Ocular and systemic markers for vascular function in those at risk of type 2 diabetes mellitus and cardiovascular disease

The devastating impact of Type 2 Diabetes Mellitus (T2DM) -related morbidity and mortality on global healthcare is escalating with higher prevalences of obesity, poor diet, and sedentary lifestyles. Therefore, the clinical need for early diagnosis and prevention in groups of high-risk individuals is necessary. The purpose of this thesis was to investigate the use of surrogate markers, namely retinal vascular function, to determine future vascular endothelial dysfunction, atherosclerosis, large vessel disease and cardiovascular risk in certain groups. This namely covered normoglycaemic and normotensive South Asians (SAs), those with Impaired-Glucose Tolerance (IGT) and individuals with a familial history (FH) of T2DM. Additionally the effect of overweight and obesity was studied. The techniques and modified protocols adopted for this thesis involved the investigation of endothelial function by means of vascular reactivity at the ocular and systemic level. Furthermore, the relationships between retinal and systemic function with circulating markers for endothelial cell function and cardiovascular risk markers were explored. The principal studies and findings of the research were: Vascular Function in Normoglycaemic Individuals with and without a FH of T2DM WE FH individuals exhibited higher levels of total cholesterol levels that correlated well with the retinal arterial dilation amplitude to flicker light stimulus. However this did not extend to noticeable differences in markers for endothelial cell damage and impaired retinal and systemic function. Vascular Function in Normoglycaemic South-Asians vs. White-Europeans without a FH and Vascular Disturbances Compared to healthy WEs (normo -glycaemic and -tensive), SA participants exhibited levels of dyslipidaemia and a state of oxidative stress that extended to impaired vascular function as detected by reduced brachial artery flow-mediated dilation, slower retinal arterial vessel dilation reaction times (Appendix 3) and steeper constriction profiles. Furthermore, gender sub-group analysis presented in a sub-chapter shows that SA males demonstrated 24-hour systemic blood pressure (BP) and heart rate variability (HRV) abnormalities and heightened cardiovascular disease (CVD) risk. Vascular Function in Individuals Newly Diagnosed with IGT as compared to Normoglycaemic Healthy Controls Newly-diagnosed WE and SA IGT patients showed a greater risk for CVD and T2DM progression by means of 24-hour BP abnormalities, dyslipidaemia, increased carotid artery intimal-media thickness (c-IMT), Framingham scores and cholesterol ratios. Additionally, pre-clinical markers for oxidative stress and endothelial dysfunction, as evident by significantly lower levels of plasma glutathione and increased levels of von-Willebrand factor in IGT individuals, extended to impaired vascular systemic and retinal function compared to normal controls. This originally shows retinal, systemic and biochemical disturbances in newly-diagnosed IGT not previously reported before. Vascular Function in Normal, Overweight and Obese Individuals of SA and WE Ethnicity In addition to the intended study chapters, the thesis also investigated the influence of obesity and overweight on vascular function. Most importantly, it was found for the first time that compared to lean individuals it was overweight and not obese individuals that exhibited signs of vascular systemic and ocular dysfunction that was evident alongside markers of atherosclerosis, CVD risk and endothelial damage.

Ocular and systemic vascular function in human ageing

The importance of vascular risk factors in various age-related pathologies has been extensively researched. Nevertheless, the haemodynamic disturbance occurring in various ocular and systemic vascular beds to impact upon ocular function remained largely unknown. The purpose of the following studies was to explore the presence and impact of both ocular and systemic vascular dysregulation as well as biochemical vascular risk factors in healthy elderly individuals and patients with age-related macular degeneration. Furthermore, the possible role was played by circulatory oxidative stress and its relationship with endothelial dysfunction at both ocular and systemic levels has also been investigated. There were four principal sections to the present work: 1. To assess the relationship between ocular and systemic anti-oxidative defence in healthy individuals The principal findings of this work were: -It has been shown that MPOD significantly and positively related with circulatory GSH levels. 2. To investigate macro- and microcirculation and oxidative stress in early AMD patients without overt systemic disease The principal findings of this work were: -AMD patients exhibit abnormal macrocirculation compared to the controls. -Blood GSSG level was significantly higher in early AMD patients than controls. -AMD patients showed abnormal microcirculation at retinal level compared. -In early AMD patients, retinal venous RT positively correlated with blood GSSG levels. 3. To assess the relationship between ocular vascular function and circulatory markers of endothelial dysfunction and CVD risk The principal findings of this work were: -Age had a positive effect on ET-1, vWF and slope of retinal arterial constriction in otherwise healthy individuals. -Even in otherwise healthy individuals, retinal arterial vascular function showed a significant correlation with circulatory markers of endothelial dysfunction and CVD risk. 4. To assess age-related changes in ANS and vascular function, and their relationship to retinal vascular function parameters The principal findings of this work were: -Elderly individuals demonstrated abnormal circadian changes of PSNS activity compared to middle-aged group. -Elderly groups showed higher ET-1 and vWF level as well as C-IMT and AIx, and also impaired retinal vascular function compared to the middle-aged group. -In the elderly group, impaired retinal vascular function significantly correlated with the dysregulation of PSNS activity.

Abnormal retinal vascular function and lipid levels in a sample of healthy UK South Asians

Background/aims To investigate ethnic differences in retinal vascular function and their relationship to traditional risk indicators for cardiovascular disease (CVD). Methods A total of 90 normoglycaemic subjects (45 South Asian (SA) and 45 age- and gender-matched white Europeans (WEs)) were recruited for the present study. Retinal vessel reactivity to flickering light was assessed by means of the dynamic retinal vessel analyser according to a modified protocol. Fasting plasma glucose, triglycerides (TG), total, LDL and HDL cholesterol were also measured in all individuals. Results SA individuals showed higher fasting triglyceride (p=0.001) and lower HDL levels (p=0.007), leading to a higher TG:HDL-C ratio (p=0.001) than age-matched WE subjects. Additionally, in SAs, the retinal arterial reaction time in response to flicker stimulation was significantly longer in the last flicker cycle than in the WEs (p=0.039), and this change correlated positively with measured plasma TG levels (r=0.60; p=0.01). No such relationship was observed in the WEs (p>0.05). Conclusion Even in the absence of overt vascular disease, in otherwise healthy SAs there are potential signs of retinal vascular function impairment that correlates with established plasma markers for CVD risk.

Retinal and systemic vascular function in health and disease: the effect of smoking and coronary artery disease

The purpose of the following studies was to explore the effect of systemic vascular and endothelial dysfunction upon the ocular circulation and functionality of the retina. There are 6 principal sections to the present work. Retinal vessel activity in smokers and non-smokers: the principal findings of this work were: chronic smoking affects retinal vessel motion at baseline and during stimulation with flickering light; chronic smoking leads to a vaso-constrictory shift in retinal arteriolar reactivity to flicker; retinal arteriolar elasticity is decreased in chronic smokers. The effect of acute smoking on retinal vessel dynamics in smokers and non-smokers: the principal finding of this work was that retinal reactivity in chronic smokers is blunted when exposed to clicker light provocation immediately after smoking one cigarette. Ocular blood flow in coronary artery disease: The principal findings of this work were: retrobulbar and retinal blood flow is preserved in CAD patients, despite a change pulse wave transmission; arterial retinal response to flickering light provocation is significantly delayed in CAD patients; retinal venular diameters are significantly dilated in CAD patients. Autonomic nervous system function and peripheral circulation in CAD: The principal findings in this work were: CAD patients demonstrate a sympathetic overdrive during a 24 period; a delay in peripheral vascular reactivity (nail-fold capillaries) as observed in patients suffering from CAD could be caused by either arteriosclerotic changes of the vascular walls or due to systemic haemodynamic changes. Visual function in CAD: The principal findings in this work were: overall visual function in CAD patients is preserved, despite a decrease in contrast sensitivity; applying a filtering technique selecting those with greater coefficient of variance which in turn represents a decrease in reliability, some patients appear to have an impaired visual function as assessed using FDT visual field evaluation. Multiple functional, structural and biochemical vascular endothelial dysfunctions in patients suffering from CAD: relationships and possible implications: The principal findings of this work were: BMI significantly correlated with vWF (a marker of endothelial function) in CAD patients. Retinal vascular reactivity showed a significant correlation with peripheral reactivity parameters in controls which lacked in the CAD group and could reflect a loss in vascular endothelial integrity; visual field parameters as assessed by frequency doubling technology were strongly related with systemic vascular elasticity (ambulatory arterial stiffness index) in controls but not CAD patients.

An almond-enriched diet increases plasma α-tocopherol and improves vascular function but does not affect oxidative stress markers or lipid levels

Vascular dysfunction is one of the major causes of cardiovascular (CV) mortality and increases with age. Epidemiological studies suggest that Mediterranean diets and high nut consumption reduce CV disease risk and mortality while increasing plasma α-tocopherol. Therefore, we have investigated whether almond supplementation can improve oxidative stress markers and CV risk factors over 4 weeks in young and middle-aged men. Healthy middle-aged men (56 ± 5.8 years), healthy young men (22.1 ± 2.9 years) and young men with two or more CV risk factors (27.3 ± 5 years) consumed 50 g almond/day for 4 weeks. A control group maintained habitual diets over the same period. Plasma α-tocopherol/cholesterol ratios were not different between groups at baseline and were significantly elevated by almond intervention with 50 g almond/day for 4 weeks (p < 0.05). Plasma protein oxidation and nitrite levels were not different between groups whereas, total-, HDL- and LDL-cholesterols and triglycerides were significantly higher in healthy middle-aged and young men with CV risk factors but were not affected by intake. In the almond-consuming groups, flow-mediated dilatation (FMD) improved and systolic blood pressure reduced significantly after 50 g almonds/day for 4 weeks, but diastolic blood pressure reduced only in healthy men. In conclusion, a short-term almond-enriched diet can increase plasma α-tocopherol and improve vascular function in asymptomatic healthy men aged between 20 and 70 years without any effect on plasma lipids or markers of oxidative stress. © 2014 Informa UK, Ltd.

Retinal vascular function and cardiovascular risk factors

The current platform of conventional cardiovascular risk assessments tends to forsake the importance of endothelial function - a key biological mechanism by which cardiovascular risk factors exert their propensity for adverse vascular events. Moreover, the presence and severity of endothelial dysfunction in ‘low-risk’ individuals suggests considerable variability in pre-clinical risk that could potentially be detected well before the onset of disease. The aim of the present thesis was to investigate the presence and impact of retinal vascular dysfunction, as a barometer of endothelial function, in otherwise healthy individuals with one or more cardiovascular risk factors, but low to moderate cardiovascular risk. Systemic circulatory influences on retinal vascular function were also evaluated. The principle sections and findings of this work are: 1. Ageing effect on retinal vascular function • In low-risk individuals, there are age differences in retinal vascular function throughout the entire functional response curve for arteries and veins. Gender differences mainly affect the dilatory phase and are only present in young individuals. 2. Retinal vascular function in healthy individuals with a family history of cardiovascular disease • In low-risk individuals with a family history of cardiovascular disease, impairments in microvascular function at the retinal level correlate with established plasma markers for cardiovascular risk. 3. Ethnic differences in retinal vascular function • When compared to age-matched White Europeans, in low-risk middle-aged South Asians, there are impairments in retinal vascular function that correlate with established cardiovascular risk indicators. 4. Systemic circulatory influences on retinalµvascular function • Systemic antioxidant capacity (redox index) and plasma markers for cardiovascular risk (lipids) influence retinal microvascular function at both arterial and venous levels. 5. Retinal vascular function in individuals with obstructive sleep apnoea: a preliminarystudy • Patients with moderate to severe sleep apnoea exhibit attenuated retinal vascular function.

Author response: can vascular function be assessed by the interpretation of retinal vascular diameter changes?

A protocol with repeated stimulation cycles should be analyzed stepwise, in that each stimulation is evaluated, and a reaction pattern is identified. No two subjects will react identically, in that dilation and recovery times can vary; however, this is not reason enough to abandon a multiple stimulation cycle with fixed recovery and stimulation times. Furthermore, it enables us to examine and determine the range in which a normal subject will be placed and can then be compared to different pathophysiological states (i.e., smokers and different diseases). The purpose of our paper was to highlight the importance of evaluating these different cycles and the danger of false interpretation when averaging results. There are many different ways of evaluating dilatory responses and elasticity, but each of them must be carefully evaluated and should not be overaveraged, which can result in a loss of sensitivity and specificity.

The relationship of systemic markers of renal function and vascular function with retinal blood vessel responses

Purpose: To test the hypothesis of a significant relationship between systemic markers of renal and vascular function (processes linked to cardiovascular disease and its development) and retinal microvascular function in diabetes and/or cardiovascular disease.Methods: Ocular microcirculatory function was measured in 116 patients with diabetes and/or cardiovascular disease using static and continuous retinal vessel responses to three cycles of flickering light. Endothelial function was evaluated by von Willebrand factor (vWf), endothelial microparticles and soluble E selectin, renal function by serum creatinine, creatinine clearance and estimated glomerular filtration rate (eGFR). HbA1c was used as a control index.Results: Central retinal vein equivalence and venous maximum dilation to flicker were linked to HbA1c (both p<0.05). Arterial reaction time was linked to serum creatinine (p=0.036) and eGFR (p=0.039), venous reaction time was linked to creatinine clearance (p=0.018). Creatinine clearance and eGFR were linked to arterial maximum dilatation (p<0.001 and p=0.003 respectively) and the dilatation amplitude (p=0.038 and p=0.048 respectively) responses in the third flicker cycle. Of venous responses to the first flicker cycle, HbA1c was linked to the maximum dilation response (p=0.004) and dilatation amplitude (p=0.017), vWf was linked to the maximum constriction response (p=0.016), and creatinine clearance to the baseline diameter fluctuation (p=0.029). In the second flicker cycle, dilatation amplitude was linked to serum creatinine (p=0.022). Conclusions: Several retinal blood vessel responses to flickering light are linked to glycaemia and renal function, but only one index is linked to endothelial function. Renal function must be considered when interpreting retinal vessel responses.

Abnormal retinal vascular reactivity in individuals with impaired glucose tolerance:a preliminary study

To investigate the relationship between vascular function parameters measured at the retinal and systemic level and known markers for cardiovascular risk in patients with impaired glucose tolerance (IGT). Sixty age- and gender- matched White-European adults (30 IGT and 30 normal glucose tolerance -NGT) were recruited for the study. Fasting plasma glucose, lipids and 24-hour blood pressure (BP) was measured in all subjects. Systemic vascular and endothelial function was assessed using carotid-artery intimal media thickness (cIMT) and flow mediated dilation (FMD). Retinal vascular reactivity was assessed by the Dynamic Retinal Vessel Analyser (DVA). Additionally, blood glutathione (GSH, GSSG and tGSH) and plasma von-Willebrand (vWF) factor levels were also measured. Individuals with IGT demonstrated higher BP values (p<0.001), fasting TG and TG:HDL ratios (p<0.001) than NGT subjects. Furthermore, Total:HDL-C ratios and Framingham scores were raised (p=0.010 and p<0.001 respectively). Blood glutathione levels (GSH, GSSG and tGSH) were lower (p<0.001, p=0.039 and p<0.001 respectively) while plasma vWF was increased (p=0.014) in IGT subjects compared to controls. IGT individuals also demonstrated higher IMT in right and left carotid arteries (p=0.017 and p=0.005, respectively) alongside larger brachial artery diameter (p=0.015), lower FMD% (p=0.026) and GTN induced dilation (GID) (p=0.012) than healthy controls. At the retinal arterial level, the IGT subjects showed higher baseline fluctuations (BDF) (p=0.026), longer reaction time (RT) (p=0.032) and reduced baseline-corrected flicker response (bFR) (p=0.045). In IGT subjects retinal BDF correlated with and Total:HDL (p= 0.003) and HDL-C (p= 0.004). Arterial RT also correlated with FMD (p=0.017) in IGT but not NGT subjects. In IGT individuals there is a relationship between macro- and microvascular function, as well as a direct correlation between the observed retinal microcirculatory changes and established plasma markers for CVD. Multifactorial preventive interventions to decrease vascular risk in these individuals should be considered.

Patients with early age-related macular degeneration exhibit signs of macro- and micro-vascular disease and abnormal blood glutathione levels

Background: This pilot study aimed to investigate systemic and retinal vascular function and their relationship to circulatory markers of cardiovascular risk in early age-related macular degeneration (AMD) patients without any already diagnosed systemic vascular pathologies. Methods: Fourteen patients diagnosed with early AMD and 14 age- and gender-matched healthy controls underwent blood pressure, carotid intima-media thickness (C-IMT) and peripheral arterial stiffness measurements. Retinal vascular reactivity was assessed by means of dynamic retinal vessel analysis (DVA) using a modified protocol. Blood analyses were conducted for glutathione levels and plasma levels of total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG). Results: The AMD patients showed significantly greater C-IMT (p = 0.029) and augmentation index (AIx) (p = 0.042) than the age-matched controls. In addition, they demonstrated a shallower retinal arterial dilation slope (Slope AD) (p = 0.005) and a longer retinal venous reaction time (RT) to flickering light (p = 0.026). Blood analyses also revealed that AMD patients exhibited higher oxidized glutathione (GSSG) (p = 0.024), lower redox index (p = 0.043) and higher LDL-C (p = 0.033) levels than the controls. Venous RT parameter correlated positively with blood GSSG levels (r = 0.58, p = 0.038) in AMD subjects, but not in the controls (p > 0.05). Conclusions: Patients diagnosed with early AMD exhibit signs of systemic and retinal vascular alterations that correlated with known risk markers for future cardiovascular morbidity. © 2013 Springer-Verlag Berlin Heidelberg.

Primary open-angle glaucoma vs normal-tension glaucoma:the vascular perspective

Objective: To compare and contrast the presence of ocular and systemic vascular function in newly diagnosed and previously untreated primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) patients with comparable, early stage, functional loss. Methods: The systemic vascular function of 19 POAG patients, 19 NTG patients and 20 healthy controls was assessed by means of 24 hour ambulatory blood pressure (ABPM), peripheral pulse wave analysis (PWA) and carotid intima-media thickness (IMT). Retinal vascular reactivity to flicker light was assessed using dynamic retinal vessel analysis (DVA,IMEDOS, GmbH, Jena, Germany). Results: When compared to normal controls, both POAG and NTG patients exhibited similarly increased nocturnal systemic blood pressure variability (p=0.011); peripheral arterial stiffness (p=0.015), carotid IMT (p=0.040) and reduced ocular perfusion pressure (OPP) (p<0.001). Furthermore, on DVA analysis, both groups of glaucoma patients also exhibited steeper retinal arterial constriction slopes (slope AC) following cessation of flicker (p=0.007) and a similarly increased fluctuation in arterial and venous baseline diameter (p=0.008 and p=0.009 respectively) in comparison to controls. Conclusion: POAG and NTG patients exhibit similar alterations in both ocular and systemic circulation at the early stages of their disease process. This highlights not only the importance of considering vascular risk factors in both conditions, but also raises questions about the current separation of the two conditions into completely distinct clinical entities.

Palmitate induces insulin resistance and a pro-atherogenic phenotype in monocytes

The present study investigated the effect of the two most abundant FFA in plasma – palmitate and oleate – on insulin sensitivity and vascular function (monocyte phenotype and adhesion to endothelium) using in vitro cell culture models and Wistar rats. Palmitate at 300µM for 6h induced insulin resistance in THP-1 monocytes and L6 monocytes. The ceramide synthesis pathway partly accounted for the palmitate-induced insulin resistance in THP-1 monocytes but not for L6 myotubes. Oleate treatment did not induce insulin resistance in either cell type and co-incubation with oleate protected cells from palmitate-induced insulin resistance. Palmitate at 300µN for 24h significantly increased cell surface CD11b and CD36 expression in U937 monocytes. The increase in CD11b and CD36 expression was effectively inhibited by Fumonisin B1, an inhibitor of ceramide synthesis. Oleate treatment did not show any effect on CD11b and CD36 expression and co-incubation with oleate antagonised the effect of palmitate on CD11b and CD36 expression in U937 monocytes. The increase in CD11b expression did not affect U937 monocyte adhesion to ICAM-1. Treating Wistar rats with palmitate for 6h caused a transient delay in glucose disposal and an increase in adhesion of U937 monocytes to the aortic endothelium, particularly at bifurcations. In conclusion, the present study demonstrates that the saturated free fatty acid palmitate induces insulin resistance and a pro-atherogenic phenotype for monocytes, whereas the unsaturated free fatty acid oleate does not. In vivo studies also confirmed that palmitate induces insulin resistance and an increase in monocyte adhesion to aorta.

Multiple roles of angiopoietins in atherogenesis

Purpose of review: The roles of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) during vascular development have been extensively investigated, as has been their role in controlling the responsiveness of the endothelium to exogenous cytokines. However, very little is known about the role of these vascular morphogenic molecules in the pathogenesis of atherosclerosis. Here, we summarize the recent research into angiopoietins in atherosclerosis. Recent findings: Angiopoietin-2 is a context-dependent agonist that protects against the development of arteriosclerosis in rat cardiac allograft. A recent study showed, contrary to expectations, that a single systemic administration of adenoviral Ang-2 to apoE-/- mice, fed a Western diet, reduced atherosclerotic lesion size and LDL oxidation in a nitric oxide synthase dependent manner. In contrast, overexpression of Ang-1 fails to protect from rat cardiac allograft due to smooth muscle cell activation. The potential proatherogenic effect of Ang-1 is further supported by the induction of chemotaxis of monocytes by Ang-1 in a manner that is independent of Tie-2 and integrin binding. These studies highlight the need for extensive research to better understand the role of angiopoietins in the cardiovascular setting. Summary: Ang-2 inhibits atherosclerosis by limiting LDL oxidation via stimulation of nitric oxide production. In contrast, Ang-1 can promote monocyte and neutrophil migration. The angiopoietin–Tie-2 system provides an important new target for modulating vascular function.