Biology subjects

Hidalgo, A.

Publications and source records attributed to Hidalgo, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Ultrahigh-throughput screening for sialic acid-active enzymes in the microbial genetic diversity

Sialic acids (Sias) and related nonulosonic acids are critical components of glycoconjugates involved in host-pathogen interactions, immune regulation, and cell signalling. Despite their biotechnological relevance, the diversity of enzymes involved in Sia biosynthesis remains largely underexplored due to limitations in culture-dependent methods and the lack of (ultra)high-throughput screening strategies. Here, we report the development of a highly sensitive droplet-based microfluidic screening platform enabling the functional discovery of sialic acid aldolases in environmental metagenomes. The method integrates a fluorescence-coupled enzymatic cascade compatible with fluorescence-activated droplet sorting (FADS), allowing the screening of >10 droplets per experiment, as well as a downstream validation strategy for the selected hits. Although some limitations were identified, the system demonstrated high sensitivity and was utilised for the screening of a metagenomic library from garden soil. During this campaign, a potential new sialic acid aldolase enzyme was identified. This work establishes a generalizable framework for measuring complex, multi-step enzymatic functions at ultrahigh throughput using coupled cascades in droplets

microbiology↗

bioRxiv2026

High-throughput screening in hiPSC-cardiac models reveals cardiomyocyte-specific cell cycle regulatory mechanisms

Introductory ParagraphMultiple regulatory mechanisms govern cardiomyocyte proliferation including epigenetic modifications, metabolism and mechanical load. However, it is unclear whether such mechanisms can be pharmacologically targeted to induce cardiomyocyte proliferation without affecting other cell types. Here, we develop a dual-reporter (TNNT2eGFP; PCNAmScarlet-I) and a high-throughput image-based pipeline in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, with counter screening in non-myocytes, to identify compounds that selectively promote cardiomyocyte proliferation without affecting other cell types. We identify the PIM kinase inhibitor GDC-0339 as a cardiomyocyte-selective pro-proliferative compound. GDC-0339 induced proliferation of hiPSC-derived cardiomyocytes without activity in non-myocytes, non-cardiac fibroblasts or epithelial cells. Phosphoproteomic profiling of GDC-0339 in cardiomyocytes and non-cardiac fibroblasts revealed a cardiomyocyte-specific mechanism of action involving sarcomere disassembly via remodelling of the F-actin cytoskeleton and metabolic reprogramming to anaerobic metabolism via Pyruvate Dehydrogenase Kinases (PDKs). Thus, we uncover cardiomyocyte-specific mechanisms governing the cell cycle that are potentially druggable.

cell biology↗

bioRxiv2026

Weckle is a molecular switch that diverts Toll signalling from innate immunity towards growth by engaging Yki

The mechanisms underlying how the brain can switch from a plastic to a degenerative state are unknown. Tolls and Toll-Like receptors (TLRs) could be involved since they underlie both neuroinflammation and structural brain plasticity. Here, we show that the Toll signalling adaptor Weckle (Wek) - a ZAD-Zinc-finger transcription factor - can switch the response from promoting innate immunity or cell death, to inducing growth instead. Myristoylated Wek binds Tolls at the membrane, and concerted PI3K and Toll signalling drive its nuclear translocation. Wek interacts with Yorkie (Yki) - the key regulator of growth restrained by Hippo - enabling its nuclear shuttling. Remarkably, Wek blocks innate immunity. Instead, Wek and Yki together drive glial cell proliferation and growth. Through this mechanism, Toll signalling could promote structural brain plasticity via Wek and Yki downstream. As Tolls and Yki are widespread, these findings could have important implications for understanding also regeneration and cancer.

developmental biology↗

bioRxiv2026

Reprogrammed neutrophils with impaired transit mechanics drive multi-organ capillary stalling after stroke

Beyond the focal brain lesion, stroke causes systemic complications including cardiac failure, pneumonia, renal injury, and sustained immune dysfunction. The source of this multiorgan vulnerability remains unresolved. By imaging over 16,000 vessels of healthy, inflamed and ischemic brains, we identify a circulating neutrophil subpopulation reprogrammed by stroke into a pathological stalling phenotype, occluding capillaries in the brain, heart, kidneys, retina and lungs. Combining transcriptomics, genetic models, integrated microfluidics, cell mechanics assays, and in vivo imaging, we show that this subpopulation exhibits an atypical morphology, increased actin polymerization, and heightened adhesion that impair transit through capillary networks. This phenotype is present in patients with stroke, transmissible by adoptive transfer, and selectively sensitive to inhibition of the Src-family kinase Fgr. Both pharmacological and genetic inactivation of Fgr normalize neutrophil adhesion, reduce capillary stalls, and improve neurological recovery after stroke. These findings identify immune cell transit failure as a systemic driver of post-stroke pathology and a therapeutic target to improve both cerebral and multiorgan outcomes.

neuroscience↗

bioRxiv2025

HDL-associated phosphatidylserine blunts myeloid activation and protects from atherosclerosis

Background and aimsLipids play a critical role in atherosclerosis. Low-density lipoprotein (LDL)-cholesterol and certain lipid classes like sphingomyelins are associated with inflammation and poor cardiovascular outcomes. Phosphatidylserine (PS), on the other hand, is a negatively charged anti-inflammatory phospholipid class involved in efferocytosis. In this study, we sought to investigate its anti-atherosclerotic properties through a combination of complementary human lipidomics analyses, in vitro assays and in vivo experiments in Apoe-/- mice. MethodsHuman lipidomics studies were performed on the 300OB cohort comprising 300 obese and overweight individuals at risk of cardiovascular disease. In vitro assays were carried out using human monocytes and macrophages, and in vivo experiments included histopathological, immunophenotyping and single-cell transcriptomic analyses. ResultsIn humans, we identified PS as an anti-inflammatory and atheroprotective biomarker. Hence, we developed a high-density lipoprotein (HDL)-like formulation enriched in PS to exploit its properties in a targeted fashion in mice. In vitro, this formulation potently inhibited inflammatory cytokine production on human myeloid cells. Our in-depth in vivo experiments provided evidence of the formulations potent plaque-stabilizing and anti-inflammatory actions. These effects were mediated by a shift in the monocyte/macrophage compartment toward homeostatic/repairing phenotypes. ConclusionsCollectively, our results demonstrate that HDL-associated PS potently suppresses inflammation and atheroprogression, and holds promise as a viable approach to improve immunomodulatory therapies.

immunology↗

bioRxiv2025

Microfluidics based exploration for quorum quenching genes in Antarctic microbiomes

Quorum sensing (QS) is a form of microbial communication that enables each cell in a population to sense the total population density, so that gene expression can be modified accordingly. Quorum quenching (QQ) is the antagonistic disruption of this communication by competing organisms, with potential use in the ongoing human effort to control microbial populations. Previous studies have taken advantage of functional metagenomics to retrieve new QS/QQ genes, but the frequency of obtaining positive clones remained very low, suggesting the need for increased screening efficiency. Here, a new ultrahigh-throughput screening method was developed to search for genes encoding novel QQ genes based on functional metagenomics, microfluidics, and the development of an Escherichia coli reporter strain whose fluorescence is repressed in the presence of AHLs but restored by the expression of any gene that interferes with the QS system. This strain was transformed with a metagenomic short-insert library collected from Antarctic plant rhizospheres; an understudied extreme environment. The library was encapsulated in droplets containing single clones and sorted using fluorescence-activated cell sorting (FACS). After screening approximately 7,000,000 clones, around 200 were recovered and one positive hit was confirmed, showing a previously unreported mode of action that would have been difficult to detect using rational computational methods. These findings underline the potential of microfluidics to dramatically increase screening efficiency, while reducing costs and processing time, and they act as a proof of concept for the discovery of more genes involved in QS and other molecular mechanisms of interest in microbial ecology. IMPORTANCEQuorum sensing (QS) regulates essential microbial behaviours, including virulence, biofilm formation, and symbiotic interactions, making it a key target for ecological and applied microbiology. Disrupting QS through quorum quenching (QQ) has major potential in agriculture, medicine, and biotechnology as an alternative to antibiotics or chemical treatments. However, the discovery of QQ genes through brute-force approaches is limited by large screening efforts, resulting in high costs and long processing times. Our study introduces a microfluidics-based, ultrahigh-throughput functional screening platform that strongly improves screening efficiency. It allowed us to identify a novel QQ gene, predicted to be involved in a previously undescribed mechanism of QS disruption, from an under-explored extreme environment (Antarctic rhizosphere). These results demonstrate how microfluidics can optimise screening technologies, while unlocking the hidden functional diversity of microbial communities useful for biotechnological applications in microbial control.

molecular biology↗

bioRxiv2025

The neurotrophin DNT-2 regulates cell survival and connectivity via the Toll-2 receptor during visual system development of Drosophila

During development, neurons are produced in excess and those that receive trophic support are maintained, whereas excess neurons are eliminated, enabling the establishment of appropriate neural circuits. In vertebrates, neurotrophin ligands promote cell survival during periods of naturally occurring cell death, by signalling through p75 and Trk receptors. In the Drosophila optic lobe, a wave of apoptosis occurs during neural circuit development; however, whether this also involves neurotrophism remains unresolved. Drosophila neurotrophins (DNTs) are encoded by spatzle (spz) paralogue genes and bind Toll receptors instead. Here, we focused on DNT-3 (previously known as spz-3) and DNT-2 (also known as spz-5) to ask whether they underlie neurotrophism in visual system development. We show that DNT-3 (spz-3) and DNT-2 (spz-5) are both expressed in the retina and in medulla neurons, and multiple Tolls are expressed across lamina and medulla neurons. Over-expression of DNT-3 (spz-3) and DNT-2 (spz-5) could rescue natural occurring cell death, whereas their loss of function caused cell death, showing that DNT-3 and DNT-2 can, and are required to, promote cell survival during optic lobe development. Importantly, DNT-2 is expressed in Mi1 neurons and Toll-2 in connecting L1 neurons. We show that DNT-2 functions in concert with Toll-2, as Toll-2 RNAi knock-down prevented the rescue of apoptosis by DNT-2 over-expression and all Toll-2+ neurons were lost in DNT-2 mutants. Furthermore, alterations in DNT-2 or Toll-2 expression levels impaired connectivity of L1 neurons at the M1 medulla layer and altered dendritic morphology of L1 neurons. These data suggest that L1 neurons could take up DNT-2 secreted from medulla neurons during the establishment of connectivity patterns. As DNT-3 (spz-3) and DNT-2 (spz-5) are expressed in the medulla and they could influence both lamina and medulla neurons, this suggests that their function maintaining cell survival could enable the stabilisation or alignment of connected neurons across medulla columns.

developmental biology↗

bioRxiv2025

Tumor-Initiating Cells Fine-tune the Plasticity of Neutrophils to Sculpt a Protective Niche

The abundant accumulation of neutrophils in various solid cancers has been well recognized, but the functions of tumor-associated neutrophils (TANs) remain controversial. TANs have long been believed to be immune suppressive and have thus been referred to as "myeloid-derived suppressor cells". However, effective tumor control induced by immunotherapy was recently found to be associated with strong neutrophil signatures. These seemingly contradictory findings highlight the unexpected degree of plasticity and heterogeneity unique to TANs. How the cellular plasticity and functional heterogeneity of TANs are regulated remains unknown. Here, we show that, while anti-PDL1/CD40 agonist immunotherapy can induce interferon responses to reprogram many TANs, allowing them to become plastic and regain anti-tumor activities in squamous cell carcinomas, a subset of TANs residing at the tumor-stroma interface can preserve their immune suppressive state. Importantly, by designing a reverse genetic screening, we identified a group of Sox2Hi tumor-initiating cells (TICs) at the tumor-stroma interface that could upregulate Fatty Acid Desaturase 1 (Fads1) to produce arachidonic acid. This TIC-specific pathway can disrupt the interferon responsive potentials of TANs, preventing the interferon-mediated reprogramming. Thus, by fine-tuning the plasticity of neutrophils, TICs shape neutrophil heterogeneity and sculpt a protective micro-niche to survive from immunotherapy and drive cancer relapse.

cancer biology↗

bioRxiv2025

CIRCADIAN MODULATION OF NEUTROPHIL FUNCTION DETERMINES COLLATERAL PERFUSION AND OUTCOME AFTER ISCHEMIC STROKE

Stroke is a leading cause of mortality and disability, driven by complex and time-dependent mechanisms that aggravate ischemic damage. Among them, collateral perfusion determines the initial size of the ischemic core, the rate of its expansion, and the extent of the penumbra both at stroke onset and over time. Insufficiency of collaterals may occur due to genetic factors or other determinants, such as aging or cardiovascular risk factors, which reduce the number of collaterals or the diameter of those that remain. But aspects of less structural nature could also affect the effectiveness of these pathways by decreasing their patency. We hereby show that diurnal fluctuations in infarct volume in ischemic stroke mouse models are neutrophil phenotype-dependent, since differences in infarct volumes were abolished by depleting neutrophils or blocking their circadian clock, and linked to the collateral circulation: during the inactive phase of mice (daytime), collateral perfusion in the ipsilesional hemisphere was reduced, coinciding with an increase in intravascular neutrophil accumulation, suggestive of microvascular stalling. Single-cell transcriptomics, ex vivo functional assays and in vivo pharmacological and genetic strategies confirmed enhanced neutrophil extracellular traps (NETs) formation at this time. Importantly, in a cohort of human stroke patients, we identified diurnal oscillations in neutrophil and NET-related biomarkers, peaking during the human inactive phase (evening/night), and similarly associated with reduced collateral flow and poorer clinical outcomes. These findings underscore the critical role of neutrophils, their circadian dynamics and NET release in driving collateral insufficiency and ischemic brain damage, suggesting novel personalized therapeutic strategies based on circadian rhythms for the treatment of stroke.

immunology↗

bioRxiv2025

Integrated Multi-Omics Analyses of Synaptosomes Revealed Synapse-Centered Novel Targets in Alzheimer's Disease

AO_SCPLOWBSTRACTC_SCPLOWSynapse dysfunction is an early event in Alzheimers disease (AD) caused by various factors such as Amyloid beta, p-tau, inflammation, and aging. However, the exact molecular mechanism of synapse dysfunction in AD is largely unknown. To understand this, we comprehensively analyzed the synaptosome fraction in postmortem brain samples from AD patients and cognitively normal individuals. We conducted high-throughput transcriptomic analyses to identify changes in microRNA (miRNA) and mRNA levels in synaptosomes extracted from the brains of both unaffected individuals and those with Alzheimers disease (AD). Additionally, we performed mass spectrometry analysis of synaptosomal proteins in the same sample group. These analyses revealed significant differences in the levels of miRNAs, mRNAs, and proteins between the groups. To further understand the pathways or molecules involved, we used an integrated omics approach and studied the molecular interactions of deregulated synapse miRNAs, mRNAs, and proteins in the samples from individuals with AD and the control group, which demonstrated the impact of deregulated miRNAs on their target mRNAs and proteins. Furthermore, the DIABLO analysis highlighted complex relationships between mRNAs, miRNAs, and proteins that could be key in understanding the pathophysiology of AD. Our study identified synapse-centered novel candidates that could be critical in restoring synapse dysfunction in AD.

neuroscience↗

bioRxiv2024

Efficient synthesis of arabinosyl nucleosides via active site engineering of the Leishmania mexicana purine 2'-deoxyribosyltranferase

Even though 2'-deoxyribosyltransferases (NDTs) have emerged as promising and eco-friendly catalysts for synthesizing different 2-deoxynucleoside analogs, their limited activity towards substrates featuring modifications at the 2' -C and 3' -C positions of the ribose moiety significantly restrict their potential application in the pharmaceutical industry. Herein we report two engineered variants from Leishmania mexicana purine deoxyribosyltransferase (LmPDT) with the highest reported arabinosyltransferase activity for an NDT, achieved through a semi-rational design approach. The resulting variants, LmPDTN56L and LmPDTN56C, display up to 13.5- and 30-fold enhanced activity on the synthesis of vidarabine compared to the wild-type enzyme. Moreover, thermal unfolding and thermal challenge experiments revealed that the improvement in arabinosyl transferase activity did not result in a stability trade-off, with Tm values similar to the wild-type enzyme and similar activity retention at 40 {degrees}C. After biochemical characterization, the optimal reaction parameters for variant LmPDTN56C were determined to be pH 6 and a temperature interval from 30 to 45 {degrees}C. Interestingly, under optimal operational conditions (40 {degrees}C, 50 mM MES buffer pH 6) the initial 30-fold enhancement observed for LmPDTN56C escalated to a remarkable 40-fold improvement. This comprehensive study elucidates the potential of the engineered LmPDT variants for efficient and tailored biocatalytic synthesis of arabinosyl nucleosides, paving the way for enhanced applications in nucleoside analog production.

biochemistry↗

bioRxiv2024

Histone deacetylase inhibition expands cellular proteostasis repertoires to enhance neuronal stress resilience

Neurons are long-lived, terminally differentiated cells with limited regenerative capacity. The maintenance of proteostasis and membrane protein trafficking is essential for neuronal function throughout development and aging. Cellular stressors, including endoplasmic reticulum (ER) protein folding stress and endosomal membrane trafficking stress, accumulate as neurons age and correlate with age-dependent neurodegeneration. However, how neurons coordinate stress responses across diverse cellular perturbations remains poorly understood. Here we demonstrate that histone deacetylases (HDACs) constrain the flexibility of neurons to engage distinct molecular pathways in response to different types of stress. Genetic or pharmacological inhibition of class I HDACs enhances neuronal resilience to both ER protein folding stress and endosomal membrane trafficking stress in C. elegans and mammalian neurons. RNA sequencing analyses in C. elegans, mouse, and human iPSC-derived neurons reveal that HDAC inhibition makes neurons more developmentally plastic and induces a permissive transcriptional state that likely represents partial cell fate reprogramming. These transcriptomic changes enable neurons to activate latent proteostasis pathways tailored to the specific nature of the stress. Given the growing excitement around partial reprogramming as a strategy to reset the epigenetic clock, our findings establish a direct link between epigenetic modulation and the neuronal stress response, pointing to new therapeutic strategies for enhancing neuronal resilience in aging and disease.

neuroscience↗

bioRxiv2024

Zfp263 is a transcriptional checkpoint of neutrophil development

During development neutrophils undergo extensive changes in nuclear morphology, gene expression programmes, and acquire specific effector functions. However, molecular mechanisms controlling neutrophil development remain largely unknown. Here we systematically analysed changes in gene expression and chromatin landscape across stages of neutrophil differentiation, to construct a network of transcription factors, predicted to control neutrophil development and acquisition of functions. Zinc finger protein 263 (Zfp263) emerged at the apex of this network. Neutrophil maturation ex vivo and in vivo was blocked in cells lacking Zfp263 and accelerated if its levels were induced. Mechanistically, Zfp263 acts as a direct transcriptional repressor of the ERK1/2 pathway, that supports the proliferative capacity of neutrophil progenitors and limits their differentiation into mature cells. Blocking ERK1/2 signalling in the Zfp263 deficient cells using chemical inhibitors, rescues neutrophil differentiation. Other targets of Zfp263 include genes involved in neutrophil migration, cytokine and chemokine expression, reactive oxygen species and formation of neutrophil extracellular traps. Thus, our study outlines a framework for modulation of neutrophil development and / or function, aimed at neutrophil-mediated diseases.

genomics↗

bioRxiv2024

Fungi activate Toll-1 dependent immune evasion to induce cell loss in the host brain

Fungi evolve within the host, ensuring their own nutrition and reproduction, at the expense of host health. They intervene in hosts brain function, to alter host behaviour and induce neurodegeneration. In humans, fungal infections are emerging as drivers of neuroinflammation, neurodegenerative diseases and psychiatric disorders. However, how fungi alter the host brain is unknown. Fungi trigger an innate immune response mediated by the Toll-1/TLR receptor, the adaptor MyD88 and the transcription factor Dif/NF{kappa}B, that induce the expression of antimicrobial peptides (AMPs). However, in the nervous system, Toll-1/TLR could also drive an alternative pathway involving the adaptor Sarm, which causes cell death instead. Sarm is the universal inhibitor of MyD88 and could drive immune evasion. The entomopathogenic fungus Beauveria bassiana is well-known to activate Toll-1 signalling in innate immunity in Drosophila. In fruit-flies, the adaptor Wek links Toll-1 to Sarm. Thus, here we asked whether B. bassiana could damage the Drosophila brain via Toll-1, Wek and Sarm. We show that exposure to B. bassiana reduced fly lifespan and impaired locomotion. B. bassiana entered the brain and induced the up-regulation of AMPs, as well as wek and sarm, within the brain. Exposure to B. bassiana caused neuronal and glial loss in the adult Drosophila brain. Importantly, RNAi knockdown of Toll-1, wek or sarm concomitantly with infection prevented B. bassiana induced cell loss. By contrast, over-expression of wek or sarm was sufficient to cause dopaminergic neuron loss in the absence of infection. These data show that B. bassiana caused cell loss in the host brain via Toll-1/Wek/Sarm signalling driving immune evasion. We conclude that pathogens can benefit from an innate immunity receptor to damage the host brain. A similar activation of Sarm downstream of TLRs in response to fungal infections could underlie psychiatric and neurodegenerative diseases in humans.

neuroscience↗

bioRxiv2024

Broad therapeutic benefit of myosin inhibition in hypertrophic cardiomyopathy

Myosin inhibitor mavacamten is the only targeted treatment available for hypertrophic cardiomyopathy (HCM), a disease caused by hundreds of genetic variants that affect mainly sarcomeric myosin and its negative regulator cardiac myosin-binding protein C (cMyBP-C, encoded by MYBPC3). Here, we have examined whether the reported limited efficacy of mavacamten in a fraction of HCM patients can result from dissimilar HCM pathomechanisms triggered by different genetic variants, a scenario particularly relevant for MYBPC3-associated HCM. To this aim, we have generated knock-in mice including missense pathogenic variant cMyBP-C p.R502W, which, different from patients who carry truncations in the protein, develop progressive pathogenic myocardial remodeling in the absence of alterations of cMyBP-C levels and localization. Mechanistically, we find that mutation R502W reduces the binding affinity of cMyBP-C for myosin without inducing a shift towards more active myosin conformations as observed when cMyBP-C levels are reduced. Despite these diverging molecular alterations, we show that mavacamten blunts myocardial remodeling both in R502W and cMyBP-C-deficient, knock-out hearts. These beneficial effects are accompanied by improved tolerance to exercise only in R502W animals. Hence, our results indicate that myosin inhibition is effective to treat HCM caused by both truncating and missense variants in MYBPC3 regardless of the primary pathomechanisms they elicit.

physiology↗

bioRxiv2024

Thermostable in vitro transcription-translation for enzyme screening in microdroplets

BackgroundIn vitro expression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Most in vitro expression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use of in vitro expression is its high price compared with host-based recombinant expression, there is a need to develop alternative in vitro expression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. ResultsTo this end, we produced high-expression cell-free extracts from Thermus thermophilus for in vitro translation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostable in vitro expression systems, while the preparation procedure is simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, {beta}-glucosidase and cellobiose hydrolase activities could be measured with minimal background. ConclusionsCell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

microbiology↗

bioRxiv2023

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodelling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodelling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodelling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/539799v4_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@14fa4bcorg.highwire.dtl.DTLVardef@1c86bbborg.highwire.dtl.DTLVardef@118edfcorg.highwire.dtl.DTLVardef@1b06916_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo

There is growing evidence that mutations in non-coding cis-regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1. We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer ({Delta}enFLT1) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.

genomics↗