Five-Year Outcomes of the Danish Cardiovascular Screening (DANCAVAS) Trial.

BACKGROUND: Limited data suggest a benefit of population-based screening for cardiovascular disease with respect to the risk of death. METHODS: We performed a population-based, parallel-group, randomized, controlled trial involving men 65 to 74 years of age living in 15 Danish municipalities. The participants were randomly assigned in a 1:2 ratio to undergo screening (the invited group) or not to undergo screening (the control group) for subclinical cardiovascular disease. Randomization was based on computer-generated random numbers and stratified according to municipality. Only the control group was unaware of the trial-group assignments. Screening included noncontrast electrocardiography-gated computed tomography to determine the coronary-artery calcium score and to detect aneurysms and atrial fibrillation, ankle-brachial blood-pressure measurements to detect peripheral artery disease and hypertension, and a blood sample to detect diabetes mellitus and hypercholesterolemia. The primary outcome was death from any cause. RESULTS: A total of 46,611 participants underwent randomization. After exclusion of 85 men who had died or emigrated before being invited to undergo screening, there were 16,736 men in the invited group and 29,790 men in the control group; 10,471 of the men in the invited group underwent screening (62.6%). In intention-to-treat analyses, after a median follow-up of 5.6 years, 2106 men (12.6%) in the invited group and 3915 men (13.1%) in the control group had died (hazard ratio, 0.95; 95% confidence interval [CI], 0.90 to 1.00; P = 0.06). The hazard ratio for stroke in the invited group, as compared with the control group, was 0.93 (95% CI, 0.86 to 0.99); for myocardial infarction, 0.91 (95% CI, 0.81 to 1.03); for aortic dissection, 0.95 (95% CI, 0.61 to 1.49); and for aortic rupture, 0.81 (95% CI, 0.49 to 1.35). There were no significant between-group differences in safety outcomes. CONCLUSIONS: After more than 5 years, the invitation to undergo comprehensive cardiovascular screening did not significantly reduce the incidence of death from any cause among men 65 to 74 years of age. (Funded by the Southern Region of Denmark and others; DANCAVAS ISRCTN Registry number, ISRCTN12157806.).

Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis.

AIMS: Blood eosinophil count and eosinophil cationic protein (ECP) concentration are risk factors of cardiovascular diseases. This study tested whether and how eosinophils and ECP contribute to vascular calcification and atherogenesis. METHODS AND RESULTS: Immunostaining revealed eosinophil accumulation in human and mouse atherosclerotic lesions. Eosinophil deficiency in ΔdblGATA mice slowed atherogenesis with increased lesion smooth muscle cell (SMC) content and reduced calcification. This protection in ΔdblGATA mice was muted when mice received donor eosinophils from wild-type (WT), Il4-/-, and Il13-/- mice or mouse eosinophil-associated-ribonuclease-1 (mEar1), a murine homologue of ECP. Eosinophils or mEar1 but not interleukin (IL) 4 or IL13 increased the calcification of SMC from WT mice but not those from Runt-related transcription factor-2 (Runx2) knockout mice. Immunoblot analyses showed that eosinophils and mEar1 activated Smad-1/5/8 but did not affect Smad-2/3 activation or expression of bone morphogenetic protein receptors (BMPR-1A/1B/2) or transforming growth factor (TGF)-ß receptors (TGFBR1/2) in SMC from WT and Runx2 knockout mice. Immunoprecipitation showed that mEar1 formed immune complexes with BMPR-1A/1B but not TGFBR1/2. Immunofluorescence double-staining, ligand binding, and Scatchard plot analysis demonstrated that mEar1 bound to BMPR-1A and BMPR-1B with similar affinity. Likewise, human ECP and eosinophil-derived neurotoxin (EDN) also bound to BMPR-1A/1B on human vascular SMC and promoted SMC osteogenic differentiation. In a cohort of 5864 men from the Danish Cardiovascular Screening trial and its subpopulation of 394 participants, blood eosinophil counts and ECP levels correlated with the calcification scores of different arterial segments from coronary arteries to iliac arteries. CONCLUSION: Eosinophils release cationic proteins that can promote SMC calcification and atherogenesis using the BMPR-1A/1B-Smad-1/5/8-Runx2 signalling pathway.

Eosinophils Protect Mice From Angiotensin-II Perfusion-Induced Abdominal Aortic Aneurysm.

RATIONALE: Blood eosinophil count and ECP (eosinophil cationic protein) associate with human cardiovascular diseases. Yet, whether eosinophils play a role in cardiovascular disease remains untested. The current study detected eosinophil accumulation in human and murine abdominal aortic aneurysm (AAA) lesions, suggesting eosinophil participation in this aortic disease. OBJECTIVE: To test whether and how eosinophils affect AAA growth. METHODS AND RESULTS: Population-based randomized clinically controlled screening trials revealed higher blood eosinophil count in 579 male patients with AAA than in 5063 non-AAA control (0.236±0.182 versus 0.211±0.154, 109/L, P<0.001). Univariate (odds ratio, 1.381, P<0.001) and multivariate (odds ratio, 1.237, P=0.031) logistic regression analyses indicated that increased blood eosinophil count in patients with AAA served as an independent risk factor of human AAA. Immunostaining and immunoblot analyses detected eosinophil accumulation and eosinophil cationic protein expression in human and murine AAA lesions. Results showed that eosinophil deficiency exacerbated AAA growth with increased lesion inflammatory cell contents, matrix-degrading protease activity, angiogenesis, cell proliferation and apoptosis, and smooth muscle cell loss using angiotensin-II perfusion-induced AAA in Apoe-/- and eosinophil-deficient Apoe-/-ΔdblGATA mice. Eosinophil deficiency increased lesion chemokine expression, muted lesion expression of IL (interleukin) 4 and eosinophil-associated-ribonuclease-1 (mEar1 [mouse EOS-associated-ribonuclease-1], human ECP homolog), and slanted M1 macrophage polarization. In cultured macrophages and monocytes, eosinophil-derived IL4 and mEar1 polarized M2 macrophages, suppressed CD11b+Ly6Chi monocytes, and increased CD11b+Ly6Clo monocytes. mEar1 treatment or adoptive transfer of eosinophil from wild-type and Il13-/- mice, but not eosinophil from Il4-/- mice, blocked AAA growth in Apoe-/-ΔdblGATA mice. Immunofluorescent staining and immunoblot analyses demonstrated a role for eosinophil IL4 and mEar1 in blocking NF-κB (nuclear factor-κB) activation in macrophages, smooth muscle cells, and endothelial cells. CONCLUSIONS: Eosinophils play a protective role in AAA by releasing IL4 and cationic proteins such as mEar1 to regulate macrophage and monocyte polarization and to block NF-κB activation in aortic inflammatory and vascular cells.

Cost effectiveness of population screening vs. no screening for cardiovascular disease: the Danish Cardiovascular Screening trial (DANCAVAS).

AIMS: A recent trial has shown that screening of men for cardiovascular disease (CVD) may reduce all-cause mortality. This study assesses the cost effectiveness of such screening vs. no screening from the perspective of European healthcare systems. METHODS AND RESULTS: Randomized controlled trial-based cost-effectiveness evaluation with a mean 5.7 years of follow-up. Screening was based on low-dose computed tomography to detect coronary artery calcification and aortic/iliac aneurysms, limb blood pressure measurement to detect peripheral artery disease and hypertension, telemetric assessment of the heart rhythm to detect atrial fibrillation, and measurements of the cholesterol and HgbA1c levels. Censoring-adjusted incremental costs, life years (LY), and quality-adjusted LY (QALY) were estimated and used for cost-effectiveness analysis. The incremental cost of screening for the entire health care sector was €207 [95% confidence interval (CI) -24; 438, P = 0.078] per invitee for which gains of 0.019 LY (95% CI -0.007; 0.045, P = 0.145) and 0.023 QALY (95% CI -0.001; 0.046, P = 0.051) were achieved. The corresponding incremental cost-effectiveness ratios were of €10 812 per LY and €9075 per QALY, which would be cost effective at probabilities of 0.73 and 0.83 for a willingness to pay of €20 000. Assessment of population heterogeneity showed that cost effectiveness could be more attractive for younger men without CVD at baseline. CONCLUSIONS: Comprehensive screening for CVD is overall cost effective at conventional thresholds for willingness to pay and also competitive to the cost effectiveness of common cancer screening programmes. The screening target group, however, needs to be settled.

Retinal Vascular Fractal Dimensions and Their Association with Macrovascular Cardiac Disease.

INTRODUCTION: As the only part of the human vasculature, the retina is available for direct, noninvasive inspection. Retinal vascular fractal dimension (DF) is a method to measure the structure of the retinal vascular tree, with higher noninteger values between 1 and 2 representing a more complex and dense retinal vasculature. Retinal vascular structure has been associated with a variety of systemic diseases, and this study examined the association of DF and macrovascular cardiac disease in a case-control design. METHODS: Retinal fundus photos were captured with Topcon TRC-50X in 38 persons that had coronary artery bypass grafting (CABG, cases) and 37 cardiovascular healthy controls. The semiautomatic software VAMPIRE was used to measure retinal DF. RESULTS: Patients with CABG had lower DF of the retinal main venular vessels compared to the control group (1.15 vs. 1.18, p = 0.01). In a multivariable regression model adjusted for gender and age, eyes in the fourth quartile with higher DF were less likely to have CABG compared to patients in the first (OR, 7.20; 95% confidence interval: 1.63-31.86; p = 0.009) and second (OR, 8.25; 95% confidence interval: 1.70-40.01; p = 0.009) quartiles. CONCLUSIONS: This study demonstrates that lower complexity of main venular vessels associates with higher risk of having CABG. The research supports the hypothesis that the retinal vascular structure can be used to assess nonocular macrovascular disease.

Diabetes Is Not Associated with the Risk of Rupture Among Patients with Abdominal Aortic Aneurysms - Results From a Large Danish Register Based Matched Case Control Study From 1996 to 2016.

OBJECTIVE: Numerous studies have shown a paradoxical protective effect of diabetes on the development and progression of abdominal aortic aneurysm (AAA). The aim of this study was to investigate whether the protective role of diabetes on AAA extends to rupture, given the presence of an AAA. METHODS: This was a register based case control study. Patients with ruptured AAA (RAAA) were matched 1:1 with patients undergoing elective surgery for AAA by sex, age, and year of diagnosis. Multiple conditional logistic regression was performed to estimate the odds ratio (OR) associating a diagnosis of diabetes with RAAA. No protocol was registered. RESULTS: From 1996 to 2016, there were 6293 potential people with RAAA. A total of 898 people with a RAAA were excluded since no matching controls existed. This left 5 395 cases in the study. The cases had a median age of 75, and 85.4% were men. Diabetes was defined by hospital diagnosis or the redemption of antidiabetic prescriptions within one year. Comparing cases with controls and the presence of diabetes, a significant crude OR of 0.82 (95% confidence interval [CI] 0.71-0.95) was found. When adjusting for confounders OR increased to 0.97 (CI 0.83-1.14). Stratifying by age and year of diagnosis did not change the results markedly. OR associating RAAA with diabetes was significantly elevated in women (adjusted OR 1.82 [CI 1.17-2.81]). Of the 5395 cases, the overall 30 days mortality was 58% (n = 3145). Using Cox regression, a crude hazard ratio (HR) of 1.06 (CI 0.93-1.22) was found for the 30 day mortality and having diabetes compared with not having diabetes. Adjusting for index year, male sex, and age had little effect on this estimate (HR 1.11 [CI 0.97-1.28]). CONCLUSION: Diabetes was not found to protect against RAAA, given the presence of an AAA. Furthermore, diabetes did not increase the risk of dying within 30 days of RAAA.

Periarterial fat from two human vascular beds is not a source of aldosterone to promote vasoconstriction.

Mouse adipocytes have been reported to release aldosterone and reduce endothelium-dependent relaxation. It is unknown whether perivascular adipose tissue (PVAT) releases aldosterone in humans. The present experiments were designed to test the hypothesis that human PVAT releases aldosterone and induces endothelial dysfunction. Vascular reactivity was assessed in human internal mammary and renal segmental arteries obtained at surgery. The arteries were prepared with/without PVAT, and changes in isometric tension were measured in response to the vasoconstrictor thromboxane prostanoid receptor agonist U46619 and the endothelium-dependent vasodilator acetylcholine. The effects of exogenous aldosterone and of mineralocorticoid receptor (MR) antagonist eplerenone were determined. Aldosterone concentrations were measured by ELISA in conditioned media incubated with human adipose tissue with/without angiotensin II stimulation. Presence of aldosterone synthase and MR mRNA was examined in perirenal, abdominal, and mammary PVAT by PCR. U46619 -induced tension and acetylcholine-induced relaxation were unaffected by exogenous and endogenous aldosterone (addition of aldosterone and MR blocker) in mammary and renal segmental arteries, both in the presence and absence of PVAT. Aldosterone release from incubated perivascular fat was not detectable. Aldosterone synthase expression was not consistently observed in human adipose tissues in contrast to that of MR. Thus, exogenous aldosterone does not affect vascular reactivity and endothelial function in ex vivo human arterial segments, and the tested human adipose tissues have no capacity to synthesize/release aldosterone. In perspective, physiologically relevant effects of aldosterone on vascular function in humans are caused by systemic aldosterone originating from the adrenal gland.

Proteomic Discovery and Validation of the Confounding Effect of Heparin Administration on the Analysis of Candidate Cardiovascular Biomarkers.

BACKGROUND: Several plasma proteins have been suggested as markers for a variety of cardiovascular conditions but fail to qualify in independent patient cohorts. This may relate to interference of medication on plasma protein concentrations. We used proteomics to identify plasma proteins that changed in concentration with heparin administration and therefore potentially may confound their evaluation as biomarkers in situations in which heparin is used. METHODS: We used a proteomic approach based on isobaric tagging and nano-LC-MS/MS analysis to quantify several hundred proteins in a discovery study in which individual plasma samples from 9 patients at intravascular ultrasound follow-up 12 months after an acute myocardial infarction before heparin administration and 2, 15, and 60 min after heparin administration; we validated our findings in 500 individual plasma samples obtained at admission from patients with suspected ST segment elevation myocardial infarction (STEMI), of whom 363 were treated with heparin before admission. RESULTS: In the discovery study, 25 of 653 identified plasma proteins displayed a changed concentration after heparin administration (Bonferroni-corrected P value at P < 7.66 × 10-5). Fourteen of the proteins changed significantly among heparin-treated patients in the validation study (nominal significance level of P < 6.92 × 10-5). Among heparin-affected proteins in both the discovery study and the validation study were midkine, spondin 1, secreted frizzled-like protein 1, lipoprotein lipase, and follistatin, all previously associated with STEMI. CONCLUSIONS: Medications such as heparin administration given before blood sampling may confound biomarker discovery and should be carefully considered in such studies.

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2 ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies.NEW & NOTEWORTHY This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.

Metformin treatment does not affect the risk of ruptured abdominal aortic aneurysms.

OBJECTIVE: Diabetes counteracts formation and rupture of abdominal aortic aneurysms, possibly through arterial matrix accumulation. Use of metformin, on the other hand, reduces arterial accumulation of matrix molecules. Consequently, we hypothesized that metformin treatment may reverse the protective role of diabetes on the development and course of aneurysms, that is, that metformin would be associated with aneurysm rupture among individuals with diabetes. METHODS: Using nationwide Danish registry data, we performed a nested case-control study on the association between long-term use of metformin and ruptured abdominal aortic aneurysm (RAAA). The source population was defined as all individuals in Denmark with diabetes. Cases were all individuals within the source population who were hospitalized with a primary diagnosis of RAAA. For each case, 10 controls matched by age and sex were randomly selected from the source population by risk set sampling. The main exposure measure was a cumulative dispensing of 1000 g of metformin between January 1995 and the index date. RESULTS: We identified 362 cases of RAAA during 1998 to 2013, of which 83.7% occurred in men with a median age of 74 years. In total, 22.4% of the case population were long-term metformin users compared with 28.8% of the controls. We found a statistically nonsignificant protective effect of long-term metformin use toward RAAA with crude odds ratio (OR) of 0.74 (confidence interval, 0.54-1.00). When adjusted for covariates, OR increased to 0.84 (confidence interval, 0.61-1.17). None of the subgroups had ORs deviating substantially from the main result. CONCLUSIONS: Metformin use does not increase the risk of RAAA among individuals with diabetes.

First-trimester multimarker prediction of gestational diabetes mellitus using targeted mass spectrometry.

AIMS/HYPOTHESIS: Gestational diabetes mellitus (GDM) is associated with an increased risk of pre-eclampsia, macrosomia and the future development of type 2 diabetes mellitus in both mother and child. Although an early and accurate prediction of GDM is needed to allow intervention and improve perinatal outcome, no single protein biomarker has yet proven useful for this purpose. In the present study, we hypothesised that multimarker panels of serum proteins can improve first-trimester prediction of GDM among obese and non-obese women compared with single markers. METHODS: A nested case-control study was performed on first-trimester serum samples from 199 GDM cases and 208 controls, each divided into an obese group (BMI ≥27 kg/m(2)) and a non-obese group (BMI <27 kg/m(2)). Based on their biological relevance to GDM or type 2 diabetes mellitus or on their previously reported potential as biomarkers for these diseases, a number of proteins were selected for targeted nano-flow liquid chromatography (LC) MS analysis. This resulted in the development and validation of a 25-plex multiple reaction monitoring (MRM) MS assay. RESULTS: After false discovery rate correction, six proteins remained significantly different (p<0.05) between obese GDM patients (n=135) and BMI-matched controls (n=139). These included adiponectin, apolipoprotein M and apolipoprotein D. Multimarker models combining protein levels and clinical data were then constructed and evaluated by receiver operating characteristic (ROC) analysis. For the obese, non-obese and all GDM groups, these models achieved marginally higher AUCs compared with adiponectin alone. CONCLUSIONS/INTERPRETATION: Multimarker models combining protein markers and clinical data have the potential to predict women at a high risk of developing GDM.

Cutaneous Porphyrias: Causes, Symptoms, Treatments and the Danish Incidence 1989-2013.

Porphyrias are rare diseases caused by altered haem synthesis leading to the accumulation of different haem intermediates. Neurovisceral attacks may occur in acute porphyrias, while photosensitivity is the presenting symptom in cutaneous porphyrias. We present here an overview of symptoms and a flowchart for the diagnosis of cutaneous porphyrias, with recommendations for monitoring and an update of treatment options. From the Danish Porphyria Register, we present the incidences and approximate prevalences of cutaneous porphyrias within the last 25 years. A total of 650 patients with porphyria cutanea tarda were identified, 73 with erythropoietic protoporphyria, 9 with variegate porphyria, 4 with hereditary coproporphyria and one with congenital erythropoietic porphyria. The total incidence of all porphyrias was ~0.52/100,000 per year.

Extracellular matrix biomarker, fibulin-1 and its association with soluble uPAR in a bi-ethnic South African population: the SAfrEIC study.

BACKGROUND: Fibulin-1 and soluble urokinase-type plasminogen activator receptor (suPAR) emerged as mediators in the development of sclerotic disease. SuPAR along with C-reactive protein (CRP) and albumin delineate inflammatory processes associated with extracellular matrix turnover in atherosclerosis. We explored the independent relationship of fibulin-1 with these inflammatory markers in a bi-ethnic South African population. METHODS: This study included 290 Africans (men: n=130 and women: n=160) and 343 sex- and age-matched Caucasians (men: n=160 and women: n=183). Serum fibulin-1, suPAR, CRP and albumin levels were measured along with conventional cardiovascular and metabolic variables. RESULTS: In both single and age-adjusted regression analyses, fibulin-1 correlated with both suPAR and albumin in African men and with suPAR in Caucasian men. These findings were absent in women. In multivariate regression analysis, these associations were confirmed in African men (R(2)=0.22; ß=0.329; p<0.001) and Caucasian men (R(2)=0.14; ß=0.234; p=0.008). Fibulin-1 independently associated positively with suPAR in all men, but inversely with albumin in African men only. CONCLUSIONS: These results are indicating the presence of potential subclinical inflammation (suPAR) within the extracellular matrix of endothelial tissue, contributing to the potential onset of cardiac fibrosis or vascular sclerosis among these South African men with lower albumin levels.

Lipocalin-type prostaglandin D synthase is not a biomarker of atherosclerotic manifestations.

OBJECTIVE: Over the last decades Lipocalin-type prostaglandin D synthase (L-PGDS), Osteoprotegerin (OPG), Osteopontin (OPN) and Pregnancy associated plasma protein A (PAPP-A) have been reported to be associated with coronary artery disease, and L-PGDS has been proposed as a potential new diagnostic tool in the setting of stable coronary artery disease. We set out to investigate if measurement of concentrations of these biomarkers could be used to differentiate between four groups of individuals with different atherosclerotic manifestations. METHODS: A total of 120 individuals from four equal gender- and age-matched groups were studied: (i) no previous cardiovascular disease (CVD) and no coronary calcifications [CAC-negative group], (ii) no previous CVD but evidence of severe coronary calcifications [CAC-positive group], (iii) acute coronary syndrome [ACS-group], and (iv) clinical stable patients with CVD, who were referred for cardiovascular surgery [CVD-group]. Concentrations of L-PGDS, OPG, OPN and PAPP-A were analyzed and compared between the four groups. RESULTS: We did not find any significant differences in L-PGDS concentrations between the four groups (p = 0.32). OPG concentrations differed significantly (p = 0.003), with the highest concentration observed in ACS patients. Considering OPN (p = 0.12) and PAPP-A (p = 0.53) their concentrations between groups did not differ significantly. CONCLUSION: The main message from this study is the observation that L-PGDS based on a single blood test appears to be less valuable than previously proposed in identification of patients with coronary artery disease. However, ACS patients have higher OPG concentrations than patients with different manifestations of stable atherosclerosis. Neither OPN nor PAPP-A concentrations differed between groups.

The influence of vitamin D analogs on calcification modulators, N-terminal pro-B-type natriuretic peptide and inflammatory markers in hemodialysis patients: a randomized crossover study.

BACKGROUND: The risk of cardiovascular disease is tremendously high in dialysis patients. Dialysis patients treated with vitamin D analogs show decreased cardiovascular morbidity and mortality compared with untreated patients. We examined the influence of two common vitamin D analogs, alfacalcidol and paricalcitol, on important cardiovascular biomarkers in hemodialysis patients. Anti-inflammatory effects and the influence on regulators of vascular calcification as well as markers of heart failure were examined. METHODS: In 57 chronic hemodialysis patients enrolled in a randomized crossover trial comparing paricalcitol and alfacalcidol, we examined the changes in osteoprotegerin, fetuin-A, NT-proBNP, hs-Crp, IL-6 and TNF-α, during 16 weeks of treatment. RESULTS: NT-proBNP and osteoprotegerin increased comparably in the paricalcitol and alfacalcidol-treated groups. Fetuin-A increased significantly in the alfacalcidol-treated group compared with the paricalcitol-treated group (difference 32.84 µmol/l (95% C.I.; range 0.21-67.47)) during the first treatment period. No difference was found between the groups during the second treatment period, and IL-6, TNF-α and hs-Crp were unchanged in both treatment groups. CONCLUSIONS: Paricalcitol and alfacalcidol modulate regulators of vascular calcification. Alfacalcidol may increase the level of the calcification inhibitor fetuin-A. We did not find any anti-inflammatory effect or difference in changes of NT-proBNP. TRIAL REGISTRY: ClinicalTrials.gov NCT00469599 May 3 2007.

Hepatocyte growth factor pathway upregulation in the bone marrow microenvironment in multiple myeloma is associated with lytic bone disease.

Lytic bone disease (LBD) in multiple myeloma (MM) is caused by osteoclast hyperactivation and osteoblast inhibition. Based on in vitro studies, the hepatocyte growth factor (HGF) pathway is thought to be central in osteoblast inhibition. We evaluated the gene expression of the HGF pathway in vivo using bone marrow biopsies (BMBs) of patients with MM and monoclonal gammopathy of undetermined significance (MGUS), and healthy volunteers (HV). BMBs (N = 110) obtained at diagnosis were snap-frozen and used to evaluate gene expression by quantitative reverse transcription polymerase chain reaction. LBD was evaluated using standard radiographs. Enzyme-linked immunosorbent assay (ELISA) was performed on matched bone marrow plasma and immunohistochemistry on matched formalin-fixed paraffin-embedded biopsies. Gene expression of HGF, SDC1, and MET in BMBs were significantly altered in MM versus HV and MGUS, and HGF and MET correlated with the extent of LBD. A significant correlation between gene and protein expression levels was observed for SDC1 (Syndecan-1) and HGF. The HGF bone marrow plasma level was significantly lower in MM patients with no/limited versus advanced LBD. Our novel approach using snap-frozen BMBs seems generally applicable because it allows evaluation of gene expression independent of the extent of MM plasma-cell infiltration. Our study highlights the importance of the HGF pathway in MM LBD.

Plasma concentrations of extracellular matrix protein fibulin-1 are related to cardiovascular risk markers in chronic kidney disease and diabetes.

BACKGROUND: Fibulin-1 is one of a few extracellular matrix proteins present in blood in high concentrations. We aimed to define the relationship between plasma fibulin-1 levels and risk markers of cardiovascular disease. METHODS: Plasma fibulin-1 was determined in subjects with chronic kidney disease (n = 32; median age 62.5, inter-quartile range 51 - 73 years) and 60 age-matched control subjects. Among kidney disease patients serological biomarkers related to cardiovascular disease (fibrinogen, interleukin 6, C-reactive protein) were measured. Arterial applanation tonometry was used to determine central hemodynamic and arterial stiffness indices. RESULTS: We observed a positive correlation of fibulin-1 levels with age (r = 0.38; p = 0.033), glycated hemoglobin (r = 0.80; p = 0.003), creatinine (r = 0.35; p = 0.045), and fibrinogen (r = 0.39; p = 0.027). Glomerular filtration rate and fibulin-1 were inversely correlated (r = -0.57; p = 0.022). There was a positive correlation between fibulin-1 and central pulse pressure (r = 0.44; p = 0.011) and central augmentation pressure (r = 0.55; p = 0.001). In a multivariable regression model, diabetes, creatinine, fibrinogen and central augmentation pressure were independent predictors of plasma fibulin-1. CONCLUSION: Increased plasma fibulin-1 levels were associated with diabetes and impaired kidney function. Furthermore, fibulin-1 levels were associated with hemodynamic cardiovascular risk markers. Fibulin-1 is a candidate in the pathogenesis of cardiovascular disease observed in chronic kidney disease and diabetes.

Decorin is down-regulated in multiple myeloma and MGUS bone marrow plasma and inhibits HGF-induced myeloma plasma cell viability and migration.

OBJECTIVES: Decorin is a stromal-produced small leucine-rich proteoglycan known to attenuate tumour pro-survival, migration, proliferation and angiogenic signalling pathways. Recent studies have shown that decorin interacts with the hepatocyte growth factor (HGF) receptor c-Met, a potential key pathway in multiple myeloma (MM). METHODS: Decorin levels in paired peripheral blood and bone marrow plasma samples from healthy volunteers (HV) (n = 23), and patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 41) and MM (n = 19) were determined by ELISA. Further, the ability of decorin to inhibit HGF-induced effects on MM cell lines were analysed in vitro using cell viability and Transwell migration assays. RESULTS: We found that decorin concentrations were significantly higher (P < 0.05) in bone marrow (BM) plasma from HVs (median 35.2 ng/mL; range, 15.3-99.1) compared with MGUS (median 22.5 ng/mL; range, 11.1-59.5) and patients with MM (median 21.5 ng/mL; range, 10.6-35.9). Decorin levels were higher in BM plasma than in peripheral blood in all groups, with a BM/PB ratio of 3.9, 3.4 and 2.5 for HV, MGUS and MM, respectively. A positive correlation (Spearman's ρ = 0.51, P < 0.05) was found between simultaneously measured levels of HGF and decorin in BM plasma in HVs, but not in MGUS or MM samples. Functionally, decorin inhibited HGF-induced migration and viability of INA-6 and ANBL-6 MM cell lines, independent of c-Met down-regulation. CONCLUSION: Our results show that decorin is down-regulated in MGUS and MM bone marrow plasma and that it inhibits HGF-induced viability and migration of myeloma cell lines in vitro.

Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer.

A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15-0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by T(m) versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.

Angiotensin II regulates microRNA-132/-212 in hypertensive rats and humans.

MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are emerging as key regulators in cardiovascular development and disease. MiRNAs are involved in cardiac hypertrophy, heart failure and remodeling following cardiac infarction; however, miRNAs involved in hypertension have not been thoroughly investigated. We have recently reported that specific miRNAs play an integral role in Angiotensin II receptor (AT1R) signaling, especially after activation of the Gαq signaling pathway. Since AT1R blockers are widely used to treat hypertension, we undertook a detailed analysis of potential miRNAs involved in Angiotensin II (AngII) mediated hypertension in rats and hypertensive patients, using miRNA microarray and qPCR analysis. The miR-132 and miR-212 are highly increased in the heart, aortic wall and kidney of rats with hypertension (159 ± 12 mm Hg) and cardiac hypertrophy following chronic AngII infusion. In addition, activation of the endothelin receptor, another Gαq coupled receptor, also increased miR-132 and miR-212. We sought to extend these observations using human samples by reasoning that AT1R blockers may decrease miR-132 and miR-212. We analyzed tissue samples of mammary artery obtained from surplus arterial tissue after coronary bypass operations. Indeed, we found a decrease in expression levels of miR-132 and miR-212 in human arteries from bypass-operated patients treated with AT1R blockers, whereas treatment with ß-blockers had no effect. Taken together, these data suggest that miR-132 and miR-212 are involved in AngII induced hypertension, providing a new perspective in hypertensive disease mechanisms.