Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Senescence of endothelial cells promotes phenotypic changes in adventitial fibroblasts: possible implications for vascular aging.

Aging is the most important risk factor for the development of cardiovascular diseases. Senescent cells release plethora of factors commonly known as the senescence-associated secretory phenotype, which can modulate the normal function of the vascular wall. It is currently not well understood if and how endothelial cell senescence can affect adventitial niche. The aim of this study was to characterize oxidative stress-induced endothelial cells senescence and identify their paracrine effects on the primary cell type of the adventitia, the fibroblasts. Human aortic endothelial cells (HAEC) were treated with hydrogen peroxide to induce premature senescence. Mass spectrometry analysis identified several proteomic changes in senescent HAEC with top upregulated secretory protein growth differentiation factor 15 (GDF-15). Treatment of the human adventitial fibroblast cell line (hAdv cells) with conditioned medium (CM) from senescent HAEC resulted in alterations in the proteome of hAdv cells identified in mass spectrometry analysis. Majority of differentially expressed proteins in hAdv cells treated with CM from senescent HAEC were involved in the uptake and metabolism of lipoproteins, mitophagy and ferroptosis. We next analyzed if some of these changes and pathways might be regulated by GDF-15. We found that recombinant GDF-15 affected some ferroptosis-related factors (e.g. ferritin) and decreased oxidative stress in the analyzed adventitial fibroblast cell line, but it had no effect on erastin-induced cell death. Contrary, silencing of GDF-15 in hAdv cells was protective against this ferroptotic stimuli. Our findings can be of importance for potential therapeutic strategies targeting cell senescence or ferroptosis to alleviate vascular diseases.

Sclerostin in chronic kidney disease-mineral bone disorder think first before you block it!

Canonical Wnt signalling activity is a major player in physiological and adaptive bone metabolism. Wnt signalling is regulated by soluble inhibitors, with sclerostin being the most widely studied. Sclerostin's main origin is the osteocyte and its major function is blockade of osteoblast differentiation and function. Therefore, sclerostin is a potent inhibitor of bone formation and mineralization. Consequently, blocking sclerostin via human monoclonal antibodies (such as romosozumab) represents a promising perspective for the treatment of (postmenopausal) osteoporosis. However, sclerostin's physiology and the effects of sclerostin monoclonal antibody treatment are not limited to the skeleton. Specifically, the potential roles of sclerostin in chronic kidney disease (CKD) and associated pathologies covered by the term chronic kidney disease and mineral bone disorder (CKD-MBD), which also includes accelerated cardiovascular calcification, warrant specific attention. CKD-MBD is a complex disease condition in which sclerostin antibodies may interfere at different levels and influence the multiform interplay of hyperparathyroidism, renal osteodystrophy and vascular calcification, but the clinical sequelae remain obscure. The present review summarizes the potential effects of sclerostin blockade in CKD-MBD. We will address and summarize the urgent research targets that are being identified and that need to be addressed before a valid risk-benefit ratio can be established in the clinical setting of CKD.

Analysis of Ebola Virus Entry Into Macrophages.

Ebolaviruses constitute a public health threat, particularly in Central and Western Africa. Host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. Lectins, TAM receptor tyrosine kinases (Tyro3, Axl, Mer), T cell immunoglobulin and mucin domain (TIM) proteins, integrins, and Niemann-Pick C1 (NPC1) have been reported to promote entry of ebolaviruses into certain cellular systems. However, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. Here, we show that mannose-specific lectins, TIM-1 and Axl augment entry into certain cell lines but do not contribute to Ebola virus (EBOV)-glycoprotein (GP)-driven transduction of macrophages. In contrast, expression of Mer, integrin αV, and NPC1 was required for efficient GP-mediated transduction and EBOV infection of macrophages. These results define cellular factors hijacked by EBOV for entry into macrophages and, considering that Mer and integrin αV promote phagocytosis of apoptotic cells, support the concept that EBOV relies on apoptotic mimicry to invade target cells.

Cell surface serine protease matriptase-2 suppresses fetuin-A/AHSG-mediated induction of hepcidin.

Matriptase-2 is a type II transmembrane serine protease controlling the expression of hepcidin, the key regulator of iron homeostasis. By cleaving hemojuvelin, matriptase-2 suppresses bone morphogenetic protein/sons of mothers against decapentaplegic signaling. So far, the only known putative substrates of matriptase-2 are hemojuvelin and matriptase-2 itself. In this study, fetuin-A (α2-Heremans-Schmid glycoprotein) was identified in vitro as a substrate of matriptase-2. The protease-substrate interaction was validated by isolating matriptase-2 via the affinity to fetuin-A. Fetuin-A is a liver-derived plasma protein with multiple functions, which is proteolytically processed to yield a disulfide-linked two-chain form. In co-transfected cells, a matriptase-2-dependent conversion of unprocessed fetuin-A into a two-chain form was detected. Conversely, downregulation of endogenously expressed matriptase-2 stabilized fetuin-A. Arg and Lys residues located within the 40 residue spanning connecting peptide of fetuin-A were identified as cleavage sites for matriptase-2. Analysis of hepcidin expression revealed an inductive effect of fetuin-A, which was abolished by matriptase-2. Fetuin-A deficiency in mice resulted in decreased hepcidin mRNA levels. These findings implicate a role of fetuin-A in iron homeostasis and provide new insights into the mechanism of how matriptase-2 might modulate hepcidin expression.

Mapping cardiac remodeling in chronic kidney disease.

Patients with advanced chronic kidney disease (CKD) mostly die from sudden cardiac death and recurrent heart failure. The mechanisms of cardiac remodeling are largely unclear. To dissect molecular and cellular mechanisms of cardiac remodeling in CKD in an unbiased fashion, we performed left ventricular single-nuclear RNA sequencing in two mouse models of CKD. Our data showed a hypertrophic response trajectory of cardiomyocytes with stress signaling and metabolic changes driven by soluble uremia-related factors. We mapped fibroblast to myofibroblast differentiation in this process and identified notable changes in the cardiac vasculature, suggesting inflammation and dysfunction. An integrated analysis of cardiac cellular responses to uremic toxins pointed toward endothelin-1 and methylglyoxal being involved in capillary dysfunction and TNFα driving cardiomyocyte hypertrophy in CKD, which was validated in vitro and in vivo. TNFα inhibition in vivo ameliorated the cardiac phenotype in CKD. Thus, interventional approaches directed against uremic toxins, such as TNFα, hold promise to ameliorate cardiac remodeling in CKD.

Distinct role of mitochondrial function and protein kinase C in intimal and medial calcification in vitro.

Introduction: Vascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways. Materials and methods and results: Human coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs-a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease-a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). In vitro validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP. Conclusion: Our direct comparison results of two in vitro calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification in vitro. We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in in vitro studies using different calcification protocols.

Cardiac Remodeling in Chronic Kidney Disease.

Cardiac remodeling occurs frequently in chronic kidney disease patients and affects quality of life and survival. Current treatment options are highly inadequate. As kidney function declines, numerous metabolic pathways are disturbed. Kidney and heart functions are highly connected by organ crosstalk. Among others, altered volume and pressure status, ischemia, accelerated atherosclerosis and arteriosclerosis, disturbed mineral metabolism, renal anemia, activation of the renin-angiotensin system, uremic toxins, oxidative stress and upregulation of cytokines stress the sensitive interplay between different cardiac cell types. The fatal consequences are left-ventricular hypertrophy, fibrosis and capillary rarefaction, which lead to systolic and/or diastolic left-ventricular failure. Furthermore, fibrosis triggers electric instability and sudden cardiac death. This review focuses on established and potential pathophysiological cardiorenal crosstalk mechanisms that drive uremia-induced senescence and disease progression, including potential known targets and animal models that might help us to better understand the disease and to identify novel therapeutics.

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.

Lumenal calcification and microvasculopathy in fetuin-A-deficient mice lead to multiple organ morbidity.

The plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP)-rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore pathological mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the calcification-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue calcification. Here we studied mechanisms leading to soft tissue calcification, organ damage and death in these mice. We analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice. Fetuin-A-deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature death. Importantly, early-stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the calcification was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis. Collectively, these results demonstrate that soft tissue calcification can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of calcified matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.

Fetuin-A protein distribution in mature inflamed and ischemic brain tissue.

BACKGROUND: The liver-derived plasma protein fetuin-A is strongly expressed during fetal life, hence its name. Fetuin-A protein is normally present in most fetal organs and tissues, including brain tissue. Fetuin-A was neuroprotective in animal models of cerebral ischemia and lethal chronic inflammation, suggesting a role beyond the neonatal period. Little is known, however, on the presence of fetuin-A in mature human brain tissue under different physiological and pathological conditions. METHODS: We studied by immunohistochemistry (IHC) the distribution of fetuin-A protein in mature human brain autopsy tissues from patients without neurological disease, patients with inflammatory brain disorders, and patients with ischemic brain lesions. To identify fetuin-A-positive cells in these tissues we co-localized fetuin-A with GFAP (astrocytes) and CD68 (macrophages, activated microglia). RESULTS AND DISCUSSION: Unlike previous reports, we detected fetuin-A protein also in mature human brain as would be expected from an abundant plasma protein also present in cerebrospinal fluid. Fetuin-A immunoreactivity was increased in ischemic white matter and decreased in inflamed cerebellar tissue. Fetuin-A immunostaining was predominantly associated with neurons and astrocytes. Unlike the developing brain, the adult brain lacked fetuin-A immunostaining in CD68-positive microglia. Our findings suggest a role for fetuin-A in tissue remodeling of neonatal brain, which becomes obsolete in the adult brain, but is re-activated in damaged brain tissue. To further assess the role of fetuin-A in the mature brain, animal models involving ischemia and inflammation need to be studied.

Cellular Clearance and Biological Activity of Calciprotein Particles Depend on Their Maturation State and Crystallinity.

Background: The liver-derived plasma protein fetuin-A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes saturated mineral solutions by forming colloidal protein-mineral complexes called calciprotein particles (CPP). CPP are initially spherical, amorphous and soft, and are referred to as primary CPP. These particles spontaneously convert into secondary CPP, which are larger, oblongate, more crystalline, and less soluble. CPP mediate excess mineral transport and clearance from circulation. Methods: We studied by intravital two-photon microscopy the clearance of primary vs. secondary CPP by injecting i.v. synthetic fluorescent CPP in mice. We analyzed CPP organ distribution and identified CPP endocytosing cells by immunofluorescence. Cellular clearance was studied using bone marrow-derived mouse wildtype and scavenger receptor A (SRA)-deficient macrophages, as well as human umbilical cord endothelial cells (HUVEC), monocyte-derived macrophages (hMDM), and human aortic endothelial cells (haEC). We employed mouse wildtype and mutant immortalized macrophages to analyze CPP-induced inflammasome activation and cytokine secretion. Results: In live mice, only primary CPP were rapidly cleared by liver sinusoidal endothelial cells (LSEC), whereas primary and secondary CPP were cleared by Kupffer cells. Scavenger receptor A (SRA)-deficient bone marrow macrophages endocytosed secondary CPP less well than did wildtype macrophages. In contrast, primary CPP endocytosis did not depend on the presence of SRA, suggesting involvement of an alternative clearance pathway. CPP triggered TLR4 dependent TNFα and IL-1ß secretion in cultured macrophages. Calcium content-matched primary CPP caused twice more IL-1ß secretion than did secondary CPP, which was associated with increased calcium-dependent inflammasome activation, suggesting that intracellular CPP dissolution and calcium overload may cause this inflammation. Conclusions: Secondary CPP are endocytosed by macrophages in liver and spleen via SRA. In contrast, our results suggest that primary CPP are cleared by LSEC via an alternative pathway. CPP induced TLR4-dependent TNFα and inflammasome-dependent IL-1ß secretion in macrophages suggesting that inflammation and calcification may be considered consequences of prolonged CPP presence and clearance.

Sclerostin deficiency modifies the development of CKD-MBD in mice.

Sclerostin is a soluble antagonist of canonical Wnt signaling and a strong inhibitor of bone formation. We present experimental data on the role of sclerostin in chronic kidney disease - bone mineral disorder (CKD-MBD). METHODS: We performed 5/6 nephrectomies in 36-week-old sclerostin-deficient (SOST-/-) B6-mice and in C57BL/6J wildtype (WT) mice. Animals received a high phosphate diet for 11weeks. The bones were analyzed by high-resolution micro-computed tomography (µCT) and quantitative bone histomorphometry. Aortic tissue was analyzed regarding the extent of vascular calcification. RESULTS: All nephrectomized mice had severe renal failure, and parathyroid hormone was highly increased compared to corresponding sham animals. All SOST-/- animals revealed the expected high bone mass phenotype. Overall, the bone compartment in WT and SOST-/- mice responded similarly to nephrectomy. In uremic WT animals, µCT data at both the distal femur and lumbar spine revealed significantly increased trabecular volume compared to non-uremic WTs. In SOST-/- mice, the differences between trabecular bone volume were less pronounced when comparing uremic with sham animals. Cortical thickness and cortical bone density at the distal femur decreased significantly and comparably in both genotypes after 5/6 nephrectomy compared to sham animals (cortical bone density -18% and cortical thickness -32%). Overall, 5/6 nephrectomy and concomitant hyperparathyroidism led to a genotype-independent loss of cortical bone volume and density. Overt vascular calcification was not detectable in either of the genotypes. CONCLUSION: Renal osteodystrophy changes were more pronounced in WT mice than in SOST-/- mice. The high bone mass phenotype of sclerostin deficiency was detectable also in the setting of chronic renal failure with severe secondary hyperparathyroidism.

Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice.

Fetuin-A / α2-Heremans-Schmid-glycoprotein (gene name Ahsg) is a systemic inhibitor of ectopic calcification. Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg-/- mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg-/- mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg-/- mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg+/- mice. Homozygous fetuin-A-deficient Ahsg-/- mice and, to a lesser degree, heterozygous Ahsg+/- mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg-/- mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg-/- mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation.

Hybrid µCT-FMT imaging and image analysis.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (µCT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal µCT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.