Influence of LRP5 polymorphisms on normal variation in BMD

Genetic studies based on cohorts with rare and extreme bone phenotypes have shown that the LRP5 gene is an important genetic modulator of BMD. Using family-based and case-control approaches, this study examines the role of the LRP5 gene in determining normal population variation of BMD and describes significant association and suggestive linkage between LRP5 gene polymorphisms and BMD in >900 individuals with a broad range of BMD. Introduction: Osteoporosis is a common, highly heritable condition determined by complex interactions of genetic and environmental etiologies. Genetic factors alone can account for 50-80% of the interindividual variation in BMD. Mutations in the LRP5 gene on chromosome 11q12-13 have been associated with rare syndromes characterized by extremely low or high BMD, but little is known about the contribution of this gene to the development of osteoporosis and determination of BMD in a normal population. Materials and Methods: To examine the entire spectrum of low to high BMD, 152 osteoporotic probands, their families (597 individuals), and 160 women with elevated BMD (T score > 2.5) were recruited. BMD at the lumbar spine, femoral neck, and hip were measured in each subject using DXA. Results: PAGE sequencing of the LRP5 gene revealed 10 single nucleotide polymorphisms (SNPs), 8 of which had allele frequencies of >5%, in exons 8, 9, 10, 15, and 18 and in introns 6, 7, and 21. Within families, a strong association was observed between an SNP at nucleotide C171346A in intron 21 and total hip BMD (p < 1 × 10-5 in men only, p = 0.0019 in both men and women). This association was also observed in comparisons of osteoporotic probands and unrelated elevated BMD in women (p = 0.03), along with associations with markers in exons 8 (C135242T, p = 0.007) and 9 (C141759T, p = 0.02). Haplotypes composed of two to three of the SNPs G121513A, C135242T, G138351A, and C141759T were strongly associated with BMD when comparing osteoporotic probands and high BMD cases (p < 0.003). An SNP at nucleotide C165215T in exon 18 was linked to BMD at the lumbar spine, femoral neck, and total hip (parametric LOD scores = 2.8, 2.5, and 2.2 and nonparametric LOD scores = 0.3, 1.1, and 2.2, respectively) but was not genetically associated with BMD variation. Conclusion: These results show that common LRP5 polymorphisms contribute to the determination of BMD in the general population.

Shared common variants in prostate cancer and blood lipids

Background Epidemiological and clinical studies suggest comorbidity between prostate cancer (PCA) and cardiovascular disease (CVD) risk factors. However, the relationship between these two phenotypes is still not well understood. Here we sought to identify shared genetic loci between PCA and CVD risk factors. Methods We applied a genetic epidemiology method based on conjunction false discovery rate (FDR) that combines summary statistics from different genome-wide association studies (GWAS), and allows identification of genetic overlap between two phenotypes. We evaluated summary statistics from large, multi-centre GWA studies of PCA (n = 50 000) and CVD risk factors (n = 200 000) [triglycerides (TG), low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol, systolic blood pressure, body mass index, waist-hip ratio and type 2 diabetes (T2D)]. Enrichment of single nucleotide polymorphisms (SNPs) associated with PCA and CVD risk factors was assessed with conditional quantile-quantile plots and the Anderson-Darling test. Moreover, we pinpointed shared loci using conjunction FDR. Results We found the strongest enrichment of P-values in PCA was conditional on LDL and conditional on TG. In contrast, we found only weak enrichment conditional on HDL or conditional on the other traits investigated. Conjunction FDR identified altogether 17 loci; 10 loci were associated with PCA and LDL, 3 loci were associated with PCA and TG and additionally 4 loci were associated with PCA, LDL and TG jointly (conjunction FDR < 0.01). For T2D, we detected one locus adjacent to HNF1B. Conclusions We found polygenic overlap between PCA predisposition and blood lipids, in particular LDL and TG, and identified 17 pleiotropic gene loci between PCA and LDL, and PCA and TG, respectively. These findings provide novel pathobiological insights and may have implications for trials using targeting lipid-lowering agents in a prevention or cancer setting.

Is there enough evidence with evolocumab and alirocumab (antibodies to proprotein convertase substilisin-kexin type, PCSK9) on cardiovascular outcomes to use them widely?

Introduction: Statins alone often do not reduce LDL cholesterol levels sufficiently to given maximum cardiovascular benefit. Thus, additional drugs are required to reduce the levels of LDL cholesterol. Monoclonal antibodies to PCSK9 have recently been shown to decrease LDL cholesterol, but it is not known whether they improve cardiovascular outcomes. Areas covered: Evaluation of two clinical trials reporting cardiovascular outcomes with antibodies to PCSK9; the OSLER extension with evolocumab and the ODYSSEY LONG TERM trial with alirocumab. Expert opinion: In OSLER and ODYSSEY LONG TERM, there were very few cardiovascular outcomes, but the trials do suggest that evolocumab and alirocumab may reduce these outcomes. However, there are also some safety concerns with both of these antibodies. Large clinical outcome trials are underway with both evolocumab and alirocumab, which will probably clarify both the safety concerns and any cardiovascular benefits with these antibodies. In our opinion, these antibodies may be suitable for use in subjects with familial hypercholesterolemia, who are uncontrolled with their present medications, provided intensive safety and cardiovascular monitoring is being undertaken. However, evolocumab and alirocumab should be used with caution in other subjects, until outcome studies in higher numbers of subjects, have shown acceptable safety and cardiovascular profiles.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.

Dyslipidaemia, glucose intolerance and cardiovascular disease mortality and morbidity in Europeans and Asians

Dyslipidaemia, a major risk factor of cardiovascular disease (CVD), is prevalent not only in diabetic patients but also in individuals with impaired glucose tolerance (IGT) or impaired fasting glucose (IFG). The aims of this study were: 1) to investigate lipid levels in relation to glucose in European (Study I) and Asian (Study II) populations without a prior history of diabetes; 2) to study the ethnic difference in lipid profiles controlling for glucose levels (Study III); 3) to estimate the relative risk for cardiovascular mortality (Study IV) and morbidity (Study V) associated with dyslipidaemia in individuals with different glucose tolerance status. Data of 15 European cohorts with 19 476 subjects (I and III) and 13 Asian cohorts with 19 763 individuals (II and III) from 21 countries aged 25-89 years, without a prior history of diabetes at enrollment, representing Asian Indian, Chinese, European, Japanese and Mauritian Indian, were compared. The lipid-CVD relationship was studied in 14 European cohorts of 17 763 men and women which provided with follow-up data on vital status, with 871 CVD deaths occurred during the average 10-year follow-up (IV). The impact of dyslipidaemia on incidence of coronary heart disease (CHD) in persons with different glucose categories (V) was further evaluated in 6 European studies, with 9087 individuals free of CHD at baseline and 457 developed CHD during follow-up. Z-scores of each lipid component were used in the data analysis (I, II, IV and V) to reduce the differences in methodology between studies. Analyses of cardiovascular mortality and morbidity were performed using Cox proportional hazards regression analysis adjusting for potential confounding factors. Within each glucose category, fasting plasma glucose (FPG) levels were correlated with increasing levels of triglycerides (TG), total cholesterol (TC), TC to high-density lipoprotein (HDL) ratio and non-HDL cholesterol (non-HDL-C) (p<0.05 in most of the ethnic groups) and inversely associated with HDL-C (p<0.05 in some, but not all, of the populations). The association of lipids with 2-h plasma glucose (2hPG) followed a similar pattern as that for the FPG, except the stronger association of HDL-C with 2hPG. Compared with Central & Northern (C & N) Europeans, multivariable adjusted odd ratios (95% CIs) for having low HDL-C were 4.74 (4.19-5.37), 5.05 (3.88-6.56), 3.07 (2.15-4.40) and 2.37 (1.67-3.35) in Asian Indian men but 0.12 (0.09-0.16), 0.07 (0.04-0.13), 0.11 (0.07-0.20) and 0.16 (0.08-0.32) in Chinese men who had normoglycaemia, prediabetes, undiagnosed and diagnosed diabetes, respectively. Similar results were obtained for women. The prevalence of low HDL-C remained higher in Asian Indians than in others even in individuals with LDL-C < 3 mmol/l. Dyslipidaemia was associated with increased CVD mortality or CHD incidence in individuals with isolated fasting hyperglycaemia or IFG, but not in those with isolated post-load hyperglycaemia or IGT. In conclusion, hyperglycaemia is associated with adverse lipid profiles in Europeans and Asians without a prior history of diabetes. There are distinct patterns of lipid profiles associated with ethnicity regardless of the glucose levels, suggesting that ethnic-specific strategies and guidelines on risk assessment and prevention of CVD are required. Dyslipidaemia predicts CVD in either diabetic or non-diabetic individuals defined based on the fasting glucose criteria, but not on the 2-hour criteria. The findings may imply considering different management strategies in people with fasting or post-load hyperglycaemia.

Inflammation and Complement Activation in Intracranial Artery Aneurysms

Intracranial artery aneurysms (IAs) are estimated to be present in 2.3% of the population. A rupture of an IA causes subarachnoid hemorrhage, with up to 50% mortality. The annual low rupture risk of an IA indicates that most IAs never rupture. The current treatment options are invasive and somewhat risky. Thus rupture-prone IAs should be identified and this requires a better understanding of the IA wall pathobiology. Inflammatory cell infiltrations have been found to precede IA rupture, indicating the role of inflammation in IA wall degeneration and rupture. The complement system is a key mediator of inflammation and house-hold processing of injured tissue. This study aimed at identifying the role of complement activation in IA wall degeneration and the complement activators involved and determining how the complement system is regulated in the IA wall. In immunostainings, the end-product of complement activation, the terminal complement complex (TCC), was located mainly in the outer part of the IA wall, in areas that had also sustained loss of cells. In electron microscopy, the area of maximum TCC accumulation contained cellular debris and evidence of both apoptotic and necrotic cell death. Complement activation correlated with IA wall degeneration and rupture, de-endothelialization, and T-cell and CD163-positive macrophage infiltration. The complement system was found to become activated in all IAs by the classical pathway, with recruitment of alternative pathway amplification. Of the potential activators immunoglobulins G and M and oxidatively modified lipids were found in large areas. Lipid accumulation was observed to clearly colocalize with TCC and C-reactive protein. In the luminal parts of the IA wall, complement activation was limited by cellular expression of protectin (CD59) and extracellular matrix-bound inhibitors, C4b binding protein and factor H whereas the outer part of the wall lacked cells expressing protectin as well as matrix-bound factor H. In single nucleotide polymorphism-analysis, age-related macular degeneration-associated factor H Y402H polymorphism did not associate with the presence of IAs or their rupture The data suggest that complement activation and TCC formation are involved in IA wall degeneration and rupture. Complement seems to become activated by more than one specific activator. The association of complement with de-endothelialization and expression of several complement activators indicate a possible role of endothelial dysfunction and/or impaired clearance mechanisms. Impaired complement regulation seems to be associated with increased complement activation in IA walls. These results stress the role of chronic inflammation in IA wall pathobiology and the regulatory role of complement within this process. Imaging inflammation would possibly enhance the diagnostics of rupture-prone IAs, and targeting IA treatment to prevent chronic inflammation might improve IA treatment in the future.

Association of a low density lipoprotein receptor microsatellite variant with obesity

OBJECTIVE: To determine whether a microsatellite polymorphism located towards the 3' end of the low density lipoprotein receptor gene (LDLR) is associated with obesity. DESIGN: A cross-sectional case-control study. SUBJECTS: One hundred and seven obese individuals, defined as a body mass index (BMI) > or = 26 kg/m2, and 163 lean individuals, defined as a BMI < 26 kg/m2. MEASUREMENTS: BMI, blood pressure, serum lipids, alleles of LDLR microsatellite (106 bp, 108 bp and 112 bp). RESULTS: There was a significant association between variants of the LDLR microsatellite and obesity, in the overall tested population, due to a contributing effect in females (chi 2 = 12.3, P = 0.002), but not in males (chi 2 = 0.3, P = 0.87). In females, individuals with the 106 bp allele were more likely to be lean, while individuals with the 112 bp and/or 108 bp alleles tended to be obese. CONCLUSIONS: These results suggest that in females, LDLR may play a role in the development of obesity.

Cross-sectional study of a microsatellite marker in the low density lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.

Diet and the development of atherosclerosis: a whole-diet approach from childhood to adulthood

Cardiovascular diseases (CVDs) are the leading cause of mortality in the world. Studies of the impact of single nutrients on the risk for CVD have often provided inconclusive results, and recent research in nutritional epidemiology with a more holistic whole-diet approach has proven fruitful. Moreover, dietary habits in childhood and adolescence may play a role in later health and disease, either independently or by tracking into adulthood. The main aims of this study were to find childhood and adulthood determinants of adulthood diet, to identify dietary patterns present among the study population and to study the associations between long-term food choices and cardiovascular health in young Finnish adults. The study is a part of the multidisciplinary Cardiovascular Risk in Young Finns study, which is an ongoing, prospective cohort study with a 21-year follow-up. At baseline in 1980, the subjects were children and adolescents aged 3 to 18 years (n included in this study = 1768), and young adults aged 24 to 39 years at the latest follow-up study in 2001 (n = 1037). Food consumption and nutrient intakes were assessed with repeated 48-hour dietary recalls. Other determinations have included comprehensive risk factor assessments using blood tests, physical measurements and questionnaires. In the latest follow-up, ultrasound examinations were performed to study early atherosclerotic vascular changes. The average intakes showed substantial changes since 1980. Intakes of fat and saturated fat had decreased, whereas the consumption of fruits and vegetables had increased. Intake of fat and consumption of vegetables in childhood and physical activity in adulthood were important health behavioural determinants of adult diet. Additionally, a principal component analysis was conducted to identify major dietary patterns at each study point. A similar set of two major patterns was recognised throughout the study. The traditional dietary pattern positively correlated with the consumption of traditional Finnish foods, such as rye, potatoes, milk, butter, sausages and coffee, and negatively correlated with fruit, berries and dairy products other than milk. This type of diet was independently associated with several risk factors of CVD, such as total and low-density lipoprotein cholesterol, apolipoprotein B and C-reactive protein concentrations among both genders, as well as with systolic blood pressure and insulin levels among women. The traditional pattern was also independently associated with intima media thickness (IMT), a subclinical predictor of CVD, in men but not in women. The health-conscious pattern, predominant among female subjects, non-smokers and urbanites, was characterised by more health-conscious food choices such as vegetables, legumes and nuts, tea, rye, fish, cheese and other dairy products, as well as by the consumption of alcoholic beverages. This pattern was inversely, but less strongly, associated with cardiovascular risk factors. Tracking of the dietary pattern scores was observed, particularly among subjects who were adolescents at baseline. Moreover, a long-term high intake of protein concurrent with a low intake of fat was positively associated with IMT. These findings suggest that food behaviour and food choices are to some extent established as early as in childhood or adolescence and may significantly track into adulthood. Long-term adherence to traditional food choices seems to increase the risk for developing CVD, especially among men. Those with intentional or unintentional low fat diets, but with high intake of protein may also be at increased risk for CVD. The findings offer practical, food-based information on the relationship between diet and CVD and encourage further use of the whole-diet approach in epidemiological research. The results support earlier findings that long-term food choices play a role in the development of CVD. The apparent influence of childhood habits is important to bear in mind when planning educational strategies for the primary prevention of CVD. Further studies on food choices over the entire lifespan are needed.

Elevated levels of PFOS and PFHxS in firefighters exposed to aqueous film forming foam (AFFF)

Exposure to aqueous film forming foam (AFFF) was evaluated in 149 firefighters working at AFFF training facilities in Australia by analysis of PFOS and related compounds in serum. A questionnaire was designed to capture information about basic demographic factors, lifestyle factors and potential occupational exposure (such as work history and self-reported skin contact with foam). The results showed that a number of factors were associated with PFAA serum concentrations. Blood donation was found to be linked to low PFAA levels, and the concentrations of PFOS and PFHxS were found to be positively associated with years of jobs with AFFF contact. The highest levels of PFOS and PFHxS were one order of magnitude higher compared to the general population in Australia and Canada. Study participants who had worked ten years or less had levels of PFOS that were similar to or only slightly above those of the general population. This coincides with the phase out of 3M AFFF from all training facilities in 2003, and suggests that the exposures to PFOS and PFHxS in AFFF have declined in recent years. Self-reporting of skin contact and frequency of contact were used as an index of exposure. Using this index, there was no relationship between PFOS levels and skin exposure. This index of exposure is limited as it relies on self-report and it only considers skin exposure to AFFF, and does not capture other routes of potential exposure. Possible associations between serum PFAA concentrations and five biochemical outcomes were assessed. The outcomes were serum cholesterol, triglycerides, high-density lipoproteins, low density lipoproteins, and uric acid. No statistical associations between any of these endpoints and serum PFAA concentrations were observed.

Antioxidative long chain alkylresorcinols. Synthesis and deuterium labelling of bioactive compounds present in whole grains

The first synthesis of long chain 5-n-alkylresorcinols (C15-C25) in whole grains and whole grain products by a novel modification of Wittig reaction is described. 5-n-Alkylresorcinols are phenolic lipids that have various effects on biological systems, such as antioxidant activity and interaction with biological membranes. These compounds are considered as biomarkers of whole grain intake, which is connected with reduced risk of cardiovascular diseases and certain cancers. Novel hapten derivatives of 5-n-alkylresorcinols, potential compounds for immunoanalytical techniques, are prepared by the same procedure utilizing microwave catalysed aqueous Wittig reaction as the key step. The synthesised analogues are required by various analytical, metabolism and bioactivity investigations. Four alternative strategies for producing deuterium polylabelled 5-n-alkylresorcinols are explored. Ring-labelled D3-alkylresorcinols were synthesized by acidic H/D exchange. Side chain -labelled D4-derivative was prepared by a total synthesis approach utilizing D2 deuterogenation of a D2-alkene derivative, and deuterogenation of alkynes was investigated in another total synthesis approach. An -D3-labelled alkylresorcinol is isotopically pure and completely stable under all relevant conditions encountered during analytical work. The labelling of another phenolic component of whole grains was explored. The preparation of D3-ferulic acid and related compounds by way of selective methylation of the precursors is described. The deuterated compounds are useful as standards in the quantification of these natural products in various substances, such as food and human fluids. The pure 5-n-alkylresorcinol analogues prepared were used in in vitro experiments on alkylresorcinol antioxidant activity and antigenotoxicity. The in vitro experiments show that alkylresorcinols act as antioxidants, especially when incorporated into biological systems, but possess lower activity in chemical tests (FRAP and DPPH assay). Whole grain alkylresorcinols are shown for the first time to have a protective effect against copper induced oxidation of LDL, and H2O2 or genotoxic faecal water induced damage on HT29 cells.

Nature of the thiamin-binding protein from chicken egg yolk

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.

The high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.

Acidic pH and acidic enzymes in atherosclerosis

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids and fibrous connective tissue in the arterial wall. Recently, it has been suggested that decrease in the pH of extracellular fluid of the arterial intima may enhance LDL accumulation by increasing binding of the LDL to matrix proteoglycans and also by making the plaque more favorable for acidic enzymes to be active. Many lysosomal acidic enzymes have been found in atherosclerotic plaques. In this thesis, we were able to induce secretion of lysosomal acidic cathepsin F from human monocyte-derived macrophages by stimulation with angiotensin II. We also showed that LDL pre-proteolyzed with cathepsin S was more prone to subsequent hydrolytic modifications by lipases. Especially acidic secretory sphingomyelinase was able to hydrolyze pre-proteolyzed LDL even at neutral pH. We also showed that the proteolyzed and lipolyzed LDL particles were able to bind more efficiently to human aortic proteoglycans. In addition, the role of extracellular acidic pH on the ability of macrophages to internalize LDL was studied. At acidic pH, the production of cell surface proteoglycans in macrophages was increased as well as the binding of native and modified LDL to cell surface proteoglycans. Furthermore, macrophages cultured at acidic pH showed increased internalization of modified and native LDL leading to foam cell formation. This thesis revealed various mechanisms by which acidic pH can increase LDL retention and accumulation in the arterial intima and has the potential to increase the progression of atherosclerosis.

Postmenopausal hot flushes, vascular health and hormone therapy

Vasomotor hot flushes are complained of by approximately 75% of postmenopausal women, but their frequency and severity show great individual variation. Hot flushes have been present in women attending observational studies showing cardiovascular benefit associated with hormone therapy use, whereas they have been absent or very mild in randomized hormone therapy trials showing cardiovascular harm. Therefore, if hot flushes are a factor connected with vascular health, they could perhaps be one explanation for the divergence of cardiovascular data in observational versus randomized studies. For the present study 150 healthy, recently postmenopausal women showing a large variation in hot flushes were studied in regard to cardiovascular health by way of pulse wave analysis, ambulatory blood pressure and several biochemical vascular markers. In addition, the possible impact of hot flushes on outcomes of hormone therapy was studied. This study shows that women with severe hot flushes exhibit a greater vasodilatory reactivity as assessed by pulse wave analysis than do women without vasomotor symptoms. This can be seen as a hot flush-related vascular benefit. Although severe night-time hot flushes seem to be accompanied by transient increases in blood pressure and heart rate, the diurnal blood pressure and heart rate profiles show no significant differences between women without and with mild, moderate or severe hot flushes. The levels of vascular markers, such as lipids, lipoproteins, C-reactive protein and sex hormone-binding globulin show no association with hot flush status. In the 6-month hormone therapy trial the women were classified as having either tolerable or intolerable hot flushes. These groups were treated in a randomized order with transdermal estradiol gel, oral estradiol alone or in combination with medroxyprogesterone acetate, or with placebo. In women with only tolerable hot flushes, oral estradiol leads to a reduced vasodilatory response and increases in 24-hour and daytime blood pressures as compared to women with intolerable hot flushes receiving the same therapy. No such effects were observed with the other treatment regimes or in women with intolerable hot flushes. The responses of vascular biomarkers to hormone therapy are unaffected by hot flush status. In conclusion, hot flush status contributes to cardiovascular health before and during hormone therapy. Severe hot flushes are associated with an increased vasodilatory, and thus, a beneficial vascular status. Oral estradiol leads to vasoconstrictive changes and increases in blood pressure, and thus to possible vascular harm, but only in women whose hot flushes are so mild that they would probably not lead to the initiation of hormone therapy in clinical practice. Healthy, recently postmenopausal women with moderate to severe hot flushes should be given the opportunity to use hormone therapy alleviate hot flushes, and if estrogen is prescribed for indications other than for the control of hot flushes, transdermal route of administration should be favored.