recent data have demonstrated better endothelial func tion in adults with CVD who have LDL levels < 80 when compared to those with LD L between 80-100mg/dl (46). Thus, adequate primary prevention of CVD in patients with T1 D may require both tight glycemic control and aggressive (i.e. pharmacologic) management of li pids and blood pressure to levels lower than those currently labeled as normal. Furthermore, to truly minimize the risk of CVD in patients with T1D, initiation of statins and ACE inhib itors may be needed shortly after diagnosis regardless of cholesterol and blood pressure values. While prospective studies are needed to determine if more aggressive management of lipids and blood pressure can d ecrease the CVD risk associated with T1D, non-invasive measures of vascular function such as Endo-PAT may provide the additional level of risk

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33 stratification needed to jus tify early initiation of pharmaco logic lipid and blood pressure lowering therapy in children with T1D. Table 4-1. Laborat ory and Endo-PAT characteristics of controls and T1D subjects. Characteristic Diabetes (n=44) Controls (n=20) P Value Endo-PAT Score 1.64 0.5 1.95 0.3 0.01 Mean intra-subject SD 0.261 --Age (years) 14.6 1.5 14.1 1.5 0.41 BMI (kg/m2) 22.6 2.7 21.5 2.6 0.34 Systolic BP (mmHg) 109.5 10 103.9 6.3 0.02 Diastolic BP (mmHg) 69.6 9.6 69.3 4.9 0.87 Glucose (mg/dl) 200.1 89.7 86.4 11.5 0.0001 HbA1c (%) 8.34 1.2 --Total cholesterol (mg/dl) 166.4 35 147.7 20 0.03 LDL(mg/dl) 87.6 26 78.1 21 0.16 HDL (mg/dl) 61.7 12.9 38.9 11.1 0.0001 Triglycerides (mg/dl) 85.5 58.7 68.6 25.8 0.22 Figure 4-1. Box Plot of Endo-PAT Score in Controls a nd Subjects with Type 1 Diabetes. ThisThe box plot demonstr ates a significantly lower Endo-PAT score in the 44 children with type 1 diabetes when compared to the 20 control children. Although there was a wide range of Endo-PAT scores, the lower Endo-PAT score is indicative of relativ e endothelial dysfunction in the cohort with type 1 diabetes.

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35 CHAPTER 5 CONCLUSIONS The risk of recurrent cardiovascular events is higher in T1D patients than in nondiabetic patients. Well established data document that the ear ly anatomical signs of atherosclerosis appear in childhood. Nevertheless, current guidelines fo r treating lipids and blood pressure in children with T1D may be underestimating the utility of more aggressive primary prevention. Noninvasive techniques such as radial tonometry and Endo-PAT can be used to demonstrate abnormal arterial stiffness and endothelial functio n in children with T1D. These non-invasive techniques may provide the extra tier of cardiovascular risk stratification information needed to design optimal cardiovascular risk reduction strategies in children with T1D.

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36 LIST OF REFERENCES 1. Laing SP, Swerdlow AJ, Slater SD, Botha JL, Burden AC, Waugh NR Smith AW, Hill RD, Bingley PJ, Patterson CC, Qiao Z, Keen H: Th e British Diabetic Association Cohort Study, I: all-cause mortality in patients with insulin-treated diabetes mellitus. Diabet Med 16:459-465, 1999 2. Pambianco G, Costacou T, Ellis D, Becker DJ, Klein R, Orchard TJ: The 30-year natural history of type 1 diabetes complications: the P ittsburgh Epidemiology of Diabetes Complications Study experience. Diabetes 55:1463-1469, 2006 3. Orchard TJ, Forrest KY, Kuller LH, Becker DJ : Lipid and blood pressure treatment goals for type 1 diabetes: 10-year incidence data fr om the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetes Care 24:1053-1059, 2001 4. The absence of a glycemic threshold for th e development of long-term complications: the perspective of the Diabetes C ontrol and Complications Trial. Diabetes 45:1289-1298, 1996 5. Soedamah-Muthu SS, Fuller JH, Mulnier HE Raleigh VS, Lawrenson RA, Colhoun HM: High risk of cardiovascular diseas e in patients with type 1 diab etes in the U.K.: a cohort study using the general practice research database. Diabetes Care 29:798-804, 2006 6. Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus. Jama 287:2563-2569, 2002 7. Nathan DM, Cleary PA, Backlund JY, Genuth SM, Lachin JM, Orchard TJ, Raskin P, Zinman B: Intensive diabetes treatment and cardiovascul ar disease in patients with type 1 diabetes. N Engl J Med 353:2643-2653, 2005 8. Effect of intensive diabet es treatment on the developmen t and progression of long-term complications in adolescents with insulin-depen dent diabetes mellitus: Diabetes Control and Complications Trial. Diab etes Control and Complicat ions Trial Research Group. J Pediatr 125:177-188, 1994 9. Soedamah-Muthu SS, Stehouwer CD: Cardiovasc ular disease morbidity and mortality in patients with type 1 diabetes mellitus: management strategies. Treat Endocrinol 4:75-86, 2005 10. Haller MJ, Samyn M, Nichols WW, Brusko T, Wasserfall C, Schwartz RF, Atkinson M, Shuster JJ, Pierce GL, Silverstein JH: Radial artery tonometry dem onstrates arterial stiffness in children with type 1 diabetes. Diabetes Care 27:2911-2917, 2004 11. Mahmud F, Earing, MG, Lee, RA, Lteif, AN, Driscoll, DJ, and A Lerman: Altered Endothelial Function in Asymptomatic MA le Adolescents with Type 1 Diabetes. Congenital Heart Disease 1:98-103, 2006

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37 12. Bonetti PO, Lerman LO, Lerman A: Endothelia l dysfunction: a marker of atherosclerotic risk. Arterioscler Thromb Vasc Biol 23:168-175, 2003 13. Clarkson P, Celermajer DS, Donald AE, Sampson M, Sorensen KE, Adams M, Yue DK, Betteridge DJ, Deanfield JE: Impaired vascular reactivity in insulin-dep endent diabetes mellitus is related to disease duration and low density lipoprotein cholesterol levels. J Am Coll Cardiol 28:573-579, 1996 14. Singh TP, Groehn H, Kazmers A: Vascular func tion and carotid intima l-medial thickness in children with insulin-dependent diabetes mellitus. J Am Coll Cardiol 41:661-665, 2003 15. Chen CH, Nevo E, Fetics B, Pak PH, Yin FC Maughan WL, Kass DA: Estimation of central aortic pressure waveform by mathematical transformation of radial tonometry pressure. Validation of generali zed transfer function. Circulation 95:1827-1836, 1997 16. Karamanoglu M, O'Rourke MF, Avolio AP, Ke lly RP: An analysis of the relationship between central aortic and peripheral upper limb pressure waves in man. Eur Heart J 14:160167, 1993 17. Pauca AL, O'Rourke MF, Kon ND: Prospec tive evaluation of a method for estimating ascending aortic pressure from the radial artery pr essure waveform. Hypertension 38:932-937, 2001 18. Wilkinson IB, MacCallum H, Flint L, Cockcr oft JR, Newby DE, Webb DJ: The influence of heart rate on augmentation index and cen tral arterial pressure in humans. J Physiol 525 Pt 1:263270, 2000 19. Boscato LM, Stuart MC: Heterophilic an tibodies: a problem for all immunoassays. Clin Chem 34:27-33, 1988 20. She JX, Ellis TM, Wilson SB, Wasserfall CH, Marron M, Reimsneider S, Kent SC, Hafler DA, Neuberg DS, Muir A, Strominger JL, Atki nson MA: Heterophile an tibodies segregate in families and are associated with protection from type 1 diabetes. Proc Natl Acad Sci U S A 96:8116-8119, 1999 21. Kuvin JT, Patel AR, Sliney KA, Pandian NG, Sheffy J, Schnall RP, Karas RH, Udelson JE: Assessment of peripheral vascular endothelial func tion with finger arterial pulse wave amplitude. Am Heart J 146:168-174, 2003 22. Bonetti PO, Pumper GM, Higano ST, Holmes DR, Jr., Kuvin JT, Lerman A: Noninvasive identification of patients with early coronary at herosclerosis by assessm ent of digital reactive hyperemia. J Am Coll Cardiol 44:2137-2141, 2004 23. Jarvisalo MJ, Raitakari M, Toikka JO, Putto-Laurila A, Rontu R, Laine S, Lehtimaki T, Ronnemaa T, Viikari J, Raitakari OT: Endothe lial dysfunction and increased arterial intimamedia thickness in children with type 1 diabetes. Circulation 109:1750-1755, 2004

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38 24. Wiltshire EJ, Gent R, Hirte C, Pena A, Thomas DW, Couper JJ: Endothelial dysfunction relates to folate status in children and adolescents with type 1 diabetes. Diabetes 51:2282-2286, 2002 25. Wilkinson IB, MacCallum H, Rooijmans DF Murray GD, Cockcroft JR, McKnight JA, Webb DJ: Increased augmentation index and systolic stress in type 1 diabetes mellitus. Qjm 93:441-448, 2000 26. Farkas K, Jermendy G, Herold M, Ruzicska E, Sasvari M, Somogyi A: Impairment of the NO/cGMP Pathway in the Fasting and Postpran dial State in Type 1 Diabetes Mellitus. Exp Clin Endocrinol Diabetes 112:258-263, 2004 27. Raitakari OT, Pitkanen OP, Lehtimaki T, Lahde npera S, Iida H, Yla-Herttuala S, Luoma J, Mattila K, Nikkari T, Taskinen MR, Viikari JS, Knuuti J: In vivo low density lipoprotein oxidation relates to coronary reactivity in young men. J Am Coll Cardiol 30:97-102, 1997 28. Toikka JO, Niemi P, Ahotupa M, Niinikoski H, Viikari JS, Ronnemaa T, Hartiala JJ, Raitakari OT: Large-artery elastic properties in young men: relationships to serum lipoproteins and oxidized low-density lipoproteins. Arterioscler Thromb Vasc Biol 19:436-441, 1999 29. Mullen MJ, Wright D, Donald AE, Thorne S, Thomson H, Deanfield JE : Atorvastatin but not L-arginine improves endothelial function in type I diabetes mellitus: a double-blind study. J Am Coll Cardiol 36:410-416, 2000 30. Nathan DM, Lachin J, Cleary P, Orchard T, Brillon DJ, Backlund JY, O'Leary DH, Genuth S: Intensive diabetes therapy and carotid intim a-media thickness in type 1 diabetes mellitus. N Engl J Med 348:2294-2303, 2003 31. Effect of intensive diabetes treatment on caro tid artery wall thickness in the epidemiology of diabetes interventions and co mplications. Epidemiology of Diabetes Interventions and Complications (EDIC) Research Group. Diabetes 48:383-390, 1999 32. The effect of intensive treatment of diab etes on the development and progression of longterm complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 329:977-986, 1993 33. Berenson GS, Srinivasan SR, Bao W, Newman WP, 3rd, Tracy RE, Wattigney WA: Association between multiple cardiovascular risk factors and atherosclerosis in children and young adults. The Bogalusa Heart Study. N Engl J Med 338:1650-1656, 1998 34. Wissler RW: An overview of th e quantitative influence of seve ral risk factors on progression of atherosclerosis in young pe ople in the United States. Path obiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Am J Med Sci 310 Suppl 1:S29-36, 1995

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39 35. Davis PH, Dawson JD, Riley WA, Lauer RM: Ca rotid intimal-medial thickness is related to cardiovascular risk factors measured from childhood through middle age: The Muscatine Study. Circulation 104:2815-2819, 2001 36. Li S, Chen W, Srinivasan SR, Bond MG Tang R, Urbina EM, Berenson GS: Childhood cardiovascular risk factors and carotid vascular changes in ad ulthood: the Bogalusa Heart Study. Jama 290:2271-2276, 2003 37. Rosenthal DN, Chin C: Brachial artery reactivity : A modified technique with applicability to children. J Am Soc Echocardiogr 12:850-852, 1999 38. Nichols WW, Singh BM: Augmentation index as a measure of peripheral vascular disease state. Curr Opin Cardiol 17:543-551, 2002 39. Wilkinson IB, Fuchs SA, Jansen IM, Spra tt JC, Murray GD, Cockcroft JR, Webb DJ: Reproducibility of pulse wave velocity and augmentation index measured by pulse wave analysis. J Hypertens 16:2079-2084, 1998 40. Siebenhofer A, Kemp C, Sutton A, Williams B: The reproducibility of central aortic blood pressure measurements in healthy su bjects using applanation tonometry and sphygmocardiography. J Hum Hypertens 13:625-629, 1999 41. Lind L, Pettersson K, Johansson K: Analysis of endothelium-depende nt vasodilation by use of the radial artery pulse wave obtained by applanation tonometry. Clin Physiol Funct Imaging 23:50-57, 2003 42. Schram MT, Chaturvedi N, Schalkwijk C, Gi orgino F, Ebeling P, Fuller JH, Stehouwer CD: Vascular risk factors and mark ers of endothelial function as determinants of inflammatory markers in type 1 diabetes: the EUR ODIAB Prospective Complications Study. Diabetes Care 26:2165-2173, 2003 43. Hope SA, Tay DB, Meredith IT, Cameron JD: Use of arterial transfer functions for the derivation of central aortic waveform characteri stics in subjects with type 2 diabetes and cardiovascular disease. Diabetes Care 27:746-751, 2004 44. Frost D, Friedl A, Beischer W: Determinants of early carotid atherosclerosis progression in young patients with type 1 diabetes mellitus. Exp Clin Endocrinol Diabetes 110:92-94, 2002 45. Schwab KO, Doerfer J, Hecker W, Grulic h-Henn J, Wiemann D, Kordonouri O, Beyer P, Holl RW: Spectrum and prevalence of atherogeni c risk factors in 27,358 children, adolescents, and young adults with type 1 diabetes: crosssectional data from the German diabetes documentation and quality management system (DPV). Diabetes Care 29:218-225, 2006 46. Kuvin JT, Patel AR, Sliney KA, Pandian NG, Karas RH: Comparison of flow-mediated dilatation of the brachial artery in coronary patients with low-density lipoprotein cholesterol levels <80 mg/dl versus patients with levels 80 to 100 mg/dl. Am J Cardiol 95:93-95, 2005

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40 BIOGRAPHICAL SKETCH Michael James Haller, M.D., was born on Ma rch 28, 1975, in Gainesville, Florida. He graduated from Gainesville High School and th en pursued his undergra duate degree at Duke University. After completing his B.S. in Biology at Duke University in 1996, Michael returned to Gainesville to attend medical school at the University of Florid a. After completing his medical school training in 2000, Michael completed both a pediatrics residency and a pediatric endocrinology fellowship at the University of Florida before accepting a position in 2006 as an assistant professor of pediatric endocrinology at the University of Florida. Michaels current research focuses on the prevention and cure of Type 1 diabetes and the primary prevention of cardiovascular disease in child ren with Type 1 diabetes. Michael is married to Allison Sherman Haller, M.D.


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Table of Contents
    Title Page
        Page 1
        Page 2
    Dedication
        Page 3
    Acknowledgement
        Page 4
    Table of Contents
        Page 5
        Page 6
    List of Tables
        Page 7
    List of Figures
        Page 8
    Abstract
        Page 9
        Page 10
    Introduction
        Page 11
        Page 12
    Materials and methods
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
    Arterial stiffness findings
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
    Endothelial dysfunction findings
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
    Conclusions
        Page 35
    References
        Page 36
        Page 37
        Page 38
        Page 39
    Biographical sketch
        Page 40
Full Text











ARTERIAL STIFFNESS AND ENDOTHELIAL DYSFUNCTION
IN CHILDREN WITH TYPE 1 DIABETES











By

MICHAEL JAMES HALLER


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2006



























Copyright 2006

By

Michael James Haller


























To the children who face the challenges of diabetes every day.


















ACKNOWLEDGMENTS

I thank the members of my supervisory committee for their mentoring, the many

participants in my research studies, and the Children's Miracle Network (CMN) and the Diabetes

Action Research and Education Foundation (DARE) for financial support. Additional funding

was provided by NIH grants 42288-05 and 39250-06 and GCRC grant MO1-RR00082. I thank

the Florida Camp for Children and Youth with Diabetes (FCCYD) and Benton Pediatrics for

allowing patient recruitment, and Kelvin Lee and Jennifer Stein for performing radial tonometry

and Endo-PAT. I thank my parents for providing unwavering support of my intellectual

curiosity and academic interests. I thank my wife for filling my life with joy and meaning every

day.










TABLE OF CONTENTS


ACKNOWLEGEMENTS ..................................................................... ........... 4

L IST O F TA B L E S ................................. ........................................................ 7

LIST OF FIGURES ............................................................................ ........... 8

A B S T R A C T .................................................................................................... 9

CHAPTER

1 INTRODUCTION................................................................... .............. 11

2 MATERIALS AND METHODS........................................ ......... ... ........ 13

Arterial Stiffness Studies ............................................................ .......... 13
Subjects ....................................................................... . ..... 13
Study Protocol ..................................... .. .................... .......... 14
Measurement of Arterial Stiffness by Radial Tonometry.......................... 15
Measurement of Lipids, HbAlc, Glucose .......................................... 16
Measurement of Cytokines .................................. .............................. 16
Measurement of Autoantibodies...................................................16
Statistical Considerations ................................. ................. .......... 17
Endothelial Dysfunction Studies ................................. ......................... ... 18
Subjects ....................................................................... . ..... 18
Stu dy P rotocol ........................................... ................ .... .. ......... 18
Measurement of Endothelial Function................................................ 18
Statistical Considerations ................................ ............... ......... ...20

3 ARTERIAL STIFFNESS FINDINGS..................................................... 22

Augmentation Index (AI) ........................................................ .......... 22
L ip id s ................................................................................................. 2 2
Blood Pressure ....................................................................... ........... 22
Length of Diabetes and Control .................................................... .......... 22
C y to k in e s ............................................................................................. 2 3
E x e rc ise ............................................................................................... 2 3
Family History ....................................................... ................. .......... 23
G e n d e r .................................... ............................................................. 2 3
D iscu ssio n ....................................................................................... ..... 2 4

4 ENDOTHELIAL DYSFUNCTION FINDINGS.............................................30









Endo-PAT Score .................................................................................. .. 30
Discussion .............................................................................................30

5 CONCLUSIONS ..................................................................... .......... 34

APPENDIX

LIST OF REFERENCES .................................................................................. 35

BIOGRAPHICAL SKETCH ............................................................................ 39












































6










LIST OF TABLES


Table Page

3-1 M watched T1D Subjects and Controls in AI Study................................... .............29

3-2 Spearman Correlations in T1D Subjects and Controls in AI Study......................... 29

4-1 Laboratory and Endo-PAT characteristics of controls and T1D subjects...................33










LIST OF FIGURES


Figure Page


2-1 R adial artery tonom etry .............. ..................................... ............2............20

2-2 Radial artery and corresponding aortic pulse waves............................................21

2-3 Schematic of Endo-PAT in use............................................................. 21

4-1 Box plot of Endo-PAT score in controls and subjects with Type 1 Diabetes...............33









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ARTERIAL STIFFNESS AND ENDOTHELIAL DYSFUNCTION
IN CHILDREN WITH TYPE 1 DIABETES

By

Michael James Haller

December 2006

Chair: Marian C. Limacher
Major Department: Medical Sciences--Clinical Investigation

To determine if children with type 1 diabetes mellitus (T1D) have increased arterial

stiffness and endothelial dysfunction, we performed two separate case-control studies using

radial artery tonometry and augmentation index to estimate arterial stiffness and peripheral artery

tonometry (PAT) and Endo-PAT score to estimate endothelial function. In our arterial stiffness

study we studied children aged 10-18 years, 98 with T1D and 57 healthy controls matched for

age, sex, race, and BMI, generating 43 matched pairs. Radial artery tonometry was performed

under basal resting conditions, immediately before a fasting blood sample was collected for

analysis of fasting lipids, HbAlc, glucose, and cytokines on all children. In our endothelial

dysfunction studies, 44 children with T1D (age 14.6 2.7 years; duration of diabetes 6.01 4

years; range of diabetes duration 1-16 years; HbAlc 8.34% + 1.2) and 20 control children (age

14.1 + 1.5 years) underwent endothelial function testing after an overnight fast using the Endo-

PAT finger tip device. Each child had height, weight, BMI, blood pressure, fasting lipid

profile, and glucose determinations. All children with T1D underwent a second Endo-PAT

study 4 weeks after their initial study in order to determine the intra-patient variability of the

technique.











We determined that children with diabetes had a significantly higher augmentation index

corrected to a heart rate of 75 (Al75) than their matched controls. Mean Al75 in T1D subjects was

1.11+10.15 versus -3.3210.36 in controls. The case-control difference was 5.20+11.02

(p=0.0031). Children with T1D had endothelial dysfunction as evidenced by lower mean Endo-

PAT scores (1.63 vs 1.95, p=0.01) when compared to control children. The mean intra-patient

standard deviation of Endo-PAT score in the children with T1D was 0.261. Children with T1D

had higher High Densisty Lipoprotein (HDL) cholesterol (p= 0.0001), mean systolic BP

(p=0.02), and mean total cholesterol (p=0.03) than control children. No significant differences in

age, body mass index (BMI), diastolic blood pressure, Low Density Lipoprotein (LDL)

cholesterol, or triglycerides were observed between the children with T1D and control children.

Using radial artery tonometry derived measures of arterial stiffness and Endo-PAT

derived measures of endothelial function, we determined that children with T1D have increased

arterial stiffness and endothelial dysfunction compared to matched controls. Such early arterial

abnormalities likely contribute to accelerated progression to cardiovascular disease. Future

studies will be able to use these noninvasive techniques to assess the impact of specific

interventions on arterial health in children with T1D. Radial artery tonometry and Endo-PAT

are promising noninvasive techniques that can be used to assess arterial stiffness and endothelial

dysfunction in children with T1D. Non-invasive measures like radial artery tonometry and Endo-

PAT may provide additional risk stratification data needed to justify more aggressive primary

prevention of CVD in children with T1D.









CHAPTER 1
INTRODUCTION

Type 1 diabetes (T1D) is a well established risk factor for the development of premature

cardiovascular disease (CVD)(1). Despite advances in medical practice over the last 25 years, the

incidence of early CVD in the T1D population remains disproportionately high (2-5). The

Diabetes Control and Complications Trial (DCCT) and its longitudinal follow up, the

Epidemiology of Diabetes Interventions and Complications Study (EDIC), have demonstrated

that the risk of TID-related microvascular and macrovascular complications is related to long-

term glycemic control (6; 7).

Unfortunately, even with intensive insulin therapy, the majority of children with T1D are

unable to maintain near-normal glycemia. Children in the intensive arm of the DCCT were only

able to achieve an average HbAlc of 8.1% (8). However, glycemic control is only one of several

important risk factors in defining CVD risk. Minimizing the long term risks for CVD in patients

with T1D may require early and aggressive management of other important CVD risk factors

such as blood pressure and lipids (9; 10). Many adult studies have demonstrated that the

incidence of cardiovascular events can be lowered through reduction of plasma cholesterol levels

and optimal management of hypertension. Unfortunately, the majority of patients who are being

treated aggressively have already manifested cardiovascular complications.

Because children rarely experience cardiovascular events, surrogate markers of CVD are

needed to provide the additional risk stratification needed to justify and monitor the effects of

more aggressive therapy (11). Brachial artery reactivity is a technique that measures the

endothelium-dependent dilation of the brachial artery in response to reactive hyperemia. In

patients with endothelial dysfunction, the ability of the artery to dilate is impaired. Endothelial

dysfunction, as measured by decreased brachial reactivity, and arterial stiffness, as measured by









pulse wave analyses, have both been shown to be independent predictors of cardiovascular

events (12). Impaired brachial reactivity has been demonstrated in adults and in a small group of

children with T1D (13; 14). Unfortunately, standard brachial reactivity studies are difficult to

perform, require expensive equipment and involve subjective analysis of the results. Several new

methods for measuring endothelial function and arterial stiffness are now available and may be

more applicable for everyday clinical use.

To date, studies of arterial stiffness and endothelial function in children with T1D using

these newer techniques have not been performed. Because radial tonometry and PAT can be

performed in nearly any clinic setting, are easy and affordable to perform, and because they

provide the user with an instant analysis of the patient's arterial stiffness or endothelial function,

tonometry and PAT have a potential advantage over standard brachial reactivity and carotid IMT

as clinically useful tools. To test the utility of radial tonometry and PAT, we studied whether

children with T1D had increased arterial stiffness and endothelial dysfunction when compared to

healthy controls.









CHAPTER 2
MATERIALS AND METHODS

Arterial Stiffness Studies

Subjects

We evaluated arterial stiffness in 98 children with type 1 diabetes and 57 control

volunteers using radial artery tonometry. Children were recruited from the Florida Diabetes

Camp, the University of Florida diabetes and primary care clinics, and general pediatrics

practices in the area. Children with T1D were recruited by letters sent to the parents of all

children registered for diabetes camp offering free lipid and HbAlc analyses in return for

participation. Controls were recruited from general pediatrics practices by letters sent to the

parents of clinic patients offering free analyses of lipids, glucose, and HbAlc in return for

participation. While children with T1D would likely have been routinely tested for lipids,

glucose, and HbAlc, healthy controls would not. Thus, excluding diabetes, there may have been

differences in background cardiovascular risk between cases and controls as controls whose

physician or family perceived them as being at increased risk may have been more inclined to

participate. Inclusion criteria for both children with diabetes and controls were age between 10

and 18 years, no known cardiovascular disease, and no history of using anti-hypertensive or lipid

lowering medications. Children with diabetes were included only if they had been diagnosed for

at least one year. Children were classified as having T1D based on a history of acute onset of

polyuria, polydipsia, polyphagia, weight loss, and ketosis. When the history was not clear, islet

cell, glutamic acid decarboxylase, or insulin autoantibody status was used to confirm T1D. There

were no children with diabetes included that did not have either a well documented history or at

least one positive diabetes-related autoantibody. From the total group, 43 matched pairs were









identified who met all inclusion criteria. The groups were matched for age (2yrs), sex, race, and

BMI (3kg/m2).

The study was approved by the Institutional Review Board of the University of Florida.

Subjects' parents provided written consent before their child was enrolled in the study and the

subjects provided assent. Subjects' parents completed a brief questionnaire that included age,

race, medications, family history, and level of exercise. Family history was specified in the

questionnaire as pertaining to only 1st and 2nd degree relatives of the child, and definitions of

hypercholesterolemia, hypertension, and early heart disease were provided. Level of exercise

was graded on a 1 to 4 scale (1 = no exercise, 2 = minimal exercise, 3 = moderate exercise, and 4

= extreme exercise). The most recent HbAlc, duration of diagnosis, and history of recent

illnesses was obtained from the medical record.

Study Protocol

Radial artery tonometry was performed and blood samples were obtained between 6 am

and 10 am on the same day, with the child supine and relaxed. Study subjects were required to

fast after midnight and to abstain from caffeine for 24 hours before the study. Height was

measured on a wall mounted, calibrated stadiometer (Genentech, San Francisco, California) and

weight was read from a calibrated digital scale. After a 5 minute rest in the supine position, the

subjects had their blood pressure measured with a digital oscillometric device (Omron model

HEM-739, Omron Healthcare Inc., Vernon Hills, Illinois). Radial tonometry was then

performed. Following the completion of the radial tonometry, a venous blood sample was

obtained for glucose, HbAlc, total cholesterol, LDL, HDL, triglycerides, hsCRP, IL-1p3, and IL-

6. Subjects with T1D had their morning insulin injection postponed until all studies were

completed.









Measurement of Arterial Stiffness by Radial Tonometry

Augmentation index (AI) and augmentation index corrected to a heart rate of 75 (Al75)

were measured using the SphygmoCor Vx version 7.01 (AtCor Medical, Sydney, Australia). In

brief, a high-fidelity micromanometer with a frequency response of > 2kHz (Millar Instruments,

Houston, Texas) was placed on the right radial artery and gentle pressure was applied until a

consistent waveform was produced. After 10-20 sequential waveforms had been acquired, the

integrated software was used to generate an averaged peripheral and corresponding central

waveform that was used for the determination of the AI and Al75 (Figure 2-1).

The algorithm used to convert the radial pulse wave to an aortic wave form was derived

from invasive arterial pressure and flow data obtained by cardiac catheterization and has been

validated in several adult studies (15-17). Validation studies are underway to confirm that the

same algorithm can be applied in children. A quality index is displayed and represents the

reproducibility of the waveform. A value greater than 70 is considered to demonstrate excellent

waveform consistency. For this study, only measurements with a quality index above 80 were

accepted. Two acceptable measurements were obtained on each subject. AI is defined as the

difference between the first and second peaks of the central arterial waveform, expressed as a

percentage of the pulse pressure and measures the contribution that the wave reflection makes to

the arterial pressure waveform. The amplitude and timing of the reflected wave depends largely

on the stiffness of the small and large arteries. Thus, AI provides a measure of systemic arterial

stiffness. Al75 allows for a true comparison of the augmentation of central pressure between

study subjects by discounting differences related to heart rate variation (18). An elevated or

positive AI suggests stiffer arteries than a low or negative AI (Figure 2-2).









Measurement of Lipids, HbAlc, Glucose

Serum was collected from study participants using standard venipuncture techniques and

Vacutainer serum separator tubes (BD Biosciences, San Diego, CA, USA). After collection,

samples to be analyzed for lipid, HbAlc, and glucose were immediately refrigerated and

transported to the Shands Hospital laboratory at the University of Florida. Samples were

analyzed in the clinical laboratory using standard technique.

Measurement of Cytokines

Serum for cytokine and autoantibody analysis was separated into serial aliquots and

frozen at -80 C within 1 hour of the blood draw. All serum analyses were conducted following a

single freeze-thaw cycle. Cytokine measurements from serum were performed using a

commercially available multiplexed kit (Beadlyte Human Multi-Cytokine Detection System 3,

Upstate, Lake Placid, NY) and the Luminex100 LabMAPTM System. Quantitative evaluation of

the serum cytokines IL-13P and IL-6 was performed. Serum samples were subjected to a 1:2

dilution in serum diluent provided by the manufacturer in order to reduce the effects of

interfering heterophile species (19; 20). High sensitivity CRP (Alpco, Windham NH, USA)

levels were measured by standard sandwich ELISA techniques according to manufacturer's

instructions. Serum analyte concentrations were calculated using 4-parameter analysis utilizing

SoftMax Pro Software, Ver. 2.2.1 (Molecular Devices Corp., Sunnyvale, CA, USA).

Measurement of Autoantibodies

Autoantibodies against two TID-associated autoantigens were tested from serum

obtained from all study participants including those against GAD65 and IA-2. Assays were

performed as previously described (20). The investigators are regular participants in workshops

and proficiency tests sponsored by the Immunology of Diabetes Society and CDC to validate









assay performance. At the most recent effort (DASP 2003), our performance for GADA assay

indicated 80% sensitivity/95% specificity for type 1 diabetes, while our IA-2A assay provided

64% sensitivity/100% specificity.

Statistical Considerations

As described above, the study was planned as a matched pair design. However, if

matching factors are treated as covariates all conclusions qualitatively remained the same when

analysis was performed on all entrants, independent of match availability. When analyzing the

matched pairs, case-control comparisons were assessed with one sample paired t-tests for the

following dependent variables: AI and Al75 (primary) and total cholesterol, HDL, LDL,

triglycerides, blood pressure, HbAlc, and glucose (secondary). All p-values were two-sided. The

original study was planned for a sample size of 100 matched pairs. We were unable to recruit

the anticipated numbers of matched controls within the planned time-frame due to strict

matching criteria and unwillingness of control subjects to participate in a blood draw, However,

the matched variations in the variables AI and Al75 were smaller than originally anticipated, and

a retrospective power calculation, using matched standard deviations of 11.6 and 11.0

respectively, demonstrates that the actual sample size of 43 matched pairs yields sensitivity to

differences in the paired means of 5.6 and 5.3 respectively at p=0.025 two-sided and 80% power.

As a secondary objective, separate analyses for associations with AI and Al75 were

conducted within controls (n=57) and T1D cases (n=98). Due to the potential for outliers in

hsCRP and the qualitative nature of some of the variables, Spearman's correlation was utilized to

examine the relationship between AI, Al75 and total cholesterol, LDL, HDL, triglycerides, blood

pressure, HbAlc, glucose, hsCRP, IL-1 IL-6, family history, and exercise regimen.









Endothelial Dysfunction Studies


Subjects

Forty-four children (22 Male, 22 Female) with T1D for at least 1 year were recruited

from the University of Florida pediatric endocrinology clinic for this study. Twenty control

children (12 Male, 8 Female) were recruited from the Mayo Clinic, Rochester, MN. Control

subjects were non-smokers and community based, and did not have any co-existing medical

conditions or any family history of premature cardiovascular disease or hyperlipidemia. All T1D

and control patients who participated in this study were Caucasian.

Study Protocol

Following an overnight fast and using identical protocols, endothelial function was

assessed in all children using the Endo-PAT device (Itamar Medical Ltd, Caesarea, Israel).

Height, weight, and blood pressure were recorded before Endo-PAT testing. Fasting blood

work was performed immediately following the Endo-PAT assessment. Following Endo-PAT

testing, blood was obtained for lipid profile and glucose determinations in all subjects and also

for HbAlc determination in children with T1D. Laboratory analysis of the blood samples was

performed at the separate sites using identical laboratory platforms. Four weeks after their initial

test, T1D subjects had repeat Endo-PAT testing to determine intra-patient variability.

Measurement of Endothelial Function

Endo-PAT is a non-invasive device that combines the traditional flow mediated

dilatation technique with pneumatic finger-tip probes to measure arterial pulse wave amplitude

and provide an objective measure of endothelial function. The Endo-PAT device is an operator

independent device that allows affordable and objective measurements of endothelial function

and eliminates the need for an ultrasound technician or interpretation of the ultrasound signal.









The Endo-PAT system uses the identical arterial occlusive procedure used to induce

reactive hyperemia in standard brachial reactivity measurements of endothelial function.

However, instead of measuring brachial artery diameter, Endo-PAT uses finger-tip

plethysmography probes to measure the changes in pulse wave amplitude observed before and

after the period of reactive hyperemia. An Endo-PAT score is then calculated to provide a

measure of endothelial function. In adults, the Endo-PAT score has shown excellent correlation

with measures of coronary and peripheral endothelial dysfunction (21; 22). Correlations of Endo-

PAT score with invasive measures of endothelial function have not been performed in children.

Only one previous study has used the Endo-PAT device to demonstrate endothelial

dysfunction in children with T1D(11). This study was designed to confirm the usefulness of

Endo-PAT as a surrogate measure of CVD risk in children with T1D. We expanded the scope

of the initial trial by studying a larger group of children with T1D and healthy controls and

measuring the reproducibility of Endo-PAT with serial testing. We hypothesized that children

with T1D would have endothelial dysfunction (decreased Endo-PAT score) when compared to

healthy controls and that Endo-PAT would have acceptable intra-patient variability.

To perform Endo-PAT, the patient sits in a reclining chair with the hands at heart level

and propped in a comfortable position such that the fingers are hanging freely. Fingertip probes

are placed on both index fingers and pulse wave amplitudes are recorded for the duration of the

study. After 5 minutes of baseline measurement, arterial flow to the non-dominant arm is

occluded for 5 minutes using a blood pressure cuff inflated to 40mmHg above systolic pressure.

After the 5 minute occlusion, the cuff is rapidly deflated to allow for reactive or flow-mediated

hyperemia. Pulse wave amplitudes are recorded for at least 5 minutes after the cuff is deflated.

The Endo-PAT system compares the ratio of arterial pressure in the two fingers before and after









the occlusion to calculate the Endo-PAT score. The Endo-PAT score is calculated as the ratio

of the average pulse wave amplitude measured over 60 seconds starting 1 minute after cuff

deflation divided by the average pulse wave amplitude measured at baseline. This ratio is

normalized to the concurrent signal from the contra-lateral finger to correct for changes in

systemic vascular tone (Figure 2-3).

Statistical Considerations

The primary endpoint for this study was the Endo-PAT score. HbAlc, LDL, HDL, total

cholesterol, triglycerides, glucose, systolic and diastolic blood pressure, and BMI were analyzed

as secondary endpoints. The two groups of patients (TID and control) were compared using a

two-sided two sample t-test. Intra-patient standard deviations for Endo-PAT were estimated

from the repeated measures and averaged over the patients within each subgroup. With a sample

of 44 TID patients and 20 controls, a two sided, two-sample t-test had 80% power at P=0.05 to

detect a difference of 0.78 standard deviations between the two groups. Assuming a standard

deviation of 0.4, this corresponds to sensitivity to an Endo-PAT score difference of 0.32 units.
















Figure 2-1. Radial artery tonometry. A high fidelity tonometer is placed on the radial artery until
a consistent waveform is generated by the attached software program.




















Figure 2-2. Radial artery and corresponding aortic pulse waves. Augmentation index increases
with age as arteries stiffen.


pr I


-F


Occluded Arm


Control Arm


^.me-c-


Figure 2-3. Schematic of Endo-PAT in use. This figure demonstrates the set up and use of the
Endo-PAT. The probes are placed on the index fingers of both arms. A reactive
hyperemia procedure is performed by occluding the brachial artery of one arm for 5
minutes. Data are automatically analyzed by the software package and the Endo-
PAT score is provided. (With permission from Itamar Medical, Ltd.)









CHAPTER 3
MATERIAL STIFFNESS FINDINGS

Augmentation Index (AI)

Laboratory and tonometry characteristics of the matched diabetes and control groups are

shown in Table 3-1. Spearman correlations of Al and Al75 are shown in Table 3-2. T1D was

associated with increased arterial stiffness as evidenced by the higher Al75. The mean Al75 in

T1D subjects was 1.8910.75 whereas the Al75 for controls was -3.32 10.36. Using a paired

difference two-sided t-test, the AL75 demonstrated a case-control difference of 5.2+11.0

(p=0.003). There was no significant difference in the uncorrected AI (p=0.37).

Lipids

Total cholesterol, LDL, and HDL analyses did not reveal any significant case-control

differences. Triglyceride levels, however, were significantly higher in the control population

with a case-control difference of -25.062.6 (p=0.012). Even when lipid values among only the

T1D subjects were analyzed, no significant associations were noted between total cholesterol,

LDL, HDL, or triglycerides and either AI or Al75 (Table 2-2).

Blood Pressure

Systolic and diastolic blood pressure analysis revealed significantly higher values in the

controls, with differences of -5.214.6 (p=0.025) and -3.679.9 (p=0.019), respectively.

However, no significant associations were noted between systolic or diastolic blood pressure and

AI or Al75 (Table 2-2).

Length of Diabetes and Control

Duration of diabetes, HbAlc, and blood glucose did not demonstrate a significant

association with AI or Al75 (Table 2-2). Due to the lack of substantive variation in ages, the









study was not adequately powered to determine if the difference in AI or AI75 between controls

and T1D subjects became apparent at a specific age.

Cytokines

There were no significant associations between AI or Al75 and IL-1, IL-6, or hsCRP

(Table 2-2).

Exercise

Level of exercise was graded on a 1 to 4 scale (1 = no exercise, 2 = minimal exercise, 3 =

moderate exercise, and 4 = extreme exercise). Differences in the reported exercise level between

T1D cases (2.89) and controls (2.79) were not statistically significant. Reported exercise level

among T1D cases failed to demonstrate a significant correlation with AI or Al75. Reported

exercise level among controls alone did, however, reveal a significant association with both AI

(r = -0.44 p<.001) and Al75 (r = -0.33 p=0.015)(Table 2-2).

Family History

Among T1D cases, family history failed to demonstrate a significant correlation with

either AI or Al75. Among controls significant associations were seen between AI and family

history of hypercholesterolemia (r = 0.29, p = 0.030 for AI) and early heart disease (r = 0.38, p =

0.004 for AI, r = 0.42, p = 0.001 for A175), but not family history of hypertension (Table 2-2).

Gender

There were 26 male matched pairs and 17 female matched pairs for a total of 43 matched

pairs in the case-control analysis. The case-control difference among paired females

demonstrated a significant difference in mean Al75. Case-control difference of mean Al75 among

paired males did not reveal any significant differences. Although the case-control difference

between Al75 in matched females reached significance and the difference between matched









males did not, there was no significant difference between males and females. Hence, the overall

conclusion should take precedence over the gender-specific conclusion.

Discussion

This study confirms that children as young as 10 years of age with T1D have increased

arterial stiffness when compared to matched controls. Although several recent studies present

similar findings (14; 23-25), this is the first study to use radial artery tonometry and

augmentation index to demonstrate increased arterial stiffness in children with T1D.

Arterial stiffness as a result of endothelial dysfunction is an early sign of cardiovascular

disease occurring more often and at an earlier age in patients with T1D compared to those without

diabetes. Endothelial dysfunction in T1D is primarily due to increased production of advanced

glycation end products (AGEs) causing decreased production and action of nitric oxide and a

concomitant decrease in arterial compliance (26). The resultant endothelial dysfunction allows

for premature integration of lipid laden macrophages in arterial walls. In addition, the

hyperglycemic environment results in qualitative changes in LDL particle size, oxidation, and

glycation that have been implicated in early increases in carotid artery IMT and endothelial

dysfunction (27; 28). Despite the fact that most T1D patients in reasonable metabolic control

have normal cholesterol profiles, decreasing these modified LDLs may be a plausible

intervention to reduce cardiovascular disease in T1D. Decreasing LDL values in relatively

young T1D patients (average age 34) with normal initial LDL values resulted in improved

endothelial function after just 6 weeks of station therapy (29).

Because management of hyperglycemia is the cornerstone of therapy in T1D, optimal

glucose control has been suggested as the primary target to minimize the risk of macrovascular

disease. However, even the DCCT's intensive therapy group (average HbAlc of 7.2%), while









showing improved carotid IMT when compared to the conventional treatment group (average

HbAlc 9.1%), had significantly increased carotid IMT compared to controls (30; 31). This

difference correlated significantly with blood pressure and cholesterol levels (32). Optimal

management of all co-morbidities is needed to minimize risk.

Nevertheless, many clinicians believe that diabetes related cardiovascular risk is not

sufficiently affected by metabolic derangements in childhood to pursue optimal management of

lipids and hypertension in children with T1D. Data from the Pittsburgh Epidemiology of

Complications Study, a 10 year follow up of patients who developed T1D before the age of 17,

showed that high blood pressure and increased LDL were independent risk factors for

microvascular disease, macrovascular disease, and mortality (3). Autopsy studies of over 3000

children found that children have evidence of aortic fatty streaks as early as 3 years of age and

raised fibrous plaques as early as 8 years of age (33; 34).The Muscatine Heart Study and the

Bogalusa Heart Study have demonstrated that cardiovascular risk factors apparent in childhood

predict future coronary artery disease (35; 36).

Radial artery tonometry is an easily learned, affordable, noninvasive, reproducible and

accurate technique that can be used to monitor arterial stiffness and, therefore, future

cardiovascular risk in both high-risk children and adults (13; 37-39). Radial artery tonometry

has been shown to have excellent intra-observer and intra-patient reproducibility and to provide a

reliable assessment of endothelial dysfunction (40; 41). In adults with T1D, augmentation index

is increased prematurely (38).

This is the first study to evaluate radial artery tonometry in children with T1D. Although

we were unable to show any significant correlations between AI in children with T1D and

established cardiovascular risk factors, it is possible that the T1D children with the most elevated









AIs might have demonstrated correlations if the study had been adequately powered to evaluate

these relationships. Nevertheless, we did demonstrate a clear difference in AI75 between controls

and subjects with T1D. While the exact mechanisms remain unclear, the observed difference in

AI75 documents that there are identifiable abnormalities in vessel stiffness in children with T1D.

Using a combination of flow-mediated dilatation and carotid IMT, Jarvisalo, et al. and

Singh et al., demonstrated endothelial dysfunction in children with T1D (14; 23). Both studies

revealed correlations with endothelial dysfunction and LDL cholesterol. The two studies

disagreed, however, when describing the relationship between increased carotid IMT and T1D.

While the disparate findings may be explained by differences in technique, it is possible that the

differences are related to the age and pubertal status of the patient populations. The average age

of the T1D children in the two studies was 11 and 15 years, respectively. Although endothelial

dysfunction is present in young children with T1D, perhaps the correlations with endothelial

function and other cardiovascular risk factors do not become apparent after puberty.

In our study, the average age of the T1D children was 12.9 years. The differences in age

and pubertal status, in addition to the different methods used to measure endothelial function,

could explain differences in between various studies. Unfortunately, the age distribution of our

study population was too narrow to determine at what age the difference between controls and

children with T1D becomes apparent. Studies with broader age distributions are needed to

determine the effects of age and puberty.

Adult and pediatric studies of endothelial dysfunction in diabetes patients have

demonstrated significant associations with high LDL, low HDL, hypertension, glycemic control,

duration of disease, folic acid status, exercise regimen, and gender (13; 23; 24; 42). Although we

did demonstrate that T1D was associated with increased arterial stiffness, this study did not find









any significant associations with lipids, blood pressure, glycemic control, cytokines, family

history, or gender in the children with diabetes. The inability to demonstrate a correlation

between established cardiovascular risk factors and increased Al75 in children with diabetes is

most likely related to the small sample size, narrow age range and lack of longitudinal follow-up.

Nonetheless, the risk factors measured are well established as being relevant to the development

of atherosclerosis. In children, however, it may be necessary to measure variables that may be

more directly related to arterial stiffness such as AGEs, qualitative lipids, nitrite, and superoxide

dismutase.

In control children, we found a significant association between elevated AI and family

history of hypercholesterolemia. This may have represented a volunteer bias as the control

families who volunteered for the study were more likely to have a positive family history of heart

disease. This could explain why the control children had significantly higher triglycerides and

blood pressures than the T1D subjects. A bias towards increased cardiovascular risk in controls

would have decreased the difference in AI75, between controls and children with diabetes.

Nonetheless, T1D children demonstrated greater arterial stiffness than controls. That higher

exercise levels were associated with decreased arterial stiffness in the controls but not in the T1D

subjects suggests that the metabolic derangements of diabetes obscure some of the advantages of

exercise.

Despite the novel findings provided by this study, there are several important limitations.

The Sphygmocor Vx software used to analyze the wave reflections has not been fully validated

in children. The transfer function used to calculate the aortic pressure wave was validated using

directly measured aortic and radial pressure waves in adults. While age and AI are linearly

associated in adults, such that the same transfer function could be used at all ages, no childhood









data are yet available to confirm the transfer function's validity. Although case-control

comparisons eliminated the need for known age adjusted normal values, the argument could be

made that the transfer function may not accurately determine aortic pressure waves in children.

In a recent study of adults with type 2 diabetes, Hope et al. suggested that generalized transfer

functions may not be valid at all in patients with diabetes (43). While their concerns about the

applicability of transfer functions to specific populations is valid, they also found no significant

differences between directly measured and transfer function derived AI in their diabetes

population.

The strict matching criteria that were employed resulted in a lower than expected total

number of matches. With a larger study, associations between AI and lipids, blood pressure, and

cytokines might have been uncovered. Another weakness of this study was the omission of

Tanner staging which may have resulted in significant pubertal differences between age-matched

pairs. Pubertal status may be an important determinant in deciphering when children with T1D

develop increased arterial stiffness and should be included in all future studies. Finally, the

failure to include albuminuria as a study variable is a recognized limitation. Several other studies

have demonstrated the importance of microalbuminuria in predicting atherosclerosis in T1D

patients (44).

Despite these limitations, the study does provide additional evidence that children with

T1D have endothelial dysfunction. Further studies are needed to confirm the relationship

between arterial stiffness and other modifiable cardiovascular risk factors. Future interventional

studies should be developed to provide longitudinal comparisons of optimal blood pressure,

lipid, and glucose control with endothelial function in T1D children.









Table 3-1. Matched TID Subjects and Controls in AI Study. (n=43) Means, standard
deviations, paired differences of the means, and p values are shown.
T1D Controls
Mean + SD Mean + SD Paired Difference SD P value
AI 1.11 + 10.15 -0.47 9.79 1.59 11.61 0.37
AI75 1.88 10.75 -3.31 + 10.36 5.20 11.02 0.0031
HbAlc 8.41 + 1.29 5.2 0.25 3.17 1.23 <0.0001
T Cholesterol 151.19 29.46 152.66 36.1 -5.12 35.72 0.35
Triglycerides 61.05 26.32 89.36 54.72 -25.0 62.60 0.012
HDL 56.67 8.2 53.09 12.26 4.05 15.43 0.093
LDL 85.18 34.1 86.02 25.25 -4.09 32.31 0.41
Glucose 159.67 68.78 85.22 9.58 75.89 70.18 <0.001
SBP 109.93 13.6 115.02 10.31 -5.09 14.51 0.025
DBP 67.45 8.8 71.18 7.96 -3.73 9.80 0.015

Table 3-2. Spearman Correlations in T1D Subjects and Controls in AI Study. Spearman
values and p-values are shown for AI and AI75 in all T1D (n=98) subjects and
Controls (n=57).
T1DM (n=98) Controls (n=57)
AI (p value) AI75 (p value) AI (p value) AI75 (p value)
HbAlc 0.076 (0.46) 0.089 (0.39) 0.060 (0.66) 0.094 (0.49)
T Cholesterol 0.055 (0.60) 0.056 (0.59) 0.150 (0.27) 0.198 (0.14)
Triglycerides 0.096 (0.35) 0.061 (0.55) 0.018 (0.89) 0.046 (0.73)
HDL -0.027 (0.80) 0.086 (0.41) 0.192 (0.15) 0.126 (0.35)
LDL 0.062 (0.54) -0.013 (0.89) 0.071 (0.16) 0.076 (0.58)
Glucose 0.164 (0.11) 0.132 (0.20) 0.004 (0.97) -0.0004 (0.997)
SBP -0.132 (0.20) -0.144 (0.15) -0.289 (0.03) -0.169 (0.21)
DBP 0.094 (0.36) 0.094 (0.35) -0.059 (0.66) 0.058 (0.67)
IL-1j3 -0.190 (0.06) -0.079 (0.44) 0.210 (0.12) 0.010 (0.46)
IL-6 -0.150 (0.14) -0.126 (0.22) -0.152 (0.26) -0.074 (0.59)
hsCRP -0.005 (0.96) 0.004 (0.96) 0.059 (0.66) 0.133 (0.33)
FHofHTN 0.120 (0.24) 0.149 (0.14) -0.114 (0.44) -0.066 (0.63)
FH of Early CVD -0.031 (0.44) -0.078 (0.45) 0.345 (0.0086) 0.381 (0.0034)
FH of Cholesterol -0.132 (0.20) -0.101 (0.33) 0.303 (0.02) 0.218 (0.10)
Exercise 0.027 (0.79) 0.032 (0.76) -0.346 (0.0083) -0.449 (0.0005)
Years of Diabetes 0.041 (0.44) 0.080 (0.44) N/A N/A









CHAPTER 4
ENDOTHELIAL DYSFUNCTION FINDINGS

Endo-PAT Score

The summary statistics for the comparisons are presented in Table 4-1. The mean intra-

patient standard deviation of Endo-PAT in the T1D patients was 0.261. Children with T1D

(n=44) had endothelial dysfunction as evidenced by lower mean Endo-PAT scores (1.63 vs

1.95, p=0.01) when compared to control children (n=20). However, the range of Endo-PAT

scores was wide. Children with T1D had a range of Endo-PAT scores of 1.01 to 3.01 while

control children had a range of 1.5 to 2.5 (Figure 4-1). Children with T1D had higher mean

systolic BP (p=0.02), mean total cholesterol (p=0.03), and mean HDL (p=0.0001) than control

children. It was not clear why there were such large differences in blood pressure or HDL

between the two groups. No significant differences in age, BMI, diastolic blood pressure, LDL,

or triglycerides were observed between the children with T1D and control children.

Discussion

This study confirms that Endo-PAT can reliably evaluate endothelial function in

children with T1D. Significant differences in Endo-PAT score, consistent with endothelial

dysfunction, were observed between children with T1D and controls.

Several characteristics of our study population warrant further comment. The two study

populations compared were of similar age and gender, racially homogeneous, and tested using an

identical protocol and technique despite the fact that they derived from different locations in

North America (Florida and Minnesota). Our T1D population had higher systolic BP, HDL, and

total cholesterol. In addition to the hyperglycemia associated with T1D, these important CVD

risk factors could explain some of the difference in Endo-PAT score between the two groups.

We must accept, therefore, that the difference in endothelial dysfunction seen in the two groups









may be partially explained by selection bias and not by CVD risk associated solely with the

disease state of TID. In addition, our study was not designed to determine the relative

importance of glycemic control, blood pressure, cholesterol, BMI, family history, or any other

CVD risk factors on Endo-PAT score. Additional studies will be needed to further investigate

the relative importance of other traditional CVD risk factors on Endo-PAT score. Finally, Endo-

PAT has not yet been used to evaluate the risk of future cardiovascular events in adult or

pediatric populations.

Despite the limitations of this particular study, we believe that Endo-PAT has several

potential advantages over traditional reactive hyperemia measurements. Specifically, Endo-

PAT testing is affordable, reproducible, and operator independent. Therefore, the procedure is

not subject to the subjective interpretations of blood vessel diameter associated with brachial

artery ultrasound imaging methods. While larger studies are needed to confirm the association

between low Endo-PAT score and traditional measures of endothelial dysfunction, Endo-PAT

may be useful as both a research and clinical tool in assessing endothelial function in populations

at high risk for developing premature CVD.

Recent Epidemiology of Diabetes Interventions and Complicaitons (EDIC) study data

demonstrate that intensive diabetes management significantly decreases cardiovascular events

amongst patients with T1D (7). Unfortunately, the overall risk of CVD in T1D patients remains

disproportionately high. Because T1D is still associated with a 2-3 fold increased risk of

premature CVD, further efforts are needed to accurately identify and treat those patients at high

risk for CVD. CVD is a process rooted in early childhood. A multitude of studies now

demonstrate that risk factors present in childhood predict cardiovascular event rates in adulthood

(33; 35). Furthermore, autopsy studies have shown that permanent plaques associated with CVD









appear in the arteries of children as young as 8 years of age (36). Still, outside of striving for

tight glycemic control in children with T1D, many pediatric endocrinologists struggle with

which measures to adopt to appropriately identify and manage comorbidities associated with

CVD risk (45).

The average HbAlc in our T1D study population (8.34%) approached the HbAlc of the

adolescents in the intensive therapy arm of the DCCT and yet endothelial dysfunction was easily

identified in our T1D study population. Thus, until technological advances provide for marked

improvements in glycemic control for all children with T1D, we are likely to continue to observe

average HbAlc levels well above those that eliminate risk of complications even in relatively

compliant patients.

Similarly, while slightly higher than those seen in the control population, the average

LDL cholesterol (LDL) in our T1D population (87.6 mg/dl) and systolic blood pressure (BP)

(109 mmHg) easily met American Diabetes Association and American Heart Association goals.

Recent data have demonstrated better endothelial function in adults with CVD who have LDL

levels < 80 when compared to those with LDL between 80-100mg/dl (46). Thus, adequate

primary prevention of CVD in patients with T1D may require both tight glycemic control and

aggressive (i.e. pharmacologic) management of lipids and blood pressure to levels lower than

those currently labeled as "normal." Furthermore, to truly minimize the risk of CVD in patients

with T1D, initiation of stations and ACE inhibitors may be needed shortly after diagnosis

regardless of cholesterol and blood pressure values.

While prospective studies are needed to determine if more aggressive management of

lipids and blood pressure can decrease the CVD risk associated with T1D, non-invasive

measures of vascular function such as Endo-PAT may provide the additional level of risk










stratification needed to justify early initiation of pharmacologic lipid and blood pressure

lowering therapy in children with T1D.


Table 4-1. Laboratory and Endo-PAT characteristics of controls and T1D subjects.
Characteristic Diabetes (n=44) Controls (n=20) P Value
Endo-PAT Score 1.64 + 0.5 1.95 + 0.3 0.01
Mean intra-subject SD 0.261 -- --
Age (years) 14.6 1.5 14.1 + 1.5 0.41
BMI (kg/m2) 22.6 2.7 21.5 +2.6 0.34
Systolic BP (mmHg) 109.5 10 103.9 + 6.3 0.02
Diastolic BP (mmHg) 69.6 + 9.6 69.3 + 4.9 0.87
Glucose (mg/dl) 200.1 + 89.7 86.4 + 11.5 0.0001
HbAlc (%) 8.34 + 1.2 -- --
Total cholesterol (mg/dl) 166.4 + 35 147.7 + 20 0.03
LDL(mg/dl) 87.6 26 78.1 +21 0.16
HDL (mg/dl) 61.7 + 12.9 38.9+ 11.1 0.0001
Triglycerides (mg/dl) 85.5 58.7 68.6+ 25.8 0.22


3.25-
3.00-
2.75-
2.50-
2.25-
2.00-
1.75-
1.50-
1.25-
1.00-
0.75-
0.50-
0.25-
0-n


N=20


N=44


Type I Diabetics Controls
Figure 4-1. Box Plot of Endo-PAT Score in Controls and Subjects with Type 1
Diabetes. ThisThe box plot demonstrates a significantly lower Endo-PAT
score in the 44 children with type 1 diabetes when compared to the 20 control
children. Although there was a wide range of Endo-PAT scores, the lower
Endo-PAT score is indicative of relative endothelial dysfunction in the
cohort with type 1 diabetes.


}I









CHAPTER 5
CONCLUSIONS

The risk of recurrent cardiovascular events is higher in T1D patients than in nondiabetic

patients. Well established data document that the early anatomical signs of atherosclerosis appear

in childhood. Nevertheless, current guidelines for treating lipids and blood pressure in children

with T1D may be underestimating the utility of more aggressive primary prevention. Non-

invasive techniques such as radial tonometry and Endo-PAT can be used to demonstrate

abnormal arterial stiffness and endothelial function in children with T1D. These non-invasive

techniques may provide the extra tier of cardiovascular risk stratification information needed to

design optimal cardiovascular risk reduction strategies in children with T1D.









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BIOGRAPHICAL SKETCH

Michael James Haller, M.D., was born on March 28, 1975, in Gainesville, Florida.

He graduated from Gainesville High School and then pursued his undergraduate degree at

Duke University. After completing his B.S. in Biology at Duke University in 1996, Michael

returned to Gainesville to attend medical school at the University of Florida. After completing his

medical school training in 2000, Michael completed both a pediatrics residency and a pediatric

endocrinology fellowship at the University of Florida before accepting a position in 2006 as

an assistant professor of pediatric endocrinology at the University of Florida. Michael's

current research focuses on the prevention and cure of Type 1 diabetes and the primary

prevention of cardiovascular disease in children with Type 1 diabetes. Michael is married to

Allison Sherman Haller, M.D.