Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function.

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 µm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Biomarkers of systemic inflammation in farmers with musculoskeletal disorders; a plasma proteomic study.

BACKGROUND: Farmers have an increased risk for musculoskeletal disorders (MSD) such as osteoarthritis of the hip, low back pain, and neck and upper limb complaints. The underlying mechanisms are not fully understood. Work-related exposures and inflammatory responses might be involved. Our objective was to identify plasma proteins that differentiated farmers with MSD from rural referents. METHODS: Plasma samples from 13 farmers with MSD and rural referents were included in the investigation. Gel based proteomics was used for protein analysis and proteins that differed significantly between the groups were identified by mass spectrometry. RESULTS: In total, 15 proteins differed significantly between the groups. The levels of leucine-rich alpha-2-glycoprotein, haptoglobin, complement factor B, serotransferrin, one isoform of kininogen, one isoform of alpha-1-antitrypsin, and two isoforms of hemopexin were higher in farmers with MSD than in referents. On the other hand, the levels of alpha-2-HS-glycoprotein, alpha-1B-glycoprotein, vitamin D- binding protein, apolipoprotein A1, antithrombin, one isoform of kininogen, and one isoform of alpha-1-antitrypsin were lower in farmers than in referents. Many of the identified proteins are known to be involved in inflammation. CONCLUSIONS: Farmers with MSD had altered plasma levels of protein biomarkers compared to the referents, indicating that farmers with MSD may be subject to a more systemic inflammation. It is possible that the identified differences of proteins may give clues to the biochemical changes occurring during the development and progression of MSD in farmers, and that one or several of these protein biomarkers might eventually be used to identify and prevent work-related MSD.

Levels of perfluoroalkyl substances and risk of coronary heart disease: Findings from a population-based longitudinal study.

BACKGROUND: Cross-sectional studies have shown an association between exposure to perfluoroalkyl substances (PFASs) and coronary heart disease (CHD). These findings need to be evaluated in longitudinal settings. OBJECTIVES: To investigate the risk of CHD in relation to PFAS levels in a longitudinal setting among Swedish rural residents. METHODS: In a population-based prospective cohort of male farmers and rural residents recruited in 1990-1991, all men who received a CHD diagnosis between 1992 and 2009 were identified from national registers (n=253). For each CHD case, one control, matched for age, was chosen randomly from the cohort. For all cases and controls, levels of eight PFASs at baseline were measured in stored blood samples. In addition, for a subsample, PFAS levels were also measured in serum samples collected at a follow-up in 2002-2003. RESULTS: There were no statistically significant associations between levels of seven of the eight PFASs at baseline and risk for developing CHD. There was a significant association between perfluoroheptanoic acid (PFHpA) and CHD (OR=2.72; 95% CI: 1.52, 4.84) for the 3rd quartile and (OR=2.45; 95% CI: 1.40, 4.29) for the 4th quartile compared to the lowest quartile. Changes in levels of PFCs between baseline and follow-up did not differ systematically between cases and controls. CONCLUSIONS: This longitudinal study does not lend support to the previously reported cross-sectional relationship between PFAS levels and CHD risk. We found a significant association with PFHpA, but this could be a chance finding, considering its chemical resemblance to other PFASs.

High dairy fat intake related to less central obesity: a male cohort study with 12 years' follow-up.

OBJECTIVE: To study associations between dairy fat intake and development of central obesity. DESIGN: A prospective population-based cohort study with two surveys 12 years apart. SETTING: Nine municipalities selected from different parts of Sweden representing the rural areas in the country. SUBJECTS: 1782 men (farmers and non-farmers) aged 40-60 years at baseline participated in a baseline survey (participation rate 76%) and 1589 men participated at the follow-up. 116 men with central obesity at baseline were excluded from the analyses. MAIN OUTCOME MEASURES: Central obesity at follow-up defined as waist hip ratio ≥ 1. RESULTS: 197 men (15%) developed central obesity during follow-up. A low intake of dairy fat at baseline (no butter and low fat milk and seldom/never whipping cream) was associated with a higher risk of developing central obesity (OR 1.53, 95% CI 1.05-2.24) and a high intake of dairy fat (butter as spread and high fat milk and whipping cream) was associated with a lower risk of central obesity (OR 0.52, 95% CI 0.33-0.83) as compared with medium intake (all other combinations of spread, milk, and cream) after adjustment for intake of fruit and vegetables, smoking, alcohol consumption, physical activity, age, education, and profession. The associations between dairy fat intake and central obesity were consistent across body mass index categories at baseline. CONCLUSION: A high intake of dairy fat was associated with a lower risk of central obesity and a low dairy fat intake was associated with a higher risk of central obesity.

Mortality and morbidity among farmers, nonfarming rural men, and urban referents: a prospective population-based study.

A cohort of 1,220 farmers, 1,130 nonfarming rural men, and 1,087 urban referents from Sweden were monitored for 12 years. Farmers had lower mortality than urban referents for all causes of death (hazard ratio [HR] = 0.51; 95% confidence interval [CI], 0.37-0.71), cancer (HR = 0.44; 95% CI, 0.24-0.78) and cardiovascular diseases (HR = 0.60; 95% CI, 0.36-0.99). Nonfarming rural men had lower mortality than urban referents for all causes of deaths (HR = 0.81; 95% CI, 0.70-0.94). Farmers and nonfarming rural men had significantly lower morbidity risks of cancer and of psychiatric disorders than urban referents. Farmers had significantly lower risk of endocrine disorders, cardiovascular disorders, and respiratory disorders. In general, morbidity was lower among nonfarming rural men compared with urban referents and was even lower among farmers. Urban referents had, however, significantly less musculoskeletal disorder morbidity. An urban-rural factor and a farming occupational or lifestyle factor results in lower mortality and morbidity rates except concerning musculoskeletal disorders.

Functioning in neck and low back pain from a 12-year perspective: a prospective population-based study.

OBJECTIVE: The aim of this study was to evaluate the effects of unspecific neck pain and low back pain at a given time (1990-91) with respect to physical and social functioning and role limitations due to emotional problems 12 years later. METHODS: A rural male study population (2351 individuals) was established in 1989 and a first survey conducted in 1990-91. A follow-up survey was performed in 2002-03. A total of 1405 persons participated in both surveys. Functioning and role limitations in 2002-03 were evaluated using the SF-36 instrument. Several possible confounders were included in the analyses. RESULTS: Unspecific neck pain or low back pain in 1990-91 was shown in a multivariate longitudinal regression model to be significantly related to limited physical (odds ratio (OR)=2.08; 95% confidence interval (CI) 1.51-2.87) and social (OR=1.92; 95% CI 1.33-2.75) functioning 12 years later. The effects were only slightly modified by the confounders analysed. However, higher education independently and significantly predicted a low risk for functional limitations. CONCLUSION: Non-specific neck pain and low back pain at a given time impacted on the risk of limited physical and social functioning many years later. Current symptoms of depression and anxiety at the time for the second survey had a high impact on functional limitations.

Nasal complaints and signs of disease in farmers--a methodological study.

CONCLUSION: The methods used in this study are suitable for field studies that involve examinations of groups of workers. For individual examinations, there is no gold standard method that can discriminate work-related discomfort from other causes of rhinitis. OBJECTIVES: Studies of the effects of occupation on farmers' health have mainly focused on lower airways; few studies have examined effects on upper airways. This study investigated nasal functions in three groups of farmers (swine, milk and grain producers) and a control group using different methods, suitable for field studies. SUBJECTS AND METHODS: Health-related complaints were examined and several functional tests, such as expirogram, olfactory threshold test, acoustic rhinometry, nasal lavage with biomarkers of inflammation (eosinophilic cationic proteins (ECP), myeloperoxidase (MPO), tryptase, albumin) and allergy tests were performed. The different tests were correlated to nasal complaints and to each other. RESULTS: Nasal blockage complaints were more common among farmers; overall, nasal polyps were more frequent in grain producers. Objective parameters showed more pronounced mucosal swelling in farmers and higher concentrations of ECP in nasal lavage compared with controls. Lung function, olfactory threshold, atopy frequency and allergen-specific IgE to the storage mite Lepidoglyphus destructor did not differ between farmers and controls. Mucosal swelling measured with acoustic rhinometry was more pronounced in subjects with nasal complaints, hypersensitivity, nasal polyps and symptoms from lower airways. There was a correlation between biomarkers in nasal lavage (MPO, albumin and ECP).

Role of ADAMTS-1 in atherosclerosis: remodeling of carotid artery, immunohistochemistry, and proteolysis of versican.

OBJECTIVE: We investigated the potential role of ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motif type I) in atherogenesis. METHODS AND RESULTS: ADAMTS-1 is expressed at the highest levels in the aorta when compared with other human tissues examined. Immunolocalization studies in human aorta and coronary artery indicate that ADAMTS-1 expression is mainly seen at low levels in the medial layer, but upregulated in the intima when plaque is present. We found that ADAMTS-1 mRNA levels are significantly higher in proliferating/migrating cultured primary aortic vascular smooth muscle cells (VSMCs) compared with resting/confluent cells. Using the mouse carotid artery flow cessation model, we show that there are differences in vessel remodeling in ADAMTS-1 transgenic/apoE-deficient mice compared with apoE deficiency alone, particularly a significant increase in intimal hyperplasia. We show that ADAMTS-1 can cleave the large versican containing proteoglycan population purified from cultured human aortic VSMCs. Finally, using versican peptide substrates, we show data suggesting that ADAMTS-1 cleaves versican at multiple sites. CONCLUSIONS: We hypothesize that ADAMTS-1 may promote atherogenesis by cleaving extracellular matrix proteins such as versican and promoting VSMC migration.

Primary care consultation, hospital admission, sick leave and disability pension owing to neck and low back pain: a 12-year prospective cohort study in a rural population.

BACKGROUND: Neck and low back pain are common musculoskeletal complaints generating large societal costs in Western populations. In this study we evaluate the magnitude of long-term health outcomes for neck and low back pain, taking possible confounders into account. METHOD: A cohort of 2,351 Swedish male farmers and rural non-farmers (40-60 years old) was established in 1989. In the first survey, conducted in 1990-91, 1,782 men participated. A 12-year follow-up survey was made in 2002-03 and 1,405 men participated at both times. After exclusion of 58 individuals reporting a specific back diagnosis in 1990-91, the study cohort encompassed 1,347 men. The health outcomes primary care consultation, hospital admission, sick leave and disability pension were assessed in structured interviews in 2002-03 (survey 2). Symptoms and potential confounders were assessed at survey 1, with the exception of rating of depression and anxiety, which was assessed at survey 2. Multiple logistic regression generating odds ratios (OR) with 95% confidence intervals (95% CI) was performed to adjust the associations between reported symptoms and health outcomes for potential confounders (age, farming, workload, education, demand and control at work, body mass index, smoking, snuff use, alcohol consumption, psychiatric symptoms and specific back diagnoses during follow up). RESULTS: Of the 836 men reporting current neck and/or low back pain at survey 1, 21% had had at least one primary care consultation for neck or low back problems, 7% had been on sick leave and 4% had disability pension owing to the condition during the 12 year follow up. Current neck and/or low back pain at survey 1 predicted primary care consultations (OR = 4.10, 95% CI 2.24-7.49) and sick leave (OR = 3.22, 95% CI 1.13-9.22) after potential confounders were considered. Lower education and more psychiatric symptoms were independently related to sick leave. Lower education and snuff use independently predicted disability pension. CONCLUSION: Few individuals with neck or low back pain were on sick leave or were granted a disability pension owing to neck or low back problems during 12 years of follow up. Symptoms at baseline independently predicted health outcomes. Educational level and symptoms of depression/anxiety were important modifiers.

Gene expression analysis suggests that EBF-1 and PPARgamma2 induce adipogenesis of NIH-3T3 cells with similar efficiency and kinetics.

Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPARgamma2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPARgamma2 induce adipocyte differentiation with comparable kinetics and efficiency.

Low back pain comorbidity among male farmers and rural referents: a population-based study.

Farmers report more low back pain (LBP) than rural referents. We have previously demonstrated that the difference in reporting rate cannot be fully explained by known risk factors such as physical work exposures, psychosocial factors and lifestyle. Other etiological factors must be involved. In this cross-sectional population-based study, we investigate LBP comorbidity in terms of coexistent symptoms. A total of 1,013 male farmers, 40-60 years old, and 769 matched rural referents participated in an extensive health survey. Information on causes of primary health care and hospital admissions, symptoms, lifestyle factors, physical work exposures and psychosocial factors were gathered through standardized interviews and questionnaires. In the combined farmer-referent group, the prevalence of LBP was associated with musculoskeletal symptoms other than LBP, chest discomfort, dyspepsia, symptoms from eyes, nose and throat mucous membranes, skin problems, work-related fever attacks, and primary care appointments due to digestive disorders. The associations were independent of age, educational level, smoking habits, body mass index, physical work exposures and psychosocial factors. Presence of both respiratory and digestive disorders doubled the LBP prevalence. Significant associations between LBP and digestive and respiratory disorders were revealed, indicating that LBP and these disease entities may have etiological factors in common.

A comparison of oligonucleotide and cDNA-based microarray systems.

Large-scale public data mining will become more common as public release of microarray data sets becomes a corequisite for publication. Therefore, there is an urgent need to clarify whether data from different microarray platforms are comparable. To assess the compatibility of microarray data, results were compared from the two main types of high-throughput microarray expression technologies, namely, an oligonucleotide-based and a cDNA-based platform, using RNA obtained from complex tissue (human colonic mucosa) of five individuals. From 715 sequence-verified genes represented on both platforms, 64% of the genes matched in "present" or "absent" calls made by both platforms. Calls were influenced by spurious signals caused by Alu repeats in cDNA clones, clone annotation errors, or matched probes that were designed to different regions of the gene; however, these factors could not completely account for the level of call discordance observed. Expression levels in sequence-verified, platform-overlapping genes were not related, as demonstrated by weakly positive rank order correlation. This study demonstrates that there is only moderate overlap in the results from the two array systems. This fact should be carefully considered when performing large-scale analyses on data originating from different microarray platforms.

Shotgun sequencing and microarray analysis of RDA transcripts.

Monitoring of differential gene expression is an important step towards understanding of gene function. We describe a comparison of the representational difference analysis (RDA) subtraction process with corresponding microarray analysis. The subtraction steps are followed in a quantitative manner using a shotgun cloning and sequencing procedure that includes over 1900 gene sequences. In parallel, the enriched transcripts are spotted onto microarrays facilitating large scale hybridization analysis of the representations and the difference products. We show by the shotgun procedure that there is a high diversity of gene fragments represented in the iterative RDA products (92-67% singletons) with a low number of shared sequences (<9%) between subsequent subtraction cycles. A non redundant set of 1141 RDA clones were immobilized on glass slides and the majority of these clones (97%) gave repeated good fluorescent signals in a subsequent hybridization of the labelled and amplified original cDNA. We observed only a low number of false positives (<2%) and a more than twofold differential expression for 32% (363) of the immobilized RDA clones. In conclusion, we show that by random sequencing of the difference products we obtained an accurate transcript profile of the individual steps and that large-scale confirmation of the obtained transcripts can be achieved by microarray analysis.

Psychosocial factors and low back pain, consultations, and sick leave among farmers and rural referents: a population-based study.

Farmers have more low back pain (LBP) than nonfarmers. In a previous report, we found that differences between farmers and nonfarmers in physical work exposure did not explain the LBP differences. In this report, we tested the hypothesis that psychosocial factors might explain the differences in LBP reporting, medical consultation, and sick leave. A cross-sectional population-based survey of 1,013 middle-aged farmers and 769 matched referents was performed. Data on LBP, consultations, and sick leave during lifetime was obtained along with information on psychosocial, social network, and lifestyle variables. Several of the psychosocial variables were associated with LBP but the difference in LBP prevalence between farmers and nonfarmers could be explained only marginally. Farmers and self-employed referents tended to have lower odds of sick leave because of LBP than employed referents after adjustment for psychosocial factors.

Musculoskeletal symptoms among farmers and non-farmers: a population-based study.

Farming has been considered a high-risk occupation for musculoskeletal disorders. However, documentation of the increased risk is weak except for hip osteoarthritis. 1,013 full-time farmers in nine Swedish municipalities and 769 non-farmers (referents), matched for age, sex, and residential area, participated in the study. Information about medical history, consultations, and sick leave for musculoskeletal disorders was obtained by questionnaire and interview. The farmers reported significantly more symptoms affecting the hands and forearms, low back, and hips than did the non-farmers, and a non-significant trend in the same direction was found for symptoms from the neck, shoulders, and knees. However, the farmers did not seek medical advice more often than the referents, and they reported significantly less sick leave for these problems. Thus, farmers appear to have more musculoskeletal symptoms than do non-farmers.

The impact of physical work exposure on musculoskeletal symptoms among farmers and rural non-farmers.

In order to evaluate the impact of physical work exposure on differences in musculoskeletal symptom reported among Swedish farmers and referents, a cross-sectional, population-based cohort study was performed. Male farmers (N = 1221) and matched non-farmers (N = 1130) were invited to take part a survey in which 76 % participated. The analyses were based on 657 matched pairs. Lifetime incidence of musculoskeletal symptoms, information on work exposure, physical workload and leisure time physical activity were assessed by questionnaire and structured interview. Physical work capacity and muscle strength were measured. Farmers reported more low back and hip problems than the referents. After adjustment for the influence of work exposure factors, farmers still had a significant excess rate of low back and hip symptoms compared to the referents, and a significantly lower rate of neck and shoulder problems. In conclusion, work exposure explained some but not all of the farmer-referent differences in musculoskeletal symptom rates.

Type 2 diabetes among farmers and rural and urban referents: cumulative incidence over 20 years and risk factors in a prospective cohort study.

BACKGROUND: Few studies have examined the risk of type 2 diabetes in various occupational groups. Farmers in Sweden have a low risk of coronary heart disease, but less is known about diabetes. OBJECTIVE: To analyze the cumulative incidence and relative risk of type 2 diabetes among farmers and referents taking lifestyle factors and components of the metabolic syndrome into account. METHODS: In a longitudinal observational cohort study we followed 1,220 farmers, 1,130 rural non-farmer referents and 1,219 urban referents over 20 years. Outcomes were generated from national registers and from two surveys 12 years apart. Baseline data were assessed at the first survey conducted in 1990-91. RESULTS: Farmers had a significantly lower risk of all diabetes compared with urban and rural referents (p<0.05). A total of 91 farmers (8.4%) and 102 non-farming rural referents (11.5%) were identified with type 2 diabetes over the 20 year study period (OR=0.70; 95% CI 0.52-0.95). Fractional analyses of lifestyle factors and components of the metabolic syndrome showed that the low risk of type 2 diabetes among farmers was explained in terms of physical activity and meal quality. Farmers had significantly higher physical capacity (p<0.001) and scored higher in a meal quality index than rural referents (p<0.001). CONCLUSIONS: The prevalence of type 2 diabetes was significantly lower among farmers. The low relative risk was explained by high physical activity and better meal quality, indicating that farmers' lifestyles and their work environment are health-promoting.

Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via Interference of a Protein-Protein Interaction.

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein-protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC50 of 0.44 µM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

Potent fibrinolysis inhibitor discovered by shape and electrostatic complementarity to the drug tranexamic acid.

Protein-protein interfaces provide an important class of drug targets currently receiving increased attention. The typical design strategy to inhibit protein-protein interactions usually involves large molecules such as peptides and macrocycles. One exception is tranexamic acid (TXA), which, as a lysine mimetic, inhibits binding of plasminogen to fibrin. However, the daily dose of TXA is high due to its modest potency and pharmacokinetic properties. In this study, we report a computational approach, where the focus was on finding electrostatic potential similarities to TXA. Coupling this computational technique with a high-quality low-throughput screen identified 5-(4-piperidyl)-3-isoxazolol (4-PIOL) as a potent plasminogen binding inhibitor with the potential for the treatment of various bleeding disorders. Remarkably, 4-PIOL was found to be more than four times as potent as the drug TXA.