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FoxO1-zDHHC4-CD36 S-Acylation Axis Drives Metabolic Dysfunction in Diabetes.
BACKGROUND: The fatty acid (FA) transporter CD36 (FA translocase/cluster of differentiation 36) is the gatekeeper of cardiac FA metabolism. Preferential localization of CD36 to the sarcolemma is one of the initiating cellular responses in the development of muscle insulin resistance and the type 2 diabetic heart. Posttranslational S-acylation controls protein trafficking, and in this study, we hypothesized that increased CD36 S-acylation may underpin the preferential sarcolemmal localization of CD36, driving metabolic and contractile dysfunction in diabetes. METHODS AND RESULTS: Type 2 diabetes increased cardiac CD36 S-acylation, CD36 sarcolemmal localization, FA oxidation rates, and triglyceride storage in the diabetic heart. CD36 S-acylation was increased in diabetic rats, db/db mice, diabetic pigs, and insulin-resistant human iPSC-derived cardiomyocytes, demonstrating conservation between species. The enzyme responsible for S-acylating CD36, zDHHC4, was transcriptionally upregulated in the diabetic heart, and genetic silencing of zDHHC4 using siRNA or lentiviral shRNA decreased CD36 S-acylation. We identified that zDHHC4 expression is under the regulation of the transcription factor FoxO (forkhead box O) 1, as FoxO1 binds to the promotor of zDHHC4 and induces its transcription, as assessed using chromatin immunoprecipitation-seq, chromatin immunoprecipitation-quantitative PCR, luciferase assays, and siRNA silencing. Diabetic mice with cardiomyocyte-specific FoxO1 deletion had decreased cardiac zDHHC4 expression and decreased CD36 S-acylation, which was further confirmed using diabetic mice treated with the FoxO1 inhibitor AS1842856. Pharmacological inhibition of zDHHC enzymes in diabetic hearts decreased CD36 S-acylation, sarcolemmal CD36 content, FA oxidation rates, and triglyceride storage, culminating in improved cardiac function in diabetes. Conversely, inhibiting the deacylating enzymes in control hearts increased CD36 S-acylation, sarcolemmal CD36 content, and FA metabolic rates in control hearts, recapitulating the metabolic phenotype seen in diabetic hearts. CONCLUSIONS: Activation of the FoxO1-zDHHC4-CD36 S-acylation axis in diabetes drives metabolic and contractile dysfunction in type 2 diabetic heart.
Working memory filtering at encoding and maintenance in healthy ageing, Alzheimer's and Parkinson's disease.
The differential impact on working memory (WM) performance of distractors presented at encoding or during maintenance was investigated in Alzheimer's Disease (AD), Parkinson's Disease (PD) patients, elderly (EHC) and young healthy controls (YHC), (n = 28 per group). Participants reported the orientation of an arrow from a set of either two or three items, with a distractor present either at encoding or at maintenance. MRI data with hippocampal volumes was also acquired. Mean absolute error and mixture model metrics i.e., memory precision, target detection, misbinding (swapping the features of an object with another probed item) and guessing were computed. EHC and PD patients showed good filtering abilities both at encoding and maintenance. However, AD patients exhibited significant filtering deficits specifically when the distractor appeared during maintenance. In healthy ageing there was a prominent decline in WM memory precision, whilst in AD lower target detection and higher guessing were the main sources of error. Conversely, PD was associated only with higher guessing rates. Hippocampal volume was significantly correlated with filtering during maintenance - but not at encoding. These findings demonstrate how healthy ageing and neurodegenerative diseases exhibit distinct patterns of WM impairment, including when filtering irrelevant material either at encoding and maintenance.
Cholinergic degeneration in prodromal and early Parkinson's: a link to present and future disease states.
The neuropathological process in Parkinson's disease (PD) and Lewy body disorders has been shown to extend well beyond the degeneration of the dopaminergic system, affecting other neuromodulatory systems in the brain which play crucial roles in the clinical expression and progression of these disorders. Here, we investigate the role of the macrostructural integrity of the nucleus basalis of Meynert (NbM), the main source of cholinergic input to the cerebral cortex, in cognitive function, clinical manifestation, and disease progression in non-demented subjects with PD and individuals with isolated REM sleep behaviour disorder (iRBD). Using structural MRI data from 393 early PD patients, 128 iRBD patients, and 186 controls from two longitudinal cohorts, we found significantly lower NbM grey matter volume in both PD (β=-12.56, p=0.003) and iRBD (β=-16.41, p=0.004) compared to controls. In PD, higher NbM volume was associated with better higher-order cognitive function (β=0.10, p=0.045), decreased non-motor (β=-0.66, p=0.026) and motor (β=-1.44, p=0.023) symptom burden, and lower risk of future conversion to dementia (Hazard ratio (HR)<0.400, p<0.004). Higher NbM volume in iRBD was associated with decreased future risk of phenoconversion to PD or dementia with Lewy bodies (DLB) (HR<0.490, p<0.016). However, despite similar NbM volume deficits to those seen in PD, associations between NbM structural deficits and current disease burden or clinical state were less pronounced in iRBD. These findings identify NbM volume as a potential biomarker with dual utility: predicting cognitive decline and disease progression in early PD, while also serving as an early indicator of phenoconversion risk in prodromal disease. The presence of structural deficits before clear clinical correlates in iRBD suggests complex compensatory mechanisms may initially mask cholinergic dysfunction, with subsequent failure of these mechanisms potentially contributing to clinical conversion.
Automated quality control of T1-weighted brain MRI scans for clinical research: methods comparison and design of a quality prediction classifier
T1-weighted (T1w) MRI is widely used in clinical neuroimaging for studying brain structure and its changes, including those related to neurodegenerative diseases, and as anatomical reference for analysing other modalities. Ensuring high-quality T1w scans is vital as image quality affects reliability of outcome measures. However, visual inspection can be subjective and time-consuming, especially with large datasets. The effectiveness of automated quality control (QC) tools for clinical cohorts remains uncertain. In this study, we used T1w scans from elderly participants within ageing and clinical populations to test the accuracy of existing QC tools with respect to visual QC and to establish a new quality prediction framework for clinical research use. Four datasets acquired from multiple scanners and sites were used (N = 2438, 11 sites, 39 scanner manufacturer models, 3 field strengths – 1.5T, 3T, 2.9T, patients and controls, average age 71 ± 8 years). All structural T1w scans were processed with two standard automated QC pipelines (MRIQC and CAT12). The agreement of the accept-reject ratings was compared between the automated pipelines and with visual QC. We then designed a quality prediction framework that combines the QC measures from the existing automated tools and is trained on clinical research datasets. We tested the classifier performance using cross-validation on data from all sites together, also examining the performance across diagnostic groups. We then tested the generalisability of our approach when leaving one site out and explored how well our approach generalises to data from a different scanner manufacturer and/or field strength from those used for training, as well as on an unseen new dataset of healthy young participants with movement related artefacts. Our results show significant agreement between automated QC tools and visual QC (Kappa=0.30 with MRIQC predictions; Kappa=0.28 with CAT12’s rating) when considering the entire dataset, but the agreement was highly variable across datasets. Our proposed robust undersampling boost (RUS) classifier achieved 87.7% balanced accuracy on the test data combined from different sites (with 86.6% and 88.3% balanced accuracy on scans from patients and controls respectively). This classifier was also found to be generalisable on different combinations of training and test datasets (average balanced accuracy of leave-one-site-out = 78.2%; exploratory models on field strengths and manufacturers = 77.7%; movement related artefact dataset when including 1% scans in the training = 88.5%). While existing QC tools may not be robustly applicable to datasets comprised of older adults, they produce quality metrics that can be leveraged to train a more robust quality control classifiers for ageing and clinical cohorts.
Integrative role of CTPS cytoophidia in polyploid tissue growth and nutrient adaptation
Tissue growth and development are fundamental to organismal survival, requiring precise coordination of metabolic processes, nutrient availability, and signaling pathways. Cytidine triphosphate synthase (CTPS) is a rate-limiting enzyme in nucleotide biosynthesis and assembles filamentous cytoophidia, conserved across species. Despite increasing interest in cytoophidia, how CTPS filaments integrate metabolic and signaling cues to drive cell size and tissue growth remains incompletely understood. Using RNA interference and clustered regularly interspaces short palindromic repeats (CRISPR) / CRISPR-associate nuclease 9 gene editing, we generated CTPS-knockdown and point-mutated mutants to investigate the role of cytoophidia in cell growth. Specifically, we introduced the H355A mutation, which disrupts CTPS filament formation without affecting its enzymatic activity. Our findings revealed that CTPS depletion or filament disruption significantly impairs growth in polyploid organs, such as the fat body and salivary glands, underscoring the pivotal role of CTPS cytoophidia in cell growth regulation. Mutants lacking cytoophidia exhibited reduced DNA replication activity and smaller cell sizes compared to wild-type controls. Mechanistically, we found that nutrient-sensing pathways, particularly insulin-PI3K-Akt signaling pathway, regulate CTPS expression and cytoophidia formation in response to nutrient availability. Activation of the sterol regulatory element-binding protein partially rescued the growth defects caused by CTPS depletion. These findings provide new insights into the molecular mechanisms of the regulation of CTPS filaments, highlighting their role as critical mediators of tissue growth by integrating environmental demands, metabolism, and signaling pathways to regulate cell size and nutrient adaptation.
Distinct epicardial gene regulatory programs drive development and regeneration of the zebrafish heart.
Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart fully regenerates following injury. Reactivation of cardiac developmental programs is considered key to successfully regenerating the heart, yet the regulation underlying the response to injury remains elusive. Here, we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardia. We identified epicardial enhancer elements with specific activity during development or during adult heart regeneration. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programs driving each of these processes, which were largely distinct. Loss of Hif1ab, Nrf1, Tbx2b, and Zbtb7a, central regulators of the regenerating epicardial network, in injured hearts resulted in elevated epicardial cell numbers infiltrating the wound and excess fibrosis after cryoinjury. Our work identifies differences between the regulatory blueprint deployed during epicardial development and regeneration, underlining that heart regeneration goes beyond the reactivation of developmental programs.
Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants.
Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.
Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.
COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.
N-terminal cysteine acetylation and oxidation patterns may define protein stability.
Oxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.
Comparative analysis of N-terminal cysteine dioxygenation and prolyl-hydroxylation as oxygen-sensing pathways in mammalian cells.
In animals, adaptation to changes in cellular oxygen levels is coordinated largely by 2-oxoglutarate-dependent prolyl-hydroxylase domain (PHD) dioxygenase family members, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of non-transcription factor substrates; the regulators of G-protein signaling 4, 5. and 16 and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their co-evolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2-sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO, and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia. Collectively, our comparative analysis provides further insight into ADO/N-degron-mediated oxygen sensing and its integration into established mechanisms of oxygen homeostasis.
Monitoring ADO dependent proteolysis in cells using fluorescent reporter proteins.
2-Aminoethanethiol dioxygenase (ADO) is the mammalian orthologue of the plant cysteine oxidases and together these enzymes are responsible for catalysing dioxygenation of N-terminal cysteine residues of certain proteins. This modification creates an N-degron motif that permits arginylation and subsequent proteasomal degradation of such proteins via the Arg-branch of the N-degron pathway. In humans 4 proteins have been identified as substrates of ADO; regulators of G-protein signalling (RGS) 4, 5 and 16, and interleukin-32 (IL-32). Nt-cysteine dioxygenation of these proteins occurs rapidly under normoxic conditions, but ADO activity is very sensitive to O2 availability and as such the stability of substrate proteins is inversely proportional to cellular O2 levels. Much is still to understand about the biochemistry and physiology of this pathway in vitro and in vivo, and Cys N-degron targeted fluorescent proteins can provide a simple and effective tool to study this at both subcellular and high-throughput scales. This chapter describes the design, production and implementation of a fluorescent fusion protein proteolytically regulated by ADO and the N-degron pathway.
Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.
Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.
Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation.
Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O2 levels. Using an O2-sensitive phosphorescent nanoparticle probe, we measured an intracellular O2 level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O2. Induction of HO-1 and GCLM by SFN (2.5 μM) was significantly attenuated in cells adapted to 5 kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O2 and subjected to hypoxia (1 kPa O2, 1 h) and reoxygenation. In cells adapted to 18 kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 μM). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX™ Red and FeRhoNox™-1, was diminished in cells cultured under 5 kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O2 levels.
Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans.
The extensive oxygen gradient between the air we breathe (Po2 ~21 kPa) and its ultimate distribution within mitochondria (as low as ~0.5-1 kPa) is testament to the efforts expended in limiting its inherent toxicity. It has long been recognized that cell culture undertaken under room air conditions falls short of replicating this protection in vitro. Despite this, difficulty in accurately determining the appropriate O2 levels in which to culture cells, coupled with a lack of the technology to replicate and maintain a physiological O2 environment in vitro, has hindered addressing this issue thus far. In this review, we aim to address the current understanding of tissue Po2 distribution in vivo and summarize the attempts made to replicate these conditions in vitro. The state-of-the-art techniques employed to accurately determine O2 levels, as well as the issues associated with reproducing physiological O2 levels in vitro, are also critically reviewed. We aim to provide the framework for researchers to undertake cell culture under O2 levels relevant to specific tissues and organs. We envisage that this review will facilitate a paradigm shift, enabling translation of findings under physiological conditions in vitro to disease pathology and the design of novel therapeutics.
Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.
Unregulated increases in cellular Ca2+ homeostasis are a hallmark of pathophysiological conditions and a key trigger of cell death. Endothelial cells cultured under physiologic O2 conditions (5% O2) exhibit a reduced cytosolic Ca2+ response to stimulation. The mechanism for reduced plateau [Ca2+]i upon stimulation was due to increased sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated reuptake rather than changes in Ca2+ influx capacity. Agonist-stimulated phosphorylation of the SERCA regulatory protein phospholamban was increased in cells cultured under 5% O2. Elevation of cytosolic and mitochondrial [Ca2+] and cell death after prolonged ionomycin treatment, as a model of Ca2+ overload, were lower when cells were cultured long-term under 5% compared with 18% O2. This protection was abolished by cotreatment with the SERCA inhibitor cyclopiazonic acid. Taken together, these results demonstrate that culturing cells under hyperoxic conditions reduces their ability to efficiently regulate [Ca2+]i, resulting in greater sensitivity to cytotoxic stimuli.-Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.
A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.
Intracellular O2 is a key regulator of NO signaling, yet most in vitro studies are conducted in atmospheric O2 levels, hyperoxic with respect to the physiologic milieu. We investigated NO signaling in endothelial cells cultured in physiologic (5%) O2 and stimulated with histamine or shear stress. Culture of cells in 5% O2 (>5 d) decreased histamine- but not shear stress-stimulated endothelial (e)NOS activity. Unlike cells adapted to a hypoxic environment (1% O2), those cultured in 5% O2 still mobilized sufficient Ca2+ to activate AMPK. Enhanced expression and membrane targeting of PP2A-C was observed in 5% O2, resulting in greater interaction with eNOS in response to histamine. Moreover, increased dephosphorylation of eNOS in 5% O2 was Ca2+-sensitive and reversed by okadaic acid or PP2A-C siRNA. The present findings establish that Ca2+ mobilization stimulates both NO synthesis and PP2A-mediated eNOS dephosphorylation, thus constituting a novel negative feedback mechanism regulating eNOS activity not present in response to shear stress. This, coupled with enhanced NO bioavailability, underpins differences in NO signaling induced by inflammatory and physiologic stimuli that are apparent only in physiologic O2 levels. Furthermore, an explicit delineation between physiologic normoxia and genuine hypoxia is defined here, with implications for our understanding of pathophysiological hypoxia.-Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.
Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels.
The effects of physiological oxygen tension on Nuclear Factor-E2-Related Factor 2 (Nrf2)-regulated redox signaling remain poorly understood. We report the first study of Nrf2-regulated signaling in human primary endothelial cells (EC) adapted long-term to physiological O2 (5%). Adaptation of EC to 5% O2 had minimal effects on cell ultrastructure, viability, basal redox status or HIF1-α expression. Affymetrix array profiling and subsequent qPCR/protein validation revealed that induction of select Nrf2 target genes, HO-1 and NQO1, was significantly attenuated in cells adapted to 5% O2, despite nuclear accumulation and DNA binding of Nrf2. Diminished HO-1 induction under 5% O2 was stimulus independent and reversible upon re-adaptation to air or silencing of the Nrf2 repressor Bach1, notably elevated under 5% O2. Induction of GSH-related genes xCT and GCLM were oxygen and Bach1-insensitive during long-term culture under 5% O2, providing the first evidence that genes related to GSH synthesis mediate protection afforded by Nrf2-Keap1 defense pathway in cells adapted to physiological O2 levels encountered in vivo.
Extracellular Matrix Protein-1 as a Mediator of Inflammation-Induced Fibrosis After Myocardial Infarction.
Irreversible fibrosis is a hallmark of myocardial infarction (MI) and heart failure. Extracellular matrix protein-1 (ECM-1) is up-regulated in these hearts, localized to fibrotic, inflammatory, and perivascular areas. ECM-1 originates predominantly from fibroblasts, macrophages, and pericytes/vascular cells in uninjured human and mouse hearts, and from M1 and M2 macrophages and myofibroblasts after MI. ECM-1 stimulates fibroblast-to-myofibroblast transition, up-regulates key fibrotic and inflammatory pathways, and inhibits cardiac fibroblast migration. ECM-1 binds HuCFb cell surface receptor LRP1, and LRP1 inhibition blocks ECM-1 from stimulating fibroblast-to-myofibroblast transition, confirming a novel ECM-1-LRP1 fibrotic signaling axis. ECM-1 may represent a novel mechanism facilitating inflammation-fibrosis crosstalk.