Optimized workflow to modify microRNA expression in primary human intravascular cells

Background - A comprehensive dissection of the role of microRNAs (miRNAs) in gene regulation and subsequent cell functions requires a specific and efficient knockdown or overexpression of the miRNA of interest; these are achieved by transfecting the cell of interest with a miRNA inhibitor or a miRNA mimic, respectively. Inhibitors and mimics of miRNAs with a unique chemistry and/or structural modifications are available commercially and require different transfection conditions. Here, we aimed to investigate how various conditions affect the transfection efficacy of two miRNAs with high and low endogenous expression, miR-15a-5p and miR-20b-5p respectively, in human primary cells. Results - MiRNA inhibitors and mimics from two commonly used commercial vendors were employed, i.e., mirVana (Thermo Fisher Scientific) and locked nucleic acid (LNA) miRNA (Qiagen). We systematically examined and optimized the transfection conditions of such miRNA inhibitors and mimics to primary endothelial cells and monocytes using either a lipid-based carrier (lipofectamine) for delivery or an unassisted uptake. Transfection of LNA inhibitors with either phosphodiester (PE)- or phosphorothioate (PS)-modified nucleotide bonds, delivered using a lipid-based carrier, efficiently downregulated the expression levels of miR-15a-5p already 24 h following transfection. MirVana miR-15a-5p inhibitor displayed a less efficient inhibitory effect, which was not improved 48 h following a single transfection or two consecutive transfections. Interestingly, LNA-PS miR-15a-5p inhibitor efficiently reduced the levels of miR-15a-5p when delivered without a lipid-based carrier in both ECs and monocytes. When using a carrier, mirVana and LNA miR-15a-5p and miR-20b-5p mimics showed similar efficiency 48 h following transfection to ECs and monocytes. None of the miRNA mimics effectively induced overexpression of the respective miRNA when given to primary cells without a carrier. Conclusion - LNA miRNA inhibitors efficiently downregulated the cellular expression of miRNA, such as miR-15a-5p. Furthermore, our findings suggest that LNA-PS miRNA inhibitors can be delivered in the absence of a lipid-based carrier, whereas miRNA mimics need the aid of a lipid-based carrier to achieve sufficient cellular uptake

Serum osteoprotegerin levels are related to height loss: The Tromsø Study

Severe loss of body height is often a consequence of osteoporotic vertebral fractures. Osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) are cytokines essential for the regulation of bone resorption. The aim of this study was to assess the relationship between the OPG/RANKL system and height loss. A total of 4,435 inhabitants from the municipality of Tromsø, Norway (2,169 men and 2,266 women) were followed for 6 years. Baseline measurements included height, weight, bone mineral density, OPG, RANKL, serum parathyroid hormone and information about lifestyle, prevalent diseases and use of medication. Height was measured again at follow-up, and the loss of height was categorized into 4 groups: ≤1, 1.1–2, 2.1–3, >3 cm. We found increasing height loss with increasing baseline OPG levels in both men and women (P trend = 0.02 and 0.001, respectively), after adjustments for age and other confounders. However, when the women were stratified according to menopausal status and use of hormone replacement therapy (HRT), a significant relationship was present only among postmenopausal women not using HRT (P trend = 0.02). No relations between OPG and height loss were found in post-menopausal HRT-users and premenopausal women (P trend ≥0.39). We conclude that height loss is positively associated with OPG in men and in postmenopausal women not using HRT. No relationship was found between RANKL and height loss

Cardiorespiratory fitness and future risk of venous thromboembolism

This is the peer reviewed version of the following article: Evensen, L. H., Isaksen, T., Brækkan, S. K. & Hansen, J.-B. (2019). Cardiorespiratory fitness and future risk of venous thromboembolism. Journal of Thrombosis and Haemostasis, 17(12), 2160-2168., which has been published in final form at https://doi.org/10.1111/jth.14619. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.Background - Cardiorespiratory fitness (CRF) is a strong predictor of future arterial cardiovascular disease and premature mortality. However, there are limited data on the association between CRF and the risk of incident venous thromboembolism (VTE). Objectives - To investigate whether estimated CRF (eCRF) was associated with the risk of incident VTE in a cohort recruited from the general population. Methods - Participants (n = 10 393) from the sixth survey of the Tromsø Study (2007—08) were included, and incident VTEs were recorded up to 31 December 2016. CRF was estimated in sex‐specific algorithms based on age, waist circumference, resting heart rate, and self‐reported physical activity. Hazard ratios (HRs) with 95% confidence intervals (CIs) of VTE according to categories of eCRF were estimated in Cox regression models adjusted for sex with age as timescale. The impact of weight status was evaluated in analyses stratified by weight category. Results - There were 176 incident VTEs during follow‐up. Compared with individuals with eCRF 100% of age‐predicted had 46% (HR 0.54; 95% CI 0.39‐0.77) and 67% (HR 0.33; 95% CI 0.20‐0.54) lower VTE risk, respectively. Compared with overweight/obese individuals with eCRF Conclusions - Higher eCRF was associated with lower risk of incident VTE. The association was independent of weight categories, suggesting that higher eCRF may modify the association between obesity and VTE

Surgery As a Trigger for Incident Venous Thromboembolism: Results from a Population-Based Case-Crossover Study

Background Surgery is a major transient risk factor for venous thromboembolism (VTE). However, the impact of major surgery as a VTE trigger has been scarcely investigated using a case-crossover design. Aim To investigate the role of major surgery as a trigger for incident VTE in a population-based case-crossover study while adjusting for other concomitant VTE triggers. Methods We conducted a case-crossover study with 531 cancer-free VTE cases derived from the Tromsø Study cohort. Triggers were registered during the 90 days before a VTE event (hazard period) and in four preceding 90-day control periods. Conditional logistic regression was used to estimate odds ratios (ORs) with 95% confidence intervals (CIs) for VTE according to major surgery and after adjustment for other VTE triggers. Results Surgery was registered in 85 of the 531 (16.0%) hazard periods and in 38 of the 2,124 (1.8%) control periods, yielding an OR for VTE of 11.40 (95% CI: 7.42–17.51). The OR decreased to 4.10 (95% CI: 2.40–6.94) after adjustment for immobilization and infection and was further attenuated to 3.31 (95% CI: 1.83–5.96) when additionally adjusted for trauma, blood transfusion, and central venous catheter. In a mediation analysis, 51.4% (95% CI: 35.5–79.7%) of the effect of surgery on VTE risk could be mediated through immobilization and infection. Conclusions Major surgery was a trigger for VTE, but the association between surgery and VTE risk was in part explained by other VTE triggers often coexisting with surgery, particularly immobilization and infection

Hand grip strength in venous thromboembolism: risk of recurrence and mortality

Background - There is limited information on the relationship between muscle strength and recurrence and mortality after incident venous thromboembolism (VTE). Objectives - To investigate whether weak hand grip strength (HGS) was associated with risk of recurrence and mortality in patients with VTE recruited from the general population. Methods - Participants from the Tromsø Study with a first-time VTE (n = 545) were included, and all VTE recurrences and deaths among the participants were recorded in the period 1994 to 2020. Weak HGS was defined as lowest 25th percentile of the general population, and incidence rates for VTE recurrence and mortality according to weak vs normal (>25th percentile) HGS, with 95% CIs, were estimated. Results - There were 90 recurrences and 350 deaths during a median of 3.7 years of follow-up. The fully adjusted hazard ratio (HR) for overall VTE recurrence for those with weak HGS vs those with normal HGS was 2.02 (95% CI, 1.23-3.30). The corresponding HRs for recurrence were 2.22 (95% CI, 1.18-4.17) in patients with a first deep vein thrombosis and 1.60 (95% CI, 0.72-3.57) in patients with a first pulmonary embolism. The cumulative 1-year survival was 74.9% and 77.8% in those with weak and normal HGS, respectively. For overall mortality after incident VTE, the fully adjusted HR for those with weak HGS was 1.34 (95% CI, 1.04-1.72). Conclusion - Weak HGS was associated with an increased risk of recurrent VTE, and the association appeared to be particularly pronounced after incident deep vein thrombosis. There was a slightly lower survival probability among those with weak HGS than among those with normal HGS

Consanguinity and rare mutations outside of MCCC genes underlie nonspecific phenotypes of MCCD.

Purpose3-Methylcrotonyl-CoA carboxylase deficiency (MCCD) is an autosomal recessive disorder of leucine catabolism that has a highly variable clinical phenotype, ranging from acute metabolic acidosis to nonspecific symptoms such as developmental delay, failure to thrive, hemiparesis, muscular hypotonia, and multiple sclerosis. Implementation of newborn screening for MCCD has resulted in broadening the range of phenotypic expression to include asymptomatic adults. The purpose of this study was to identify factors underlying the varying phenotypes of MCCD.MethodsWe performed exome sequencing on DNA from 33 cases and 108 healthy controls. We examined these data for associations between either MCC mutational status, genetic ancestry, or consanguinity and the absence or presence/specificity of clinical symptoms in MCCD cases.ResultsWe determined that individuals with nonspecific clinical phenotypes are highly inbred compared with cases that are asymptomatic and healthy controls. For 5 of these 10 individuals, we discovered a homozygous damaging mutation in a disease gene that is likely to underlie their nonspecific clinical phenotypes previously attributed to MCCD.ConclusionOur study shows that nonspecific phenotypes attributed to MCCD are associated with consanguinity and are likely not due to mutations in the MCC enzyme but result from rare homozygous mutations in other disease genes.Genet Med 17 8, 660-667

Risk factors and predictors for venous thromboembolism in people with ischemic stroke: A systematic review

Identification of individuals with ischemic stroke at particularly high risk of venous thromboembolism (VTE) is crucial for targeted thromboprophylaxis. To guide clinical decision-making and development of risk prediction models, increased knowledge on risk factors and biomarkers is needed. Therefore, we set out to identify risk factors and predictors for VTE in people with ischemic stroke by conducting a systematic review of the literature. Medline and Embase were searched from January 1990 and onwards. Studies investigating demographic, clinical, and/or laboratory factors for stroke-related VTE were considered. Two reviewers screened all retrieved records, independently and in duplicate. Risk of bias assessments were guided by a structured framework (PROSPERO-ID: CRD42020176361). Of 4674 identified records, 26 studies were included. Twenty-six demographic, clinical, and laboratory factors associated with increased risk of stroke-related VTE after multivariable adjustments were identified. The following factors were reported by ≥2 studies: prior VTE, cancer, prestroke disability, leg weakness, increasing lesion volume of the brain infarct, infection, low Barthel Index, increasing length of hospital stay, biochemical indices of dehydration, as well as elevated levels of D-dimer, C-reactive protein, and homocysteine. The majority of the studies were of poor quality with moderate or high risk of bias. In conclusion, this systematic review informs on several potential risk factors and predictors for VTE in people with ischemic stroke. To improve risk stratification and guide development of risk prediction models, further confirmation is needed because there were few high-quality studies on each factor

Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1β stimulation

Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) – a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1β. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1β alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1β on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1β, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1β intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1β-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1β was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis

Joint Effect of Multiple Prothrombotic Genotypes and Mean Platelet Volume on the Risk of Incident Venous Thromboembolism

Background - A high mean platelet volume (MPV), a marker of increased platelet reactivity, is a risk factor for venous thromboembolism (VTE). Whether established prothrombotic single nucleotide polymorphisms (SNPs) further increase the VTE risk in subjects with high MPV because of biological interaction remains unknown. Aim - To investigate the joint effect of high MPV and prothrombotic genotypes, comprising a 5-SNP genetic risk score (GRS), on the risk of VTE in a population-based case–cohort. Methods - Incident VTE cases (n = 653) and a subcohort (n = 1,774) were derived from the Tromsø Study (1994–2012). DNA was genotyped for rs8176719 (ABO), rs6025 (F5), rs1799963 (F2), rs2036914 (F11), and rs2066865 (FGG). Hazard ratios (HRs) for VTE with 95% confidence intervals (CIs) were estimated according to predefined MPV-strata ( Results - The combination of high MPV and risk alleles, either as individual SNPs or the GRS, had an additive effect on VTE risk. Compared with subjects with MPV Conclusion - The combination of high MPV and prothrombotic genotypes had an additive effect on VTE risk, suggesting there is no biological interaction between these risk factors in the pathogenesis of VTE

Impact of chronic inflammation, assessed by hs-CRP, on the association between red cell distribution width and arterial cardiovascular disease: the Tromso Study

Red cell distribution width (RDW), a measure of variability in size of circulating erythrocytes, is associated with arterial cardiovascular disease (CVD), but the underlying mechanism remains unclear. We aimed to investigate the impact of chronic inflammation as measured by high-sensitivity C-reactive protein (hs-CRP) on this relationship, and explore whether RDW could be a mediator in the causal pathway between inflammation and arterial CVD. Baseline characteristics, including RDW and hs-CRP, were obtained from 5,765 individuals attending a population-based cohort study. We followed up participants from inclusion in the fourth survey of the Tromsø Study (1994/1995) until December 31, 2012. Multivariable Cox-regression models were used to calculate hazard ratios (HR) with 95% confidence intervals (CI) for incident myocardial infarction (MI) and ischemic stroke across quintiles of hs-CRP and RDW. Subjects with hs-CRP in the highest quintile had 44% higher risk of MI (HR: 1.44, 95% CI: 1.14–1.80), and 64% higher risk of ischemic stroke (HR: 1.64, 95% CI: 1.20–2.24) compared with subjects in the lowest quintile. RDW mediated 7.2% (95% CI: 4.0–30.8%) of the association between hs-CRP and ischemic stroke. Subjects with RDW in the highest quintile had 22% higher risk of MI (HR: 1.22, 95% CI: 0.98–1.54) and 44% higher risk of ischemic stroke (HR: 1.44, 95% CI: 1.06–1.97) compared with subjects in the lowest quintile. These risk estimates were slightly attenuated after adjustments for hs-CRP. Our findings suggest that chronic inflammation is not a primary mechanism underlying the relationship between RDW and arterial CVD