Apolipoprotein Proteomics for Residual Lipid-Related Risk in Coronary Heart Disease.

BACKGROUND: Recognition of the importance of conventional lipid measures and the advent of novel lipid-lowering medications have prompted the need for more comprehensive lipid panels to guide use of emerging treatments for the prevention of coronary heart disease (CHD). This report assessed the relevance of 13 apolipoproteins measured using a single mass-spectrometry assay for risk of CHD in the PROCARDIS case-control study of CHD (941 cases/975 controls). METHODS: The associations of apolipoproteins with CHD were assessed after adjustment for established risk factors and correction for statin use. Apolipoproteins were grouped into 4 lipid-related classes [lipoprotein(a), low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides] and their associations with CHD were adjusted for established CHD risk factors and conventional lipids. Analyses of these apolipoproteins in a subset of the ASCOT trial (Anglo-Scandinavian Cardiac Outcomes Trial) were used to assess their within-person variability and to estimate a correction for statin use. The findings in the PROCARDIS study were compared with those for incident cardiovascular disease in the Bruneck prospective study (n=688), including new measurements of Apo(a). RESULTS: Triglyceride-carrying apolipoproteins (ApoC1, ApoC3, and ApoE) were most strongly associated with the risk of CHD (2- to 3-fold higher odds ratios for top versus bottom quintile) independent of conventional lipid measures. Likewise, ApoB was independently associated with a 2-fold higher odds ratios of CHD. Lipoprotein(a) was measured using peptides from the Apo(a)-kringle repeat and Apo(a)-constant regions, but neither of these associations differed from the association with conventionally measured lipoprotein(a). Among HDL-related apolipoproteins, ApoA4 and ApoM were inversely related to CHD, independent of conventional lipid measures. The disease associations with all apolipoproteins were directionally consistent in the PROCARDIS and Bruneck studies, with the exception of ApoM. CONCLUSIONS: Apolipoproteins were associated with CHD independent of conventional risk factors and lipids, suggesting apolipoproteins could help to identify patients with residual lipid-related risk and guide personalized approaches to CHD risk reduction.

Proteomic Atlas of Atherosclerosis: The Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes, and Outcomes.

BACKGROUND: Using proteomics, we aimed to reveal molecular types of human atherosclerotic lesions and study their associations with histology, imaging, and cardiovascular outcomes. METHODS: Two hundred nineteen carotid endarterectomy samples were procured from 120 patients. A sequential protein extraction protocol was employed in conjunction with multiplexed, discovery proteomics. To focus on extracellular proteins, parallel reaction monitoring was employed for targeted proteomics. Proteomic signatures were integrated with bulk, single-cell, and spatial RNA-sequencing data, and validated in 200 patients from the Athero-Express Biobank study. RESULTS: This extensive proteomics analysis identified plaque inflammation and calcification signatures, which were inversely correlated and validated using targeted proteomics. The inflammation signature was characterized by the presence of neutrophil-derived proteins, such as S100A8/9 (calprotectin) and myeloperoxidase, whereas the calcification signature included fetuin-A, osteopontin, and gamma-carboxylated proteins. The proteomics data also revealed sex differences in atherosclerosis, with large-aggregating proteoglycans versican and aggrecan being more abundant in females and exhibiting an inverse correlation with estradiol levels. The integration of RNA-sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages, and the calcification and sex signatures to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in plaques, such as fetuin-A. Dimensionality reduction and machine learning techniques were applied to identify 4 distinct plaque phenotypes based on proteomics data. A protein signature of 4 key proteins (calponin, protein C, serpin H1, and versican) predicted future cardiovascular mortality with an area under the curve of 75% and 67.5% in the discovery and validation cohort, respectively, surpassing the prognostic performance of imaging and histology. CONCLUSIONS: Plaque proteomics redefined clinically relevant patient groups with distinct outcomes, identifying subgroups of male and female patients with elevated risk of future cardiovascular events.

Neutrophil-Derived Protein S100A8/A9 Alters the Platelet Proteome in Acute Myocardial Infarction and Is Associated With Changes in Platelet Reactivity.

OBJECTIVE: Platelets are central to acute myocardial infarction (MI). How the platelet proteome is altered during MI is unknown. We sought to describe changes in the platelet proteome during MI and identify corresponding functional consequences. Approach and Results: Platelets from patients experiencing ST-segment-elevation MI (STEMI) before and 3 days after treatment (n=30) and matched patients with severe stable coronary artery disease before and 3 days after coronary artery bypass grafting (n=25) underwent quantitative proteomic analysis. Elevations in the proteins S100A8 and S100A9 were detected at the time of STEMI compared with stable coronary artery disease (S100A8: FC, 2.00; false discovery rate, 0.05; S100A9: FC, 2.28; false discovery rate, 0.005). During STEMI, only S100A8 mRNA and protein levels were correlated in platelets (R=0.46, P=0.012). To determine whether de novo protein synthesis occurs, activated platelets were incubated with 13C-labeled amino acids for 24 hours and analyzed by mass spectrometry. No incorporation was confidently detected. Platelet S100A8 and S100A9 was strongly correlated with neutrophil abundance at the time of STEMI. When isolated platelets and neutrophils were coincubated under quiescent and activated conditions, release of S100A8 from neutrophils resulted in uptake of S100A8 by platelets. Neutrophils released S100A8/A9 as free heterodimer, rather than in vesicles or extracellular traps. In the community-based Bruneck study (n=338), plasma S100A8/A9 was inversely associated with platelet reactivity-an effect abrogated by aspirin. CONCLUSIONS: Leukocyte-to-platelet protein transfer may occur in a thromboinflammatory environment such as STEMI. Plasma S100A8/A9 was negatively associated with platelet reactivity. These findings highlight neutrophils as potential modifiers for thrombotic therapies in coronary artery disease.

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

BACKGROUND: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. METHODS: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. RESULTS: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin ß1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-blockers had a distinct cardiac ECM profile. CONCLUSIONS: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited transcriptomes.

Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.

Pentachlorophenol and nine other chlorophenols in urine of children and adolescents in Germany - Human biomonitoring results of the German Environmental Survey 2014-2017 (GerES V).

Chlorophenols comprise of a large group of chemicals used inter alia for the production of biocides, pharmaceuticals, other industrial products and are used e.g. as antiseptics or wood preservatives due to their biocidal properties. Several of them are classified as toxic to aquatic life and harmful to humans by ingestion, inhalation, or dermal contact, causing skin and eye irritation. Moreover, chlorophenols are possibly carcinogenic to humans. The most prominent chlorophenol - pentachlorophenol - is carcinogenic to humans, was banned in Germany in 1989 and further regulated by the European Commission in 2006 and included in the Stockholm Convention in 2017. Some chlorophenols are persistent in the environment and are also biodegradation products of precursor substances. To evaluate the health-relevance of recent exposure and monitor the effectiveness of regulatory measures, chlorophenols were analysed in the population-representative German Environmental Survey on Children and Adolescents 2014-2017 (GerES V). First-morning void urine samples of 485 3-17-year-old children and adolescents were analysed for ten chlorophenols. Pentachlorophenol was still quantified in 87% of the children and adolescents with a geometric mean (GM) concentration of 0.19 µg/L (0.16 µg/gcrea) and a maximum concentration of 6.7 µg/L (5.4 µg/gcrea). The maximum concentration was well below the health-based guidance value HBM-I of 25 µg/L (20 µg/gcrea). 4-Monochlorophenol was quantified in all samples with a GM concentration of 1.38 µg/L (1.14 µg/gcrea). 2-Monochlorophenol, 2,4-dichlorophenol, and 2,5-dichlorophenol were quantified in 97%, 98%, and 95% of the samples, with GMs of 0.26 µg/L (0.21 µg/gcrea), 0.24 µg/L (0.20 µg/gcrea), and 0.26 µg/L (0.21 µg/gcrea). 2,6-dichlorophenol, 2,3,4-trichlorophenol, and 2,4,5-trichlorophenol were quantified in 17-25% of the samples with GMs below the limit of quantification (LOQ) of 0.1 µg/L 2,4,6-trichlorophenol was quantified in 72% of the samples (GM: 0.13 µg/L, 0.11 µg/gcrea), 2,3,4,6-tetrachlorophenol in 44% of the samples (GM < LOQ). Comparison to previous cycles of GerES revealed substantially lower exposure to most of the chlorophenols in GerES V. Exposure levels found in Germany were comparatively low in contrast to North American results.

R-Limonene metabolism in humans and metabolite kinetics after oral administration.

We studied the R-limonene (LMN) metabolism and elimination kinetics in a human in vivo study. Four volunteers were orally exposed to a single LMN dose of 100-130 µg kg-1 bw. In each case, one pre-exposure and subsequently all 24 h post-exposure urine samples were collected. From two subjects, blood samples were drawn up to 5 h after exposure. The parent compound was analysed in blood using headspace GC-MS. The metabolites cis- and trans-carveol (cCAR), perillyl alcohol (POH), perillic acid (PA), limonene-1,2-diol (LMN-1,2-OH), and limonene-8,9-diol (LMN-8,9-OH) were quantified in both blood and urine using GC-PCI-MS/MS. Moreover, GC-PCI-MS full-scan experiments were applied for identification of unknown metabolites in urine. In both matrices, metabolites reached maximum concentrations 1-2 h post-exposure followed by rapid elimination with half-lives of 0.7-2.5 h. In relation to the other metabolites, LMN-1,2-OH was eliminated slowest. Nonetheless, overall renal metabolite elimination was completed within the 24-h observation period. The metabolite amounts excreted via urine corresponded to 0.2 % (cCAR), 0.2 % (tCAR), <0.1 % (POH), 2.0 % (PA), 4.3 % (LMN-1,2-OH), and 32 % (LMN-8,9-OH) of the orally administered dose. GC-PCI-MS full-scan analyses revealed dihydroperillic acid (DHPA) as an additional LMN metabolite. DHPA was estimated to account for 5 % of the orally administered dose. The study revealed that human LMN metabolism proceeds fast and is characterised by oxidation mainly of the exo-cyclic double bond but also of the endo-cyclic double bond and of the methyl side chain. The study results may support the prediction of the metabolism of other terpenes or comparable chemical structures.

Human metabolism of α-pinene and metabolite kinetics after oral administration.

We studied the human in vivo metabolism and the elimination kinetics of α-pinene (αPN), a natural monoterpene which commonly occurs in the environment. Four volunteers were exposed to a single oral dose of 10 mg αPN. Each subject provided one pre-exposure and subsequently all post-exposure urine samples up to 24 h after administration. Additionally, blood samples were drawn hourly from two volunteers for 5 h. The analysis of the parent compound in blood was performed by a headspace GC-MS procedure, whereas the proposed αPN metabolites myrtenol (MYR) and cis- and trans-verbenol (cVER; tVER) were quantified in blood and urine using GC-PCI-MS/MS. Unknown metabolites were investigated using GC-PCI-MS full-scan analyses. The urinary concentration of the metabolites reached their maxima 1.6 h after exposure. Afterwards, they declined to the pre-exposure levels within the 24-h observation period with elimination half-lives of 1.5 h (MYR) and 1.6 h (cVER and tVER). The total eliminated amounts corresponded to 1.5 % (MYR), 5.6 % (cVER), and 4.1 % (tVER) of the orally applied dose. The GC-PCI-MS full-scan analyses identified three novel metabolites, of which one conforms to myrtenic acid (MYRA). A re-analysis of MYRA in urine showed maximum elimination 1.6 h after αPN ingestion, an elimination half-life of 1.4 h, and a share of the oral dose of 6.7 %. The study revealed that the human in vivo metabolism of αPN proceeds fast and elimination of metabolites takes places rapidly. The metabolism of αPN is dominated by extensive oxidation reactions at the methyl side-chains yielding in carboxylic acid structures as well as by allylic oxidation of the cyclohexenyl backbone, whereas predicted products of a double-bond oxidation were not detected.

Human metabolism of Δ3-carene and renal elimination of Δ3-caren-10-carboxylic acid (chaminic acid) after oral administration.

We studied the human in vivo metabolism of Δ(3)-carene (CRN), a natural monoterpene which commonly occurs in the human environment. Four healthy human volunteers were orally exposed to a single dose of 10 mg CRN. Each volunteer gave one urine sample before administration and subsequently collected each urine sample within 24 h after administration. The concentration of the proposed CRN metabolites Δ(3)-caren-10-ol (CRN-10-OH), Δ(3)-caren-10-carboxylic acid (chaminic acid, CRN-10-COOH), and Δ(3)-caren-3,4-diol (CRN-3,4-OH) were determined using a very specific and sensitive GC-MS/MS procedure. Other CRN metabolites were investigated using GC-PCI-MS Q1 scan analyses. CRN-10-COOH was detected in each urine sample with maximum concentration (113.0-1,172.9 µg L(-1)) 2-3 h after administration, whereas CRN-10-OH and CRN-3,4-OH were not detected in any of the samples. The renal excretion kinetics of CRN-10-COOH showed an elimination half-life of about 3 h. The cumulative excretion of CRN-10-COOH within 24 h after exposure correlated with about 2 % of the applied dose. The GC-PCI-MS Q1 scan analysis indicated several additional human CRN metabolites; thereof, six spectra enabled the prediction of the corresponding chemical structure. The results of the study indicate that CRN-10-COOH is a relevant product of the human in vivo metabolism of CRN. The oxidation of its allylic methyl group proceeds until the acidic structure without interruption. Thus, the generation of the alcoholic intermediate appeared to be the rate-determining step of this metabolic route. Nevertheless, the proportion of CRN-10-COOH in the CRN metabolism is low, and other oxidative metabolites are likely. This hypothesis was confirmed by the discovery of additional human CRN metabolites, whose predicted chemical structures fit in with further oxidative products of CRN metabolism.

Oxidative DNA damage promotes vascular aging associated with changes in extracellular matrix-regulating proteins.

AIMS: Vascular aging is characterized by vessel stiffening, with increased deposition of extracellular matrix (ECM) proteins including collagens. Oxidative DNA damage occurs in vascular aging, but how it regulates ECM proteins and vascular stiffening is unknown. We sought to determine the relationship between oxidative DNA damage and ECM regulatory proteins in vascular aging. METHODS AND RESULTS: We examined oxidative DNA damage, the major base excision repair (BER) enzyme 8-Oxoguanine DNA Glycosylase (Ogg1) and its regulators, multiple physiological markers of aging, and ECM proteomics in mice from 22-72w. Vascular aging was associated with increased oxidative DNA damage, and decreased expression of Ogg1, its active acetylated form, its acetylation regulatory proteins P300 and CBP, and the transcription factor Foxo3a. Vascular stiffness was examined in vivo in control, Ogg1-/-, or mice with vascular smooth muscle cell-specific expression of Ogg1+ (Ogg1) or an inactive mutation (Ogg1KR). Ogg1-/- and Ogg1KR mice showed reduced arterial compliance and distensibility, and increased stiffness and pulse pressure, whereas Ogg1 expression normalised all parameters to 72w. ECM proteomics identified major changes in collagens with aging, and downregulation of the ECM regulatory proteins Protein 6-lysyl oxidase (LOX) and WNT1-inducible-signaling pathway protein 2 (WISP2). Ogg1 overexpression upregulated LOX and WISP2 both in vitro and in vivo, and downregulated Transforming growth factor ß1 (TGFb1) and Collagen 4α1 in vivo compared with Ogg1KR. Foxo3a activation induced Lox, while Wnt3 induction of Wisp2 also upregulated LOX and Foxo3a, and downregulated TGFß1 and fibronectin 1. In humans, 8-oxo-G increased with vascular stiffness, while active OGG1 reduced with both age and stiffness. CONCLUSIONS: Vascular aging is associated with oxidative DNA damage, downregulation of major BER proteins, and changes in multiple ECM structural and regulatory proteins. Ogg1 protects against vascular aging, associated with changes in ECM regulatory proteins including LOX and WISP2.

Simultaneous monitoring of seven phenolic metabolites of endocrine disrupting compounds (EDC) in human urine using gas chromatography with tandem mass spectrometry.

A gas chromatographic-tandem mass spectrometric (GC-MS/MS) method for the simultaneous determination of the three well-known endocrine disruptors, bisphenol A, daidzein and genistein, as well as of four human pesticide metabolites which are supposed to have proper endocrine activity or which are metabolites of endocrine-disrupting compounds, viz., 1- and 2-naphthol, 2-isopropoxyphenol and 3,5,6-trichloropyridinol, has been developed and validated. The method involves enzymatic cleavage of the conjugates using ß-glucuronidase/arylsulfatase followed by solid-phase extraction and derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Isotopically labelled internal standards were used for all analytes, to achieve best analytical error correction. The method proved to be both sensitive and reliable in human urine with detection limits ranging from 0.1 to 0.6 µg/L for all analytes. Precision and repeatability was determined to range from 1 to 15 %. Compared with other published analytical procedures, the present method enables the simultaneous determination of a couple of phenolic agents with competitive or improved analytical reliability. Thus, the present method is suitable for a combined monitoring of the exposure to prominent xenobiotics with effects on the human endocrine system (bisphenol A, carbaryl, chlorpyrifos, chlorpyrifos-methyl, naphthalene, propoxur, triclopyr) and phytoestrogens (daidzein, genistein) in population studies.

Vascular smooth muscle cell senescence accelerates medin aggregation via small extracellular vesicle secretion and extracellular matrix reorganization.

Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid peptide. Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid accumulation in the extracellular matrix (ECM). To determine the mechanisms of AMA formation with age, we explored the impact of vascular smooth muscle cell (VSMC) senescence, EV secretion, and ECM remodeling on medin accumulation. Medin was detected in EVs secreted from primary VSMCs. Small, round medin aggregates colocalized with EV markers in decellularized ECM in vitro and medin was shown on the surface of EVs deposited in the ECM. Decreasing EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age and VSMC senescence increased EV secretion, increased EV medin loading and triggered deposition of fibril-like medin. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition, which led to enhanced EV-ECM binding and accelerated medin aggregation. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalized with EVs and medin. Isolated EVs selectively bound to HSPG2 in the ECM and its knock-down decreased formation of fibril-like medin structures. These data identify VSMC-derived EVs and HSPG2 in the ECM as key mediators of medin accumulation, contributing to age-associated AMA development.

Aberrant DNA Methylation, Expression, and Occurrence of Transcript Variants of the ABC Transporter ABCA7 in Breast Cancer.

The ABC transporter ABCA7 has been found to be aberrantly expressed in a variety of cancer types, including breast cancer. We searched for specific epigenetic and genetic alterations and alternative splicing variants of ABCA7 in breast cancer and investigated whether these alterations are associated with ABCA7 expression. By analyzing tumor tissues from breast cancer patients, we found CpGs at the exon 5-intron 5 boundary aberrantly methylated in a molecular subtype-specific manner. The detection of altered DNA methylation in tumor-adjacent tissues suggests epigenetic field cancerization. In breast cancer cell lines, DNA methylation levels of CpGs in promoter-exon 1, intron 1, and at the exon 5-intron 5 boundary were not correlated with ABCA7 mRNA levels. By qPCR involving intron-specific and intron-flanking primers, we identified intron-containing ABCA7 mRNA transcripts. The occurrence of intron-containing transcripts was neither molecular subtype-specific nor directly correlated with DNA methylation at the respective exon-intron boundaries. Treatment of breast cancer cell lines MCF-7, BT-474, SK-BR3, and MDA-MB-231 with doxorubicin or paclitaxel for 72 h resulted in altered ABCA7 intron levels. Shotgun proteomics revealed that an increase in intron-containing transcripts was associated with significant dysregulation of splicing factors linked to alternative splicing.

Mapping artisanal and small-scale mines at large scale from space with deep learning.

Artisanal and small-scale mines (asm) are on the rise. They represent a crucial source of wealth for numerous communities but are rarely monitored or regulated. The main reason being the unavailability of reliable information on the precise location of the asm which are mostly operated informally or illegally. We address this issue by developing a strategy to map the asm locations using a convolutional neural network for image segmentation, aiming to detect surface mining with satellite data. Our novel dataset is the first comprehensive measure of asm activity over a vast area: we cover 1.75 million km2 across 13 countries in Sub-Tropical West Africa. The detected asm activities range from 0.1 ha to around 2, 000 ha and present a great diversity, yet we succeed in hitting acceptable compromises of performance, as achieving 70% precision while maintaining simultaneously 42% recall. Ultimately, the remarkable robustness of our procedure makes us confident that our method can be applied to other parts of Africa or the world, thus facilitating research and policy opportunities in this sector.

Reduced secretion of neuronal growth regulator 1 contributes to impaired adipose-neuronal crosstalk in obesity.

While the endocrine function of white adipose tissue has been extensively explored, comparatively little is known about the secretory activity of less-investigated fat depots. Here, we use proteomics to compare the secretory profiles of male murine perivascular depots with those of canonical white and brown fat. Perivascular secretomes show enrichment for neuronal cell-adhesion molecules, reflecting a higher content of intra-parenchymal sympathetic projections compared to other adipose depots. The sympathetic innervation is reduced in the perivascular fat of obese (ob/ob) male mice, as well as in the epicardial fat of patients with obesity. Degeneration of sympathetic neurites is observed in presence of conditioned media of fat explants from ob/ob mice, that show reduced secretion of neuronal growth regulator 1. Supplementation of neuronal growth regulator 1 reverses this neurodegenerative effect, unveiling a neurotrophic role for this protein previously identified as a locus associated with human obesity. As sympathetic stimulation triggers energy-consuming processes in adipose tissue, an impaired adipose-neuronal crosstalk is likely to contribute to the disrupted metabolic homeostasis characterising obesity.

Extracellular Matrix Profiling and Disease Modelling in Engineered Vascular Smooth Muscle Cell Tissues.

Aortic smooth muscle cells (SMCs) have an intrinsic role in regulating vessel homeostasis and pathological remodelling. In two-dimensional (2D) cell culture formats, however, SMCs are not embedded in their physiological extracellular matrix (ECM) environment. To overcome the limitations of conventional 2D SMC cultures, we established a 3D in vitro model of engineered vascular smooth muscle cell tissues (EVTs). EVTs were casted from primary murine aortic SMCs by suspending a SMC-fibrin master mix between two flexible silicon-posts at day 0 before prolonged culture up to 14 days. Immunohistochemical analysis of EVT longitudinal sections demonstrated that SMCs were aligned, viable and secretory. Mass spectrometry-based proteomics analysis of murine EVT lysates was performed and identified 135 matrisome proteins. Proteoglycans, including the large aggregating proteoglycan versican, accumulated within EVTs by day 7 of culture. This was followed by the deposition of collagens, elastin-binding proteins and matrix regulators up to day 14 of culture. In contrast to 2D SMC controls, accumulation of versican occurred in parallel to an increase in versikine, a cleavage product mediated by proteases of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family. Next, we tested the response of EVTs to stimulation with transforming growth factor beta-1 (TGFß-1). EVTs contracted in response to TGFß-1 stimulation with altered ECM composition. In contrast, treatment with the pharmacological activin-like kinase inhibitor (ALKi) SB 431542 suppressed ECM secretion. As a disease stimulus, we performed calcification assays. The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. We compared the onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions for 7 days. Calcified EVTs displayed increased tissue stiffness by up to 30 % compared to non-calcified controls. Unlike the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor matrix Gla protein and allowed for better discrimination of the calcification propensity between independent biological replicates. In summary, EVTs are an intuitive and versatile model to investigate ECM synthesis and turnover by SMCs in a 3D environment. Unlike conventional 2D cultures, EVTs provide a more relevant pathophysiological model for retention of the nascent ECM produced by SMCs.

Association of cardiometabolic microRNAs with COVID-19 severity and mortality.

AIMS: Coronavirus disease 2019 (COVID-19) can lead to multiorgan damage. MicroRNAs (miRNAs) in blood reflect cell activation and tissue injury. We aimed to determine the association of circulating miRNAs with COVID-19 severity and 28 day intensive care unit (ICU) mortality. METHODS AND RESULTS: We performed RNA-Seq in plasma of healthy controls (n = 11), non-severe (n = 18), and severe (n = 18) COVID-19 patients and selected 14 miRNAs according to cell- and tissue origin for measurement by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of mild (n = 6), moderate (n = 39), and severe (n = 16) patients. Candidates were then measured by RT-qPCR in longitudinal samples of ICU COVID-19 patients (n = 240 samples from n = 65 patients). A total of 60 miRNAs, including platelet-, endothelial-, hepatocyte-, and cardiomyocyte-derived miRNAs, were differentially expressed depending on severity, with increased miR-133a and reduced miR-122 also being associated with 28 day mortality. We leveraged mass spectrometry-based proteomics data for corresponding protein trajectories. Myocyte-derived (myomiR) miR-133a was inversely associated with neutrophil counts and positively with proteins related to neutrophil degranulation, such as myeloperoxidase. In contrast, levels of hepatocyte-derived miR-122 correlated to liver parameters and to liver-derived positive (inverse association) and negative acute phase proteins (positive association). Finally, we compared miRNAs to established markers of COVID-19 severity and outcome, i.e. SARS-CoV-2 RNAemia, age, BMI, D-dimer, and troponin. Whilst RNAemia, age and troponin were better predictors of mortality, miR-133a and miR-122 showed superior classification performance for severity. In binary and triplet combinations, miRNAs improved classification performance of established markers for severity and mortality. CONCLUSION: Circulating miRNAs of different tissue origin, including several known cardiometabolic biomarkers, rise with COVID-19 severity. MyomiR miR-133a and liver-derived miR-122 also relate to 28 day mortality. MiR-133a reflects inflammation-induced myocyte damage, whilst miR-122 reflects the hepatic acute phase response.

Protein Aggregation Is an Early Manifestation of Phospholamban p.(Arg14del)-Related Cardiomyopathy: Development of PLN-R14del-Related Cardiomyopathy.

BACKGROUND: The p.(Arg14del) pathogenic variant (R14del) of the PLN (phospholamban) gene is a prevalent cause of cardiomyopathy with heart failure. The exact underlying pathophysiology is unknown, and a suitable therapy is unavailable. We aim to identify molecular perturbations underlying this cardiomyopathy in a clinically relevant PLN-R14del mouse model. METHODS: We investigated the progression of cardiomyopathy in PLN-R14Δ/Δ mice using echocardiography, ECG, and histological tissue analysis. RNA sequencing and mass spectrometry were performed on cardiac tissues at 3 (before the onset of disease), 5 (mild cardiomyopathy), and 8 (end stage) weeks of age. Data were compared with cardiac expression levels of mice that underwent myocardial ischemia-reperfusion or myocardial infarction surgery, in an effort to identify alterations that are specific to PLN-R14del-related cardiomyopathy. RESULTS: At 3 weeks of age, PLN-R14Δ/Δ mice had normal cardiac function, but from the age of 4 weeks, we observed increased myocardial fibrosis and impaired global longitudinal strain. From 5 weeks onward, ventricular dilatation, decreased contractility, and diminished ECG voltages were observed. PLN protein aggregation was present before onset of functional deficits. Transcriptomics and proteomics revealed differential regulation of processes involved in remodeling, inflammation, and metabolic dysfunction, in part, similar to ischemic heart disease. Altered protein homeostasis pathways were identified exclusively in PLN-R14Δ/Δ mice, even before disease onset, in concert with aggregate formation. CONCLUSIONS: We mapped the development of PLN-R14del-related cardiomyopathy and identified alterations in proteostasis and PLN protein aggregation among the first manifestations of this disease, which could possibly be a novel target for therapy.