The Evolving Role of Fetuin-A in Nonalcoholic Fatty Liver Disease: An Overview from Liver to the Heart.

Nonalcoholic fatty liver disease (NAFLD) is strongly associated to the features of metabolic syndrome which can progress to cirrhosis, liver failure and hepatocellular carcinoma. However, the most common cause of mortality in people with NAFLD is not liver-related but stems from atherosclerotic cardiovascular disease (CVD). The prevalence of NAFLD is on the rise, mainly as a consequence of its close association with two major worldwide epidemics, obesity and type 2 diabetes (T2D). The exact pathogenesis of NAFLD and especially the mechanisms leading to disease progression and CVD have not been completely elucidated. Human fetuin-A (alpha-2-Heremans Schmid glycoprotein), a glycoprotein produced by the liver and abundantly secreted into the circulation appears to play a role in insulin resistance, metabolic syndrome and inflammation. This review discusses the links between NAFLD and CVD by specifically focusing on fetuin-A's function in the pathogenesis of NAFLD and atherosclerotic CVD.

Evaluation of the pathophysiological role of Fetuin A levels in adolescents with polycystic ovary syndrome.

OBJECTIVES: Polycystic ovary syndrome (PCOS) is an endocrinopathy, in which hyperandrogenism and hyperinsulinism have both occurred. Fetuin-A, a natural inhibitor of tyrosine kinase, leads to insulin resistance. The aim was to evaluate the relationship between fetuin-A and hyperandrogenism and hyperinsulinism and the role of fetuin-A in the pathophysiology of PCOS. METHODS: Thirty-eight cases with PCOS and 40 healthy adolescents were included in the study. PCOS and controls were divided into obese/non-obese subgroups. LH, FSH, total and free testosterone (TT, FT), SHBG, androstenedione, DHEAS were measured in patients with PCOS. Fasting glucose, insulin, lipid profile, AST, ALT, HsCRP, and fetuin levels of PCOS patients and healthy controls were also measured. RESULTS: Fetuin-A levels were higher in PCOS patients than in controls. In the obese-PCOS group, when compared to non-obese PCOS patients; the levels of SHBG and HDL were low while cholesterol, LDL, triglyceride, HOMA-IR, FT, FAI, and HSCRP levels were high, but Fetuin-A levels were similar. In the obese-PCOS group, fetuin-A levels were higher than in obese-controls. HOMA-IR and fetuin-A levels were higher in non-obese PCOS patients than in non-obese controls. In the PCOS group, fetuin-A was positively correlated with TT, FT, FAI and androstenedione and negatively correlated with SHBG. Regression analysis demonstrated that FT, SHBG, and androstenedione significantly predicted fetuin-A levels (R2=54%). In non-obese PCOS patients and controls, fetuin-A was positively correlated with insulin and HOMA-IR. CONCLUSIONS: These results suggest a relationship between androgen levels and fetuin-A in PCOS cases, independent of insulin resistance, and may shed light on further studies.

Microvasculopathy and soft tissue calcification in mice are governed by fetuin-A, magnesium and pyrophosphate.

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.

Epicardial adipose tissue volume and annexin A2/fetuin-A signalling are linked to coronary calcification in advanced coronary artery disease: Computed tomography and proteomic biomarkers from the EPICHEART study.

BACKGROUND & AIMS: The role of epicardial adipose tissue (EAT) in the pathophysiology of late stage-coronary artery disease (CAD) has not been investigated. We explored the association of EAT volume and its proteome with advanced coronary atherosclerosis. METHODS: The EPICHEART Study prospectively enrolled 574 severe aortic stenosis patients referred to cardiac surgery. Before surgery, EAT volume was quantified by computed tomography (CT). During surgery, epicardial, mediastinal (MAT) and subcutaneous (SAT) adipose tissue samples were collected to explore fat phenotype by analyzing the proteomic profile using SWATH-mass spectrometry; pericardial fluid and peripheral venous blood were also collected. CAD presence was defined as coronary artery stenosis ≥50% in invasive angiography and by CT-derived Agatston coronary calcium score (CCS). RESULTS: EAT volume adjusted for body fat was associated with higher CCS, but not with the presence of coronary stenosis. In comparison with mediastinal and subcutaneous fat depots, EAT exhibited a pro-calcifying proteomic profile in patients with CAD characterized by upregulation of annexin-A2 and downregulation of fetuin-A; annexin-A2 protein levels in EAT samples were also positively correlated with CCS. We confirmed that the annexin-A2 gene was overexpressed in EAT samples of CAD patients and positively correlated with CCS. Fetuin-A gene was not detected in EAT samples, but systemic fetuin-A was higher in CAD than in non-CAD patients, suggesting that fetuin-A was locally downregulated. CONCLUSIONS: In an elderly cohort of stable patients, CCS was associated with EAT volume and annexin-A2/fetuin-A signaling, suggesting that EAT might orchestrate pro-calcifying conditions in the late phases of CAD.

Hepatokines and non-alcoholic fatty liver disease.

Nowadays non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver pathology both in adults and children. NAFLD manifestation ranges from a simple liver steatosis to steatohepatitis (nonalcoholic steatohepatitis - NASH), which may progress to advanced fibrosis, cirrhosis and end-stage liver disease. Due to the coexistence of visceral obesity, insulin resistance and dyslipidemia, NAFLD is considered to be the hepatic manifestation of metabolic syndrome. In recent years, in the pathogenesis of metabolic syndrome, type 2 diabetes mellitus, cardiovascular disease and also NAFLD, more and more attention has been paid to the so-called organokines, proteins with both paracrine or/and endocrine activities. These include most known adipokines (mainly produced by adipose tissue), myokines (mainly produced by skeletal muscles) and hepatokines exclusively or predominantly produced by the liver. It was shown that the liver may affect the lipids and glucose metabolism by hepatokines released into the blood and NAFLD seems to be associated with altered hepatokines production. Fetuin-A, fibroblast growth factor-21 (FGF-21), selenoprotein P, sex hormone-binding globulin (SHBG), angiopoietin-related growth factor (also known as angiopoietin-related protein 6) and leukocyte derived chemotaxin 2 (LECT2) are considered as the most important hepatokines. In this review, we provide an overview of the main hepatokines and we summarize the association of liver-derived proteins with the development and progression of NAFLD.

Fetuin A promotes lipotoxicity in ß cells through the TLR4 signaling pathway and the role of pioglitazone in anti-lipotoxicity.

OBJECTIVE: Fetuin A (FetA), a secreted glycoprotein, is known to affect inflammation and insulin resistance (IR) in obese humans and animals. Lipotoxicity from chronic hyperlipidemia damages pancreatic ß cells, hastening the onset of diabetes. We sought to determine whether FetA promotes lipotoxicity through modulation of the toll-like receptor 4 (TLR4) inflammatory signaling pathway as well as the protective effect of pioglitazone(PIO) on lipotoxicity. METHODS: ßTC6, a glucose-sensitive mouse pancreatic ß cell line, and Sprague-Dawley rats with diet-induced obesity, were used to investigate FetA-mediated lipotoxicity. Protein expression/activation were measured by Western blotting. Small interfering (si)RNAs for TLR4 were used. Cell apoptosis was quantified by TUNEL analysis or flow cytometry, respectively. Insulin release was assessed with an insulin ELISA. RESULTS: FetA dose-dependently aggravated palmitic acid (PA)-induced ßTC6 cell apoptosis, insulin secretion impairment, and inhibition of the expression of G-protein-coupled receptor 40 (GPR40) and pancreatic duodenal homeobox-1(PDX-1). Combined FetA + PA induced TLR4 expression, and subsequent inhibition of TLR4 signaling or expression was shown to prevent the strengthening effect of FetA on PA-induced lipotoxicity in ßTC6 cells. FetA + PA induced p-JNK and nuclear factor-κB (NF-κB) subunit P65 expression, and inhibition of this activity reduced PA+ FetA lipotoxicity in ßTC6 cells. PIO could ameliorate PA+ FetA-induced damage to ßTC6 cells. Similarly, PIO improved insulin secretion disorder, reduced apoptosis, decreased FetA, TLR4, p-JNK, NF-κB subunit P65 and cleaved caspase 3 expression, and increased GPR40 and PDX-1 expression in islet ß cells of diet-induced obese rats. The correlative bivariate analysis showed that increases in Fetuin A were directly proportional to the development of ß cell injury. CONCLUSIONS: FetA can promote lipotoxicity in ß cells through the TLR4-JNK-NF-κB signaling pathway. The protective effects of PIO on lipotoxicity in ß cells may involve the inhibition of the activation of the FetA and TLR4 signaling pathway.

Fetuin-A influences vascular cell growth and production of proinflammatory and angiogenic proteins by human perivascular fat cells.

AIMS/HYPOTHESIS: Fetuin-A (alpha2-Heremans-Schmid glycoprotein), a liver-derived circulating glycoprotein, contributes to lipid disorders, diabetes and cardiovascular diseases. In a previous study we found that perivascular fat cells (PVFCs) have a higher angiogenic potential than other fat cell types. The aim was to examine whether fetuin-A influences PVFC and vascular cell growth and the expression and secretion of proinflammatory and angiogenic proteins, and whether TLR4-independent pathways are involved. METHODS: Mono- and co-cultures of human PVFCs and endothelial cells were treated with fetuin-A and/or palmitate for 6-72 h. Proteins were quantified by ELISA and Luminex, mRNA expression by real-time PCR, and cell growth by BrDU-ELISA. Some PVFCs were preincubated with a nuclear factor κB NFκBp65 inhibitor, or the toll-like receptor 4 (TLR4) inhibitor CLI-095, or phosphoinositide 3-kinase (PI3K)/Akt inhibitors and/or stimulated with insulin. Intracellular forkhead box protein O1 (FoxO1), NFκBp65 and inhibitor of κB kinase ß (IKKß) localisation was visualised by immunostaining. RESULTS: PVFCs expressed and secreted IL-6, IL-8, plasminogen activator inhibitor 1 (PAI-1), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-BB, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), placental growth factor (PLGF) and hepatocyte growth factor (HGF). Fetuin-A upregulated IL-6 and IL-8, and this was potentiated by palmitate and blocked by CLI-095. Immunostaining and electrophoretic mobility shift assay (EMSA) showed partial NFκBp65 activation. MCP-1 was upregulated and blocked by CLI-095, but not by palmitate. However, HGF was downregulated, which was slightly potentiated by palmitate. This effect persisted after TLR4 pathway blockade. Stimulation of insulin-PI3K-Akt signalling by insulin resulted in nuclear FoxO1 extrusion and HGF upregulation. Fetuin-A counteracted these insulin effects. CONCLUSIONS/INTERPRETATION: Fetuin-A and/or palmitate influence the expression of proinflammatory and angiogenic proteins only partially via TLR4 signalling. HGF downregulation seems to be mediated by interference with the insulin-dependent receptor tyrosine kinase pathway. Fetuin-A may also influence angiogenic and proinflammatory proteins involved in atherosclerosis.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) modulates signaling pathways in head and neck squamous cell carcinoma cell line SQ20B.

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-ß was examined to determine whether levels of the TGF-ß binding AHSG influenced the effect of TGF-ß on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-ß influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis.

Fetuin-A triggers the secretion of a novel set of exosomes in detached tumor cells that mediate their adhesion and spreading.

Our goal in this study was to define the mechanisms by which fetuin-A mediates the adhesion of tumor cells. The data show that in the absence of fetuin-A, detached tumor cells secrete exosomes that contain most of the known exosomal associated proteins but lack the capacity to mediate cellular adhesion. In the presence of fetuin-A, the cells secrete exosomes, which contain, in addition to the other exosomal proteins, fetuin-A, plasminogen and histones. These exosomes mediate adhesion and cell spreading. Plasminogen is a participant in this novel adhesion mechanism. The data suggest that these exosomes play a role in tumor progression.

Fetuin-A: a multifunctional protein.

Sixty-six years have elapsed since the discovery of fetuin in 1944, but its importance in mammalian physiology has only recently been appreciated. Fetuin, first isolated from fetal bovine serum and now most commonly known as either fetuin-A, alpha-2-HS-glycoprotein (recommended name by UniprotKB and PIR), or α2-Heremans-Schmid glycoprotein, functions as an important component of diverse normal and pathological processes, including vascular calcification and bone metabolism regulation, insulin resistance, protease activity control, keratinocytes migration, and breast tumor cell proliferative signaling. Fetuin-A has also been identified as a biomarker for neurodegenerative disease. Here, we summarize recent publications focusing on the structural and functional properties of fetuin-A. The emerging importance of fetuin-A for both diagnosis and therapeutics has come to the attention of the pharmaceutical industry. Therefore, we will discuss the status of patents based on fetuin-A.

Inhibitors of vascular calcification as potential therapeutic targets.

Vascular calcification is frequent in the general population. Its incidence increases with age. It contributes to cardiovascular morbidity and mortality in patients with advanced atherosclerosis, in the presence or absence of diabetes mellitus and chronic kidney disease (CKD). Both diabetes and CKD aggravate its degree of severity and accelerate its progression. Vascular calcification is the result of both passive and active processes of calcium phosphate deposition in the arterial wall. These processes are more or less successfully opposed by inhibitory proteins and nonpeptidic factors. In the present overview we discuss the roles of several among these vascular calcification inhibitors which represent potential therapeutic targets.