Heritability of Retinal Vascular Morphology. in a Danish Twin Population

Transcription

1 Heritability of Retinal Vascular Morphology in a Danish Twin Population A Ph.D. Thesis by Nina Charlotte Bille Brahe Taarnhøj, MD Department of Ophthalmology Glostrup Hospital Denmark

2 The present Ph.D. thesis was handed in December 30 th, 2007 The thesis was accepted by The Faculty of Health Science, University of Copenhagen for public defence. The thesis will be defended on March 11 th, 2008 in Auditorium C, Glostrup Hospital, Denmark. The supervisors are Michael Larsen, Professor, MD, Line Kessel, MD, Ph.D., Department of Ophthalmology, Glostrup Hospital, Knut Borch-Johnsen, Professor, MD, Steno Diabetes Centre and Thomas Martini Jørgensen, Risøe, Denmark. The official opponents are: Fridbert Jonasson, Professor, MD, Reykjavik, Iceland, Peter Bjerre Toft, Associate professor, MD, Department of Ophthalmology, Rigshospitalet, Denmark and Hans Ibsen, Professor, MD, Medicinsk afdeling, Holbæk, Denmark. Cover photo: Digital grey-scale fundus photograph of Nina Taarnhøj s left eye. Nina Taarnhøj Printed in Denmark - 2 -

3 Preface The present Ph.D. thesis was written during my position as a research fellow at the Department of Ophthalmology, Herlev/Glostrup Hospital from 2004 to The principal purpose of the thesis was to examine the genetic influence on retinal vascular morphology. I would like to express my sincere thanks to all who have helped and inspired me during my work on this thesis. Thanks to Professor Henrik Lund-Andersen, MD, DMsc, for creating a very encouraging and pleasant atmosphere in which research at a highly international level is made possible. Thanks to Line Kessel, MD, Ph.D., Jesper Leth Hougaard MD, Ph.D., nurse Jette Kvistgaard and photographer Hans Henrik Pedersen for conducting the twin study. Thanks to Kirsten Ohm Kyvik, MD, PhD, University of Odense, scientific co-head and administrative leader of the Danish Twin Registry and professor Thorkild I.A. Sørensen, MD, DMsc. and colleagues at Institute of Preventive Medicine, Copenhagen University Hospital, Denmark, for making the twin study possible and helping me with twin analysis. For her, statistical guidance and inspiring discussions during this project I would like to warmly thank Birgit Sander, Ph.D. I am also very grateful to Dorte Nellemann Thornit, MD, for being a fun and inspiring colleague and friend. To my primary supervisor Professor Michael Larsen, MD, DMsc, I want to express my special gratitude for his interesting research ideas and experience which has provided me with a great foundation for my research. Also special thanks to my co-supervisor Line Kessel, who has been very engaged in my work and has been a really good support. Finally, I want to thank Inger Christine Munch, MD, for great cooperation with the vessel analysis. I am very appreciative to Professor Bjørn Nicolaissen, who invited me to complete my theoretical work at the Department of Ophthalmology, Ullevål University Hospital, Oslo, when I moved to Norway in Finally, I would like to express my warmest thanks and deepest affection to my husband, Frode and our son, Alexander for their patience, love and support. This Ph.D. project was made possible thanks to the financial support from The Danish Medical Research Council, The Danish Eye Research Foundation, The Danish Eye Health Society, Centre for Biomedical Optics and New Laser Systems Graduate School, The Danish Diabetes Association, The Norwegian Association of the Blind and Partially Sighted, and The Research Career Award Grant from The Juvenile Diabetes Research Foundation (Michael Larsen). Nina Taarnhøj, Copenhagen, March

4 The present thesis is based on the following papers: Paper 1: Nina C.B.B. Taarnhøj, Inger C. Munch, Kirsten Kyvik, Birgit Sander, Line Kessel, Thorkild I.A. Sørensen, Jesper L. Hougaard, and Michael Larsen, Heritability of Cilioretinal Arteries: A Twin Study. Invest Ophthalmol Vis Sci, 2005, Vol. 46, p Paper 2: Nina C.B.B. Taarnhøj, Michael Larsen, Birgit Sander, Kirsten Kyvik, Line Kessel, Jesper L. Hougaard, and Thorkild I.A. Sørensen, Heritability of Retinal Vessel Diameters and Blood Pressure: A Twin Study. Invest Ophthalmol Vis Sci, 2006, Vol. 47, p Paper 3: Nina C.B.B. Taarnhøj, Inger C. Munch, Kirsten Kyvik, Birgit Sander, Line Kessel, Thorkild I.A. Sørensen, Jesper L. Hougaard, and Michael Larsen. Straight Versus Tortuous Retinal Arteries in Relation to Blood Pressure and Genetics. (Accepted for publication in British Journal of Ophthalmology). Paper 4: Nina C.B.B. Taarnhøj and Line Kessel. Review of Twin Studies in Ophthalmology. (In revision)

5 Contents: 1. Introduction Background The eye Anatomy and physiology of the retinal vascular system Retinal vessel abnormalities Hypertensive retinopathy Branch retinal vein occlusion Retinal vessel tortuosity Computer assisted methods for analysis of retinal vessel calibres Fundus photographic retinal vessel calibre studies Introduction Associations of retinal arterial and venous calibres with eye disease Associations between retinal arterial calibre and systemic disease Associations between retinal venous calibre and systemic disease Influence of environmental factors on retinal calibres Discussion Clinical studies Purpose Methods Subjects Fundus photography and digital imaging Computerized image measurement software Visual grading of retinal vessel tortuosity and cilioretinal arteries Twin analysis

6 4.3. Results Paper 1 (Heritability of cilioretinal arteries: A twin study) Paper 2 (Heritability of retinal vessel diameters and blood pressure: A twin study) Paper 3 (Straight versus tortuous retinal arteries in relation to blood pressure and... genetics) Paper 4 (Review of twin studies in ophthalmology) Discussion Conclusions Summary in English Summary in Danish Reference list Papers

7 1. Introduction The purpose of the present Ph.D. thesis was to examine the relative importance of genetic and environmental factors on the morphology of retinal blood vessels in a population of young healthy normotensive twins, the clinical perspective being to evaluate whether a single fundus photograph may be of value in assessing an individual s likelihood of having or developing cardiovascular disease in the future. It is also potentially important to know if retinal vessel layout is influenced by genetic factors when evaluating if there is a genetic susceptibility to how some diseases affect the eye. The thesis is based on four papers; three which are covering heritability analysis of retinal blood vessel calibres, retinal arterial tortuosity, and the presence of cilioretinal arteries and one review which covers twin studies in ophthalmology. The first three studies derive from a clinical cross-sectional twin study including 58 monozygotic (MZ) and 54 dizygotic (DZ) same-sex healthy twin pairs, aged 20 to 46 years, recruited from the Danish Twin Register. The thesis covers aspects of digital fundus imaging, retinal vessel diameter measurement techniques, twin analysis, epidemiologic studies on retinal blood vessel diameters and their associations with ocular and systemic diseases, and finally a summary and discussion of the papers. Clinical signs of systemic vascular diseases are reflected in the eye, and are easily observed with ophthalmoscopy. The eye is like a window to the systemic microvasculature, and is the only place in the body where it is possible to examine blood vessels of the central nervous system noninvasively and non-destructively. Large epidemiologic studies have demonstrated associations between retinal vessel abnormalities and glaucoma, cataract and age-related macular degeneration and systemic diseases such as hypertension, cardiovascular disease, diabetes mellitus, and stroke. These associations explain only a small part of the variation in retinal vessel morphology therefore examining the genetic and environmental influence on retinal vessel layout may provide additional insights into the aetiology and pathogenesis of these complex vascular diseases. The classic twin model is a superb method for assessing the relative influence of genetic and environmental factors on retinal vessel morphology. Monozygotic twins are 100% genetically identical for which reason we assume that all observed phenotypic differences between the two twins in a pair must be attributable to environmental factors, whereas dizygotic twins share only 50% of their genes and the extent to which MZ twins are more alike than DZ twins is therefore assumed to reflect a genetic influence on the phenotype in question

8 2. Background 2.1. The eye The eye, bulbus oculi, is a ball-shaped organ, consisting of clear refractive media surrounded by a tough fibrous tissue, the sclera, lined inside by the choroid and retina (figure 1). Light passes through the refractive media: cornea, anterior chamber, lens, and vitreous, to reach the photoreceptors in the retina where it is transformed into neural impulses. The neural impulses are transported from the retina through the optic nerve and optic radiation to the primary visual cortex in the occipital pole of the brain, where they are perceived as images. 1 Figure 1. Cross sectional schematic drawing of the human eye. From: Anatomy and physiology of the retinal vascular system The retina is supplied by the ophthalmic artery by two different systems: the choroidal circulation that supplies the outer third of the retina and the branches from the central retinal vessels that supply the inner two thirds of the retina. The blood-retinal barrier has tight junctions and is highly selective. It serves to regulate the passage of pathogens and intravascular leukocytes, thus protecting the eye from surveillance by immune cells and infections. 2, 3-8 -

9 Figure 2. A cross-section of the retina, showing the 10 layers of the retina and the blood supply, with the retinal arteries and veins to the left and the choroid to the right. From a Ciba-Geigy magazine. The central retinal artery and vein The central retinal artery and vein are branches from the ophthalmic artery and vein. Approximately one centimetre behind the eye, they pierce the optic nerve, and travel forward in the centre of the optic nerve, to enter the eye through a gap in the lamina cribrosa. They both branch into one superior and one inferior branch, which subdivide into nasal and temporal branches. The four major arterial branches are functional end-arteries and they supply a quadrant of the retina each, with no overlaps. The large arterial and venous branches travel in the nerve fibre layer just beneath the inner limiting membrane. They branch into two different capillary networks, one plexus within the ganglion cell layer and one plexus within the inner nuclear layer (figure 2). The fovea is a 500!m large capillary-free zone, allowing for light to pass through undisturbed; instead the fovea is nourished from the choriocapillaris. 2, 3 The retinal blood flow is autoregulated, and is believed to be a combination of myogenic and metabolic mechanisms. Autoregulation of intraocular blood flow serves to ensure a constant level of blood flow, despite moderate variations in perfusion pressure. As a result of the autoregulation of retinal blood flow, the oxygen tension in the inner retina can be maintained at a normal level during variations in blood pressure or intra ocular pressure (IOP). But, - 9 -

10 there is an upper limit for autoregulation, therefore a sufficient increase in arterial blood pressure can overwhelm the autoregulation and result in increased retinal blood flow. 3 The Choroid The choroid is a highly pigmented, vascular, loose connective tissue that supplies the outer third of the retina and also has a thermoregulatory role. It is supplied by the posterior ciliary arteries and is situated between the sclera and the retina and consists of 5 layers: Bruch s membrane, the choriocapillaris with fenestrated capillaries, two vessel layers, and the suprachoroid. 2 Choroidal blood vessels do not respond to elevated IOP and significant reductions in choroidal blood flow cause no measurable effect on choroidal blood flow, therefore they are believed to not have autoregulation. 3 Cilioretinal arteries Occasionally the central retinal artery is assisted by one or more cilioretinal arteries, emerging from the rim of the optic disc (figure 3). These arteries are derived directly from the circle of Zinn, which is formed by small branches from the short posterior ciliary arteries, which also supply the choroid. It is of clinical relevance that a temporal cilioretinal artery supplying the fovea may spare the fovea in cases of central retinal artery occlusion. 4 Figure 3. Fundus photograph with two temporal cilioretinal arteries (CRA) supplying the fovea

11 2.3. Retinal vessel abnormalities Hypertensive retinopathy Hypertensive retinopathy represents the ocular end-organ damage secondary to arterial hypertension. Pathological changes primarily affect the retina, but changes in the choroid and optic nerve may also be involved. Essential arterial hypertension is a very common disease and is defined as having mean systolic blood pressure " 140mmHg and mean diastolic blood pressure " 90mmHg. Hypertension is associated with increased risk of stroke and cardiovascular disease. Hypertensive retinopathy is usually asymptomatic and rarely results in loss of vision. The primary pathologic change in hypertensive retinopathy is a protective vasoconstriction, seen clinically as focal arterial narrowing, generalized arterial narrowing and arterial straightening. Physiologically, retinal arteries constrict in response to elevated blood pressure, as a result of autoregulation. This is best seen in young people, because sclerosis in older patients arteries prevents the same degree of narrowing. 5 A further increase in blood pressure can break down the autoregulation, and eventually result in arterial dilation and tortuosity. 6, 7 Long-standing hypertension can cause arteriosclerosis with thickening of the arterial walls resulting in luminal narrowing. Arteriosclerotic vessel walls thicken because of intimal hyalinization, medial hypertrophy, and endothelial hyperplasia. 8 Arteriosclerotic changes manifest as copper wiring and silver wiring of the retinal arteries. The age of the patient may complicate interpretation of the fundus changes, because arterial sclerosis both occurs in hypertensive patients and in the normal ageing population. Arteriovenous crossings in the retina share a common adventitial sheath. When arteriosclerotic arteries cross over veins the vein distal to the constriction can appear dilated, darker and more tortuous. Arteriovenous nicking (AV-nicking) is diagnosed when the underlying vein becomes less apparent or maybe even disappears on either side of the artery. AV-nicking is an unambiguous finding and is a characteristic of chronic hypertensive retinopathy. Sustained hypertension leads to disruption of the blood-retinal barrier and vascular leakage resulting in haemorrhages, retinal oedema, and formation of hard exudates. Occlusions in the small end arterioles lead to retinal ischemia and is seen as cotton wool spots. In very severe hypertension the optic nerve becomes oedematous, and the patient s vision is threatened. 5, 9 A classification system for hypertensive retinopathy was introduced in 1939 by Keith, Barker, and Wagener, based on the status of retinal arteries, and systemic arteries from pectoral muscle biopsies (table 1). 10 A classification that kept the vascular changes of hypertensive retinopathy, and

12 arterial sclerosis separate was introduced in 1952 by Scheie (table 2). 5 The Keith-Barker-Wagener classification is still used in the clinic today. In Western countries, hypertensive retinopathy group 1 and 2 are common, but patients that are treated with anti-hypertensive medicine seldom develop retinopathy group 3 and 4 (table 1). Patients with hypertensive retinopathy group 3 and 4 are still seen in the clinic on rare occasions. 5, 9 Patients in group 4 are in a very critical state. KEITH-BARKER-WAGENER CLASSIFICATION Group 1 Group 2 Group 3 Group 4 Mild to moderate narrowing or sclerosis of the arteries Moderate to marked generalized arterial narrowing, focal arterial narrowing, AVnicking, and exaggeration of the light reflex As for Group 2, plus retinal oedema, cotton-wool spots, and retinal haemorrhages As for Group 3, plus papilledema Table 1. The Keith-Barker-Wagener classification of hypertensive retinopathy. 5 SCHEIE CLASSIFICATION HYPERTENSION Grade 1 Grade 2 Grade 3 Grade 4 Arterial narrowing Grade 1, plus AV-nicking and focal arterial narrowing Grade 2, plus retinal haemorrhages, hard exudates, and cotton-wool spots Grade 3, plus papilledema ARTERIAL SCLEROSIS Grade 1 Grade 2 Grade 3 Grade 4 Barely detectable light reflex changes Obvious increased light reflex changes Copper-wire arteries Silver-wire arteries Table 2. The Scheie classification of hypertensive retinopathy

13 Branch retinal vein occlusion Branch retinal vein occlusion (BRVO) is an important cause of unilateral quadrant vision loss. In The Blue Mountains Eye Study, BRVO was associated with advancing age (" 70years), increasing mean arterial blood pressure and arteriosclerotic retinal vessel signs (AV-nicking, focal arterial narrowing, arterial wall opacification) and had a cumulative 10-year incidence of 1.2%. 11 Branch retinal vein occlusions are seen at arteriovenous crossings. When an arteriosclerotic artery compresses a vein, the vein loses endothelial cells and thrombus formation leads to occlusion of the vein, stagnation of blood flow and local retinal hypoxia in the area that is drained by the vein. The clinical signs are leakage, haemorrhages, cotton wool spots, hard exudates, and oedema in the affected zone. The vein dilates and becomes tortuous distal to the site of occlusion, because of congestion and increased transmural pressure. The retinal artery supplying the affected area becomes narrowed and sheathed. The patient complains of a sudden blurred vision or a visual field defect. 5, Retinal vessel tortuosity Studies on the variation in retinal vessel tortuosity and its association with various systemic parameters in adults are limited. Some forms of arterial tortuosity are dominantly genetically inherited and are caused by weakness in the vessel wall Diabetic macular oedema 16 and branch retinal vein occlusion 5 are associated with retinal venous tortuosity. Severe hypertension, 6, 7, 17 and 9, 18 retinopathy of prematurity (ROP) are associated with retinal arterial tortuosity. Increased transmural pressure results in dilation and elongation of arteries 19 and tortuosity is observed when a critical transmural pressure threshold of a passive tube is reached. 20 This may also append to retinal vessels when pathological stresses, such as severe hypertension and retinal hypoxia exceed the autoregulatory capacity. This was observed in retinal vessels of human newborns with acute perinatal distress, where systemic metabolic distress (hypoxia and lactic acidosis) caused retinal arterial dilation and tortuosity Computer assisted methods for analysis of retinal vessel calibres Historically, we can divide research on retinal vessel morphology into three eras: the visual impression era, the quantitative era, and the digital imaging era. The first era of basic research used visual impression and yielded limited data (figure 4). Retinal arterial narrowing in hypertensive

14 retinopathy was first described by Gowers in Arteriovenous nicking as a sign of hypertensive retinopathy was described by Marcus Gunn in Figure 4. Old fashioned funduscopy with a candle as light source. In the 1970s we moved into a quantitative era, thanks to the introduction of the wide-angle fundus camera. Diameters were measured manually with a dial caliper based on visual identification of the vessel outline on a magnified projected image on a white screen or by direct visual measurement through a microscope with a screw-micrometer eye-piece. 24, 25 In 1974 Parr and Spears presented a more precise vessel measurement method. Negatives were mounted as slides between two glass plates. The widths of the parent trunk, and of its two branches, were measured with a measuring projector. They developed the Parr-Spears formula for the central retinal artery equivalent by empirically measuring the relation between branches and their trunk vessels in healthy eyes. 26 In 1986, Olaf Brinchman-Hansen introduced a better technique where the fundus photographs were measured with a computer controlled microphotometer. 27 The above mentioned methods were troublesome and time-consuming and had low intra- and inter-operator reproducibility. A new era of digital imaging began in the 1990s. Like the rest of the imaging world today, fundus photography also participated in the digital revolution. Advances in digital image analysis have allowed objective, fast, and accurate semi-automatic quantitative measurements of retinal vessel calibres. 28 The advantages of these methods are the power to measure retinal vessel diameters in eyes of large study populations and the ability to follow them over time. Another advantage is the ability to use statistics in order to adjust for important systemic and environmental

15 factors when analyzing the associations between retinal vessel calibre and ocular and systemic disease. A computer assisted semi-automatic method has been used in recent large population studies. 28 Completely automatic fundus photographic vessel measurement programs that are quicker and more accurate have recently been presented, but have not yet been tested in large population studies The Zeiss Retinal Vessel Analyser is a commercially available system which comprises a fundus camera, a video camera, a real time monitor and a computer with analyzing software for the accurate measurement of real time retinal vessel diameters. Twenty five vessel diameter readings can be obtained per second hence the vessel diameter can be measured as a function of time and can therefore be used to assess the effect of different stimuli on vessel calibres. In contrast to other vessel measurement software used in fundus photographs, the Retinal Vessel Analyser can only measure the vessel diameter in relative units, ie the relative change in diameter over time It is a good tool in physiological studies of retinal vessel diameter changes

16 3. Fundus photographic retinal vessel calibre studies 3.1. Introduction Photographic assessment of retinal trunk vessel calibres has demonstrated significant statistical effects of systemic variables, environmental conditions, and genetic factors. The aim of this chapter is to give a short summary of the most important findings of the associations between retinal vessel calibres and various systemic and environmental factors. Arteriovenous ratio (AVR) being a dimensionless figure does not vary with magnification. It is also robust toward apparent vessel broadening in blurred images and can be made using objective, automated image processing algorithms. Researchers used to interpret smaller AVR as an indicator of arterial narrowing in patients with hypertension, assuming that veins were not influenced by elevated blood pressure. Recent empirical studies of AVR and vessel diameters however have shown that artery and vein diameters provide distinctly different correlations with systemic diseases. The field of quantitative fundus photographic vessel studies is extensive. In terms of numbers of participating subjects, most of the research has been made in six major studies, covering a total of subjects, each of which has given rise to a multitude of publications: a. The Atherosclerosis Risk in Communities Study (ARIC) is a prospective cohort study of persons aged years, selected from 4 US communities, initiated in 1987 through Fundus photos were taken during the third examination in 1993 through It investigated cardiovascular disease and its risk factors among middle-aged people. b. The Blue Mountains Eye Study (BMES) is an Australian population based cohort study of common eye diseases that included 3355 participants at baseline in , when mydriatic 30 degree fundus photos were taken. The study examined microvascular characteristics and their association with ocular and systemic diseases. c. The Beaver Dam Eye Study includes 4926 persons aged years living in Beaver Dam, Wisconsin. Baseline examinations were performed in It investigated relationships between microvascular characteristics and ocular and systemic disease

17 d. The Cardiovascular Health Study (CHS) is a prospective cohort study initiated in , in 4 communities in the USA. In fundus photos were taken of 2405 people, aged years. It is a study of coronary heart disease and stroke among older people. e. The Wisconsin Epidemiologic Study of Diabetic Retinopathy (XVIII and XIX ) included 996 persons diagnosed with diabetes before age 30 years who were taking insulin, and 225 controls without diabetes, all from South Central Wisconsin. Baseline examinations were made in The study examined diabetic retinopathy, retinal vein and artery diameters and other ocular characteristics. f. The Rotterdam Study is a population-based cohort study on chronic disease in the elderly, including 7983 persons over age 55 years, from a district near the city of Rotterdam persons underwent eye examination at baseline in The following sections summarize the findings of these large-scale population studies Associations of retinal arterial and venous calibres with eye disease End-stage glaucoma has long been described as being associated with narrow retinal vessels. It remains unknown whether vessel narrowing precedes or follows retinal nerve fibre and ganglion cell loss. In the BMES, eyes with glaucomatous damage had significantly narrower retinal arteries (183±2.6!m) than eyes without glaucoma (194±0.4!m, p=0.0001) and also narrower arteries than eyes with ocular hypertension (195±1.6!m, p=0.0002, after adjusting for age, gender, body mass index (BMI), smoking, blood glucose level, mean arterial blood pressure (MAP), and refraction). Eyes with open angle glaucoma were 2.7 times more likely to have generalized arterial narrowing than eyes without glaucoma. A similar pattern was found for retinal vein diameters. There was also a strong trend for narrower retinal arteries with increasing cup-to-disc ratios. Generalized retinal arterial narrowing is an indicator of vascular abnormality, and thus appears to be associated with optic nerve damage caused by open-angle glaucoma. It is not clear whether such retinal arterial abnormality reflects an ischemic process leading to optic nerve damage, or whether it results from an autoregulatory response to the loss of retinal neurons in glaucoma. 37 A study of 64 patients with primary open-angle glaucoma found a strong association between peripapillary arterial narrowing and visual field defects in the corresponding hemifield. No statistical association was seen with retinal vein narrowing or dilation

18 In the Beaver Dam Eye Study generalized arterial and venous narrowing were significantly associated with the prevalence of nuclear cataract, defined as the odds ratio (OR) of the first versus the fifth quintile of vessel calibres (OR 0.68; CI , 0.93) and (OR 0.72; CI , 0.98), for arteries and veins, respectively. Smaller retinal artery diameter values at baseline were directly correlated with the 10-year incidence of retinal pigment epithelium (RPE) depigmentation (OR 1.93; CI , 3.34) and inversely correlated with the 10-year incidence of nuclear cataract (OR 0.71; CI , 0.99, after controlling for age and gender). Larger retinal vein diameter values at baseline were associated with the 10-year incidence of nuclear cataract (relative risk 0.60; CI , 0.84). 39 In contrast to the findings of the BMES 37 arterial narrowing in the Beaver Dam Eye Study was not associated with a large cup-to-disc ratio, high IOP, or glaucoma. 39 The relation between vessel diameters and nuclear cataract was unexpected and remains unexplained. Age-related macular degeneration and its precursor age-related maculopathy (ARM) are related to arterial hypertension. Hence, it would appear likely that retinal arterial narrowing would be associated with incident age-related maculopathy (ARM), but no such association was found in the Beaver Dam Eye Study, only an association with incident RPE depigmentation was found. 39 The BMES Study also found no significant association between artery diameter and 5-year incident late or early stage ARM, but its findings suggested that other structural changes such as AV-nicking and focal arterial narrowing could either contribute to ARM progression or share common pathologies with ARM Associations between retinal arterial calibre and systemic disease Association with age and gender Generalized retinal arterial narrowing was associated with increasing age as has uniformly been demonstrated in four different study populations. The arterial diameters decreased by 2.3 to 4.8!m per decade, 28, the correlation persisting after adjustment for correlation between artery and vein diameters. 44 Data on the association with gender have been inconsistent. In the BMES 41 and the CHS, 45 mean retinal arterial diameters were higher in women than in men across all age groups, but 42, 46 this was not corroborated by the Beaver Dam Eye Study

19 Association with current blood pressure Recent data from eight large population studies showed that narrow retinal arteries were associated with high blood pressure in adults 28, 41-43, and in children. 52 The retinal artery diameter was found to decrease by 2.8!m (p<0.0001) to 5.5!m (CI , 7.6!m) for each 10mmHg increase in MAP , 50 In the Rotterdam Study the association between current systolic blood pressure and narrower arteries was strongest in the youngest age category (55-60 years), narrowing by 3.8!m (CI , 4.7!m) per standard deviation increase in systolic blood pressure, yet becoming nonsignificant in persons above age 80 years. This may reflect that older people s vessels are sclerotic and have lost the ability to contract as a reaction to elevated blood pressure levels. 49 People with the highest quintile of current blood pressure were twice as likely to have generalized arterial narrowing than those with the lowest quintile of blood pressure. 48, 51 In children from Sydney and Singapore, each 10 mmhg increase in systolic blood pressure was associated with narrowing of the arteries by 2.1!m (CI , 2.79!m) and 1.43!m (CI , 2.59!m), respectively. 52 Association with past blood pressure In the ARIC Study, blood pressure measured 3 to 6 years before fundus photography was significantly associated with smaller artery diameters (as reflected by a lower AVR) in both men and women, whether treated or untreated for hypertension. The association with previous blood pressure was found after adjustment for current blood pressure, age, race, and smoking. 47 In the CHS, smaller arterial calibre was significantly associated with past systolic blood pressure measured 8 years before fundus photography (OR 1.11, CI , 1.27) per 10 mmhg increase in blood pressure, after adjustment for current blood pressure and use of antihypertensive medication. 51 The findings were supported by the BMES, blood pressure measured 5 years prior to fundus photography being independently associated smaller artery diameters. 8 These findings supported that generalized arterial narrowing represents cumulative effects from long-standing arterial hypertension. Association with future blood pressure The ARIC study reported that normotensive individuals with the lowest quintile of AVR at baseline were 60% more likely to develop hypertension than individuals with the highest quintile of AVR within a 3-year period of observation. 53 This was confirmed by prospective data from the BMES,

20 where narrow arteries were associated with increased risk of incident severe hypertension within a 5-year period (OR 2.6, CI , 3.9, comparing the lowest and highest AVR quintiles). 54 These studies provided evidence that generalized arterial narrowing may precede the development of severe hypertension. Association with diabetes mellitus and diabetic retinopathy In the ARIC study, lower AVR was associated with increased risk of diabetes mellitus, subjects in the lowest AVR quartile being 71% more likely to develop diabetes than subjects in the highest AVR quartile (OR, 1.71, CI , 2.57), independently of known risk factors. 55 However, it has later been documented that the association with lower AVR was largely explained by venous dilation in subjects with diabetes. 56 Larger arterial calibre was independently associated with the 4-year progression of retinopathy. Smaller arterial calibre at baseline was associated with greater 4-year and 14-year incidence of macular oedema. Arterial calibre was not associated with the incidence of proliferative retinopathy. The findings suggested that retinal vascular calibre measurements in subjects without retinopathy had little prognostic value for the development of retinopathy in persons with type 1 diabetes. 57 In diabetic subjects, mean arterial calibre decreased by 3.23!m per increase in retinopathy severity scale level and was smallest in eyes that had been treated with panretinal photocoagulation for proliferative retinopathy. This was hypothesized to result from lower volumetric flow secondary to lower metabolic needs and improved oxygenation after the destruction of ischemic retinal tissue. 58 Association with measures of atherosclerosis In the ARIC Study generalised arterial narrowing, as reflected by lower mean AVR values, was associated, after adjustment for past and present blood pressure, with carotid plaque 59 and increased carotid stiffness, 60 but not with any other clinical indicator of atherosclerosis (cardiovascular disease or stroke) or subclinical indicator of atherosclerosis (carotid or popliteal artery wall thickness, popliteal plaque, or lower limb obstructive disease) or with plasma cholesterol. Lower mean AVR was associated with higher plasma triglycerides, elevated white cells, higher fibrinogen levels, reduced albumin, smoking, greater body mass index, and elevated blood pressure. Thus, general retinal arterial narrowing shared many risk factors with atherosclerosis. Klein et al

21 suggested that generalized arterial narrowing, defined by a lower mean AVR, occurred independently of atherosclerosis and could therefore provide independent prediction for ischemic diseases of the heart, brain and other organs. 59 In the Rotterdam Study there was no association between retinal arterial narrowing and any markers of atherosclerosis except increased carotid intima-media thickness. Larger retinal venous diameters, on the other hand, were associated with several markers of atherosclerosis after adjustment for blood pressure. The relationship between AVR and atherosclerosis was mainly driven by larger vein diameters. 49 Associations with stroke and sub-clinical cerebrovascular disease There is evidence that small-vessel disease is related to both clinical and sub-clinical stroke but the underlying mechanisms are not fully understood. 61 Cerebral and retinal microcirculations share similar anatomical, embryological, and physical characteristics. 62 White matter lesions, which are caused by small vessel abnormalities, were associated with increased risk of clinical stroke. 63 In the ARIC study, the relative risk of stroke increased with decreasing quintiles of AVR, with a relative risk of 2.35 for the first versus the fifth quintile of AVR, independent of hypertension, in both men and women. 64 The Rotterdam study analyzed artery and vein diameters separately and found that generalized arterial narrowing was not associated with an increased risk of stroke. The association between smaller AVR and stroke was confirmed and the data revealed that venous dilation rather 65, 66 than arterial narrowing explained this association. Associations with coronary heart disease and cardiovascular mortality In the ARIC Study, lower AVR was associated with increased risk of incident coronary heart disease (CHD) and acute myocardial infarction in women, but not in men. Every 1 standard deviation decrease in AVR was associated with a 39% increase in incident CHD of any type. This association was independent of known risk factors such as mean arterial blood pressure, diabetes, smoking, and plasma lipid levels and was seen in women with and without hypertension and/or diabetes. Retinopathy, a marker of severe microvascular damage from hypertension, was associated with an 83% increased risk of incident CHD in women, not in men. This study supported that microvascular disease may play a more prominent role in the development of cardiovascular disease in women than in men. 67 The CHS reported that both smaller retinal arterial diameters (rate-ratio 2.0, CI , 3.7, comparing largest and smallest arterial calibre quartiles) and larger vein diameters

22 (rate-ratio 3.0, CI , 5.8, comparing largest with smallest vein calibre quartiles) were independently associated with incident coronary heart disease in elderly men and women. 68 Association with mortality In the light of the many studies showing a relationship between retinal arterial narrowing and various diseases and disease markers, an association would be expected between increased mortality and retinal arterial narrowing. The ARIC Study found no association of smaller AVR with either all-cause mortality, vascular-disease, or non-vascular-disease-related mortality. 69 Pooled analysis of data from the Beaver Dam and Blue Mountains Eye studies found that both smaller arterial and larger venous diameters were associated with increased risk of CHD and stroke mortality, independent of age, gender and vascular risk factors. 70 Generalized arterial narrowing was significantly associated with 10-year cardiovascular death in the Beaver Dam Study, with multivariable adjusted odds ratio of 1.5 (CI , 2.1), comparing presence versus absence of arterial narrowing (defined as lowest quintile of AVR). 71 Summary In summary there is evidence suggesting that retinal arterial narrowing is associated with increasing age, elevated blood pressure (past, current, and future), increasing degrees of macular diabetic oedema, increasing severity of diabetic retinopathy, increased risk of coronary heart disease, and increased mortality from CHD and stroke. Generalized arterial narrowing seems to be related to chronically high blood pressure and may therefore reflect persistent structural damage from hypertension, but there is also evidence that generalized arterial narrowing may precede the development of hypertension. It remains to be shown whether increased blood pressure leads to arterial contraction, as an autoregulatory response, which in turn leads to additional increases in blood pressure, or vice versa. Lower mean AVR shared some risk factors with atherosclerosis, but it was not directly associated with clinical and subclinical indicators of atherosclerosis. Low mean AVR seemed to occur independently of atherosclerosis. Lower mean AVR was associated with increased risk of diabetes mellitus and stroke, but it appeared that this association may be driven mostly by venous dilation rather than arterial narrowing, reflecting the importance of analyzing the effect of artery and vein diameters separately

23 3.4. Associations between retinal venous calibre and systemic disease Association with age Generalized retinal venous narrowing was associated with increasing age in four different study populations. Venous calibre decreased with 2.3!m (CI , 2.9!m) to 4.5!m (CI , 8.45!m) 28, per decade. Association with current blood pressure Retinal venous dilation was linearly and significantly associated with elevated blood pressure in participants under the age of 80 years, when adjusting for arterial calibre. After age 80 the association was insignificant, possibly due to the rigidity of the vessel walls or because of an effect of survival. 44 Association with future blood pressure In the Rotterdam Study, smaller vein calibres were found to be significantly associated with 6-year incidence of hypertension, but when adjusting for retinal arterial calibre, this association became insignificant In the BMES, retinal venous diameters were not associated with 5-year incident hypertension in the first analysis, 54 but when adjusting for retinal arterial calibres, incident hypertension was independently associated with wider venous calibres (OR 1.28, CI , 1.50). 74 This finding was consistent with a study suggesting that prehypertensive damage to the microcirculation leads to chronic retinal hypoperfusion, ischemia and venous dilation. 75 The correlation between retinal artery and vein diameters was 0.59 in The Rotterdam Study and the BMES, and persons with narrower arteries were more likely to have narrower veins. 72, 74 This confounding effect may explain the counterintuitive association between narrow veins and hypertension in the Rotterdam Study. 72 Association with diabetes mellitus and diabetic retinopathy Wider retinal venous calibre has been reported to be independently associated with progression of diabetic retinopathy, diabetic macular oedema, and incidence of proliferative diabetic retinopathy in 56-58, 76, 77 several studies

24 Association with dyslipidemia and obesity In three different study populations, dyslipidemia, such as high triglycerides and low HDL levels were associated with venous dilation The relationship between dyslipidemia and venous dilation may hypothetically involve inflammatory factors and endothelial dysfunction, which also 59, 79 play a role in the development of venous dilation in people with impaired glucose tolerance. Increasing venous diameters were also related to obesity both in persons with diabetes and in the general population during a 5-year period. Persons in the highest quintile of BMI were 70% more likely than those in the lowest quintile to have generalized venous dilation (OR 1.7, CI , 2.4). 80 Association with direct measures of atherosclerosis Larger venous diameter was associated with markers of atherosclerosis, inflammation and higher cholesterol levels in the Rotterdam Eye Study. 49 Retinal venous dilation was associated with increased carotid intima-media thickness, a marker of early atherosclerosis, but the association was largely explained by fasting insulin level in the Hoorn Study. 81 Associations with stroke and sub-clinical cerebrovascular disease Prospective data have demonstrated that larger retinal venous calibre was associated with an increased risk of stroke, cerebral infarction, white matter lesions, and lacunar infarction Each standard deviation increase in venous diameter resulted in a 13% increased risk of stroke and a 15% increased risk of cerebral infarction after adjustment for cardiovascular risk factors. 65 Retinal venous dilation was related to progression of cerebral small vessel disease, each standard deviation increase in venous diameter resulting in a 1.8 times increased risk of marked periventricular white matter lesion progression and in a 2.8 times increased risk of marked subcortical white matter lesion progression. 66 The CHS found that participants with larger retinal venous diameters were more likely to develop incident stroke during a 5-year period (rate ratio, 2.2, CI , 4.3, comparing largest with smallest venous calibre quartile, after multivariable adjustment)

25 Associations with coronary heart disease In the CHS, participants with higher retinal venous diameter had an increased 5-year risk of CHD, 11.7% (CI , 15.8%) versus 8.1% (CI , 11.6%, comparing largest with smallest venous calibre quartile). 68 Association with mortality Larger venous calibres were significantly associated with CHD and stroke mortality, in a pooled analysis of the Beaver Dam and the Blue Mountains Eye Studies. 70 Summary In summary, increasing age was associated with narrow retinal veins. Increased retinal vein diameter was associated with elevated current and future blood pressure, increased risk of diabetes mellitus, progression of diabetic retinopathy, dyslipidemia, obesity, markers of atherosclerosis and inflammation, increased risk of stroke, white matter lesions, 5-year risk of coronary heart disease, and CHD and stroke mortality. Venous dilation may be caused by the endothelial dysfunction associated with microvascular disease processes in these systemic conditions. Venous dilation has also been hypothesized to occur in response to lactic acidosis associated with retinal hypoxia in eyes with severe diabetic retinopathy. 58 These data indicated that retinal vein diameters may play their own independent role in predicting cardiovascular diseases and risk of stroke. The mechanisms underlying venous dilatation deserve more attention, as they may provide new clues into the pathophysiology of small vessel disease in the heart and brain Influence of environmental factors on retinal calibres Smoking was related to larger artery and vein diameters in three large population studies of vascular abnormalities. 49, 58, 68 Such a tendency was also found in the Danish Twin Study. 43 The mechanism may be that high carbon monoxide levels in smokers cause decreased oxygen delivery to the retina, resulting in retinal hypoxia, which causes vessel dilation. There are no conclusive data on the relation between retinal vessel calibres and alcohol consumption, oestrogen replacement therapy in women, the use of ocular betablockers, or the use of systemic antihypertensive medications

26 3.6. Discussion This review describes associations between quantitative retinal vascular calibre characteristics and systemic and ocular diseases and disease markers. The studies substantiate the idea that fundus photography may be used to advance our understanding of the pathogenesis of systemic and ocular vascular disease. There is clear evidence of involvement of aging, hypertension, arteriosclerosis, inflammation, endothelial dysfunction, smoking, and blood lipids in retinal vascular characteristics, to the extent that retinal vessel abnormalities may precede arterial hypertension, stroke and coronary heart disease. The clinical utility of using vascular calibres in cardiovascular risk prediction and in disease management requires further evaluation. Previous studies have not established specific clinical protocols or reference values graded by age, gender or blood pressure, and basic statistical measures of applicability in individuals such as positive and negative predictive values for specific outcomes have not been calculated. Because of various systemic, environmental and genetic effects it is challenging to determine uniform reference values that can be applied across populations. It has already been shown that separate reference values are needed for children, 52 It remains to be examined whether specific treatment can reduce retinal arterial narrowing and whether this will result in reduced cardiovascular risk and mortality. More precise predictions for use in individuals may potentially be derived by applying methods that enable absolute quantitative measurement of fundus dimensions. A system that could measure absolute vessel diameters in the retina, while factoring in magnification would be a potential advantage. This could be done by combining modern technologies, for example optical or ultrasonographic measurements of corneal curvature and axial length together with fundus photography to scale fundus images to true dimensions. Currently, retinal vessel measurements are based on the assumption of a standard vertical disc diameter, such as the standard 1800!m used in recent studies. Furthermore, electrocardiographic gating of fundus cameras could synchronize the 82, 83 photograph to a standard phase of the cardiac cycle, thus reducing frame-to-frame variation. The results of the Danish Twin Study 43 suggests that genetic factors determine much of the variation in retinal vessel calibres. A genome-wide linkage study of the Beaver Dam population found that retinal artery and vein diameters were linked to multiple gene loci in regions associated with hypertension, endothelial dysfunction, and vasculogenesis. 84 In conclusion, it is likely that

27 genetic, systemic and environmental factors interact to contribute to variations in retinal artery and vein calibres. Assessing these specific factors in new studies may allow for greater understanding, prevention and treatment of complex vascular diseases in humans in the future

28 4. Clinical studies 4.1. Purpose The purpose of the clinical studies that form the integral part of this thesis was to estimate the relative influence of genetic and environmental factors on retinal artery and vein calibres, retinal arterial tortuosity and the presence of cilioretinal arteries. This was done in a cross-sectional twin study, using structural equation modelling Methods Subjects We examined 55 MZ and 54 DZ healthy same-sex, normotensive twin pairs aged 20 to 46 years, recruited from the population-based Danish Twin Register Fundus photography and digital imaging We used a digital fundus camera (figure 5) to capture 50 digital grey-scale (red-free) fundus photographs centred on the macula and optic disc and 50 colour fundus photographs on transparency film. A green filter for red-free photographs was used to enhance the sharpness and contrast of the blood vessels. The advantage of digital photography is immediate feedback control of image focus and luminosity, enabling immediate adjustment of exposure settings. The clarity of the fundus photograph is related to the focus of the camera, the clarity of the patient s tear film, anterior chamber, lens, and vitreous, since the bundle of illuminating and imaging rays pass twice through these structures. Dry or swollen corneas, cataracts, and vitreous haemorrhages are examples of conditions that can blur the image. Sharpness in a photograph is a visual phenomenon that is difficult to quantify. An image is described as sharp when the borders defining the blood vessels are distinct and clear. The sharpness of a final fundus photograph is also limited by the sharpness of the specific camera s optical system. 86 Digital technology has revolutionized the way in which we can acquire, store, and share fundus images, and it gives us considerably many possibilities for quantitative assessment of retinal pathological changes. The major advantage of digital imaging in ophthalmology is the ability to conveniently enhance and adjust the photos in order to see small pathologic changes in the fundus. The first step in digital imaging is digital image capture, which is the electronic recording of light information, and its conversion to a set of numbers which are stored in an image file. Resolution