Cell-free circulating ALU repeats in serum have a prognostic value for colorectal cancer patients.

BACKGROUND: Carcinoembryonic antigen (CEA) is the only established serum biomarker for colorectal cancer (CRC). To facilitate therapy decisions and improve the overall survival of CRC patients, prognostic biomarkers are required. OBJECTIVE: We studied the prognostic value of five different cell free circulating DNA (fcDNA) fragments. The potential markers were ALU115, ALU247, LINE1-79, LINE1-300 and ND1-mt. METHODS: The copy numbers of the DNA fragments were measured in the peripheral blood serum of 268 CRC patients using qPCR, the results were compared to common and previously described markers. RESULTS: We found that ALU115 and ALU247 fcDNA levels correlate significantly with several clinicopathological parameters. An increased amount of ALU115 and ALU247 fcDNA fragments coincides with methylation of HPP1 (P< 0.001; P< 0.01), which proved to be a prognostic marker itself in former studies and also with increased CEA level (both P< 0.001). ALU115 and ALU247 can define patients with poor survival in UICC stage IV (ALU115: HR = 2.9; 95% Cl 1.8-4.8, P< 0.001; ALU247: HR = 2.2; 95% Cl 1.3-3.6; P= 0.001). Combining ALU115 and HPP1, the prognostic value in UICC stage IV is highly significant (P< 0.001). CONCLUSIONS: This study shows that an increased level of ALU fcDNA is an independent prognostic biomarker for advanced colorectal cancer disease.

Uncovering the molecular identity of cardiosphere-derived cells (CDCs) by single-cell RNA sequencing.

Cardiosphere-derived cells (CDCs) generated from human cardiac biopsies have been shown to have disease-modifying bioactivity in clinical trials. Paradoxically, CDCs' cellular origin in the heart remains elusive. We studied the molecular identity of CDCs using single-cell RNA sequencing (sc-RNAseq) in comparison to cardiac non-myocyte and non-hematopoietic cells (cardiac fibroblasts/CFs, smooth muscle cells/SMCs and endothelial cells/ECs). We identified CDCs as a distinct and mitochondria-rich cell type that shared biological similarities with non-myocyte cells but not with cardiac progenitor cells derived from human-induced pluripotent stem cells. CXCL6 emerged as a new specific marker for CDCs. By analysis of sc-RNAseq data from human right atrial biopsies in comparison with CDCs we uncovered transcriptomic similarities between CDCs and CFs. By direct comparison of infant and adult CDC sc-RNAseq data, infant CDCs revealed GO-terms associated with cardiac development. To analyze the beneficial effects of CDCs (pro-angiogenic, anti-fibrotic, anti-apoptotic), we performed functional in vitro assays with CDC-derived extracellular vesicles (EVs). CDC EVs augmented in vitro angiogenesis and did not stimulate scarring. They also reduced the expression of pro-apoptotic Bax in NRCMs. In conclusion, CDCs were disclosed as mitochondria-rich cells with unique properties but also with similarities to right atrial CFs. CDCs displayed highly proliferative, secretory and immunomodulatory properties, characteristics that can also be found in activated or inflammatory cell types. By special culture conditions, CDCs earn some bioactivities, including angiogenic potential, which might modify disease in certain disorders.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Mechanistic investigation of thermosensitive liposome immunogenicity and understanding the drivers for circulation half-life: A polyethylene glycol versus 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol study.

Various thermosensitive liposome (TSL) formulations have been described to date and it is currently unclear which are optimal for solid tumor treatment. Sufficient circulation half-life is important and most liposomes obtain this by polyethylene glycol (PEG) surface modification. 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) has been described as a promising alternative which increases TSL circulation half-life and facilitates rapid drug release under mild hyperthermia at 20-30 mol%. The present work describes an investigation of the DPPG2-TSL protein corona, blood cell interactions, complement activation in human plasma/blood and hypersensitivity reactions in rats. Furthermore, accelerated blood clearance (ABC) was investigated to obtain a complete assessment of DPPG2-TSL interactions with components of the blood and identify drivers for circulation half-life. A higher mol% DPPG2 increased Apolipoprotein E (ApoE) adsorption and decreased complement activation and granulocyte interaction in vitro. In contrast to PEG-TSL, DPPG2-TSL showed no ABC effect. In vivo hypersensitivity assessment by eicosanoid measurements, platelet and lymphocyte counting resembled the results of in vitro complement activation assays although here all DPPG2-TSL formulations induced hypersensitive responses upon i.v. administration. Prolonged circulation half-life of DPPG2-TSL may be ApoE-induced and the absent ABC effect demonstrates an advantage over PEG-TSL. Low complement activation in human plasma and blood for 20-30 mol% DPPG2-TSL presents a unique formulation attribute with the potential to strengthen clinical evaluation.

Head-to-head study of oxelumab and adalimumab in a mouse model of ulcerative colitis based on NOD/Scid IL2Rγnull mice reconstituted with human peripheral blood mononuclear cells.

This study's aim was to demonstrate that the combination of patient immune profiling and testing in a humanized mouse model of ulcerative colitis (UC) might lead to patient stratification for treatment with oxelumab. First, immunological profiles of UC patients and non-UC donors were analyzed for CD4+ T cells expressing OX40 (CD134; also known as TNFRSF4) and CD14+ monocytes expressing OX40L (CD252; also known as TNFSF4) by flow cytometric analysis. A significant difference was observed between the groups for CD14+ OX40L+ (UC: n=11, 85.44±21.17, mean±s.d.; non-UC: n=5, 30.7±34.92; P=0.02), whereas no significant difference was detected for CD4+ OX40+. CD14+ OX40L+ monocytes were correlated significantly with T helper 1 and 2 cells. Second, NOD/Scid IL2Rγ null mice were reconstituted with peripheral blood mononuclear cells from UC donors exhibiting elevated levels of OX40L, and the efficacy of oxelumab was compared with that of adalimumab. The clinical, colon and histological scores and the serum concentrations of IL-6, IL-1ß and glutamic acid were assessed. Treatment with oxelumab or adalimumab resulted in significantly reduced clinical, colon and histological scores, reduced serum concentrations of IL-6 and reduced frequencies of splenic human effector memory T cells and switched B cells. Comparison of the efficacy of adalimumab and oxelumab by orthogonal partial least squares discrimination analysis revealed that oxelumab was slightly superior to adalimumab; however, elevated serum concentrations of glutamic acid suggested ongoing inflammation. These results suggest that oxelumab addresses the pro-inflammatory arm of inflammation while promoting the remodeling arm and that patients exhibiting elevated levels of OX40L might benefit from treatment with oxelumab.

Circulating Metabolites Differentiate Acute Ischemic Stroke from Stroke Mimics.

OBJECTIVE: Early discrimination of patients with ischemic stroke (IS) from stroke mimics (SMs) poses a diagnostic challenge. The circulating metabolome might reflect pathophysiological events related to acute IS. Here, we investigated the utility of early metabolic changes for differentiating IS from SM. METHODS: We performed untargeted metabolomics on serum samples obtained from patients with IS (N = 508) and SM (N = 349; defined by absence of a diffusion weighted imaging [DWI] positive lesion on magnetic resonance imaging [MRI]) who presented to the hospital within 24 hours after symptom onset (median time from symptom onset to blood sampling = 3.3 hours; interquartile range [IQR] = 1.6-6.7 hours) and from neurologically normal controls (NCs; N = 112). We compared diagnostic groups in a discovery-validation approach by applying multivariable linear regression models, machine learning techniques, and propensity score matching. We further performed a targeted look-up of published metabolite sets. RESULTS: Levels of 41 metabolites were significantly associated with IS compared to NCs. The top metabolites showing the highest value in separating IS from SMs were asymmetrical and symmetrical dimethylarginine, pregnenolone sulfate, and adenosine. Together, these 4 metabolites differentiated patients with IS from SMs with an area under the curve (AUC) of 0.90 in the replication sample, which was superior to multimodal cranial computed tomography (CT; AUC = 0.80) obtained for routine diagnostics. They were further superior to previously published metabolite sets detected in our samples. All 4 metabolites returned to control levels by day 90. INTERPRETATION: A set of 4 metabolites with known biological effects relevant to stroke pathophysiology shows unprecedented utility to identify patients with IS upon hospital arrival, thus encouraging further investigation, including multicenter studies. ANN NEUROL 2020;88:736-746.

Ligands and receptors of the TNF superfamily are decreased in major depression and during early antidepressant therapy.

BACKGROUND: The up-regulation of pro-inflammatory agents, amongst them tumor necrosis factor (TNF), may represent low-grade inflammation in major depression. To further elucidate inflammatory mechanisms related to TNF in depression, the aim of the current study was to investigate the involvement of ligands and receptors of the TNF/TNF-receptor-superfamily yet un- or little explored in major depression. METHODS: Serum levels of ligands (TNF, TNF-related weak inducer of apoptosis [TWEAK], B-cell activating factor [BAFF], tumor necrosis factor superfamily member 14 [TNFSF14; LIGHT], A proliferation-inducing ligand [APRIL]) and receptor molecules (TNF receptor superfamily member 8 [TNFRSF8; sCD30], soluble TNF receptor type 1 [sTNFR1] and type 2 [sTNFR2]) of the TNF/TNF-receptor-superfamily were measured in 50 unmedicated patients suffering from major depression and 48 healthy controls and were reassessed in 37 of the depressed patients two weeks after the initiation of antidepressive treatment. RESULTS: In comparison to the healthy controls, the interrelated serum levels of TWEAK, BAFF, TNFSF8, sTNFR1 and sTNFR2 were reduced both in the unmedicated and medicated depressed patients. Serum levels of BAFF and TNF significantly increased during the initiation of antidepressive treatment. In the combined sample of unmedicated depressed and healthy controls, but not the separate groups, scores of the BDI-II inversely correlated with levels of TWEAK, BAFF, sTNFR1, sTNFR2 and TNFSF8. CONCLUSION: The current findings give evidence for a role of the TNF/TNF-receptor-superfamily in the pathophysiology of major depression that may involve reduced tissue regeneration and neurogenesis rather than an acceleration of pro-inflammatory pathways.

Long Noncoding RNA ANRIL: Lnc-ing Genetic Variation at the Chromosome 9p21 Locus to Molecular Mechanisms of Atherosclerosis.

Ever since the first genome-wide association studies (GWAS) on coronary artery disease (CAD), the Chr9p21 risk locus has emerged as a top signal in GWAS of atherosclerotic cardiovascular disease, including stroke and peripheral artery disease. The CAD risk SNPs on Chr9p21 lie within a stretch of 58 kilobases of non-protein-coding DNA, containing the gene body of the long noncoding RNA (lncRNA) antisense non coding RNA in the INK4 locus (ANRIL). How risk is affected by the Chr9p21 locus in molecular detail is a matter of ongoing research. Here we will review recent advances in the understanding that ANRIL serves as a key risk effector molecule of atherogenesis at the locus. One focus of this review is the shift in understanding that genetic variation at Chr9p21 not only affects the abundance of ANRIL, and in some cases expression of the adjacent CDKN2A/B tumor suppressors, but also impacts ANRIL splicing, such that 3'-5'-linked circular noncoding ANRIL RNA species are produced. We describe how the balance of linear and circular ANRIL RNA, determined by the Chr9p21 genotype, regulates molecular pathways and cellular functions involved in atherogenesis. We end with an outlook on how manipulating circular ANRIL abundance may be exploited for therapeutic purposes.

Selective agonist of TRPML2 reveals direct role in chemokine release from innate immune cells.

Cytokines and chemokines are produced and secreted by a broad range of immune cells including macrophages. Remarkably, little is known about how these inflammatory mediators are released from the various immune cells. Here, the endolysosomal cation channel TRPML2 is shown to play a direct role in chemokine trafficking and secretion from murine macrophages. To demonstrate acute and direct involvement of TRPML2 in these processes, the first isoform-selective TRPML2 channel agonist was generated, ML2-SA1. ML2-SA1 was not only found to directly stimulate release of the chemokine CCL2 from macrophages but also to stimulate macrophage migration, thus mimicking CCL2 function. Endogenous TRPML2 is expressed in early/recycling endosomes as demonstrated by endolysosomal patch-clamp experimentation and ML2-SA1 promotes trafficking through early/recycling endosomes, suggesting CCL2 being transported and secreted via this pathway. These data provide a direct link between TRPML2 activation, CCL2 release and stimulation of macrophage migration in the innate immune response.

CXCL8 hyper-signaling in the aortic abdominal aneurysm.

There are indications for elevated CXCL8 levels in abdominal aortic aneurysm disease (AAA). CXCL8 is concurrently involved in neutrophil-mediated inflammation and angiogenesis, two prominent and distinctive characteristics of AAA. As such we considered an evaluation of a role for CXCL8 in AAA progression relevant. ELISA's, real time PCR and array analysis were used to explore CXCL8 signaling in AAA wall samples. A role for CXCL8 in AAA disease was tested through the oral CXCR1/2 antagonist DF2156A in the elastase model of AAA disease. There is an extreme disparity in aortic wall CXCL8 content between AAA and aortic atherosclerotic disease (median [IQR] aortic wall CXCL8 content: 425 [141-1261] (AAA) vs. 23 [2.8-89] (atherosclerotic aorta) µg/g protein (P < 1 ·â€¯10-14)), and abundant expression of the CXCR1 and 2 receptors in AAA. Array analysis followed by pathway analysis showed that CXCL8 hyper-expression in AAA is followed increased by IL-8 signaling (Z-score for AAA vs. atherosclerotic control: 2.97, p < 0.0001). Interference with CXCL8 signaling through DF2156A fully abrogated AAA formation and prevented matrix degradation in the murine elastase model of AAA disease (p < 0.001). CXCL8-signaling is a prominent and distinctive feature of AAA, interference with the pathway constitutes a promising target for medical stabilization of AAA.

Poly(lactic-co-glycolic) Acid as a Slow-Release Drug-Carrying Matrix for Methotrexate Coated onto Intraocular Lenses to Conquer Posterior Capsule Opacification.

PURPOSE: Posterior capsule opacification (PCO) still represents the main long-term complication of cataract surgery. Research into pharmacologic PCO prophylaxis is extensive. One promising candidate drug is methotrexate (MTX). Our aim is to determine the in vitro feasibility of MTX-loaded poly(lactic-co-glycolic) (PLGA) biomatrices sprayed on intraocular lenses (IOLs) as a drug-delivery implant. METHODS: Hydrophilic and hydrophobic acrylic IOLs were spray-coated with MTX-loaded PLGA. Unsprayed, solvent only, and solvent-PLGA-sprayed IOLs served as controls. All IOLs were evaluated for their growth-inhibiting properties in an in vitro anterior segment model and the ex vivo human capsular bag. The release kinetics of MTX from the IOLs was determined. The toxicity of MTX on corneal endothelial cells was evaluated by using a dye reduction colorimetric assay. MTX was also used in a scratch assay. RESULTS: MTX-PLGA-IOL showed a significant difference in cell proliferation and migration compared with all controls in the anterior segment model (p < 0.001) and in the human capsular bag model (p = 0.04). No difference in viability was observed on corneal endothelial cells (p = 0.43; p = 0.61). MTX significantly inhibited cells in the scratch assay (p = 0.02). At all measured points, the released MTX dose remained above EC50 and below the toxic dose for the endothelium. CONCLUSIONS: In view of the strong inhibition of PCO in vitro with the lack of toxic effects on a corneal cell line, MTX encapsulating microspheres seem to be a promising method for modifying IOL.

Adventitial adipogenic degeneration is an unidentified contributor to aortic wall weakening in the abdominal aortic aneurysm.

OBJECTIVE: The processes driving human abdominal aortic aneurysm (AAA) progression are not fully understood. Although antiinflammatory and proteolytic strategies effectively quench aneurysm progression in preclinical models, so far all clinical interventions failed. These observations hint at an incomplete understanding of the processes involved in AAA progression and rupture. Interestingly, strong clinical and molecular associations exist between popliteal artery aneurysms (PAAs) and AAAs; however, PAAs have an extremely low propensity to rupture. We thus reasoned that differences between these aneurysms may provide clues toward (auxiliary) processes involved in AAA-related wall debilitation. A better understanding of the pathophysiologic processes driving AAA growth can contribute to pharmaceutical treatments in the future. METHODS: Aneurysmal wall samples were collected during open elective and emergency repair. Control perirenal aorta was obtained during kidney transplantation, and reference popliteal tissue obtained from the anatomy department. This study incorporates various techniques including (immuno)histochemistry, Western Blot, quantitative polymerase chain reaction, microarray, and cell culture. RESULTS: Histologic evaluation of AAAs, PAAs, and control aorta shows extensive medial (PAA) and transmural fibrosis (AAA), and reveals abundant adventitial adipocytes aggregates as an exclusive phenomenon of AAAs (P < .001). Quantitative polymerase chain reaction, immunohistochemistry, Western blotting, and microarray analysis showed enrichment of adipogenic mediators (C/EBP family P = .027; KLF5 P < .000; and peroxisome proliferator activated receptor-γ, P = .032) in AAA tissue. In vitro differentiation tests indicated a sharply increased adipogenic potential of AAA adventitial mesenchymal cells (P < .0001). Observed enrichment of adipocyte-related genes and pathways in ruptured AAA (P < .0003) supports an association between the extent of fatty degeneration and rupture. CONCLUSIONS: This translational study identifies extensive adventitial fatty degeneration as an ignored and distinctive feature of AAA disease. Enrichment of adipocyte genesis and adipocyte-related genes in ruptured AAA point to an association between the extent of fatty degeneration and rupture. This observation may (partly) explain the failure of medical therapy and could provide a lead for pharmaceutical alleviation of AAA progression.

Molecular roles and function of circular RNAs in eukaryotic cells.

Protein-coding and noncoding genes in eukaryotes are typically expressed as linear messenger RNAs, with exons arranged colinearly to their genomic order. Recent advances in sequencing and in mapping RNA reads to reference genomes have revealed that thousands of genes express also covalently closed circular RNAs. Many of these circRNAs are stable and contain exons, but are not translated into proteins. Here, we review the emerging understanding that both, circRNAs produced by co- and posttranscriptional head-to-tail "backsplicing" of a downstream splice donor to a more upstream splice acceptor, as well as circRNAs generated from intronic lariats during colinear splicing, may exhibit physiologically relevant regulatory functions in eukaryotes. We describe how circRNAs impact gene expression of their host gene locus by affecting transcriptional initiation and elongation or splicing, and how they partake in controlling the function of other molecules, for example by interacting with microRNAs and proteins. We conclude with an outlook how circRNA dysregulation affects disease, and how the stability of circRNAs might be exploited in biomedical applications.

Molecular Fingerprint for Terminal Abdominal Aortic Aneurysm Disease.

BACKGROUND: Clinical decision making in abdominal aortic aneurysms (AAA) relies completely on diameter. At this point, improved decision tools remain an unmet medical need. Our goal was to identify changes at the molecular level specifically leading up to AAA rupture. METHODS AND RESULTS: Aortic wall tissue specimens were collected during open elective (eAAA; n=31) or emergency repair of ruptured AAA (rAAA; n=17), and gene expression was investigated using microarrays. Identified candidate genes were validated with quantitative real-time polymerase chain reaction in an independent sample set (eAAA: n=46; rAAA: n=18). Two gene sets were identified, 1 set containing 5 genes linked to terminal progression, that is, positively associated with progression of larger AAA, and with rupture (HILPDA, ANGPTL4, LOX, SRPX2, FCGBP), and a second set containing 5 genes exclusively upregulated in rAAA (ADAMTS9, STC1, GFPT2, GAL3ST4, CCL4L1). Genes in both sets essentially associated with processes related to impaired tissue remodeling, such as angiogenesis and adipogenesis. In gene expression experiments we were able to show that upregulated gene expression for identified candidate genes is unique for AAA. Functionally, the selected upregulated factors converge at processes coordinated by the canonical HIF-1α signaling pathway and are highly expressed in fibroblasts but not inflammatory cells of the aneurysmatic wall. Histological quantification of angiogenesis and exploration of the HIF-1α network in rAAA versus eAAA shows enhanced microvessel density but also clear activation of the HIF-1α network in rAAA. CONCLUSIONS: Our study shows a specific molecular fingerprint for terminal AAA disease. These changes appear to converge at activation of HIF-1α signaling in mesenchymal cells. Aspects of this cascade might represent targets for rupture risk assessment.

CD27 co-stimulation increases the abundance of regulatory T cells and reduces atherosclerosis in hyperlipidaemic mice.

Aims: The co-stimulatory receptor CD27 modulates responses of T cells, B cells, and NK cells. Various T cell subsets participate in atherogenesis. However, the role of CD27 in atherosclerosis remains unexplored. Methods and results: Here we investigated the effect of bone marrow-derived and systemic CD27 deficiency in Apolipoprotein E-deficient (Apoe-/-) mice in early and advanced stages of atherosclerosis. Lethally-irradiated Apoe-/- mice reconstituted with Cd27-/-Apoe-/- bone marrow and consuming an atherogenic diet displayed a markedly increased plaque size and lesional inflammation compared to mice receiving Cd27+/+Apoe-/- bone marrow. Accordingly, chow diet-fed Cd27-/-Apoe-/- mice showed exacerbated lesion development and increased inflammation at the age of 18 weeks. At a more advanced stage of atherosclerosis (28 weeks), lesion size and phenotype did not differ between the two groups. Systemic and bone marrow-derived CD27 deficiency reduced the abundance of regulatory T cells (Treg) in blood, lymphoid organs, and the aorta. Numbers of other immune cells were not affected while expression of inflammatory cytokine genes (e.g. IL-1ß and IL-6) was increased in the aorta when haematopoietic CD27 was lacking. In vitro, Tregs of CD27-deficient mice showed similar suppressive capacity compared with their wild-type controls and migrated equally towards CCL19 and CCL21. However, thymic Cd27-/- Tregs underwent increased apoptosis and expressed fewer markers of proliferation in vivo. Reconstitution of Cd27-/-Apoe-/- mice with Cd27+/+Apoe-/- Tregs reversed the increase in atherosclerosis. Conclusion: We demonstrate that CD27 co-stimulation increases the number of Tregs and limits lesion development and inflammation in experimental atherosclerosis, particularly during early stages of disease. Thus, our study suggests that promotion of CD27 function may mitigate atherosclerosis.

Modulation of Glutathione Hemostasis by Inhibition of 12/15-Lipoxygenase Prevents ROS-Mediated Cell Death after Hepatic Ischemia and Reperfusion.

BACKGROUND: Reactive oxygen species- (ROS-) mediated ischemia-reperfusion injury (IRI) detrimentally impacts liver transplantation and resection. 12/15-Lipoxygenase (12/15-LOX), an antagonistic protein of the glutathione peroxidase 4 (GPX4) signaling cascade, was proven to mediate cell death in postischemic cerebral and myocardial tissue. The aim of this study was to investigate the impact of 12/15-LOX inhibition on hepatic IRI. METHODS: Livers of C57BL/6 mice were exposed to 60 minutes of partial warm ischemia and 90 minutes of reperfusion after previous Baicalein administration, an inhibitor of 12/15-LOX. Tissue samples were analyzed by TUNEL assay, Western blot, and spectral photometry. RESULTS: TUNEL labeling showed a significant reduction of hepatic cell death following baicalein pretreatment. Western Blot analysis revealed a significant downregulation of Jun-amino-terminal-kinase (JNK), caspase-3, and poly-ADP-ribose-polymerase (PARP), besides considerably lowered p44/42-MAP-kinase (ERK1/2) expression after Baicalein administration. A significant elevation of glutathione oxidation was measured in Baicalein pretreated livers. CONCLUSION: Our data show that inhibition of 12/15-lipoxygenase causes significant cell death reduction after hepatic ischemia and reperfusion by enhancing glutathione metabolism. We conclude that GPX4-dependent cell death signaling cascade might play a major role in development of hepatic IRI, in which the investigated proteins JNK, caspase-3, ERK1/2, and PARP might contribute to tissue damage.

Methylated free-circulating HPP1 DNA is an early response marker in patients with metastatic colorectal cancer.

Detection of methylated free-circulating DNA (mfcDNA) for hyperplastic polyposis 1 (HPP1) in blood is correlated with a poor prognosis for patients with metastatic colorectal cancers (mCRC). Here, we analyzed the plasma levels of HPP1 mfcDNA in mCRC patients treated with a combination therapy containing a fluoropyrimidine, oxaliplatin and bevacizumab to test whether HPP1 mfcDNA is a suitable prognostic and response biomarker. From 467 patients of the prospective clinical study AIO-KRK-0207, mfcDNA was isolated from plasma samples at different time points and bisulfite-treated mfcDNA was quantified using methylation specific PCR. About 337 of 467 patients had detectable levels for HPP1 mfcDNA before start of treatment. The detection was significantly correlated with poorer overall survival (OS) (HR = 1.86; 95%CI 1.37-2.53). About 2-3 weeks after the first administration of combination chemotherapy, HPP1 mfcDNA was reduced to non-detectable levels in 167 of 337 patients. These patients showed a better OS compared with patients with continued detection of HPP1 mfcDNA (HR HPP1(sample 1: pos/ sample 2: neg) vs. HPP1(neg/neg) = 1.41; 95%CI 1.00-2.01, HPP1(neg,pos/pos) vs. HPP1(neg/neg) = 2.60; 95%CI 1.86-3.64). Receiver operating characteristic analysis demonstrated that HPP1 mfcDNA discriminates well between patients who do (not) respond to therapy according to the radiological staging after 12 or 24 weeks (AUC = 0.77 or 0.71, respectively). Detection of HPP1 mfcDNA can be used as a prognostic marker and an early marker for response (as early as 3-4 weeks after start of treatment compared with radiological staging after 12 or 24 weeks) to identify patients who will likely benefit from a combination chemotherapy with bevacizumab.

Adam17 Deficiency Promotes Atherosclerosis by Enhanced TNFR2 Signaling in Mice.

OBJECTIVE: ADAM17 (a disintegrin and metalloproteinase 17) is a sheddase releasing different types of membrane-bound proteins, including adhesion molecules, cytokines, and their receptors as well as inflammatory mediators. Because these substrates modulate important mechanisms of atherosclerosis, we hypothesized that ADAM17 might be involved in the pathogenesis of this frequent disease. APPROACH AND RESULTS: Because Adam17-knockout mice are not viable, we studied the effect of Adam17 deficiency on atherosclerosis in Adam17 hypomorphic mice (Adam17ex/ex), which have low residual Adam17 expression. To induce atherosclerosis, mice were crossed onto the low-density lipoprotein receptor (Ldlr)-deficient background. We found that Adam17ex/ex.Ldlr-/- mice developed ≈1.5-fold larger atherosclerotic lesions, which contained more macrophages and vascular smooth muscle cells than wild-type littermate controls (Adam17wt/wt.Ldlr-/-). Reduced Adam17-mediated shedding led to significantly increased protein levels of membrane-resident TNFα (tumor necrosis factor) and TNFR2 (tumor necrosis factor receptor 2), resulting in a constitutive activation of TNFR2 signaling. At the same time, Adam17 deficiency promoted proatherosclerotic cellular functions, such as increased proliferation and reduced apoptosis in cultured macrophages and vascular smooth muscle cells and increased adhesion of macrophages to vascular endothelial cells. Because siRNA (small interfering RNA)-mediated knockdown of Tnfr2 rescued from aberrant proliferation and from misregulation of apoptosis in Adam17-depleted cells, our data indicate that TNFR2 is an important effector of ADAM17 in our mouse model. CONCLUSIONS: Our results provide evidence for an atheroprotective role of ADAM17, which might be mediated by cleaving membrane-bound TNFα and TNFR2, thereby preventing overactivation of endogenous TNFR2 signaling in cells of the vasculature.