Calcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms.
Small RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves.
The data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition.
miR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.

UNASSIGNED: The glycoprotein fetuin-A has anti-inflammatory effects, increases insulin resistance and plays an important role in calcium metabolism. The aim of our study was to assess the predictive value of fetuin-A on atherosclerotic plaque progression in comparison to the established cardiovascular biomarker high sensitivity C-reactive protein (hsCRP).
UNASSIGNED: In this prospective, single center-, cohort study, we included 194 patients with at least one cardiovascular risk factor or established cardiovascular disease (CVD). Over a period of 4 years, each patient underwent 3D plaque volumetry of the carotid and femoral arteries on a yearly basis. To evaluate the predictive value of biomarkers in terms of plaque progression, the baseline values of fetuin-A and hsCRP were correlated with the plaque progression from baseline to the last follow up visit.
UNASSIGNED: 171 patients were included in the final analysis. Baseline fetuin-A levels showed a significant negative correlation with plaque progression (r = -0.244; p = 0.001). In contrast, baseline hsCRP levels showed no correlation with plaque progression (r = 0.096, p = 0.20). In the ROC-analysis, fetuin-A had a significantly better predictive value than hsCRP (fetuin-A AUC 0.67; p = 0.001 vs hsCRP AUC 0.49; p = 0.88) with an optimal cut-off value at 712 μg/ml. In patients with high fetuin A levels (>712 μg/ml), a significantly lower plaque progression was observed compared to the group with low fetuin-A levels <712 μg/ml (high fetuin-A 197 mm3 vs. low fetuin-A 279 mm3; p = 0.01).
UNASSIGNED: Higher fetuin-A levels appear to predict lower atherosclerotic plaque progression in patients with or at risk of cardiovascular disease.

Adipokines are signaling molecules involved in the integration of metabolism. Changes in their concentrations were observed in obesity, metabolic syndrome, diabetes mellitus and cardiovascular diseases, as well as endocrine disorders. Cushing\'s syndrome is associated with metabolic dysregulation, but the significance of adipokines in this entity and related complications is largely unknown. The aim of our study was to determine the concentrations of adipokines: fetuin A, fatty acid binding protein 4 (FABP4) and retinol binding protein 4 (RBP4) in Cushing\'s syndrome and to assess their relation to established cardiovascular and diabetes risk markers.
We examined 21 subjects with Cushing\'s syndrome and 24 healthy controls in a cross-sectional manner. Venous blood samples were analysed for adipokines, cortisol, adrenocorticotrophin, glucose, insulin, glycated haemoglobin (HbA1c), triglycerides, cholesterol fractions, thyrotropin and free thyroid hormones concentrations. Patients\' body mass index (BMI) was evaluated, homeostatic model assessment-insulin resistance and Systematic Coronary Risk Evaluation (SCORE) were calculated.
We found that the concentration of fetuin A was lower, while FABP4 and RBP4 concentrations were higher in Cushing\'s syndrome compared to controls [156.4 ± 60.0 µg/ml vs 260.7 ± 49.6 µg/ml; 79.8 (35.2-156.1) ng/ml vs 27.9 (17.1-36.7) ng/ml and 34 (30-37.7) mg/l vs 25.8 (23.6-27.7) mg/l, respectively]. Fetuin A correlated inversely, while FABP4 and RBP4 positively, with the concentrations of urinary free cortisol and adrenocorticotrophin. Fetuin A was positively related to LDL-cholesterol, and negatively to SCORE and HbA1c. FABP4 was associated positively with BMI, HbA1c and triglycerides, while RBP4 correlated positively with triglycerides and systolic blood pressure.
Adipokines\' concentrations change in hypercortisolism. Further research is needed to ascertain whether adipokines are involved in the development of metabolic complications accompanying Cushing\'s syndrome or secondarily reflect metabolic dysregulation.

Ectopic calcification is an important contributor to chronic diseases, such as osteoarthritis. Currently, no effective therapies exist to counteract calcification. We developed peptides derived from the calcium binding domain of human Alpha-2-HS-Glycoprotein (AHSG/Fetuin A) to counteract calcification.
A library of seven 30 amino acid (AA) long peptides, spanning the 118 AA Cystatin 1 domain of AHSG, were synthesized and evaluated in an in vitro calcium phosphate precipitation assay. The best performing peptide was modified (cyclic, retro-inverso and combinations thereof) and evaluated in cellular calcification models and the rat Medial Collateral Ligament Transection + Medial Meniscal Tear (MCLT + MMT) osteoarthritis model.
A cyclic peptide spanning AA 1-30 of mature AHSG showed clear inhibition of calcium phosphate precipitation in the nM-pM range that far exceeded the biological activity of the linear peptide variant or bovine Fetuin. Biochemical and electron microscopy analyses of calcium phosphate particles revealed a similar, but distinct, mode of action in comparison with bFetuin. A cyclic-inverso variant of the AHSG 1-30 peptide inhibited calcification of human articular chondrocytes, vascular smooth muscle cells and during osteogenic differentiation of bone marrow derived stromal cells. Lastly, we evaluated the effect of intra-articular injection of the cyclic-inverso AHSG 1-30 peptide in a rat osteoarthritis model. A significant improvement was found in histopathological osteoarthritis score and animal mobility. Serum levels of IFNγ were found to be lower in AHSG 1-30 peptide treated animals.
The cyclic-inverso AHSG 1-30 peptide directly inhibits the calcification process and holds the potential for future application in osteoarthritis.

Cardiovascular disorders represent the leading cause of death in dialysis patients. Alterations of bone and mineral metabolism (BMM) and vascular calcifications play a fundamental role in it. The objective of this study was to evaluate the predictive role on cardiovascular mortality of the measurement of biomarkers of BMM and vascular calcifications. A prospective cohort study was performed. All prevalent patients on chronic dialysis in September 2009 at our institution, who completed the total of the complementary studies, were studied. BMM biomarkers were measured (FGF 23, fetuin A, PTH, calcium and phosphorus) and the vascular calcifications were evaluated using the Kauppila and Adragao scores. Follow-up was carried out until 1/1/2019, death or transplant. Of the 30 patients included, 7 (23.3%) died due to cardiovascular causes. The follow-up time was 44.1 ± 30.4 (range = 1.4-112) months. The Adragao score was the only predictive variable of long-term cardiovascular mortality (area under the curve = 0.82; 95% CI 0.64-0.94; p < 0.001). The best cut-off point was 5 (sensitivity = 85.7%; specificity = 78.3%). It was also an independent risk factor for cardiovascular mortality adjusted for age, diabetes mellitus, coronary heart disease, aortic calcifications, time spent on dialysis and follow-up time (adjusted OR = 1.77; 95% CI = 1.06-2.96; p = 0.028). The vascular calcifications quantified from the Adragao score were the only independent predictor of long-term cardiovascular mortality. This score represents a simple, useful and superior tool to the biomarkers of BMM.
Los trastornos cardiovasculares representan la primera causa de muerte en los pacientes en diálisis. Las alteraciones del metabolismo óseo y mineral (MOM) y las calcificaciones vasculares juegan un papel fundamental en la misma. El objetivo de este estudio fue evaluar el rol predictor sobre la mortalidad cardiovascular de la medición de los biomarcadores del MOM y las calcificaciones vasculares. Se realizó un estudio de cohorte prospectivo. Se estudiaron todos los pacientes prevalentes en diálisis crónica en septiembre del 2009 en nuestra institución que completaron el total de los estudios complementarios. Se midieron biomarcadores del MOM (FGF 23, fetuína A, PTH, calcio y fósforo) y se evaluaron las calcificaciones vasculares mediante los scores de Kauppila y de Adragao. Se realizó un seguimiento hasta el 1/1/2019, la muerte o el trasplante. De los 30 pacientes incluidos, 7 (23.3%) fallecieron por causa cardiovascular. El tiempo de seguimiento fue de 44.1 ± 30.4 (rango = 1.4-112) meses. El score de Adragao fue la única variable predictiva de muerte cardiovascular a largo plazo (área bajo la curva = 0.82; IC95% = 0.64-0.94; p < 0.001). El mejor punto de corte fue de 5 (sensibilidad = 85.7%; especificidad = 78.3%). Además, fue un factor de riesgo independiente de muerte cardiovascular ajustado por edad, diabetes mellitus, enfermedad coronaria, calcificaciones aorticas, tiempo de permanencia en diálisis y tiempo de seguimiento (OR ajustado = 1.77; IC95% = 1.06-2.96; p = 0.028). Las calcificaciones vasculares cuantificadas a partir del score de Adragao fueron el único predictor independiente de mortalidad cardiovascular a largo plazo. Este score representa una herramienta simple, útil y superior a los biomarcadores del MOM.

Circulating calcification inhibitors: fetuin A (FA) and osteoprotegerin (OPG) together with soluble ligand of receptor activator of nuclear factor kappa-B (sRANKL) have been linked to vascular calcifications and arterial damage. This study aimed to evaluate relationships between FA, OPG, sRANKL, and arterial damage in children with primary hypertension (PH).
In this cross-sectional single-center study, calcification inhibitors (FA, OPG, sRANKL) levels were measured in blood samples of 60 children with PH (median age 15.8, IQR: [14.5-16.8] years) and 20 age-matched healthy volunteers. In each participant, peripheral and central blood pressure evaluation (BP) and ambulatory BP monitoring (ABPM) were performed. Arterial damage was measured using common carotid artery intima media thickness (cIMT), pulse wave velocity (PWV), augmentation index (AIx75HR), and local arterial stiffness (ECHO-tracking-ET) analysis.
Children with PH had significantly higher peripheral and central BP, BP in ABPM, thicker cIMT, higher PWV, and AIx75HR. FA was significantly lower in patients with PH compared to healthy peers without differences in OPG, sRANKL, and OPG/sRANKL and OPG/FA ratios. In children with PH, FA level correlated negatively with cIMT Z-score and ET AIx; sRANKL level correlated negatively with ABPM systolic blood pressure (SBP), SBP load, diastolic BP load, and AIx75HR; OPG/sRANKL ratio correlated positively with SBP load, while OPG/FA ratio correlated positively with ET AIx. In multivariate analysis, FA was a significant determinant of cIMT (mm) and cIMT Z-score.
This study reveals that in children with primary hypertension, arterial damage is related to lower fetuin A concentrations.

The hepatokine fetuin A (Fet A) has been associated with diverse pathological states such as insulin resistance, type 2 diabetes, macrovascular disease, and systemic ectopic and vascular calcification. Fet A may also play a role in tumor growth and metastasis. The biological activity of Fet A may be affected by various modifications, including phosphorylation, O- and N-glycosylation and fatty acid binding. We developed an antibody-based assay for the detection of Fet A phosphorylated at serine 312. Fatty acid pattern was determined by gas chromatography. Using the antibody, we found that the phosphorylation was stable in human plasma or serum at room temperature for 8 h. We observed that Fet A is present in several glycosylation forms in human plasma, but the extent of Ser312 phosphorylation was not associated with glycosylation. The phosphorylation pattern did not change during an oral glucose tolerance test (0-120 min). We further found that human Fet A binds preferentially saturated fatty acids (>90%) at the expense of mono- and poly-unsaturated fatty acids. Our results indicate that different molecular species of Fet A are present in human plasma and that these different modifications may determine the different biological effects of Fet A.

Fetuin-A is a multifunctional glycoprotein that has been implicated in insulin resistance and bone metabolism. We assessed whether fetuin-A is associated with poor or excessive fetal growth. In the Shanghai Birth Cohort, we conducted a nested case-control study of 60 trios of small-for-gestational-age (SGA, birth weight <10th percentile), optimal-for-gestational-age (OGA, 25-75th, the reference) and large-for-gestational-age (LGA, >90th percentile) infants matched by sex and gestational age. Cord plasma concentrations of fetuin-A and fetal growth factors [insulin, proinsulin, insulin-like growth factor (IGF)-I and IGF-II] were measured. Cord plasma fetuin-A concentrations were higher in SGA (809.4 ± 306.9 μg/ml, P = 0.026) and LGA (924.2 ± 375.9 μg/ml, P < 0.001) relative to OGA (680.7 ± 262.1 μg/ml) newborns, and were not correlated to insulin, proinsulin, IGF-I and IGF-II (all P > 0.2). Higher fetuin-A concentrations were associated with increased risks of SGA [OR = 1.67 (1.08-2.58) per SD increment, P = 0.024] and LGA [OR = 2.36 (1.53-3.66), P < 0.001]. Adjusting for maternal and neonatal characteristics and fetal growth factors, the elevated risk changed little for LGA [adjusted OR = 2.28 (1.29-4.01), P = 0.005], but became non-significant for SGA (P = 0.202). Our study is the first to demonstrate that fetuin-A may be involved in excessive fetal growth. This association is independent of fetal growth factors.

UNASSIGNED: Vascular calcification and stiffness are associated with higher mortality and cardiovascular disease in hemodialysis patients, but the underlying mechanism is not well elucidated and previous studies have been contradictory. We sought to determine the association of circulating calcification biomarkers with calcification, stiffness, and mortality in a multiethnic incident dialysis population.
UNASSIGNED: Among 391 incident hemodialysis participants enrolled in the Predictors of Arrhythmic and Cardiovascular Risk in End Stage Renal Disease (PACE) study, we examined the cross-sectional associations of baseline fibroblast growth factor 23 (FGF23), desphospho-uncarboxylated matrix Gla protein (dp-ucMGP), fetuin-A, and osteoprotegerin (OPG) according to total coronary artery calcium score (CAC, using the Agatston calcification criteria) at baseline, vascular stiffness (pulse wave velocity [PWV]) over 4 study visits, and all-cause mortality.
UNASSIGNED: Patients\' mean age was 55 years; 40% were female, 72% were African American, and 58% had diabetes. Higher OPG and FGF23 were associated with a 1.09-fold (per 5-pmol/l increase in OPG; 95% confidence interval [CI]: 1.01-1.17) and 1.12-fold (per increase of 100 log RU/ml in FGF23; 95% CI: 1.02‒1.34) higher prevalence of CAC, independent of demographics, comorbidities, dialysis factors, and serum klotho levels. Higher OPG was associated with higher baseline PWV. Higher FGF23 was associated with lower PWV over follow-up. dp-ucMGP and fetuin-A were not associated with either CAC or vascular stiffness. After adjustment, circulating biomarkers were not associated with mortality risk.
UNASSIGNED: Several circulating calcification biomarkers were only modestly associated with subclinical cardiovascular disease in an incident multiethnic hemodialysis population; none were associated with mortality. Understanding whether these associations persist in larger, diverse hemodialysis populations is warranted before planning trials.

Background: The fibroblast growth factors (FGF) 19 subfamily, also referred to as endocrine FGFs, includes FGF19, FGF21, and FGF23 are metabolic hormones involved in the regulation of glucose and lipid metabolism. Fetuin-A is a hepatokine involved in the regulation of beta-cell function and insulin resistance. Endocrine FGFs and fetuin-A are dysregulated in metabolic disorders including obesity, type 2 diabetes, non-alcoholic fatty liver disease and polycystic ovary syndrome (PCOS). Our study was designed to examine the response of endocrine FGFs and fetuin-A to an acute intralipid, insulin infusion and exercise in PCOS and healthy women. Subjects and Measurements: Ten healthy and 11 PCOS subjects underwent 5-h saline infusions with a hyperinsulinemic-euglycemic clamp (HIEC) performed during the final 2 h. One week later, intralipid infusions were undertaken with a HIEC performed during the final 2 h. After an 8 week of exercise intervention the saline, intralipid, and HIEC were repeated. Plasma levels of endocrine FGFs and fetuin-A were measured. Results: Baseline fetuin-A was higher in PCOS women but FGF19, FGF21, and FGF23 did not differ and were unaffected by exercise. Insulin administration elevated FGF21 in control and PCOS, suppressed FGF19 in controls, and had no effects on FGF23 and fetuin-A. Intralipid infusion suppressed FGF19 and increased FGF21. Insulin with intralipid synergistically increased FGF21 and did not have effects on lipid-mediated suppression of FGF19 in both groups. Conclusion: Our study provides evidence for insulin and lipid regulation of endocrine FGFs in healthy and PCOS women, suggesting that FGF family members play a role in lipid and glucose metabolism. Clinical Trial Registration: www.isrctn.org, Identifier: ISRCTN42448814.