Biology subjects

Kim, H.

Publications and source records attributed to Kim, H..

At least 235 records · Page 13Linked to original sources

bioRxiv2024

COmapper: High-resolution mapping of meiotic crossovers by long-read sequencing in Arabidopsis

O_LIMeiotic crossovers rearrange existing genetic variation between homologous chromosomes, profoundly affecting genomic diversity. Crossovers are typically constrained to 1-3 events per chromosome pair, and their distribution is shaped by chromatin accessibility and DNA polymorphisms. Genome-wide crossover maps can be generated in plants by high-throughput short-read sequencing or linked-read sequencing. C_LIO_LIHere, we use long-read nanopore sequencing technology to develop a crossover mapping pipeline, COmapper, for high-resolution mapping of genome-wide crossovers from pooled DNA of F1 hybrid pollen and F2 recombinant seedlings derived from a cross between Arabidopsis thaliana accessions Col and Ler. We validate the high accuracy of COmapper by applying nanopore long-read sequencing to pooled DNA of Arabidopsis F2 individuals with crossovers mapped by short-read sequencing. C_LIO_LIUsing the COmapper, we constructed high-resolution genomic maps of crossovers using F1 hybrid pollen and F2 seedlings in wild type and crossover-elevated recq4a recq4b mutant, showing results comparable to short-read sequencing. Crossovers were enriched at gene-proximal promoters in wild type and increased but reshaped by high polymorphism density in recq4a recq4b. C_LIO_LIWe propose that COmapper will be widely applicable for exploring the effects of genetic, epigenetic and environmental changes on the crossover patterns across diverse plant species. C_LI

plant biology↗

bioRxiv2024

Engineered Bxb1 variants improve integrase activity and fidelity

Many current genome editing technologies rely on the action of large serine integrases (LSIs) to insert gene-sized DNA sequences into the genome. Bxb1 is the most commonly used LSI for therapeutic efforts, including PASTE, PASSIGE and I-PGI. While Bxb1 demonstrated good activity in vitro in cycling cells, the activity in non-dividing hepatocytes was significantly less efficient. Further, wild-type Bxb1 is known to have detectable off-target activity at cryptic genomic sites, which presents a potential safety risk for therapeutic development. To address these issues, we developed Bxb1 variants that demonstrate increased specificity and potency in vitro and have engineered stabilized Bxb1 variants that increase in vivo activity over 25-fold enabling targeted integration at therapeutically relevant levels.

biochemistry↗

bioRxiv2024

Benchmarking Sequence-Based and AlphaFold-Based Methods for pMHC-II Binding Core Prediction: Distinct Strengths and Consensus Approaches

BackgroundInteractions between peptide and MHC class II (pMHC-II) are crucial for T-cell recognition and immune responses, as MHC-II molecules present peptide fragments to T cells, enabling the distinction between self and non-self antigens. Accurately predicting the pMHC-II binding core is particularly important because it provides insights into pMHC-II interactions and T-cell receptor engagement. Given the high polymorphism and peptide-binding promiscuity of MHC-II molecules, computational prediction methods are essential for understanding pMHC-II interactions. While sequence-based methods are widely used, recent advances in AlphaFold-based structure prediction have opened new possibilities for improving pMHC-II binding core predictions. ResultsWe benchmarked four recent pMHC-II prediction methods with a focus on binding core prediction: two sequence-based methods, NetMHCIIpan and DeepMHCII, and two AlphaFold-based structure prediction methods, AlphaFold2 fine-tuned for peptide interactions (AF2-FT) and AlphaFold3 (AF3). The AlphaFold-based methods showed strong performance in predicting positive binders, with AF3 achieving the highest positive recall (0.86) and AF2-FT performing similarly (0.81). However, both methods frequently misclassified unbound peptides as binders. NetMHCIIpan excelled at identifying non-binders, achieving the highest negative recall (0.93), but had lower positive recall (0.44). In contrast, DeepMHCII demonstrated moderate performance without any notable strength. Consensus approaches combining AlphaFold-based methods for binder identification with filtering using NetMHCIIpan improved overall prediction precision (0.94 and 0.87 for known and unknown binding status, respectively). ConclusionsThis study highlights the complementary strengths of AlphaFold-based and sequence-based methods for predicting pMHC-II binding core regions. AlphaFold-based methods excel in predicting positive binders, while NetMHCIIpan is highly effective at identifying non-binders. Future research should focus on improving the prediction of unbound peptides for AlphaFold-based models. Since NetMHCIIpans binding core predictive ability is already high, future efforts should concentrate on enhancing its binding prediction to further improve overall accuracy.

bioinformatics↗

bioRxiv2024

A feedforward loop between STAT1 and YAP1 stimulates lipid biosynthesis, accelerates tumor growth, and promotes chemotherapy resistance in mutant KRAS colorectal cancer.

In tumorous conditions, the transcription factor STAT1, traditionally recognized for its anti-tumor role in immunology, exhibits pro-survival characteristics, though the underlying mechanisms remain unclear. Investigating STAT1s function in isogenic colorectal tumor cells with wild-type or mutant KRAS, we found that STAT1 specifically promotes the survival and proliferation of cells with mutant KRAS. Through gene expression profiling, we discovered a previously unknown role of STAT1 in upregulating sterol and lipid biosynthetic genes specifically in mutant KRAS cells. This effect is driven by STAT1s phosphorylation at serine 727 and its cooperation with STAT3 and STAT5 for the transcriptional upregulation of sterol regulatory element-binding proteins (SREBP) 1 and 2, which boost de novo sterol and lipid biosynthesis. In mutant KRAS cells, STAT1 amplifies the mevalonate pathway, maintaining its serine 727 phosphorylation through RHO GTPase signaling and establishing a positive feedback loop through the transcription factors YAP1 and TEAD4, further driving lipid biosynthesis and tumor growth. Through xenograft tumor assays in mice, we discovered that the STAT1-YAP1 axis plays a role in mutant KRAS tumor cells resistance to mevalonate pathway inhibitors, which can be overcome by pharmacologically targeting the YAP1-TEAD interaction. Additionally, the STAT1-YAP1 arm is essential for the intrinsic resistance to EGFR-targeting therapy in the mutant KRAS colon cancer cells. These findings indicate that the STAT1-YAP1 pathway plays a significant role in therapy resistance and presents a potential therapeutic target in mutant KRAS colorectal cancer.

cancer biology↗

bioRxiv2024

Diversified, miniaturized and ancestral parts for mammalian genome engineering and molecular recording

As the synthetic biology and genome engineering fields mature and converge, there is a clear need for a "parts list" of components that are diversified with respect to both functional activity (to facilitate design) and primary sequence (to facilitate assembly). Here we designed libraries composed of extant, ancestral, mutagenized or miniaturized variants of Pol III promoters or guide RNA (gRNA) scaffolds and quantified their ability to mediate precise edits to the mammalian genome via multiplex prime editing. We identified thousands of parts that reproducibly drive a range of editing activities in human and mouse stem cells and cancer cell lines, including hundreds exhibiting similar or greater activity than the sequences used in conventional genome engineering constructs. We further conducted saturation mutagenesis screens of canonical Pol III promoters (U6p, 7SKp, H1p) and the prime editing guide RNA (pegRNA) scaffold, which identified tolerated variants that can be superimposed on baseline parts to further enhance sequence diversity. While characterizing thousands of orthologous promoters from hundreds of extant or ancestral genomes, we incidentally mapped the functional landscape of mammalian Pol III promoter evolution. Finally, to showcase the usefulness of these parts, we designed a "ten key" molecular recording array that lacks repetitive subsequences in order to facilitate its one-step assembly in yeast. Upon delivering this 15.8 kb tandem array of promoters and guides to mammalian cells, individual pegRNAs exhibited balanced activities as predicted by the activity of component parts, despite their relocation to a single locus. Looking forward, we anticipate that the diversified parts and variant effect maps reported here can be leveraged for the design, assembly and deployment of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity, which will be useful for multiplex perturbation, advanced biological recorders and complex genetic circuits.

synthetic biology↗

bioRxiv2024

Cross-species evidence for a developmental origin of adult hypersomnia with loss of synaptic adhesion molecules beat-Ia/CADM2

Idiopathic hypersomnia (IH) is a poorly-understood sleep disorder characterized by excessive daytime sleepiness despite normal nighttime sleep. Combining human genomics with behavioral and mechanistic studies in fish and flies, we uncover a role for beat-Ia/CADM2, synaptic adhesion molecules of the immunoglobulin superfamily, in excessive sleepiness. Neuronal knockdown of Drosophila beat-Ia results in sleepy flies and loss of the vertebrate ortholog of beat-Ia, CADM2, results in sleepy fish. We delineate a developmental function for beat-Ia in synaptic elaboration of neuropeptide F (NPF) neurites projecting to the suboesophageal zone (SEZ) of the fly brain. Brain connectome and experimental evidence demonstrate these NPF outputs synapse onto a subpopulation of SEZ GABAergic neurons to stabilize arousal. NPF is the Drosophila homolog of vertebrate neuropeptide Y (NPY), and an NPY receptor agonist restores sleep to normal levels in zebrafish lacking CADM2. These findings point towards NPY modulation as a treatment target for human hypersomnia.

neuroscience↗

bioRxiv2024

Galvanin is an electric-field sensor for directed cell migration

Directed cell migration of immune and epithelial cells is critical for their rapid response to tissue injury or infection. Endogenous electric fields generated by disruption of the transepithelial potential across the skin have been postulated to play an important role in guiding cells to wound sites, though how individual cells sense these tissue-scale physical cues remains largely unknown. We have identified Galvanin (TMEM154), a previously uncharacterized single-pass transmembrane protein, as being required for electric-field-guided migration of individual rapidly moving cells. Galvanin functions in both immune and epithelial cell types. Upon exposure of cells to an electric field, Galvanin rapidly relocalizes to the anodal side of a cell, and the net charge on its extracellular domain is necessary and sufficient to drive this spatial relocalization. Furthermore, expression of Galvanin is sufficient to confer electric field-guided migration on otherwise non-responsive epithelial cells. In human neutrophils, we show that Galvanin relocalization is immediately followed by changes in the spatial pattern of cellular protrusion and retraction. The strong directional response of these cells is lost upon truncation of Galvanins intracellular domain, suggesting that Galvanin acts as a direct sensor of the electric field, transducing spatial information about a cells electrical environment to the intracellular migratory apparatus. This sensor relocalization mechanism of cell steering defines a new paradigm for directed cell migration.

cell biology↗

bioRxiv2024

Deleting PTEN, but not SOCS3 or myelin inhibitors, robustly boosts BRAF-elicited intraspinal regeneration of peripheral sensory axons

Primary sensory axons fail to regenerate into the spinal cord after dorsal root (DR) injury, resulting in persistent sensory deficits. This regenerative failure occurs at the dorsal root entry zone (DREZ), the CNS-PNS interface where injured sensory axons encounter both extrinsic inhibitory cues and a limited intrinsic growth state. Although several approaches have promoted partial DR regeneration across the DREZ, sustained long-distance regeneration, particularly of large-diameter myelinated axons, remains a major challenge. We previously showed that induced expression of constitutively active B-RAF (kaBRAF) increases the regenerative competence of injured adult DRG neurons. Here, we tested whether robust intraspinal regeneration after cervical DR injury could be achieved by selective kaBRAF expression alone or in combination with removal of myelin-associated inhibitors or deletion of neuron-intrinsic growth suppressors, PTEN or SOCS3. kaBRAF promoted reproducible but limited regeneration across the DREZ and did not produce significant functional recovery by two months. Additional deletion of Nogo, MAG, and OMgp produced only a modest improvement in kaBRAF-mediated regeneration. Deletion of PTEN or SOCS3, either alone or together, failed to promote meaningful growth across the DREZ. In contrast, PTEN deletion dramatically enhanced kaBRAF-mediated regeneration, enabling many axons to penetrate the DREZ and grow deep into the spinal cord, whereas SOCS3 deletion provided little additional benefit. These findings identify combined activation of BRAF-MEK-ERK and PI3K-Akt-mTOR signaling as a powerful strategy for stimulating robust intraspinal regeneration of injured DR axons.

neuroscience↗

bioRxiv2024

SOX2 drives fetal reprogramming and reversible dormancy in colorectal cancer

Cellular plasticity plays critical roles in tissue regeneration, tumour progression and therapeutic resistance. However, the mechanism underlying this cell state transition remains elusive. Here, we show that the transcription factor SOX2 induces fetal reprogramming and reversible dormancy in colorectal cancer (CRC). SOX2 expression correlates with fetal reprogramming and poor prognosis in human primary and metastatic colorectal adenocarcinomas. In mouse CRC models, rare slow-cycling SOX2+SCA1+ cells are detected in early Apc-deleted mouse tumours that undergo slow clonal expansion over time. In contrast, the SOX2+ clones were found proliferative in advanced Apc-/-;KrasG12D/+;p53-/-;Tgfbr2-/-(AKPT) tumours, accompanied by dynamic cell state reprogramming from LGR5+ to LGR5-SCA1+ cells. Using transgenic mouse models, we demonstrate that ectopic expression of SOX2 inhibits intestinal lineage differentiation and induces fetal reprogramming. SOX2+ cells adopt dynamic cell cycle states depending on its expression level. High SOX2 expression results in hyperproliferation, whereas low SOX2 levels induce senescence-mediated dormancy. We find that loss of p53 can reverse SOX2-induced senescence, in line with the dormant cell state exit of the SOX2+ cells observed in advanced tumours. Finally, SOX2 expression is induced by 5-FU treatment in CRC. SOX2-expressing organoids exhibit increased tolerance to chemotherapy treatment, whilst deletion of SOX2 in AKPT tumour organoids sensitises drug responses. We propose that SOX2-induced plasticity and reversible dormancy promotes tumour progression and drug tolerance in CRC.

cancer biology↗

bioRxiv2024

Intracellular dry mass density increases under growth-induced pressure

Cells that proliferate in confined environments develop mechanical compressive stress, referred to as growth-induced pressure, which inhibits growth and division across various organisms. Recent studies have shown that in these confined spaces, the diffusivity of intracellular nanoparticles decreases. However, the physical mechanisms behind this reduction remain unclear. In this study, we use quantitative phase imaging to measure the refractive index and dry mass density of Saccharomyces cerevisiae cells proliferating under confinement in a microfluidic bioreactor. Our results indicate that the observed decrease in diffusivity can be at least attributed to the intracellular accumulation of macromolecules. Furthermore, the linear scaling between cell content and growth-induced pressure suggests that the concentrations of macromolecules and osmolytes are maintained proportionally under such pressure in S. cerevisiae.

biophysics↗

bioRxiv2024

Community-wide interactions sustain life in geothermal spring habitats

We investigated an alga-dominated geothermal spring community in Yellowstone National Park, USA. Our goal was to determine how cells cope with abiotic stressors during diurnal sampling that spanned over two orders of magnitude in solar irradiance. We report a community level response to toxic metal resistance and energy cycling that spans the three domains of life. Arsenic detoxification is accomplished via complementary gene expression by different lineages. Photosynthesis is dominated by Cyanidioschyzon, with the mixotroph, Galdieria, relegated to nighttime heterotrophy. Many key functions, including the cell cycle, are strongly regulated by diurnal light fluctuations. These results demonstrate that biotic interactions are highly structured in extreme habitats. We suggest this was also the case on the early Earth when geothermal springs were cradles of microbial life, prior to the origin of eukaryotes.

evolutionary biology↗

bioRxiv2024

Connectivity between supplementary motor complex and primary motor cortex: a dual-coil paired-pulse TMS study

In recent years, dual-coil paired-pulse transcranial magnetic stimulation (ppTMS) has garnered interest for its potential in elucidating neural circuit dynamics. In this study, the dual-coil ppTMS was utilized to assess the effective connectivity between the supplementary motor complex (SMC) and the primary motor cortex (M1) in humans. A robust facilitatory connection between the SMC and M1 was observed, manifested as a 19% increase in mean peak-to-peak motor-evoked potentials following preconditioning of SMC 7 ms prior to M1 stimulation. Importantly, the facilitatory influence of SMC only occurred when the preconditioning stimulation was administered 4 cm anterior to Cz but not when applied at 5-cm, 6-cm, or 7-cm distance. While previous work has focused on demonstrating important temporal dynamics for SMC-M1 plasticity, the present findings highlight a critical contribution of spatial specificity for modulation of SMC-M1 circuitry. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/610643v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@12fb447org.highwire.dtl.DTLVardef@e4bfacorg.highwire.dtl.DTLVardef@1e3d262org.highwire.dtl.DTLVardef@93984_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe conditioned stimulus induced larger MEPs compared to the unconditioned stimulus C_LIO_LIThe connectivity between SMC and M1 diminished when the SMC coil was moved forward C_LIO_LIFindings highlight the importance of spatial specificity for modulation of SMC-M1 circuitry C_LI

neuroscience↗

bioRxiv2024

Mult-scale and multi-context mapping of cell states across heterogenous spatial samples.

In clinical applications, spatial data collected under varying conditions, time points, or patients often lack discernible structural alignment. Computational tools designed to align adjacent tissue sections are unsuited for dealing with this structural heterogeneity. There is a growing demand for methods that can effectively align and compare spatial data in the absence of obvious visual correspondence. To address this challenge, we developed an interpretable cell mapping strategy by considering spatial context at various scales. Our approach outperforms existing mapping tools in dealing with heterogeneous samples and is flexible enough to map cells across samples, technologies, resolutions, developmental and regenerative time. Using our approach, we showed spatiotemporal decoupling of cells during development. We even performed alignment for a population of spatial data from cancer patients to identify sub- populations. Our interpretable mapping approach facilitates systemic comparison and analysis of heterogeneous spatial data.

bioinformatics↗

bioRxiv2024

A cup of coffee can boost your motor performance: Enhanced motor sequence learning with caffeine

Caffeine is consumed in various beverages, such as coffee, energy drinks, soda, and tea. The effects of caffeine have been shown to manifest in various ways across different studies. Some studies suggest that caffeine can enhance motor performance. Despite this, numerous motor learning studies do not control for participants caffeine intake or do not record whether participants consumed caffeine. The purpose of this study was to compare the motor learning performance (i.e., online improvement) of individuals who consumed caffeine and those who did not before practice with a novel motor sequence task. Sixty-five right-handed healthy undergraduate students participated in this study. Individuals reported their caffeine consumption through a pre-experiment questionnaire prior to performing an eight-item serial reaction time task. The participants who consumed caffeine reported drinking one cup of coffee on average 158.8 minutes before performing the sequential task. The motor performance of individuals who consumed caffeine was compared to those who did not, with the caffeine-consuming group (n = 13) exhibiting faster response times during online learning than the non-caffeine group (n = 52). The findings of this study suggest that caffeine should be controlled for in future motor learning research.

neuroscience↗

bioRxiv2024

MYH11 rare variant augments aortic growth and induces cardiac hypertrophy and heart failure with pressure overload

Smooth muscle cell-specific myosin heavy chain, encoded by MYH11, is selectively expressed in smooth muscle cells (SMCs). Pathogenic variants in MYH11 predispose to a number of disorders, including heritable thoracic aortic disease associated with patent ductus arteriosus, visceral myopathy, and megacystis-microcolon-intestinal hypoperistalsis syndrome. Rare variants of uncertain significance occur throughout the gene, including MYH11 p.Glu1892Asp, and we sought to determine if this variant causes thoracic aortic disease in mice. Genomic editing was used to generate Myh11E1892D/E1892D mice. Wild-type (WT) and mutant mice underwent cardiovascular phenotyping and with transverse aortic constriction (TAC). Myh11E1892D/E1892D and WT mice displayed similar growth, blood pressure, root and ascending aortic diameters, and cardiac function up to 13 months of age, along with similar contraction and relaxation on myographic testing. TAC induced hypertension similarly in Myh11E1892D/E1892D and WT mice, but mutant mice showed augmented ascending aortic enlargement and increased elastic fragmentation on histology. Unexpectedly, male Myh11E1892D/E1892D mice two weeks post-TAC had decreased ejection fraction, stroke volume, fractional shortening, and cardiac output compared to similarly treated male WT mice. Importantly, left ventricular mass increased significantly due to primarily posterior wall thickening, and cardiac histology confirmed cardiomyocyte hypertrophy and increased collagen deposition in the myocardium and surrounding arteries. These results further highlight the clinical heterogeneity associated with MYH11 rare variants. Given that MYH11 is selectively expressed in SMCs, these results implicate a role of vascular SMCs in the heart contributing to cardiac hypertrophy and failure with pressure overload. Author SummaryIn this study, we explore the impact of a specific genetic variant, MYH11 p.Glu1892Asp, on the heart and blood vessels in mice. The MYH11 gene is crucial for smooth muscle cells, which are found in the walls of blood vessels and play an important role in various vascular diseases. We created mice with this genetic variant to see if it would lead to thoracic aortic disease, a condition affecting the main artery from the heart. We found that mice with the variant were similar to normal mice in many aspects, such as growth, blood pressure, and heart function, for up to 13 months. However, when we induced high blood pressure in the mice, the mutant mice showed more significant enlargement of the aorta and damage to the elastic fibers in the aortic walls. Interestingly, male mutant mice also developed heart problems, such as reduced heart pumping ability and increased heart muscle thickness, after the high blood pressure challenge. This was accompanied by signs of heart muscle cell enlargement and increased tissue stiffness. These findings suggest that this rare MYH11 variant can contribute to a range of heart and vascular issues, particularly under conditions of pressure overload, and highlight the importance of smooth muscle cells in the development of these problems.

genetics↗

bioRxiv2024

Serpina1e mediates the exercise-induced enhancement of hippocampal memory

The exercise-induced enhancement of learning and memory is thought to be regulated by body{square}brain interactions via secretory proteins in the blood plasma1,2. Given the prominent role that skeletal muscle plays during exercise, the beneficial effects of exercise on cognitive functions appear to be mediated by muscle-derived secretory factors including myokines3,4. However, the specific myokines that exert beneficial effects on cognitive functions remain to be elucidated. Here, we reveal that a novel myokine, Serpina1e, acts a molecular mediator that directly supports long-term memory formation in the hippocampus. Using an in vivo myokine-labeling mouse model, proteomic analysis revealed that the Serpina1 family of proteins are the myokines whose levels increased the most in plasma after chronic aerobic exercise for 4 weeks. Systemic delivery of recombinant Serpina1e into sedentary mice was sufficient for reproducing the beneficial effect of exercise on hippocampus-associated cognitive functions. Moreover, plasma Serpina1e can cross the blood-cerebral spinal fluid (CSF) barrier and blood-brain barrier to reach the brain, thereby influencing hippocampal function. Indeed, an increase in the plasma level of Serpina1e promoted hippocampal neurogenesis, increased the levels of brain-derived neurotrophic factor (BDNF) and induced neurite growth. Our findings reveal that Serpina1e is a myokine that migrates to the brain and mediates exercise-induced memory enhancement by triggering neurotrophic growth signaling in the hippocampus. This discovery elucidates the molecular mechanisms underlying the beneficial effects of exercise on cognitive function and may have implications for the development of novel therapeutic interventions for alleviating cognitive disorders.

neuroscience↗

bioRxiv2024

Combined effects of pharmacological interventions and intermittent theta-burst stimulation on motor sequence learning

Drugs that modulate N-methyl-D-aspartate (NMDA) or {gamma}-Aminobutyric acid type A (GABAA) receptors can shed light on their role in synaptic plasticity mechanisms underlying the effects of non-invasive brain stimulation. However, research on the combined effects of these drugs and exogenous stimulation on motor learning is limited. This study aimed to investigate the effects of pharmacological interventions combined with intermittent theta-burst stimulation (iTBS) on human motor learning. Nine right-handed healthy subjects (mean age {+/-} SD: 31.56 {+/-} 12.96 years; 6 females) participated in this double-blind crossover study. All participants were assigned to four drug conditions in a randomized order: (1) D-cycloserine (partial NMDA receptor agonist), (2) D-cycloserine + dextromethorphan (NMDA receptor agonist + antagonist), (3) lorazepam (GABAA receptor agonist), and (4) placebo (identical microcrystalline cellulose capsule). After drug intake, participants practiced the 12-item keyboard sequential task as a baseline measure. Two hours after drug intake, iTBS was administered at the primary motor cortex. Following iTBS, the retention test was performed in the same manner as the baseline measure. Our findings revealed that lorazepam combined with iTBS impaired motor learning during the retention test. Future studies are still needed for a better understanding of the mechanisms through which TMS may influence human motor learning.

neuroscience↗

bioRxiv2024

Inhibiting monocyte migration reduces arterial thrombosis and facilitates thrombolysis

BackgroundMonocytes contribute to the initiation and propagation of venous thrombosis. Little is known about the roles monocytes play in arterial thrombosis, the cause of stroke and MI. MethodsWe investigated how chemokine receptor 2 (CCR2) knockout (-/-) affects platelet function, blood coagulation, thrombus volume, and thrombolytic susceptibility in 627 mice with FeCl3-mediated carotid arterial thrombosis: three CX3CR1-GFP mice, 326 C57BL/6 mice, and 288 CCR2-/- mice. We performed i) intravital microscopy imaging of leukocyte recruitment to carotid thrombus, ii) flow cytometry-based quantification of leukocytes in blood vs. thrombus and leukocyte-platelet aggregates in blood, iii) platelet aggregometry, iv) coagulation assays, v) micro-computed tomography (microCT)-based thrombus imaging using gold nanoparticles after tissue plasminogen activator (tPA) therapy with or without either a) CCR2 siRNA pretreatment (for 3 days) or post-treatment or b) clopidogrel pretreatment (for 7 days), and vi) histology including scanning electron microscopy. ResultsIntravital microscopy and flow cytometry showed that both neutrophils and monocytes were recruited to the acute arterial thrombus, as observed 30 minutes post-thrombosis. Platelet function tests demonstrated platelet aggregation to be lower in the whole blood of CCR2-/- mice (vs. C57BL/6 mice) but not in their leukocyte-free platelet rich plasma, suggesting this platelet dysfunction is cell-mediated. Flow cytometry experiments revealed lower numbers of monocyte - platelet aggregates (MPAs), a marker of platelet activity, in the blood of CCR2-/- mice, compared to C57BL/6 mice. Blood levels of FXIII and monocyte levels of FXIII-A were increased after carotid thrombosis in C57BL/6 mice but not CCR2-/- mice. Further, in vivo microCT and histology, respectively, showed that CCR2-/- mice had smaller and more porous thrombi with less fibrin cross-linking, compared with C57BL/6 mice. MicroCT also demonstrated that tPA-mediated thrombolysis was faster in CCR2-/- mice and CCR2 siRNA-treated mice, compared to C57BL/6 mice. In addition, clopidogrel had a greater effect on inducing thrombus formation with smaller sizes after FeCl3 application, while CCR2-/- had a greater effect on dissolving thrombus faster after tPA administration. ConclusionsCCR2 antagonism decreases platelet aggregation and reduces FXIII levels in blood and monocytes, thus driving arterial thrombosis towards the generation of a relatively small, porous, more lysable clot. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIn venous thrombosis, monocytes cooperate with platelets and neutrophils to promote thrombus formation; however, few studies have investigated the roles of monocytes in arterial thrombosis. C_LIO_LIAn in vitro study using arterial blood samples from patients with acute coronary syndrome showed that P-selectin on activated platelets upregulated monocyte chemokine receptor 2 (CCR2) expression, which was higher in monocyte-platelet aggregates (MPAs) than in monocytes without platelets. C_LIO_LIIn a mouse study using an in vivo model of FeCl3-induced arterial thrombosis, the time to thrombotic occlusion was prolonged in CC chemokine ligand 2 (CCL2) knockout (-/-) mice compared with wild-type mice. C_LI What New Information Does This Article Contribute?O_LIThis study is the first to demonstrate that CCR2-/--mediated monocyte deficiency exerts antithrombotic and tissue plasminogen activator (tPA)-facilitating effects in acute arterial thrombosis. C_LIO_LICCR2-/- mice had a) decreased platelet aggregability, probably due in part to reduced post-thrombosis MPA numbers, and b) lower blood and monocyte levels of Factor XIII (FXIII), compared to those of C57BL/6 mice. C_LIO_LIMicro-computed tomography (microCT) thrombus imaging showed that i) CCR2-/- mice, compared to C57BL/6 mice, had smaller thrombi, with more porosity and less fibrin cross-linking on histology and ii) CCR2-/- or CCR2 siRNA facilitates tPA-mediated thrombolysis. C_LI The majority of ischemic strokes occur due to thromboembolic occlusion of cerebral arteries. Tissue plasminogen activator (tPA), which is the only FDA-approved drug for acute ischemic stroke, has only a moderate efficacy rate regarding early recanalization, which is closely associated with thrombus volume and thrombolytic resistance. Monocytes, in cooperation with platelets and neutrophils, contribute to thrombus formation in venous thrombosis. To date, only a few studies have detailed potential prothrombotic roles for monocytes in arterial thrombosis. In the present study, using a mouse model of FeCl3-mediated carotid artery thrombosis and high-resolution microCT thrombus imaging, we demonstrate that CCR2-/--mediated monocyte deficiency exerts antithrombotic and tPA-facilitating effects. Compared to C57BL/6 mice, CCR2-/- mice exhibited decreased platelet aggregability along with lower numbers of circulating MPAs (a marker of platelet activity) and reduced monocyte recruitment to arterial thrombi. Moreover, CCR2-/- mice had lower levels of both circulating FXIII and monocyte FXIII-A, which are known to strengthen and stabilize thrombi. In line with these findings, i) CCR2-/- mice had smaller, more porous thrombi with less fibrin cross-linking and ii) tPA-mediated thrombolysis was faster in CCR2-/- mice or with CCR2 siRNA.

immunology↗