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The molecular structures, electronic properties, and the UV–vis absorption spectra of the oxides of mineral green pigment (MGP) have been theoretically calculated by using the density-functional theory (DFT) method. Our results reveal that the ground-state structure (isomer- I ) of the oxides takes on an O-bridged bond with the polycyclic ring. The chemical stabilities of the oxides ( I - V ) are gradually reduced, of which the I isomer has the largest gap (2.50 eV). Moreover, the adsorption of the two oxygen atoms tailors the electronic structures of oxides, and the electronic properties are keys to understand the structural stabilization of the complexes. Additionally, the strongest UV–vis absorption band of the I isomer has been assigned; e.g., the crucial excitation originates from a HOMO-17→LUMO (32%) transition at ~ 187.3 nm. Graphical abstract

Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound’s therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme. miR-132 was shown to drive pathological cardiac remodeling, a hallmark of heart failure. Here, the authors show that an antisense inhibitor of miR-132 has favourable pharmacokinetics, safety-tolerability and preclinical efficacy in mouse and porcine models of heart failure.

The shear-responsive transcription factor Krüppel-like factor 2 (KLF2) is a critical regulator of endothelial gene expression patterns induced by atheroprotective flow. As microRNAs (miRNAs) post-transcriptionally control gene expression in many pathogenic and physiological processes, we investigated the regulation of miRNAs by KLF2 in endothelial cells. KLF2 binds to the promoter and induces a significant upregulation of the miR-143/145 cluster. Interestingly, miR-143/145 has been shown to control smooth muscle cell (SMC) phenotypes; therefore, we investigated the possibility of transport of these miRNAs between endothelial cells and SMCs. Indeed, extracellular vesicles secreted by KLF2-transduced or shear-stress-stimulated HUVECs are enriched in miR-143/145 and control target gene expression in co-cultured SMCs. Extracellular vesicles derived from KLF2-expressing endothelial cells also reduced atherosclerotic lesion formation in the aorta of ApoE −/−  mice. Combined, our results show that atheroprotective stimuli induce communication between endothelial cells and SMCs through an miRNA- and extracellular-vesicle-mediated mechanism and that this may comprise a promising strategy to combat atherosclerosis. Dimmeler and colleagues show that the atheroprotective transcription factor KLF2 activates expression of the microRNAs miR-143/145 in endothelial cells. miR-143/145 are subsequently enriched in secreted microvesicles and taken up by smooth muscle cells to elicit anti-atherogenic responses.

Chronic cardiac stress induces pathologic hypertrophy and fibrosis of the myocardium. The microRNA-29 (miR-29) family has been found to prevent excess collagen expression in various organs, particularly through its function in fibroblasts. Here, we show that miR-29 promotes pathologic hypertrophy of cardiac myocytes and overall cardiac dysfunction. In a mouse model of cardiac pressure overload, global genetic deletion of miR-29 or antimiR-29 infusion prevents cardiac hypertrophy and fibrosis and improves cardiac function. Targeted deletion of miR-29 in cardiac myocytes in vivo also prevents cardiac hypertrophy and fibrosis, indicating that the function of miR-29 in cardiac myocytes dominates over that in non-myocyte cell types. Mechanistically, we found cardiac myocyte miR-29 to de-repress Wnt signaling by directly targeting four pathway factors. Our data suggests that, cell- or tissue-specific antimiR-29 delivery may have therapeutic value for pathological cardiac remodeling and fibrosis. MicroRNA-29 is known to reduce collagen production in fibroblasts thereby inhibiting fibrosis in various organs. Here, Sassi et al. show that miR-29 can also enhance fibrotic signalling and pathological hypertrophy of the heart through its action in cardiomyocytes.

Transforming growth factor-β1 (TGF-β1) plays a premier role in fibrosis. To understand the molecular events underpinning TGF-β1-induced fibrogenesis, we examined the proteomic profiling of a TGF-β1-induced in vitro model of fibrosis in NRK-49F normal rat kidney fibroblasts. Mass spectrometric analysis indicated that 628 cell-lysate proteins enriched in 44 cellular component clusters, 24 biological processes and 27 molecular functions were regulated by TGF-β1. Cell-lysate proteins regulated by TGF-β1 were characterised by increased ribosomal proteins and dysregulated proteins involved in multiple metabolic pathways, including reduced Aldh3a1 and induced Enpp1 and Impdh2, which were validated by enzyme-linked immunosorbent assays (ELISA). In conditioned media, 62 proteins enriched in 20 cellular component clusters, 40 biological processes and 7 molecular functions were regulated by TGF-β1. Secretomic analysis and ELISA uncovered dysregulated collagen degradation regulators (induced PAI-1 and reduced Mmp3), collagen crosslinker (induced Plod2), signalling molecules (induced Ccn1, Ccn2 and Tsku, and reduced Ccn3) and chemokines (induced Ccl2 and Ccl7) in the TGF-β1 group. We conclude that TGF-β1-induced fibrogenesis in renal fibroblasts is an intracellular metabolic disorder and is inherently coupled with inflammation mediated by chemokines. Proteomic profiling established in this project may guide development of novel anti-fibrotic therapies in a network pharmacology approach.

Reactive protein cysteine thiols are critical to sensing and transducing oxidant signals, often by induction of disulfide bonds that alter their activity or interactions. Identifying such redox active proteins nowadays is mostly achieved using thiol redox proteomics with such datasets increasingly available. Subsequently, we are challenged with determining how changes in the redox state of a protein of interest alters its activity or interactions and how this affects physiology or disease progression including in vivo scenarios. Such studies necessitate the measurement of how the protein redox state changes with health or disease-related interventions, with it not always being practicable to resort back to resource-intensive proteomics to achieve this. In some proteins, oxidation to a disulfide state causes a non-reducing gel-shift, but this is mostly not the case and so other efficient approaches are required to index changes in redox state. Here we assessed the utility of immobilized, solid-phase phenylarsine oxide (PAO-Sepharose) as a tool for indexing the thiol redox state of candidate proteins in cardiac samples from in vivo interventions associated with oxidative stress. PAO-Sepharose, which binds proteins with proximal reduced thiol pairs but not when they form a disulfide, was also used to identify proteins that that are oxidised in isolated perfused mouse hearts exposed to hydrogen peroxide or diamide using proteomics. This together with complementary studies using a cardiac-specific FLAG-Thioredoxin-1C35S-HA transgenic ‘trap-mutant’ mouse model allowed identification of heart proteins susceptible to oxidant-induced disulfide bond formation using proteomics. Thus, two in vitro approaches identified putative cardiac thiol redox sensor proteins that were then assessed with in vivo follow-up studies for their susceptibility to oxidation during endotoxemia induced by lipopolysaccharide or type I diabetes induced by streptozotocin in mice. Of five proteins selected for further analysis by PAO-Sepharose binding, two, namely apoptotic protease activating factor 1 interacting protein (APIP) and γ-glutamylcyclotransferase (GGCT), displayed significantly lower affinity capture from hearts from lipopolysaccharide- or streptozotocin-treated mice, consistent with oxidation of their vicinal thiols. We conclude that PAO-Sepharose is an effective and accessible tool for identifying oxidant-sensitive protein thiols in both ex vivo and in vivo models of oxidative stress. As increasing numbers of thiol redox proteins are identified, PAO-Sepharose binding is an efficient method to determine if they change their oxidation state during interventions relevant to health and disease.

The microvasculature plays a key role in tissue perfusion and exchange of gases and metabolites. In this study we use human blood vessel organoids (BVOs) as a model of the microvasculature. BVOs fully recapitulate key features of the human microvasculature, including the reliance of mature endothelial cells on glycolytic metabolism, as concluded from metabolic flux assays and mass spectrometry-based metabolomics using stable tracing of 13 C-glucose. Pharmacological targeting of PFKFB3, an activator of glycolysis, using two chemical inhibitors results in rapid BVO restructuring, vessel regression with reduced pericyte coverage. PFKFB3 mutant BVOs also display similar structural remodelling. Proteomic analysis of the BVO secretome reveal remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovers microvessel structure. In this work we demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity. The microvasculature is critical for delivery of oxygen and metabolites throughout tissues. Here they use human blood vessel organoids to show that CTGF is a critical paracrine regulator of microvascular integrity that can restore pericyte coverage and vessel structure.

The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Here we identify phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor of the CCCCGG antisense repeat RNA in cytosol. The aminoacylation of tRNA Phe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNA Phe . Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this study reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology. GGGGCC repeat expansion in the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia. Here the authors show that CCCCGG antisense repeat RNA binds and inhibits phenylalanine-tRNA synthetase resulting in decreased levels of tRNAphe and phenylalanine rich proteins.

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca 2+ transient decay time, Ca 2+ ‐load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow‐up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end‐stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non‐failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca 2+ ‐binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca 2+ ‐scavenging, suggesting impaired local Ca 2+ cycling as an important disease culprit. Synopsis The disease mechanism linking the phospholamban (PLN) p.Arg14del mutation to dilated cardiomyopathy is incompletely understood. In this study, patient‐derived human induced pluripotent stem cell‐cardiomyocytes were used to elucidate this molecular mechanism. Sarcoplasmic reticulum function remained unaltered in PLN p.Arg14del human cardiomyocytes. Impairment of the interface between endoplasmic reticulum and mitochondria was discovered as a novel disease phenotype. Cytoplasmic calcium‐scavenging improved the cardiomyopathy phenotype and revealed the role for cytoplasmic calcium in disease development. Graphical Abstract The disease mechanism linking the phospholamban (PLN) p.Arg14del mutation to dilated cardiomyopathy is incompletely understood. In this study, patient‐derived human induced pluripotent stem cell‐cardiomyocytes were used to elucidate this molecular mechanism.

Plasma high‐density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR‐33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short‐term anti‐miR‐33 treatment has been previously studied, the long‐term effect of miR‐33 antagonism in vivo remains to be elucidated. Here, we show that long‐term therapeutic silencing of miR‐33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high‐fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR‐33 increases the expression of genes involved in fatty acid synthesis such as acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR‐33 antisense oligonucleotides. We also report that anti‐miR‐33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP‐responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR‐33 when mice are fed a high‐fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR‐33 in non‐human primates before we can translate this therapy to humans. Synopsis Although short‐term anti‐miR‐33 therapy was reported to increase circulating HDL‐cholesterol and reduce atherosclerosis, long‐term adverse effects are here shown for the first time in mice fed a high‐fat diet to result in hypertriglyceridemia and moderate hepatic steatosis. The effect of long‐term inhibition of miR‐33 was determined in mice fed a chow diet and high‐fat diet. Chronic therapeutic silencing of miR‐33 increased circulating triglycerides and lipid accumulation in the livers of mice fed a high‐fat diet. miR‐33 inhibition raised the expression of genes involved in fatty acid synthesis and lipid metabolism. Further studies are warranted to understand the complex gene regulatory network controlled by miR‐33. Graphical Abstract Although short‐term anti‐miR‐33 therapy was reported to increase circulating HDL‐cholesterol and reduce atherosclerosis, long‐term adverse effects are here shown for the 1st time in mice fed a high‐fat‐diet to result in hypertriglyerimedia and moderate hepatic steatosis.