This thesis aims to evaluate the blood flow velocity in the Brachial artery during reactive hyperemia. Primarily to appraise the information it might contain regarding cardiovascular function and cardiovascular risk. Ultrasonographic doppler measurements of the Brachial artery were made on the 1016 men and women aged 70 included in the prospective investigation of the vasculature in Uppsala seniors (PIVUS) study. Analysis of the blood flow velocity in the forearm was made in comparison to established methods of estimating endothelial function, clinical markers of cardiovascular risk, the Framingham risk score and global atherosclerosis determined by whole body magnetic resonance angiography. Systolic blood flow velocity was positively related to cardiovascular risk whereas the diastolic velocity was inversely correlated. However, the systolic to diastolic blood flow velocity (SDFV) ratio was more closely associated with cardiovascular risk than its components apart. Ultrasonographic markers of Carotid atherosclerosis were related to the SDFV ratio. Concentric left ventricular remodeling and left ventricular mass index were also associated with the SDFV ratio, but not to its numerator or denominator separately. A similar pattern was found when assessing SDFV ratio in relation to global atherosclerosis, as well as to established markers of arterial compliance and vasodilation. In conclusion, during reactive hyperemia of the Brachial artery, the systolic to diastolic blood flow velocity ratio appears to contain information of additional value than its components separately, independently of established cardiovascular risk factors. Possibly, the SDFV ratio could offer a promising means to estimate cardiovascular risk in aging populations. / PIVUS

Apolipoprotein (apo) A-V is the most recently discovered member of a protein family responsible for the structure and metabolic fate of plasma lipoproteins. While most of the apolipoproteins are well characterized with regard to structure, interactions and function, the role of apoA-V is not well understood. ApoA-V is synthesized only in liver and is present in blood at much lower concentration than the other apolipoproteins. Although apoA-V is firmly established as an important determinant for plasma triacylglycerol (TG) metabolism, the mechanism is unclear. ApoA-V has been suggested to act through 1) an intracellular mechanism affecting lipoprotein assembly and secretion, 2) direct or indirect activation of lipoprotein lipase (LPL), or 3) interaction with endocytotic lipoprotein receptors. Two other novel players involved in the clearance of lipoproteins are angiopoietin-like protein (ANGPTL) 3 and 4. Previous studies have shown that the coiled-coil domain (ccd) of ANGPTL3 and -4 can inactivate lipoprotein lipase (LPL). The functional site of action of LPL is at the capillary endothelium, but the enzyme is synthesized mostly in adipocytes and myocytes and has to be transported by trancytosis to the luminal side of endothelial cells. Both ANGPTLs are present in tissues and in the circulating blood, but it is not known were the inactivation of LPL normally takes place. The aim of this thesis was to investigate the mechanism by which apoA-V exerts its effect on TG metabolism and to investigate in further detail how ANGPTLs act on the LPL system. Binding of apoA-V to receptors involved in lipoprotein metabolism was investigated by surface plasmon resonance technique (SPR). ApoA-V was found to bind to the LDL receptor related protein 1 (LRP1) and to the mosaic type 1 receptor sorLA. Binding could be competed by receptor associated protein (RAP) or by heparin, and was calcium dependent. We concluded that apoA-V binds to the LA-repeats of these receptors. In further experiments apoA-V was shown to increase binding of TG-rich chylomicrons to the receptors. This demonstrated a possible mechanism for the TG-lowering effect of apoA-V in vivo. A putative binding region in apoA-V for heparin and receptors was investigated by site-directed mutagenesis. Two positively charged amino acid residues were changed (Arg210Glu/Lys211Gln), resulting in decreased binding to heparin and to LRP1 and thus the localization of one important functional region in apoA-V. Since the receptor sorLA also contains a Vsp10p domain, another Vsp10p domain family member, sortilin, was investigated. ApoA-V was found to interact also with this receptor. In experiments with human embryonic kidney cells transfected with sorLA or sortilin, apoA-V was found to bind to cell surfaces and to be rapidly internalized while co-localized with the receptors on the way to lysosomes for degradation. Additional apoA-V mutants, identified in patients with severe hypertriglyceridemia, were investigated with regard to effects in vitro on LPL activity and receptor binding. The most severe mutants displayed null binding to LRP1, whereas the effect on LPL activity was retained. These results suggest that lack of receptor interaction mirrors the loss of biological function in a better way than the in vitro effect on LPL activity. We noted that ccd-ANGPTL3 and -4 did not prevent the LPL-mediated uptake of chylomicron-like lipoproteins in primary murine hepatocytes. Therefore LPL activity was measured after pre-incubation with ccd-ANGPTL3 or 4 in the presence or absence of TG-rich lipoproteins. Physiological concentrations of lipoproteins were found to protect LPL from inactivation by ccd-ANGPTLs. Investigation by SPR demonstrated that the ccd-ANGPTLs did not bind to the lipoproteins. Other experiments showed that less than 1% of ANGPTL4 in human serum was bound to TG-rich lipoproteins. This implies that the known binding of LPL to TG-rich lipoproteins stabilizes the enzyme and protects it from inactivation by ANGPTLs. We conclude that the normal levels of ANGPTLs in plasma are too low to affect the LPL-system and that inactivation of the enzyme by ANGPTLs is more likely to occur locally in the extracellular interstitium of tissues where LPL is en route to its endothelial binding sites and where the concentrations of the TG-rich lipoproteins are low.

<p>Consequences of atherosclerosis may result in a number of diseases of the cardiovascular system that represent serious health problems and major causes of morbidity and mortality worldwide. Although it is initially considered as disease of fibro-lipid and thrombus deposition in the arterial wall, it also involves an ongoing inflammatory response.</p><p>Normally, the inflammatory response is considered as a protective defence mechanism of the body. However, if the inflammation gets out of proportion to the threat it is dealing with, it may then result in a sustained chronic disorder and thus may underlie the initial stage of atherogenesis. The work of this thesis focuses on the expression of cytokines/chemokines and the vascular transcriptional response to inflammation, i.e. LPS in atherosclerosis. This has mainly been studied in animal models of atherosclerosis; consequently, we set out to investigate these events using human material in vitro (human carotid lesions).</p><p>Employing quantitative analysis, we were able to detect a significant induction of protein and mRNA of the cytokines IL-1β, IL-6, IL-10 and TNF-α and the chemokines IL-8 and MCP-1 by LPS in both atherosclerotic and non-atherosclerotic vessels. In contrast, LPS induction of TNF-α, IL-1β and IL-10 was solely observed in the lesions, but not in normal arteries. In addition, the impact of IL-1 gene polymorphism on the risk of myocardial infarction (MI) was estimated by DNA genotyping of 387 survivors of a first MI and 387 sex and age-matched control subjects. We found no statistically significant differences in either genotypic distribution or allelic frequencies of IL-1β (-511) or IL-1Ra (VNTR) polymorphisms between first-time survivors of myocardial infarction and their age-matched healthy controls. Incontrast, our results demonstrated a strong association between the IL-1Ra genotype and severity of angiographically determined coronary artery disease in post-MI patients. To further investigate the vascular response to inflammation, we used gene array analysis to evaluate the human vascular transcriptional response to LPS of non-atherosclerotic human renal arteries compared to carotid lesions. In LPS treated renal arteries, 54% of the transcripts gave a detectable signal, where 4% were upregulated and 3.8% down-regulated. In the LPS stimulated carotid lesions, 44% of transcripts were detected. In this latter group, 5.1% of transcripts were increased and 3.3% decreased. Interestingly, a newly identified virus-inducible antiviral protein, CMV inducible gene ₅/viperin (Cig₅), was among the most strongly induced gene in both normal and atherosclerotic biopsies. Single gene analysis revealed viperin in the endothelium of human atherosclerotic lesions. Further, viperin was induced in vascular cells by inflammatory stimuli and CMV infection.</p><p>In conclusion we show that atherosclerotic vessels produce more proinflammatory cytokines/chemokines than normal vessels. Interestingly, our results indicate that LPS enhances the expression of cytokines/chemokines in a similar pattern both in lesions and normal arteries. However, the response is stronger in atherosclerotic lesions. Furthermore, our results suggest that genetic polymorphisms within the IL-1Ra loci may influence the severity of CAD. Finally, the CMV inducible gene ₅/viperin have been identified as a putative culprit molecule in vascular inflammation and atherosclerosis.</p>

Background: Patients with sleep apnea have frequent apneas and hypopneas during sleep. Apneas can be either central or obstructive. The apnea-hypopnea index (AHI) is the mean number of apneas and hypopneas per hour of sleep. Aims: 1) To evaluate the effect of a mandibular advancement device on obstructive apneas and sleep; 2) to evaluate the influence of body position on central apnea frequency; 3) to investigate whether obstructive or central apnea is related to mortality in patients with stroke; and 4) to investigate sleep and sleeping positions in women. Methods: Subjects were investigated during whole-night sleep respiratory recordings, either polysomnography including continuous recordings of EEG, EOG, EMG, airflow, respiratory effort, ECG, pulse oximetry and body position, or simplified sleep apnea recordings without EEG, EOG and EMG. Results: The frequency of obstructive apneas, hypopneas and arousals decreased and rapid eye movement (REM) sleep increased in patients with mild, moderate and severe sleep apnea during treatment with a mandibular advancement device. Central apneas were more prevalent in the supine position compared with the non-supine position in patients with Cheyne-Stokes respiration. The mean ± SD central AHI was 41 ± 13 in the supine position and 26 ± 12 in the non-supine position, p&lt;0.001. Stroke patients with obstructive sleep apnea ran an increased risk of death during 10 ± 0.6 years of follow-up with an adjusted hazard ratio of 1.76 (95% CI 1.05-2.95) compared with controls, independent of hypertension, age, body mass index, gender, smoking, diabetes mellitus, atrial fibrillation, Mini-Mental State Examination and Barthel-ADL. Central apnea was not related to early death. Total sleep time, sleep efficiency, rapid eye movement sleep, slow wave and time in the supine position decreased with age in women. Sleep quality in women was reduced with age, body mass index, obstructive sleep apnea, smoking, alcohol and hypertension. Conclusions: Obstructive sleep apneas and arousals are reduced and REM sleep is increased using a mandibular advancement device in patients with mild, moderate and severe sleep apnea. The frequency of central apneas and hypopneas is increased in the supine position in patients with Cheyne-Stokes respiration. Stroke patients with obstructive sleep apnea run an increased risk of early death. Central sleep apnea was not related to early death among the present patients. Normal values for sleep stages and sleeping positions are presented in a population-based sample of women. Age, body mass index, obstructive sleep apnea, smoking, alcohol and hypertension reduce sleep quality in women.

Carotid stenosis is one of several causes of ischemic stroke and entails a high risk of ischemic stroke recurrence. Removal of a carotid stenosis by carotid endarterectomy results in a risk reduction for ischemic stroke, but the magnitude of risk reduction depends on several factors. If the delay between the last symptom and carotid endarterectomy is less than 2 weeks, the absolute risk reduction is &gt;10%, regardless of age, sex, or if the degree of carotid stenosis is 50–69% or 70–99%. Thus, speed is the key. However, if many patients suffers an ischemic stroke recurrence within the first 2 weeks of the presenting event, an additional benefit is likely be obtained if carotid endarterectomy is performed even earlier than within 2 week after the presenting event. Carotid endarterectomy for asymptomatic carotid stenoses carries a small risk reduction for stroke. Screening for asymptomatic carotid stenosis requires a prevalence of &gt;5% in the examined population, i.e., higher than in the general population; however, directed screening in groups with a prevalence of &gt;5% is beneficial. The aims of this thesis were to investigate the length of the delay to carotid endarterectomy, determine the risk of recurrent stroke before carotid endarterectomy, and determine if a calcification in the area of the carotid arteries seen on dental panoramic radiographs is a valid selection method for directed ultrasound screening to detect asymptomatic carotid stenosis. Consecutive patients with a symptomatic carotid stenosis who underwent a preoperative evaluation aimed at carotid endarterectomy at Umeå Stroke Centre between January 1, 2004–March 31, 2006 (n=275) were collected retrospectively and between August 1, 2007–December 31, 2009 (n=230) prospectively. In addition, 117 consecutive persons, all preliminarily eligible for asymptomatic carotid endarterectomy and with a calcification in the area of the carotid arteries seen on panoramic radiographs, were prospectively examined with carotid ultrasound. The median delay between the presenting event and carotid endarterectomy was 11.7 weeks in the first half year of 2004, dropped to 6.9 weeks in the first quarter year of 2006, and had dropped to 3.6 weeks in the second half year of 2009. The risk of ipsilateral ischemic stroke recurrence was 4.8% within 2 days, 7.9% within 1 week, and 11.2% within 2 weeks of the presenting event. For patients with a stroke or transient ischemic attack as the presenting event, this risk was 6.0% within 2 days, 9.7% within 1 week, and 14.3% within 2 weeks of the presenting event. For the 10 patients with a near-occlusion, the risk of ipsilateral ischemic stroke recurrence was 50% at 4 weeks after the presenting event. Among the 117 persons with a calcification in the area of the carotid arteries seen on panoramic radiographs, eight had a 50–99% carotid stenosis, equalling a prevalence of 6.8% (not statistically significantly over the pre-specified 5% threshold). Among men, the prevalence of 50–99% carotid stenosis was 12.5%, which was statistically significantly over the pre-specified 5% threshold. In conclusion: The delay to carotid endarterectomy was longer than 2 weeks. Additional benefit is likely to be gained by performing carotid endarterectomy within a few days of the presenting event instead of at 2 weeks because many patients suffer a stroke recurrence within a few days; speed is indeed the key. The finding that near-occlusion entails an early high risk of stroke recurrence stands in sharp contrast to previous studies; one possible explaination is that this was a high-risk period missed in previous studies. The incidental finding of a calcification in the area of the carotid arteries on a panoramic radiograph is a valid indication for carotid ultrasound screening in men who are otherwise eligible for asymptomatic carotid endarterectomy.

Consequences of atherosclerosis may result in a number of diseases of the cardiovascular system that represent serious health problems and major causes of morbidity and mortality worldwide. Although it is initially considered as disease of fibro-lipid and thrombus deposition in the arterial wall, it also involves an ongoing inflammatory response. Normally, the inflammatory response is considered as a protective defence mechanism of the body. However, if the inflammation gets out of proportion to the threat it is dealing with, it may then result in a sustained chronic disorder and thus may underlie the initial stage of atherogenesis. The work of this thesis focuses on the expression of cytokines/chemokines and the vascular transcriptional response to inflammation, i.e. LPS in atherosclerosis. This has mainly been studied in animal models of atherosclerosis; consequently, we set out to investigate these events using human material in vitro (human carotid lesions). Employing quantitative analysis, we were able to detect a significant induction of protein and mRNA of the cytokines IL-1β, IL-6, IL-10 and TNF-α and the chemokines IL-8 and MCP-1 by LPS in both atherosclerotic and non-atherosclerotic vessels. In contrast, LPS induction of TNF-α, IL-1β and IL-10 was solely observed in the lesions, but not in normal arteries. In addition, the impact of IL-1 gene polymorphism on the risk of myocardial infarction (MI) was estimated by DNA genotyping of 387 survivors of a first MI and 387 sex and age-matched control subjects. We found no statistically significant differences in either genotypic distribution or allelic frequencies of IL-1β (-511) or IL-1Ra (VNTR) polymorphisms between first-time survivors of myocardial infarction and their age-matched healthy controls. Incontrast, our results demonstrated a strong association between the IL-1Ra genotype and severity of angiographically determined coronary artery disease in post-MI patients. To further investigate the vascular response to inflammation, we used gene array analysis to evaluate the human vascular transcriptional response to LPS of non-atherosclerotic human renal arteries compared to carotid lesions. In LPS treated renal arteries, 54% of the transcripts gave a detectable signal, where 4% were upregulated and 3.8% down-regulated. In the LPS stimulated carotid lesions, 44% of transcripts were detected. In this latter group, 5.1% of transcripts were increased and 3.3% decreased. Interestingly, a newly identified virus-inducible antiviral protein, CMV inducible gene 5/viperin (Cig5), was among the most strongly induced gene in both normal and atherosclerotic biopsies. Single gene analysis revealed viperin in the endothelium of human atherosclerotic lesions. Further, viperin was induced in vascular cells by inflammatory stimuli and CMV infection. In conclusion we show that atherosclerotic vessels produce more proinflammatory cytokines/chemokines than normal vessels. Interestingly, our results indicate that LPS enhances the expression of cytokines/chemokines in a similar pattern both in lesions and normal arteries. However, the response is stronger in atherosclerotic lesions. Furthermore, our results suggest that genetic polymorphisms within the IL-1Ra loci may influence the severity of CAD. Finally, the CMV inducible gene 5/viperin have been identified as a putative culprit molecule in vascular inflammation and atherosclerosis.

Background Cardiovascular diseases, including myocardial infarction and stroke, are the main reason of death in Sweden and Western Europe. High iron stores are believed to produce oxygen radicals, which is the presumed putative mechanism behind lipid peroxidation, atherosclerosis and subsequent cardiovascular disease. Iron levels are associated with the hemochromatosis associated HFE single nucleotide polymorphisms C282Y and H63D. Bilirubin is an antioxidant present in relatively high levels in the human body. Several previous studies have found an association between high bilirubin levels and a lower risk for cardiovascular disease. Bilirubin levels are highly influenced by the common promoter polymorphism TA-insertion UGT1A1*28, the main reason for benign hyperbilirubinemia in Caucasians. There is a lack of prospective studies on both the association of iron and bilirubin levels, and the risk for myocardial infarction and ischemic stroke. Material and methods Iron, transferrin iron saturation, TIBC, ferritin and bilirubin were analyzed and HFE C282Y, HFE H63D and UGT1A1*28 were determined in myocardial infarction and stroke cases, and their double matched referents within the Northern Sweden Health and Disease Study Cohort. Results There were no associations between iron levels in the upper normal range and risk for myocardial infarction or stroke. No associations were seen for HFE-genotypes, except for a near fivefold increase in risk for myocardial infarction in HFE H63D homozygous women. Plasma bilirubin was lower in cases vs. referents both in the myocardial infarction and the stroke cohort. Despite a strong gene-dosage effect on bilirubin levels in both cases and referents, the UGT1A1*28 polymorphism did not influence the risk for myocardial infarction or stroke. Conclusion High iron stores are not associated with increased risk for neither myocardial infarction, nor stroke. There was no association between UGT1A1*28 and the risk for myocardial infarction or stroke. Consequently data suggests that other factors, which also may lower bilirubin, are responsible for the elevated risk observed in conjunction with lower bilirubin levels.

Fibroblast growth factor-23 (FGF23) was initially identified as the causative factor of autosomal dominant hypophosphatemic rickets. Further studies confirmed that FGF23 is predominantly expressed in the osteocytes and osteoblasts of bone and that circulating FGF23 acts on the kidney to inhibit renal phosphate reabsorption and 1,25(OH)2D3 hydroxylation. With the progression of chronic kidney disease (CKD), the kidneys become insufficient to maintain a normal systemic mineral homeostasis, resulting in various abnormalities of bone and mineral metabolism, generally referred to as Chronic Kidney Disease – Mineral and Bone Disorders (CKD-MBD). FGF23 increases early in the course of CKD in order to maintain normal serum phosphate levels; long before a significant increase in serum phosphate can be detected. Recent studies suggest that increased FGF23 levels are associated with progression of CKD, mortality, and the development of refractory secondary hyperparathyroidism. Because FGF23 is the very earliest marker of CKD-MBD, it is of particular interest to evaluate the relation between FGF23 and CKD-MBD abnormalities, in the setting of early CKD and also in individuals with normal renal function. In the present work, we show that FGF23 is linked to several dynamic measurements of vascular function, including endothelial dysfunction, arterial stiffness, and atherosclerosis. FGF23 is also positively associated with left ventricular mass index and an increased risk of having left ventricular hypertrophy. All associations were independent of serum phosphate and were strengthened in subjects with diminished renal function. Furthermore, we found significant evidence for an association between higher FGF23 and increased fat mass and dyslipidemia, which could represent a novel pathway linking FGF23 to cardiovascular disease. Finally, we show that FGF23 is a significant predictor of future fracture risk. Although these associations could be reflecting the increased risk associated with hyperphosphatemia and calcitriol deficiency, current evidence points towards FGF23 being more than an innocent bystander. At the very least, FGF23 holds promise of being a bio-marker of cardiovascular status and phosphate-related toxicity both in CKD and in the general population, and might be a therapeutic target that could improve the fatal prognosis in CKD patients.