Biology subjects

Patel, S.

Publications and source records attributed to Patel, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Quiescence modulates age-related changes in the functional capacity of highly proliferative canine lung mesenchymal stromal cell populations

The functional capacity of highly proliferative cell populations changes with age. Here, we report that the proliferative capacity of canine lung mesenchymal stromal cells (LMSCs) declines with increasing age of the donor. However, other functional changes such as reduced autophagy, reduced migration/wound healing, increased production of reactive oxygen species, and increased senescence are not significantly altered with increasing age. Furthermore, transcriptomic profiling suggests minimal age-related changes. These data suggest that the reduced proliferative capacity of lung LMSCs isolated from aging donors may be associated with reversible cell cycle arrest (quiescence), rather than irreversible cell cycle arrest (senescence). Similar findings have been reported in other systems, including neural and muscle stem cells that are associated with low turnover-rate tissues.

developmental biology↗

bioRxiv2025

Cannabinoid Modulation of Central Amygdala Population Dynamics During Threat Investigation

Cannabinoids modulate innate avoidance, threat-reactivity, and stress adaptations via modulation amygdala-associated circuits; however, the mechanisms by which cannabinoids modulate amygdala representation of threat-related behavior are not known. We show that cannabinoid administration increases the activity of central amygdala (CeA) somatostatin neurons (SOM) and alters basal network dynamics in a manner supporting generation of antagonistic sub-ensembles within the SOM population. Moreover, diverging neuronal population trajectory dynamics and enhanced antagonistic sub-ensemble representation of threat-related behaviors, and enhanced threat-related location representation, were also observed. Lastly, cannabinoid administration increased the proportion of SOM neurons exhibiting multidimensional representation of threat-related behaviors and behavior-location conjunction. While cannabinoid receptor activation ex vivo suppressed excitatory inputs to SOM neurons, our data suggest preferential suppression of local GABA release subserves cannabinoid activation of CeA SOM neurons. These data provide insight into how cannabinoid-mediated presynaptic suppression transforms postsynaptic population dynamics and reveal cellular mechanisms by which cannabinoids could affect threat-reactivity.

neuroscience↗

bioRxiv2025

Fusobacteria nucleatum determines the expression of amphetamine-induced behavioral responses through an epigenetic phenomenon.

Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric disorders. They are also misused (AMPH use disorder; AUD), with devastating outcomes. Recent studies have implicated dysbiosis in the pathogenesis of AUD. However, the mechanistic roles of microbes in AUD are unknown. Fusobacterium nucleatum (Fn) is a bacterium that increases in abundance in both rats and humans upon AMPH exposure. Fn releases short-chain fatty acids (SCFAs), bacterial byproducts thought to play a fundamental role in the gut-brain axis as well as the pathogenesis of AUD. We demonstrate that in gnotobiotic Drosophila melanogaster, colonization with Fn or dietary supplementation of the SCFA butyrate, a potent inhibitor of histone deacetylases (HDACs), enhances the psychomotor and rewarding properties of AMPH as well as its ability to promote male sexual motivation. Furthermore, solely HDAC1 RNAi targeted inhibition recapitulates these enhancements, pointing to a specific process underlying this Fn phenomenon. Of note is that the expression of these AMPH behaviors is determined by the increase in extracellular dopamine (DA) levels that result from AMPH-induced reversal of DA transporter (DAT) function, termed non-vesicular DA release (NVDR). The magnitude of AMPH-induced NVDR is dictated, at least in part, by DAT expression levels. Consistent with our behavioral data, we show that Fn, butyrate, and HDAC1 inhibition enhance NVDR by elevating DAT expression. Thus, the participation of Fn in AUD stems from its ability to release butyrate and inhibit HDAC1. These data offer a microbial target and probiotic interventions for AUD treatment.

neuroscience↗

bioRxiv2025

Polymerase Eta Recruits FANCD2 to Common Fragile Sites to Maintain Genome Stability

The replicative polymerase delta is inefficient copying repetitive DNA sequences. Error-prone translesion polymerases have been shown to switch with high-fidelity replicative polymerases to help navigate repetitive DNA. We and others have demonstrated the importance of one such translesion polymerase, polymerase Eta (pol eta), in facilitating replication at genomic regions called common fragile sites (CFS), which are difficult-to-replicate genomic regions that are hypersensitive to replication stress. However, the mechanistic basis for pol etas role in facilitating DNA replication at CFS and(or) at other genomic regions is currently unclear. Importantly, the functional importance of three non-catalytic domains of pol eta, the Ubiquitin-binding Zinc finger (UBZ), PCNA interacting protein (PIP) domain, and the F1 domain which mediates its switch with replicative DNA polymerases in mediating replication stress, especially at CFS loci is not clear. Here, we report that the PIP and UBZ domains of Pol Eta are both critical for its role in mediating cellular replication stress, especially at CFS. The absence of either domain induced elevated replication stress, replication stalling and DNA damage accumulation genome wide. This effect was even more pronounced at CFS loci leading to the accumulation of under replication DNA in G2/M. Importantly, while the inactivation of the UBZ domain resulted in a robust FANCD2 monoubiquitylation (a prominent marker of FANCD2 activation), FANCD2 recruitment genome wide was significantly impacted, especially at CFSs such as FRA16D. These S-phase phenotypes result in ssDNA gap formation and the persistence of under-replicated genomic regions upon transition to G2/M. While post-replicative gap filing/ repair by Mitotic DNA synthesis is activated in the mutants, it only effectively resolves UFBs in the F1* cells. The PIP*, UBZ* and pol eta-/- cells unfortunately manifest excessive toxic cytosolic DNA that instigates a strong innate immune response. These results collectively show that translesion polymerase Eta functions in a common pathway with FANCD2 to prevent replication perturbation and instability at CFS loci.

molecular biology↗

bioRxiv2024

Engineering Multiplexed Synthetic Breath Biomarkers as Diagnostic Probes

Breath biopsy is emerging as a rapid and non-invasive diagnostic tool that links exhaled chemical signatures with specific medical conditions. Despite its potential, clinical translation remains limited by the challenge of reliably detecting endogenous, disease-specific biomarkers in breath. Synthetic biomarkers represent an emerging paradigm for precision diagnostics such that they amplify activity-based biochemical signals associated with disease fingerprints. However, their adaptation to breath biopsy has been constrained by the limited availability of orthogonal volatile reporters that are detectable in exhaled breath. Here, we engineer multiplexed breath biomarkers that couple aberrant protease activities to exogenous volatile reporters. We designed novel intramolecular reactions that leverage protease-mediated aminolysis, enabling the sensing of a broad spectrum of proteases, and that each release a unique reporter in breath. This approach was validated in a mouse model of influenza to establish baseline sensitivity and specificity in a controlled inflammatory setting and subsequently applied to diagnose lung cancer using an autochthonous Alk-mutant model. We show that combining multiplexed reporter signals with machine learning algorithms enables tumor progression tracking, treatment response monitoring, and detection of relapse after 30 minutes. Our multiplexed breath biopsy platform highlights a promising avenue for rapid, point-of-care diagnostics across diverse disease states.

bioengineering↗

bioRxiv2024

Afadin Sorts Different Retinal Neuron Types into Accurate Cellular Layers

Neurons use cell-adhesion molecules (CAMs) to interact with other neurons and the extracellular environment: the combination of CAMs specifies migration patterns, neuronal morphologies, and synaptic connections across diverse neuron types. Yet little is known regarding the intracellular signaling cascade mediating the CAM recognitions at the cell surface across different neuron types. In this study, we investigated the neural developmental role of Afadin1-4, a cytosolic adapter protein that connects multiple CAM families to intracellular F-actin. We introduced the conditional Afadin mutant5 to an embryonic retinal Cre, Six3-Cre6-8. We reported that the mutants lead to the scrambled retinal neuron distribution, including Bipolar Cells (BCs), Amacrine Cells (ACs), and retinal ganglion cells (RGCs), across three cellular layers of the retina. This scrambled pattern was first reported here at neuron-type resolution. Importantly, the mutants do not display deficits for BCs, ACs, or RGCs in terms of neural fate specifications or survival. Additionally, the displayed RGC types still maintain synaptic partners with putative AC types, indicating that other molecular determinants instruct synaptic choices independent of Afadin. Lastly, there is a significant decline in visual function and mis-targeting of RGC axons to incorrect zones of the superior colliculus, one of the major retinorecipient areas. Collectively, our study uncovers a unique cellular role of Afadin in sorting retinal neuron types into proper cellular layers as the structural basis for orderly visual processing.

neuroscience↗

bioRxiv2024

Lymphatic activation of ACKR3 signaling regulates lymphatic response after ischemic heart injury.

2.BackgroundIschemic heart disease is a prevalent cause of death and disability worldwide. Recent studies reported a rapid expansion of the cardiac lymphatic network upon ischemic heart injury and proposed that cardiac lymphatics may attenuate tissue edema and inflammatory mechanisms after ischemic heart injury. Nevertheless, the mechanisms through which hypoxic conditions affect cardiac lymphangiogenesis and function remain unclear. Here, we aimed to characterize the role of the adrenomedullin decoy receptor atypical chemokine receptor 3 (ACKR3) in the lymphatic response following ischemic heart injury. MethodsSpatial assessment of ACKR3 signaling in the heart after ischemic heart injury was conducted using ACKR3-TangoGFP reporter mice. Roles of ACKR3 after ischemic heart injury were characterized in Ackr3{Delta}Lyve1 mice and in cultured human lymphatic endothelial cells (LECs) exposed to hypoxia. ResultsUsing the novel ACKR3-Tango-GFP reporter mice, we detected activation of ACKR3 signaling in cardiac lymphatics adjacent to the site of ischemic injury of left anterior descending artery (LAD) ligation. Ackr3{Delta}Lyve1 mice exhibited better survival and were protected from the formation of acute tissue edema after ischemic cardiac injury. Ackr3{Delta}Lyve1 mice exhibited a denser cardiac lymphatic network after LAD ligation, especially in the injured tissues. Transcriptomic analysis revealed changes in cardiac lymphatic gene expression patterns that have been associated with extracellular matrix remodeling and immune activation. We also found that ACKR3 plays a critical role in the regulating continuous cell-cell junction dynamics in LECs under hypoxic conditions. ConclusionsLymphatic expression of ACKR3 governs numerous processes following ischemic heart injury, including the lymphangiogenic response, edema protection and overall survival. These results expand our understanding of how the heart failure biomarker adrenomedullin, regulated by lymphatic ACKR3, may exert its cardioprotective roles after ischemic cardiac injury.

physiology↗

bioRxiv2024

Alcohol Withdrawal Alters the Inhibitory Landscape of the Prelimbic Cortex in an Interneuron- and Sex-specific Manner

Alcohol use disorder (AUD) is highly prevalent and associated with substantial morbidity and high mortality among substance use disorders. While there are currently three FDA-approved medications for treating AUDs, none specifically target the withdrawal/negative affect stage of AUD, underscoring the need to understand the underlying neurobiology during this critical stage of the addiction cycle. One key region involved in alcohol withdrawal and negative affect is the prelimbic cortex, a subregion of the medial prefrontal cortex. While previous studies have examined alcohol-related adaptations in prefrontal cortical principal glutamatergic neurons, here we used male and female PV:Ai14, SOM:Ai14 and VIP:Ai14 mice to examine synaptic adaptations in all three major classes of prelimbic cortex interneurons following 72 hour withdrawal from a continuous access to two bottle choice model of EtOH drinking in male and female mice. We found that alcohol withdrawal increased excitability of prelimbic PV interneurons in males, but decreased excitability in prelimbic VIP interneurons in females. Additionally, alcohol withdrawal reduced GABA release onto PV interneurons in males while increasing glutamate release onto VIP interneurons in females. In SOM interneurons, alcohol withdrawal had no effect on excitability, but decreased glutamate release onto SOM interneurons in males. Together, our studies identified sex-specific alcohol withdrawal-induced synaptic plasticity in three different types of interneurons and could provide insight into the cellular substrates of negative affective states associated with alcohol withdrawal.

neuroscience↗

bioRxiv2024

Small molecule modulator of neuronal lysosome positioning and function resolves Alzheimers Disease-linked pathologies in cultured human neurons

Abnormal increase in axonal lysosome abundance is associated with multiple neurodegenerative diseases including Alzheimers disease. However, the underlying mechanisms and disease relevance are not fully understood. We have recently identified RH1115 as a small molecule modulator of the autophagy-lysosomal pathway that regulates lysosome positioning in neurons. This allowed us to manipulate neuronal lysosome distribution including in axons and interrogate its contribution to both optimal neuronal functioning and to disease pathology. We demonstrate that the small molecule not only rescues aberrant buildup of both axonal autophagic and lysosomal intermediates but also reduces secreted A{beta}42 levels in human iPSC-derived neurons lacking the lysosomal adaptor, JIP3. We thus demonstrate that restoring efficient axonal lysosome transport has an anti-amyloidogenic effect in human neurons and is a promising therapeutic strategy for Alzheimers disease. Furthermore, we show that the small molecule enhances neuronal lysosome degradation, requires the lysosomal adaptor JIP4 to rescue the axonal lysosome pathology in the JIP3 KO neurons and increases levels of the JIP4-interacting lysosomal membrane protein, TMEM55B. Lastly, treatment with the small molecule led to a striking rescue of locomotor defects in JIP3 KO zebrafish larvae. Thus, we have identified a small molecule which can be impactful in neurodegenerative diseases that have a lysosomal pathology and have determined its molecular targets in modulating axonal lysosome abundance.

cell biology↗

bioRxiv2024

The STRIPAK complex is required for radial sorting and laminin receptor expression in Schwann cells

During peripheral nervous system development, Schwann cells undergo Rac1-dependent cytoskeletal reorganization as they insert cytoplasmic extensions into axon bundles to radially sort, ensheath, and myelinate individual axons. However, our understanding of the direct effectors targeted by Rac1 is limited. Here, we demonstrate that striatin-3 and MOB4 are novel Rac1 interactors. We show that, similar to Rac1-null Schwann cells, Schwann cell specific ablation of striatin-3 causes defects in lamellipodia formation. In addition, conditional Schwann cell knockout of multiple striatin proteins presents a severe delay in radial sorting. Finally, we demonstrate here that deletion of Rac1 or striatin-1/3 in Schwann cells causes defects in Hippo pathway regulation, phosphorylation of the Hippo pathway effectors YAP and TAZ, and expression of genes co-regulated by YAP and TAZ, such as extracellular matrix receptors. In summary, our results indicate that striatin-3 is a novel Rac1 interactor, show that striatin proteins are required for peripheral nervous system development, and reveal a role for Rac1 in regulation of the Hippo pathway in Schwann cells.

neuroscience↗

bioRxiv2024

Spectral estimation at the edge

Cognitive functions depend on neuronal communication, which is subserved by the synchronization of neuronal rhythms. Rhythms are characterized by their frequency, power and phase. If the phase of a rhythm just preceding an input is predictive of the neuronal or behavioral response to the input, this provides strong evidence for a functional role of the rhythm. Yet, this requires estimating the phase of a rhythm at the edge of the epoch. This is challenging, because any phase estimation that is spectrally specific requires a finite window length often combined with tapers that de-emphasize the signal close to the edge. To overcome this, we propose a method that builds on previously described approaches based on autoregressive modeling of the data and corresponding extrapolation beyond the edge. In contrast to related previous approaches, the modeling is based on the broadband signals, avoiding filtering-related group delays, and the extrapolation is performed multiple times, allowing averaging and thereby the reduction of extrapolation noise. The new method provided more accurate phase estimation at the edge for most simulated datasets, and for an empirical dataset from awake macaque area V4. We propose that the enhanced phase estimation accuracy at the edge might help to investigate the functional roles of brain rhythms and potentially also to improve phase-specific stimulation for clinical applications.

neuroscience↗

bioRxiv2024

Mis-regulation of GSK-3β causes axonal microtubule curling through Shot and Tau

Glycogen Synthase Kinase 3{beta} (GSK-3{beta}) is a key coordinator of neuronal development and maintenance; hyperactive GSK-3{beta} is linked to neurodevelopmental and -degenerative diseases and therefore a promising therapeutic target. In neurons, GSK-3{beta} coordinates the cytoskeleton by phosphorylating microtubule-binding proteins. In this study, we found that tight regulation of GSK-3{beta} kinase activity is required for the maintenance of parallel microtubule bundles in Drosophila and rat axons. Up- or down-regulation of GSK-3{beta} led to axons forming pathological swellings in which microtubule bundles disintegrated into disorganised, curled microtubules. We identified the microtubule bundling proteins Shot and Tau as key GSK-3{beta} targets and found that GSK-3{beta} exerted its regulatory effect on microtubule bundling through them. GSK-3{beta} regulates the ability of Shot and Tau to attach to microtubules and/or the plus-end protein Eb1. Mis-regulation of GSK-3{beta} leads to the loss of Eb1-Shot-mediated guidance of polymerising microtubules into parallel bundles, thus causing disorganisation. We propose microtubule disorganisation as a new explanation for how GSK-3{beta} hyperactivity leads to neurodegeneration and why global inhibition of GSK-3{beta} has not been successful in clinical trials for neuronal disorders.

cell biology↗

bioRxiv2024

Dynamic allelic expression in mouse mammary gland across the adult developmental cycle

The mammary gland, which primarily develops postnatally, undergoes significant changes during pregnancy and lactation to facilitate milk production. Through the generation and analysis of 480 transcriptomes, we provide the most detailed allelic expression map of the mammary gland, cataloguing cell-type-specific expression from ex-vivo purified cell populations over 10 developmental stages, enabling comparative analysis. The work identifies genes involved in the mammary gland cycle, parental-origin-specific and genetic background-specific expression at cellular and temporal resolution, genes associated with human lactation disorders and breast cancer. Genomic imprinting, a mechanism regulating gene expression based on parental origin, is crucial for controlling gene dosage and stem cell potential throughout development. The analysis identified 25 imprinted genes monoallelically expressed in the mammary gland, with several showing allele-specific expression in distinct cell types. No novel imprinted genes were identified and the absence of biallelically expressed imprinted genes suggests that, unlike in brain, selective absence of imprinting does not regulate gene dosage in the mammary gland. This research highlights transcriptional dynamics within mammary gland cells and identifies novel candidate genes potentially significant in the tissue during gestation and lactation. Overall, this comprehensive atlas represents a valuable resource for future studies on expression and transcriptional dynamics in mammary cells.

developmental biology↗

bioRxiv2024

One step 4x and 12x 3D-ExM: robust super-resolution microscopy in cell biology

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared to traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4x and 12x 3D-ExM). 3D-ExM is a straightforward expansion microscopy method featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12x 3D-ExM achieves a lateral resolution of under 30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.

cell biology↗

bioRxiv2024

Biallelic Loss of Molecular Chaperone Molecule AIP Results in a Novel Severe Multisystem Disease Defined by Defective Proteostasis

Children born with deleterious biallelic variants of the chaperone aryl hydrocarbon receptor interacting protein (AIP) have a novel pediatric metabolic disease presenting a severe, complex clinical phenotype characterized by failure to develop following birth. Analysis of Aip knockout mouse embryonic fibroblasts and patient-derived dermal fibroblasts revealed that AIP was required to support proteostasis; including proteasome activity, induction of autophagy and lysosome function. aip knockout zebrafish, recapitulated the phenotype of the children; dying at an early stage of development when autophagy is required to adapt to periods of starvation. Our results demonstrate that AIP plays a crucial role in initiating autophagy and maintaining proteostasis in vitro and in vivo. One Sentence SummaryHomozygous loss of the chaperone AIP results in a novel pediatric disease exhibiting multiple features of a lysosomal storage disease.

molecular biology↗

bioRxiv2024

Enhanced MC1R-signalling and pH modulation facilitate melanogenesis within late endosomes of BLOC-1-deficient melanocytes

Photoprotective melanins in the skin are synthesised by epidermal melanocytes within specialised lysosome-related organelles called melanosomes. Melanosomes coexist with lysosomes; thus, melanocytes employ specific trafficking machineries to ensure correct cargo delivery to either the endolysosomal system or maturing melanosomes. Mutations in some of the protein complexes required for melanogenic cargo delivery, such as biogenesis of lysosome-related organelles complex 1 (BLOC-1), result in hypopigmentation due to mistrafficking of cargo to endolysosomes. We show that hypopigmented BLOC-1-deficient melanocytes retain melanogenic capacity that can be enhanced by treatment with cAMP elevating agents despite the mislocalisation of melanogenic proteins. The melanin formed in BLOC-1-deficient melanocytes is not generated in melanosomes but rather within late endosomes/lysosomes to which some cargoes mislocalise. Although these organelles generally are acidic, a cohort of late endosomes/lysosomes have a sufficiently neutral pH to facilitate melanogenesis, perhaps due to mislocalised melanosomal transporters and melanogenic enzymes. Modulation of the pH of late endosomes/lysosomes by genetic manipulation or via treatment with lysosomotropic agents significantly enhances the melanin content of BLOC-1-deficient melanocytes. Our data suggest that upregulation of mistargeted cargoes can facilitate reprogramming of a subset of endolysosomes to generate some functions of lysosome-related organelles.

cell biology↗

bioRxiv2024

Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration

This study characterizes a fluorescent Slc17a6-tdTomato neuronal reporter mouse line offering strong labeling in axons throughout the optic nerve, dendrites and soma in 99% of retinal ganglion cells (RGCs). The model facilitates neuronal assessment ex vivo with wholemounts quantified to show neurodegeneration following optic nerve crush or elevated IOP as related to glaucoma, in vitro with robust Ca2+ responses to P2X7 receptor stimulation in neuronal cultures, and in vivo using a confocal scanning laser ophthalmoscope (cSLO). While the tdTomato signal showed strong overlap with RGC markers, BRN3A and RBPMS, there was no cross-labeling of displaced amacrine cells in the ganglion cell layer. Controls indicated no impact of Slc17a6-tdTomato expression on light-dependent neuronal function, as determined with a microelectrode array (MEA), or on structure, as measured with optical coherence tomography (OCT). In summary, this novel neuronal reporter mouse model offers an effective means to increase the efficiency for real-time, specific visualization of retinal ganglion cells. It holds substantial promise for enhancing our understanding of RGC pathology in glaucoma and other diseases of the optic nerve, and could facilitate the screening of targeted therapeutic interventions for neurodegeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/599589v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@13db8dforg.highwire.dtl.DTLVardef@1345f10org.highwire.dtl.DTLVardef@14d34e1org.highwire.dtl.DTLVardef@1cacb87_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. Fluorescent identification of axons, dendrites and soma of neuronal retinal ganglion cells with a genetic marker as a tool for facilitating the study of neurodegeneration. Puttipong Sripinun, Wennan Lu, Sergei Nikonov, Suhani Patel, Sarah Hennessy, Claire H. Mitchell*. This study delves into a new mouse model, featuring a fluorescent Slc17a6-tdTomato neuronal reporter. This model effectively labels axons in the optic nerve, as well as dendrites and soma in 99% of retinal ganglion cells (RGCs). This allows for both in vitro and in vivo assessment of neurodegeneration, offering a practical tool for real-time, precise visualization of RGCs, with potential applications in various fields of neuroscience and neurology. Created by Biorender.com. C_FIG

neuroscience↗

bioRxiv2024

Multi-omic Landscape of Extracellular Vesicles in Human Carotid Atherosclerotic Plaque Reveals Endothelial Communication Networks

Background: Carotid atherosclerosis is orchestrated by cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are cell-derived nanoparticles representing a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. Methods: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into paired plaque and marginal zones (symptomatic n=16, asymptomatic n=13). EV cargos were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions, and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Results: Carotid plaques contained more EVs than adjacent marginal zones, with differential enrichment for EV-miRNAs and EV-proteins in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated an endothelial signature with roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was validated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Conclusion: Our findings indicate that EVs may drive dynamic changes in plaques through EV- vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed angiogenic processes mediated by EVs creates new therapeutic avenues for atherosclerosis.

cell biology↗