<p>Vitamin A in high doses is severely toxic to the rat skeleton, and the active metabolite retinoic acid (RA) can induce bone resorption in vitro. An excessive dietary intake of vitamin A has been associated with reduced bone mineral density and an increased risk of hip fracture. In this thesis, mechanisms of vitamin A toxicity have been investigated.</p><p>In the human osteosarcoma cell line MG-63 and in human primary osteoblast-like cultures, stimulation with RA decreased expression of osteoprotegerin (OPG), a potent inhibitor of osteoclast formation and activity. Expression of receptor activator of NF-κΒ ligand (RANKL), which stimulates osteoclastogenesis, was induced. This increase of the RANKL/OPG ratio is a likely mechanism of RA-induced bone resorption.</p><p>An interaction between vitamin A and D was demonstrated in humans for the first time. Fifteen mg retinyl palmitate antagonized the serum calcium-increasing effect of 2 μg 1,25-(OH)2-D3. This antagonism did not appear to be mediated via PTH.</p><p>Rats with subclinical hypervitaminosis A after 3 months’ exposure to approximately 9,000 IU retinyl palmitate per day had decreased bone strength, as measured by three-point-bending analysis of femur. Bone diameter and volume, but not bone mineral density, were reduced, suggesting the use of measurements other than BMD for evaluation of early hypervitaminosis A. Indirect mechanisms of toxicity may develop over time, since serum levels of other fat-soluble vitamins were decreased.</p><p>In summary, vitamin A can increase bone fragility in the rat at doses considerably lower than previously shown. The regulation of RANKL/OPG is a likely pathway for direct effects of vitamin A in bone. An antagonistic effect between vitamin A and vitamin D has been demonstrated in humans, suggesting indirect mechanisms for vitamin A toxicity.</p>

Vitamin A in high doses is severely toxic to the rat skeleton, and the active metabolite retinoic acid (RA) can induce bone resorption in vitro. An excessive dietary intake of vitamin A has been associated with reduced bone mineral density and an increased risk of hip fracture. In this thesis, mechanisms of vitamin A toxicity have been investigated. In the human osteosarcoma cell line MG-63 and in human primary osteoblast-like cultures, stimulation with RA decreased expression of osteoprotegerin (OPG), a potent inhibitor of osteoclast formation and activity. Expression of receptor activator of NF-κΒ ligand (RANKL), which stimulates osteoclastogenesis, was induced. This increase of the RANKL/OPG ratio is a likely mechanism of RA-induced bone resorption. An interaction between vitamin A and D was demonstrated in humans for the first time. Fifteen mg retinyl palmitate antagonized the serum calcium-increasing effect of 2 μg 1,25-(OH)2-D3. This antagonism did not appear to be mediated via PTH. Rats with subclinical hypervitaminosis A after 3 months’ exposure to approximately 9,000 IU retinyl palmitate per day had decreased bone strength, as measured by three-point-bending analysis of femur. Bone diameter and volume, but not bone mineral density, were reduced, suggesting the use of measurements other than BMD for evaluation of early hypervitaminosis A. Indirect mechanisms of toxicity may develop over time, since serum levels of other fat-soluble vitamins were decreased. In summary, vitamin A can increase bone fragility in the rat at doses considerably lower than previously shown. The regulation of RANKL/OPG is a likely pathway for direct effects of vitamin A in bone. An antagonistic effect between vitamin A and vitamin D has been demonstrated in humans, suggesting indirect mechanisms for vitamin A toxicity.

<p>Cerebrovascular disease (CVD) is strongly linked to hypertension and generally occurs later in life than coronary artery disease (CAD). Three quarters of the patients with symptomatic CVD are above 65 years of age. The risk factors are the same for CVD and CAD, but the relative importance of the vascular risk factors differs greatly.</p><p>Genetic causes of stroke are relatively rare. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a hereditary disease which causes CVD in young adults and middle-aged people, with migraine, stroke, psychiatric illness and dementia as clinical manifestations.</p><p>The subject of this thesis is vascular function in stroke and CADASIL. Endothelium-dependent vasodilation (EDV) and arterial stiffness were investigated by different methods in stroke patients and CADASIL patients compared with healthy controls. Venous occlusion plethysmography with intra-arterial acetylcholine was used to evaluate EDV in the forearm resistance vessels. Flow-mediated vasodilation of the brachial artery was used to evaluate EDV in a conduit artery. Stroke patients displayed reduced EDV in resistance vessels compared with a healthy control group, but this reduction was not significant when, in a larger group of stroke patients, adjustments were made for blood pressure, antihypertensive treatment and other risk factors. Flow mediated vasodilation of the brachial artery was reduced in the stroke patients even after adjustment for risk factors. </p><p>Compared with controls, the CADASIL patients showed similar EDV in the conduit artery, but reduced EDV in resistance vessels.</p><p>Arterial compliance was evaluated by augmentation index from pulse wave analysis, by a ratio of cardiac stroke volume and pulse pressure, and by the distensibility of the carotid artery in relation to pulse pressure. Stroke patients and CADASIL patients did not display any significant increase in arterial stiffness when evaluated by these methods. </p>

Cerebrovascular disease (CVD) is strongly linked to hypertension and generally occurs later in life than coronary artery disease (CAD). Three quarters of the patients with symptomatic CVD are above 65 years of age. The risk factors are the same for CVD and CAD, but the relative importance of the vascular risk factors differs greatly. Genetic causes of stroke are relatively rare. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a hereditary disease which causes CVD in young adults and middle-aged people, with migraine, stroke, psychiatric illness and dementia as clinical manifestations. The subject of this thesis is vascular function in stroke and CADASIL. Endothelium-dependent vasodilation (EDV) and arterial stiffness were investigated by different methods in stroke patients and CADASIL patients compared with healthy controls. Venous occlusion plethysmography with intra-arterial acetylcholine was used to evaluate EDV in the forearm resistance vessels. Flow-mediated vasodilation of the brachial artery was used to evaluate EDV in a conduit artery. Stroke patients displayed reduced EDV in resistance vessels compared with a healthy control group, but this reduction was not significant when, in a larger group of stroke patients, adjustments were made for blood pressure, antihypertensive treatment and other risk factors. Flow mediated vasodilation of the brachial artery was reduced in the stroke patients even after adjustment for risk factors. Compared with controls, the CADASIL patients showed similar EDV in the conduit artery, but reduced EDV in resistance vessels. Arterial compliance was evaluated by augmentation index from pulse wave analysis, by a ratio of cardiac stroke volume and pulse pressure, and by the distensibility of the carotid artery in relation to pulse pressure. Stroke patients and CADASIL patients did not display any significant increase in arterial stiffness when evaluated by these methods.

Atherosclerosis is characterized by a chronic inflammation, involving autoimmune components, in the arterial wall. An increase in proinflammatory activity relative to anti-inflammatory activity is considered to cause a progression of the disease towards plaque instability and risk of atherothrombotic events, such as acute coronary syndrome (ACS). Cortisol, the end product of the hypothalamus-pituitary-adrenal (HPA) axis, is a powerful endogenous anti-inflammatory mediator. Disturbances in the HPA axis have been reported in chronic inflammatory/autoimmune diseases, like rheumatoid arthritis. The aim of this thesis was to study various markers of systemic inflammation in patients with acute and stable conditions of coronary artery disease (CAD) and relate these findings to the cortisol response. Both patients with ACS and patients with stable CAD had high levels of C-reactive protein (CRP), interleukin (IL)-6 and IL-1 receptor antagonist, compared with healthy controls. In addition, patients with stable CAD had significantly more neutrophil-platelet aggregates than controls, as a possible indicator of neutrophil activation. The cortisol response was determined in two different cohorts of CAD patients; one consisting of patients with a first-time myocardial infarction and one consisting of patients with long-term stable CAD. From the acute phase to 3 months, the patients with a myocardial infarction showed a higher 24-h cortisol secretion and a flattened diurnal slope caused by higher cortisol levels in the evening, as compared with healthy controls. The patients with long-term stable CAD showed similarly high levels of cortisol in the evening. The levels of evening cortisol were strongly correlated with CRP and IL-6. When exposed to acute physical or acute psychological stress at 3 months, the ACS patients showed a markedly blunted cortisol response compared with healthy controls. Following the stress tests, a significant increase in CRP was observed in the patients but not in the controls, indicating a failure of the HPA axis to compensate for stress-induced inflammation in CAD. In the ACS patients, the time course of matrix metalloproteinases (MMPs) and their tissue inhibitor TIMP-1 was determined during the 3 months follow-up. A major finding was that the MMP-9 and TIMP-1 levels remained significantly higher in the patients at all time points compared to the controls. MMP-9 and TIMP-1, but not MMP-2, MMP-3 or MMP-7, were related to inflammatory activity, as assessed by CRP and IL-6. MMP-9 and TIMP-1 showed significant correlation with evening cortisol, even after adjustment for CRP and IL-6, lending further support for a link between ´high´ flat cortisol rhythm and systemic inflammatory activity. The activation status of neutrophils in stable CAD was further examined by measuring the expression, affinity state and signalling capacity of b2-integrins and the innate production of reactive oxygen species (ROS). However, the neutrophils in patients were not more activated in vivo than were cells in healthy controls, neither were they more prone to activation ex vivo. The data rather indicated an impaired function of neutrophils in stable CAD. The neutrophils in CAD patients showed a significantly lower number of total glucocorticoid receptors (GRs) and a lower GRa:GRb ratio compared to healthy controls, indicating a chronic over activation of the HPA axis and, possibly, a state of glucocorticoid resistance. Moreover, the evening cortisol levels in patients were associated with an overexpression of annexin-1, the ´second messenger´ of glucocorticoid action. In contrast to neutrophils in controls, the neutrophils in patients also showed a hyper responsiveness to exogenous annexin-1 resulting in impaired neutrophil function. To conclude, clinically stable CAD was associated with a systemic inflammatory activity, involving a high MMP-9:TIMP-1 ratio and an increased inflammatory response to acute stress but not any activation of neutrophils. This inflammatory activity was associated with a dysregulated cortisol secretion, defined by a flat diurnal rhythm and a blunted cortisol response to stress. Although the clinical relevance remains to be verified, an intriguing hypothesis is that a hyporesponsive HPA axis favours the development towards plaque instability. / On the day of the defence date the title of article III was: "A sustained elevation of serum matrix metalloproteinase-9 is associated with diurnal salivary cortisol in patients with acute myocardial infarction-a 3-month follow-up".

<p>Type 1 diabetes is caused by immuno-mediated β-cell destruction leading to insulin deficiency and hyperglycaemia. The decline in β-cell function and the clinical course after diagnosis vary. Whether the process of destruction of the β-cells is associated with markers of a non-specific inflammatory response is unknown. The aims of these studies were to identify factors of importance for clinical remission (low insulin need and normoglycaemia) and long-term β-cell function and estimate the degree of non-inflammatory response in type 1 diabetes in young adults. Clinical remission and β-cell function eight years after diagnosis were assessed and related to clinical, biochemical and immunological variables at diagnosis, including islet autoantibodies [ICA, GADA, IA-2A]. Markers of low-grade inflammation in plasma [CRP and IL-6] were estimated and the concentrations were related β-cell function [plasma C-peptide], glycaemic control and autoimmunity at diagnosis and the first year thereafter. The results showed that clinical remission occurred in about half of the patients with newly diagnosed type 1 diabetes. Preserved β-cell function eight years after diagnosis was observed in 16% of the patients classified at diagnosis as having autoimmune type 1 diabetes. Duration of remission was dependent on BMI, degree of metabolic derangement and presence of GADA at diagnosis. BMI at diagnosis was also of importance for preserved β-cell function after eight years of the disease, as were the amount of islet antibodies and presence of ICA. Elevated CRP levels were noted in the majority of cases at diagnosis and both CRP and IL-6 concentrations were stable the first year after clinical diagnosis. High concentrations of CRP and IL-6 did not relate to β-cell destruction or the degree of autoimmunity. CRP concentrations were higher in islet antibody negative than in positive patients. CRP also correlated positively to BMI, C-peptide at 12 months and to increasing HbA1c between six and 12 months. In general, females had shorter remissions, lower concentrations of serum bicarbonate and higher levels and prevalence of GADA at diagnosis, compared to males. Females also had higher HbA1c and CRP values the first year after diagnosis. In summary, BMI at diagnosis is a strong predictor of duration of remission and preservation of β-cell function. Elevated CRP concentrations are correlated to factors linked rather to insulin resistance than to β-cell destruction. Females appear to have a more acute onset and a more severe course of the disease than males.</p>

<p>Repolarization inhomogeneity is arrhythmogenic. QT dispersion (QTd) is an easily accessible ECG-variable, related to the repolarization and shown to carry prognostic information. It was originally thought to reflect repolarization inhomogeneity. Lately, arguments have been risen against this hypothesis. Other measures of inhomogeneity are being investigated, such as nondipolar components from principal component analysis (PCA) of the T-wave. In all here described populations, continuous 12-lead ECG was collected during the initial hours of observation and secondary parameters used for description of a large number of ECG-recordings.</p><p>Paper I studied QTd in 548 patients with chest pain with a median number of 985 ECG-recordings per patient. Paper II explored a spatial aspect of QTd in 276 patients with unstable coronary artery disease. QTd and a derived localized ECG-parameter were compared to angiographical measures. QTd, expressed as the mean value during the observation was a powerful marker of risk. It was however not effective in identifying high-risk patients. Variations in QTd contained no additional prognostic information. In unstable coronary artery disease, QTd was increased by a mechanism unrelated to localization of the disease.</p><p>Two relevant conditions for observing repolarization inhomogeneity might occur with conduction disturbances and during initial course of ST-elevation myocardial infarction (STEMI). Paper III compared the PCA-parameters of the T-wave in 135 patients with chest pain and conduction disturbance to 665 patients with normal conduction. Nondipolar components were quantified by medians of the nondipolar residue (TWRabsMedian) and ratio of this residue to the total power of the T-wave (TWRrelMedian). Paper IV described the changes in the nondipolar components of the T-wave in 211 patients with thrombolyzed STEMI. TWRabsMedian increased with increasing conduction disturbance and contained a moderate amount of prognostic information. In thrombolyzed STEMI, TWRabsMedian was elevated and has an increased variability. A greater decrease in absolute TWR during initial observation was seen in patients with early ST-resolution. Nondipolar components do however not reflect identical ECG-properties as the ST-elevation and their change does not occur at the same time.</p>

Type 1 diabetes is caused by immuno-mediated β-cell destruction leading to insulin deficiency and hyperglycaemia. The decline in β-cell function and the clinical course after diagnosis vary. Whether the process of destruction of the β-cells is associated with markers of a non-specific inflammatory response is unknown. The aims of these studies were to identify factors of importance for clinical remission (low insulin need and normoglycaemia) and long-term β-cell function and estimate the degree of non-inflammatory response in type 1 diabetes in young adults. Clinical remission and β-cell function eight years after diagnosis were assessed and related to clinical, biochemical and immunological variables at diagnosis, including islet autoantibodies [ICA, GADA, IA-2A]. Markers of low-grade inflammation in plasma [CRP and IL-6] were estimated and the concentrations were related β-cell function [plasma C-peptide], glycaemic control and autoimmunity at diagnosis and the first year thereafter. The results showed that clinical remission occurred in about half of the patients with newly diagnosed type 1 diabetes. Preserved β-cell function eight years after diagnosis was observed in 16% of the patients classified at diagnosis as having autoimmune type 1 diabetes. Duration of remission was dependent on BMI, degree of metabolic derangement and presence of GADA at diagnosis. BMI at diagnosis was also of importance for preserved β-cell function after eight years of the disease, as were the amount of islet antibodies and presence of ICA. Elevated CRP levels were noted in the majority of cases at diagnosis and both CRP and IL-6 concentrations were stable the first year after clinical diagnosis. High concentrations of CRP and IL-6 did not relate to β-cell destruction or the degree of autoimmunity. CRP concentrations were higher in islet antibody negative than in positive patients. CRP also correlated positively to BMI, C-peptide at 12 months and to increasing HbA1c between six and 12 months. In general, females had shorter remissions, lower concentrations of serum bicarbonate and higher levels and prevalence of GADA at diagnosis, compared to males. Females also had higher HbA1c and CRP values the first year after diagnosis. In summary, BMI at diagnosis is a strong predictor of duration of remission and preservation of β-cell function. Elevated CRP concentrations are correlated to factors linked rather to insulin resistance than to β-cell destruction. Females appear to have a more acute onset and a more severe course of the disease than males.

Repolarization inhomogeneity is arrhythmogenic. QT dispersion (QTd) is an easily accessible ECG-variable, related to the repolarization and shown to carry prognostic information. It was originally thought to reflect repolarization inhomogeneity. Lately, arguments have been risen against this hypothesis. Other measures of inhomogeneity are being investigated, such as nondipolar components from principal component analysis (PCA) of the T-wave. In all here described populations, continuous 12-lead ECG was collected during the initial hours of observation and secondary parameters used for description of a large number of ECG-recordings. Paper I studied QTd in 548 patients with chest pain with a median number of 985 ECG-recordings per patient. Paper II explored a spatial aspect of QTd in 276 patients with unstable coronary artery disease. QTd and a derived localized ECG-parameter were compared to angiographical measures. QTd, expressed as the mean value during the observation was a powerful marker of risk. It was however not effective in identifying high-risk patients. Variations in QTd contained no additional prognostic information. In unstable coronary artery disease, QTd was increased by a mechanism unrelated to localization of the disease. Two relevant conditions for observing repolarization inhomogeneity might occur with conduction disturbances and during initial course of ST-elevation myocardial infarction (STEMI). Paper III compared the PCA-parameters of the T-wave in 135 patients with chest pain and conduction disturbance to 665 patients with normal conduction. Nondipolar components were quantified by medians of the nondipolar residue (TWRabsMedian) and ratio of this residue to the total power of the T-wave (TWRrelMedian). Paper IV described the changes in the nondipolar components of the T-wave in 211 patients with thrombolyzed STEMI. TWRabsMedian increased with increasing conduction disturbance and contained a moderate amount of prognostic information. In thrombolyzed STEMI, TWRabsMedian was elevated and has an increased variability. A greater decrease in absolute TWR during initial observation was seen in patients with early ST-resolution. Nondipolar components do however not reflect identical ECG-properties as the ST-elevation and their change does not occur at the same time.

<p>Myocardial infarction (MI) is often caused by a disruption of an atherosclerotic plaque with activation of coagulation, platelets and inflammation. The aims were; to investigate whether the oral direct thrombin inhibitor, ximelagatran affected markers for coagulation, platelet and inflammation in a patient cohort with recent MI and if the coagulation markers could identify patients with increased risk of new ischemic events; to evaluate some of the mechanisms involved in formation of platelet-monocyte aggregates (PMAs). </p><p>In a biomarker substudy patients with recent MI were randomized to 24-60 mg of ximelagatran or placebo for six months. There was a persistent dose-independent reduction of coagulation markers (F1+2, D-dimer) by ximelagatran treatment. 60 % reduced their D-dimer levels after one week and that group had less ischemic events during treatment. There was an early increase of the platelet activation marker and ximelagatran in higher doses attenuated these increased levels. Both in vivo and in vitro the direct thrombin inhibitor diminished procoagulant activity and tissue factor (TF) presenting microparticles. In contrast, the inflammatory markers increased after six months of ximelagatran treatment. The PMA-levels were elevated for long-term after MI. In vitro thrombin inhibition diminished formation of PMAs. Formation of PMAs in stimulated whole blood was P-selectin dependent and induced TF expression through phosphorylation of the Src-family member Lyn in monocytes.</p><p>Addition of an oral direct thrombin inhibitor reduces coagulation and platelet activation markers for long-term after a MI together with reduced procoagulant activity which may contribute to the clinical benefit of the drug. Early reduction of D-dimer levels seems to be suitable to identify patients with reduced risk of new ischemic events independent of antithrombotic treatment. Circulating PMAs persist after a MI connecting coagulation to inflammation. Within these aggregates P-selectin induces TF, the main initiator of coagulation, partly through phosphorylation of Lyn.</p>