Vascular adhesion protein-1, its ligands and role in cardiovascular disease prediction

To study the pathogenesis and prevention of diseases, population studies in which hundreds of measurements are done from the same persons have a crucial role. A biomarker is any measurable characteristic that gives information on the biological state of the body. At its best a biomarker can predict future disease events. Cardiovascular diseases (CVDs) are chronic diseases in which one feature is harmful leukocyte trafficking. In this thesis project, I studied vascular adhesion protein-1 (VAP-1), a molecule involved in leukocyte trafficking. I wanted to identify the ligands on the surface of leukocytes to which VAP-1 binds to and thus helps the leukocytes to migrate from blood to tissues. A soluble form of VAP-1 (sVAP-1) is present in the blood. To study sVAP-1 as a biomarker, I developed two different assays to measure the sVAP-1 levels in a high throughput way. This enabled the measurements of the sVAP-1 levels in two population cohorts and examine whether sVAP-1 associates with early phases of CVDs. Furthermore, I investigated the ability of sVAP-1 levels to predict future cardiovascular events in a Finnish population. In this thesis, the first leukocyte ligands for VAP-1 were identified. Sialic acid binding Ig-like lectins Siglec-10 and Siglec-9 are expressed on different leukocytes and were able to bind to VAP-1. In addition, the sVAP-1 levels from nearly 5000 Finns were measured with the newly developed assays. The statistical analyses revealed that increased levels of serum sVAP-1 correlated with early manifestations of atherosclerosis. Additionally, sVAP-1 levels could predict future cardiovascular events and even improved the reclassification of patients to correct risk categories.Vaskulaarinen adheesioproteiini-1, sen ligandit ja rooli sydän- ja verisuonitautien ennustamisessa Sydän- ja verisuonitaudit ovat suomalaisten kansantauteja. Näille sairauksille ominaista on häiriintynyt valkosoluliikenne. Näiden tautien kehittymisestä ja ennaltaehkäisystä saadaan tietoja kansallisilla väestötutkimuksilla, joissa ihmisestä voidaan mitata niin kutsuttaja biomarkkereita, jotka antavat tietoa elimistön tilasta. Parhaimmillaan biomarkkerit ennustavat tulevia tautitapahtumia vuosien päähän näytteenottohetkestä. Tässä väitöskirjatyössä keskeisessä roolissa oli soluliikenteessä vaikuttava biomolekyyli, vaskulaarinen adheesioproteiini-1 (VAP-1). Työssä etsittiin valkosolujen pinnalla esiintyviä molekyylejä, ligandeja, joihin VAP-1 sitoutuu, ja näin auttaa valkosoluja siirtymään verestä kudoksiin. Lisäksi kahta veren liukoisen VAP-1:n määrää mittaavaa menetelmää kehitettiin väestötutkimuksiin sopivammaksi VAP-1:n biomarkkeri-roolin selvittämiseksi. Näillä highthroughput -menetelmillä oli tarkoitus selvittää assosioituuko VAP-1 sydän- ja verisuonitautien alkuvaiheisiin ja voidaanko VAP-1:n tasoilla ennustaa tulevia sydän- ja verisuonitautitapahtumia suomalaisessa väestössä. Tutkimuksissa löysimme ensimmäiset valkosoluligandit VAP-1:lle. Siaalihappoihin sitoutuvat immunoglobuliini-tyyppiset lektiinit, Siglec-10 ja Siglec-9 ilmentyvät eri valkosolujen pinnalla ja sitoutuvat VAP-1:een. Kehittämiemme menetelmien avulla määritimme VAP-1-tasot lähes 5000:lta väestötutkimuksiin osallistuneelta suomalaiselta. VAP-1-tulosten tilastollinen analyysi muiden väestötutkimuksissa määritettyjen muuttujien ohella osoitti, että VAP-1-tasot korreloivat kaulavaltimon seinämäpaksuuden kanssa. Lisäksi VAP- 1-tasoilla pystyttiin selittämään tulevien sydäntautitapahtumien riskiä ja parantamaan tilastollisen ennustemallin uudelleenluokittelukykyä

Genome-wide association study identifies seven novel loci associating with circulating cytokines and cell adhesion molecules in Finns

Background Inflammatory processes contribute to the pathophysiology of multiple chronic conditions. Genetic factors play a crucial role in modulating the inflammatory load, but the exact mechanisms are incompletely understood. Objective To assess genetic determinants of 16 circulating cytokines and cell adhesion molecules (inflammatory phenotypes) in Finns. Methods Genome-wide associations of the inflammatory phenotypes were studied in Northern Finland Birth Cohort 1966 (N=5284). A subsequent meta-analysis was completed for 10 phenotypes available in a previous genome-wide association study, adding up to 13 577 individuals in the study. Complementary association tests were performed to study the effect of the ABO blood types on soluble adhesion molecule levels. Results We identified seven novel and six previously reported genetic associations (p Conclusion The present results extend the knowledge about genetic factors contributing to the inflammatory load. Our findings suggest that two distinct mechanisms contribute to the soluble adhesion molecule levels in the ABO locus and that elevated soluble adhesion molecule levels per se may not increase risk for cardiovascular disease.Peer reviewe

First-in-Human Study of 68 Ga-DOTA-Siglec-9, PET Ligand Targeting Vascular Adhesion Protein 1

Sialic acid-binding immunoglubulin-like lectin 9 (Siglec-9) is a ligand of vascular adhesion protein 1 (VAP-1). A gallium 68-labeled peptide of Siglec-9, 68Ga-DOTA-Siglec-9, holds promise as a novel PET tracer for imaging of inflammation. This first-in-human study investigated the safety, tolerability, biodistribution, and radiation dosimetry of this radiopharmaceutical. Methods: Six healthy males underwent dynamic whole-body PET/CT. Serial venous blood samples were drawn from 1-240 min after intravenous injection of 162 ± 4 MBq of 68Ga-DOTA-Siglec-9. In addition to gamma counting, the plasma samples were analyzed by high-performance liquid chromatography to detect intact tracer and radioactive metabolites. Radiation doses were calculated using the OLINDA/EXM 2.2 software. In addition, a patient with early rheumatoid arthritis was studied with both 68Ga-DOTA-Siglec-9 and 18F-FDG PET/CT to determine the ability of the new tracer to detect arthritis. Results: 68Ga-DOTA-Siglec-9 was well tolerated by all subjects. 68Ga-DOTA-Siglec-9 was rapidly cleared from blood circulation and several radioactive metabolites were detected. The organs with the highest absorbed doses were the urinary bladder wall (0.38 mSv/MBq) and kidneys (0.054 mSv/MBq). The mean effective dose was 0.022 mSv/MBq (range 0.020-0.024 mSv/MBq). Most importantly, however, 68Ga-DOTA-Siglec-9 was able to detect arthritis comparable to 18F-FDG. Conclusion: Intravenous injection of 68Ga-DOTA-Siglec-9 was safe and biodistribution is favorable for testing of the tracer in larger group of patients with rheumatoid arthritis planned in the next phase of clinical trials. The effective radiation dose of 68Ga-DOTA-Siglec-9 was within the same range as those of other 68Ga-labeled tracers. Injection of 150 MBq of 68Ga-DOTA-Siglec-9 would expose a subject to 3.3 mSv. These findings support the possible repeated clinical use of 68Ga-DOTA-Siglec-9, e.g., in trials aiming to elucidate the treatment efficacy of novel drug candidates

The Role of Inflammatory Cytokines as Intermediates in the Pathway from Increased Adiposity to Disease

Objective This study aimed to investigate the role of cytokines as intermediates in the pathway from increased adiposity to disease.Methods BMI and circulating levels of up to 41 cytokines were measured in individuals from three Finnish cohort studies (n = 8,293). Mendelian randomization (MR) was used to assess the impact of BMI on circulating cytokines and the impact of BMI-driven cytokines on risk of obesity-related diseases. Results Observationally, BMI was associated with 19 cytokines. For every SD increase in BMI, causal effect estimates were strongest for hepatocyte growth factor, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and were as ratios of geometric means 1.13 (95% CI: 1.08-1.19), 1.08 (95% CI: 1.04-1.14), and 1.13 (95% CI: 1.04-1.21), respectively. TRAIL was associated with a small increase in the odds of coronary artery disease (odds ratio: 1.03; 95% CI: 1.00-1.06). There was inconsistent evidence for a protective role of MCP-1 against inflammatory bowel diseases.Conclusions Observational and MR estimates of the effect of BMI on cytokine levels were generally concordant. There was little evidence for an effect of raised levels of BMI-driven cytokines on disease. These findings illustrate the challenges of MR when applied in the context of molecular mediation.</p

Metabolic profiling of pregnancy : cross-sectional and longitudinal evidence

Background: Pregnancy triggers well-known alterations in maternal glucose and lipid balance but its overall effects on systemic metabolism remain incompletely understood. Methods: Detailed molecular profiles (87 metabolic measures and 37 cytokines) were measured for up to 4260 women (24-49 years, 322 pregnant) from three population-based cohorts in Finland. Circulating molecular concentrations in pregnant women were compared to those in non-pregnant women. Metabolic profiles were also reassessed for 583 women 6 years later to uncover the longitudinal metabolic changes in response to change in the pregnancy status. Results: Compared to non-pregnant women, all lipoprotein subclasses and lipids were markedly increased in pregnant women. The most pronounced differences were observed for the intermediate-density, low-density and high-density lipoprotein triglyceride concentrations. Large differences were also seen for many fatty acids and amino acids. Pregnant women also had higher concentrations of low-grade inflammatory marker glycoprotein acetyls, higher concentrations of interleukin-18 and lower concentrations of interleukin-12p70. The changes in metabolic concentrations for women who were not pregnant at baseline but pregnant 6 years later (or vice versa) matched (or were mirror-images of) the cross-sectional association pattern. Cross-sectional results were consistent across the three cohorts and similar longitudinal changes were seen for 653 women in 4-year and 497 women in 10-year follow-up. For multiple metabolic measures, the changes increased in magnitude across the three trimesters. Conclusions: Pregnancy initiates substantial metabolic and inflammatory changes in the mothers. Comprehensive characterisation of normal pregnancy is important for gaining understanding of the key nutrients for fetal growth and development. These findings also provide a valuable molecular reference in relation to studies of adverse pregnancy outcomes.Peer reviewe

Semaphorin 4D promotes bone invasion in head and neck squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCCs) frequently invade the bones of the facial skeleton. Semaphorin 4D (Sema4D) is an axon guidance molecule produced by oligodendrocytes. Sema4D was also identified in the bone microenvironment and many cancer tissues including HNSCC. To date, however, the role of Sema4D in cancer-associated bone disease is still unknown. This is the first study to demonstrate the role of Sema4D in bone invasion of cancer. In the clinical tissue samples of bone lesion of HNSCC, Sema4D was detected at high levels, and its expression was correlated with insulin-like growth factor-I (IGF-I) expression. In vitro experiments showed that IGF-I regulates Sema4D expression and Sema4D increased proliferation, migration and invasion in HNSCC cells. Sema4D also regulated the expression of receptor activator of nuclear factor κβ ligand (RANKL) in osteoblasts, and this stimulated osteoclastgenesis. Furthermore, knockdown of Sema4D in HNSCC cells inhibited tumor growth and decreased the number of osteoclasts in a mouse xenograft model. Taken together, IGF-I-driven production of Sema4D in HNSCCs promotes osteoclastogenesis and bone invasion

The Role of Inflammatory Cytokines as Intermediates in the Pathway from Increased Adiposity to Disease

Objective This study aimed to investigate the role of cytokines as intermediates in the pathway from increased adiposity to disease. Methods BMI and circulating levels of up to 41 cytokines were measured in individuals from three Finnish cohort studies (n = 8,293). Mendelian randomization (MR) was used to assess the impact of BMI on circulating cytokines and the impact of BMI-driven cytokines on risk of obesity-related diseases. Results Observationally, BMI was associated with 19 cytokines. For every SD increase in BMI, causal effect estimates were strongest for hepatocyte growth factor, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and were as ratios of geometric means 1.13 (95% CI: 1.08-1.19), 1.08 (95% CI: 1.04-1.14), and 1.13 (95% CI: 1.04-1.21), respectively. TRAIL was associated with a small increase in the odds of coronary artery disease (odds ratio: 1.03; 95% CI: 1.00-1.06). There was inconsistent evidence for a protective role of MCP-1 against inflammatory bowel diseases. Conclusions Observational and MR estimates of the effect of BMI on cytokine levels were generally concordant. There was little evidence for an effect of raised levels of BMI-driven cytokines on disease. These findings illustrate the challenges of MR when applied in the context of molecular mediation.Peer reviewe

Multivariate Genome-wide Association Analysis of a Cytokine Network Reveals Variants with Widespread Immune, Haematological, and Cardiometabolic Pleiotropy

Cytokines are essential regulatory components of the immune system, and their aberrant levels have been linked to many disease states. Despite increasing evidence that cytokines operate in concert, many of the physiological interactions between cytokines, and the shared genetic architecture that underlies them, remain unknown. Here, we aimed to identify and characterize genetic variants with pleiotropic effects on cytokines. Using three population-based cohorts (n = 9,263), we performed multivariate genome-wide association studies (GWAS) for a correlation network of 11 circulating cytokines, then combined our results in meta-analysis. We identified a total of eight loci significantly associated with the cytokine network, of which two (PDGFRB and ABO) had not been detected previously. In addition, conditional analyses revealed a further four secondary signals at three known cytokine loci. Integration, through the use of Bayesian colocalization analysis, of publicly available GWAS summary statistics with the cytokine network associations revealed shared causal variants between the eight cytokine loci and other traits; in particular, cytokine network variants at the ABO, SERPINE2, and ZFPM2 loci showed pleiotropic effects on the production of immune-related proteins, on metabolic traits such as lipoprotein and lipid levels, on blood-cell-related traits such as platelet count, and on disease traits such as coronary artery disease and type 2 diabetes.Peer reviewe