Biology subjects

Wong, S.

Publications and source records attributed to Wong, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Moving in pain - A preliminary study evaluating the immediate effects of experimental knee pain on locomotor biomechanics.

Pain changes how we move, but it is often confounded by other factors due to disease or injury. Experimental pain offers an opportunity to isolate the independent affect of pain on movement. We used cutaneous electrical stimulation to induce experimental knee pain during locomotion to study the short-term motor adaptions to pain. While other models of experimental pain have been used in locomotion, they lack the ability to modulate pain in real-time. Twelve healthy adults completed the single data collection session where they experienced six pain intensity conditions (0.5, 1, 2, 3, 4, 5 out of 10) and two pain delivery modes (tonic and phasic). Electrodes were placed over the lateral infrapatellar fat pad and medial tibial condyle to deliver the 10 Hz pure sinusoid via a constant current electrical stimulator. Pain intensity was calibrated prior to each walking bout based on the target intensity and was recorded using an 11-point numerical rating scale. Knee joint angles and moments were recorded over the walking bouts and summarized in waveform and discrete outcomes to be compared with baseline walking. Knee joint angles changed during the swing phase of gait, with higher pain intensities resulting in greater knee flexion angles. Minimal changes in joint moments were observed but there was a consistent pattern of decreasing joint stiffness with increasing pain intensity. Habituation was limited across the 30-90 second walking bouts and the electrical current needed to deliver the target pain intensities showed a positive linear relationship. Experimental knee pain shows subtle biomechanical changes and favourable habituation patterns over short walking bouts. Further exploration of this model is needed in real-world walking conditions and over longer timeframes to quantify motor adaptations.

neuroscience↗

bioRxiv2024

Closed-loop auditory stimulation targeting alpha and theta oscillations during REM sleep induces phase-dependent power and frequency changes

BackgroundAlpha and theta oscillations characterize the waking human electroencephalogram (EEG) and can be modulated by closed-loop auditory stimulation (CLAS). These oscillations also occur during rapid eye movement (REM) sleep, but whether they can be modulated by CLAS is not known. ObjectiveInvestigate whether CLAS can modulate alpha and theta oscillations during REM sleep in a targeted phase-dependent manner. MethodsWe recorded high-density EEG during an extended overnight sleep period in 18 healthy young adults. Auditory stimulation was delivered during both phasic and tonic REM sleep in alternating 6 s ON and 6 s OFF windows. During the ON windows, stimuli were phase-locked to four orthogonal phases of ongoing alpha or theta oscillations detected in a frontal electrode (Fz). ResultsDuring ON windows, the four orthogonal phases of ongoing alpha and theta oscillations were targeted with high accuracy. Alpha and theta CLAS induced phase-dependent changes in power and frequency at the target location. Frequency-specific effects were observed for alpha trough (speeding up) and rising (slowing down) and theta trough (speeding up) conditions. These phase-dependent changes of CLAS were observed during both REM sleep substages, even though the amplitude evoked by auditory stimuli which were not phase-locked was very much reduced in phasic compared to tonic REM sleep. ConclusionsThis study provides evidence that faster REM sleep rhythms can be modulated by CLAS in a phase-dependent manner. This offers a new approach to investigate how modulation of REM sleep oscillations affects the contribution of this vigilance state to brain function. Highlights- REM sleep alpha and theta oscillations can be modulated using phase-locked CLAS - Phase-dependent changes in power and frequency are observed in the target area - Phase-dependent modulation occurs in phasic and tonic REM sleep Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/582907v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@771baborg.highwire.dtl.DTLVardef@1b9b13corg.highwire.dtl.DTLVardef@22e85corg.highwire.dtl.DTLVardef@1c50edc_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Targeted Dab2 Restoration: A Novel Approach for Enhanced Wound Healing in Diabetic Conditions

Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor (VEGF)-dependent angiogenesis remain uncertain and could reveal new therapeutic targets. In our study, the molecular underpinnings of endothelial dysfunction in diabetes were investigated, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FoxM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell (EC) function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high concentrations glucose treated primary mouse skin ECs, simulating hyperglycemic conditions in diabetes mellitus. In diabetic mice with a genetic EC deficiency of Dab2 angiogenesis was reduced in vivo and in vitro when compared with wild-type mice. Restoration of Dab2 expression by injected mRNA-containing lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. At the same time, FoxM1 was downregulated in skin ECs subjected to high glucose conditions as determined by RNA-sequencing analysis. FoxM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FoxM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. These findings indicate that restoring Dab2 expression through targeted therapies can enhance angiogenesis and wound repair in diabetes. To explore this therapeutic potential, we tested LyP-1-conjugated lipid nanoparticles (LNPs) containing Dab2 or control mRNAs to target ECs and found the former significantly improved wound healing and angiogenesis in diabetic mice. This study provides evidence of the crucial roles of Dab2 and FoxM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.

cell biology↗

bioRxiv2024

Epsins promote atherosclerosis through VSMC phenotypic modulation

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA- seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

cell biology↗

bioRxiv2023

Assembly and analysis of the genome of Notholithocarpus densiflorus

Tanoak (Notholithocarpus densiflorus) is an evergreen tree in the Fagaceae family found in California and southern Oregon. Historically, tanoak acorns were an important food source for Native American tribes and the bark was used extensively in the leather tanning process. Long considered a disjunct relictual element of the Asian stone oaks (Lithocarpus spp.), phylogenetic analysis has determined that the tanoak is an example of convergent evolution. Tanoaks are deeply divergent from oaks (Quercus) of the Pacific Northwest and comprise a new genus with a single species. These trees are highly susceptible to sudden oak death (SOD), a plant pathogen (Phytophthora ramorum) that has caused widespread mortality of tanoaks. Here, we set out to assemble the genome and perform comparative studies among a number of individuals that demonstrated varying levels of susceptibility to SOD. First, we sequenced and de novo assembled a draft reference genome of N. densiflorus using co-barcoded library processing methods and an MGI DNBSEQ-G400 sequencer. To increase the contiguity of the final assembly, we also sequenced Oxford Nanopore (ONT) long reads to 30X coverage. To our knowledge, the draft genome reported here is one of the more contiguous and complete genomes of a tree species published until now, with a contig N50 of [~]1.2 Mb and a scaffold N50 of [~]2.1 Mb. In addition, we sequenced 11 genetically distinct individuals and mapped these onto the draft reference genome enabling the discovery of almost 25 million single nucleotide polymorphisms and [~]4.4 million small insertions and deletions. Finally, using co-barcoded data we were able to generate complete haplotype coverage of all 11 genomes.

genomics↗

bioRxiv2023

Single cell sequencing as a general variant interpretation assay

The human genome contains [~]70 million possible protein-altering variants, the vast majority of which are of uncertain clinical significance. Closing this gap is essential for accurate diagnosis of disease-causing variants and understanding their mechanisms of action. Towards this goal, we developed a pooled perturbation approach combining saturation mutagenesis with single cell RNA sequencing to map the effects of every single nucleotide variant in a gene. We sequenced [~]440,000 cells expressing variants in CDKN2A (p16INK4a), TP53, and SOD1, observing almost all possible protein-coding variants, with a mean of 61 cells per variant. Using single cell gene expression signatures, we show that each gene may contain multiple types of pathogenic variants that affect distinct downstream pathways. We demonstrate that single cell expression signatures outperform existing bulk experimental assays and computational models for predicting pathogenicity, and summarize both the utility and potential limitations of single cell sequencing as a general variant interpretation assay.

genomics↗

bioRxiv2023

Targeting endothelial FOXO1 protects diabetic β-cells and improves wound healing

The forkhead box O1 (FOXO1) transcription factor plays critical roles in regulating not only metabolic activity but also angiogenesis in the vascular endothelium1-4. Our previous studies show that epsin endocytic adaptors can regulate both angiogenesis and lymphangiogenesis5-7. Endothelial cells (ECs) lining the inside of blood vessels are continuously exposed to circulating insulin and insulin-like growth factors (IGFs). Emerging evidences suggest that ECs can affect {beta}-cell function8-11. Excessive IGF2, especially elevated local IGF2 levels in islets, may represent a risk factor for developing diabetes12-15; however, the underlying molecular mechanisms by which aberrant angiogenesis and endothelium-derived factors regulate pancreatic {beta}-cell function in diabetes remain unclear. Here, we report that the pancreas of diabetic patients as well as the pancreas, skin, and plasma of streptozotocin/high fat diet (STZ/HFD)-induced diabetic mice and db/db mice contains excess IGF2, which can lead to {beta}-cell dysfunction and apoptosis. Single-cell transcriptomics combined with mass spectrometry analysis reveal that endothelial-specific knockout of FOXO1 increases circulating soluble and cell-membrane or intracellular expression levels of IGF type 2 receptor (IGF2R) and CCCTC-binding factor (CTCF), while decreasing IGF2 levels in diabetes. Both IGFR215-17 and CTCF18-21 can reduce IGF2 levels and may ameliorate {beta}-cell decline associated with excess IGF2 in diabetes. Furthermore, depletion of FOXO1, epsins, or knockdown of ULK1 inhibits autophagy formation in ECs, preventing degradation of vascular endothelial growth factor receptor 2 (VEGFR2) to promote angiogenesis and improve wound healing in diabetes. Our findings reveal that endothelial FOXO1 regulates epsin-dependent angiogenesis and affects {beta}-cell function and fate through CTCF and IGF2-IGF2R, providing a potential strategy for ameliorating diabetes and accelerating cutaneous wound healing.

cell biology↗

bioRxiv2023

Single-cell multi-omics map of human foetal blood in Down's Syndrome

Downs Syndrome (DS) predisposes individuals to haematological abnormalities, such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood. To understand dysregulated hematopoiesis in DS, we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human foetal liver and bone marrow samples from three disomic and 15 trisomic foetuses. We found that differences in gene expression in DS were both cell type- and environment-dependent. Furthermore, we found multiple lines of evidence that DS haematopoietic stem cells (HSCs) are "primed" to differentiate. We subsequently established a DS-specific map of enhancer-gene relationships in disomic and trisomic HSCs using 10X Multiome data. By integrating this map with genetic variants associated with blood cell variation, we discovered that trisomy restructured enhancer-gene maps to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Further, as DS mutations display a signature of oxidative stress, we validated both increased mitochondrial mass and oxidative stress in DS, and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Altogether, our single- cell, multi-omic resource provides a high-resolution molecular map of foetal haematopoiesis in Downs Syndrome and indicates significant enhancer-gene restructuring giving rise to co- occurring haematological conditions.

genomics↗

bioRxiv2023

Kinetic evidence for multiple aggregation pathways in antibody light chain variable domains

Aggregation of antibody light chain proteins is associated with the progressive disease light chain amyloidosis. Patient-derived amyloid fibrils are formed from light chain variable domain residues in non-native conformations, highlighting a requirement that light chains unfold from their native structures in order to aggregate. However, mechanistic studies of amyloid formation have primarily focused on the self-assembly of natively unstructured peptides, and the role of native state unfolding is less well understood. Using a well-studied light chain variable domain protein known as WIL, which readily aggregates in vitro under conditions where the native state predominates, we asked how the protein concentration and addition of pre-formed fibril "seeds" alter the kinetics of aggregation. Monitoring aggregation with thioflavin T fluorescence revealed a distinctly non-linear dependence on concentration, with a maximum aggregation rate observed at 8 {micro}M protein. This behavior is consistent with formation of alternate aggregate structures in the early phases of amyloid formation. Addition of N- or C-terminal peptide tags, which did not greatly affect the folding or stability of the protein, altered the concentration dependence of aggregation. Aggregation rates increased in the presence of pre-formed seeds, but this effect did not eliminate the delay before aggregation and became saturated when the proportion of seeds added was greater than 1 in 1600. The complexity of aggregation observed in vitro highlights how multiple species may contribute to amyloid pathology in patients.

biochemistry↗

bioRxiv2023

A REM-active basal ganglia circuit that regulates anxiety

REM sleep has been hypothesized to promote emotional resilience, but any neuronal circuits mediating this have not been identified. We find that in mice, somatostatin (Som) neurons in the entopeduncular nucleus (EPSom)/internal globus pallidus are predominantly active selectively during REM sleep. This unique REM activity is necessary and sufficient for maintaining normal REM sleep. Inhibiting or exciting EPSom neurons reduced or increased REM sleep duration, respectively. Activation of the sole downstream target of EPSom neurons, Vglut2 cells in the lateral habenula (LHb), increased sleep via the ventral tegmental area (VTA). A simple chemogenetic scheme to periodically inhibit the LHb over 4 days selectively removed a significant amount of cumulative REM sleep. Chronic REM reduction correlated with mice becoming anxious and more sensitive to aversive stimuli. Therefore, we suggest that REM sleep, in part generated by the EP[->]LHb[->]VTA circuit identified here, could contribute to stabilizing reactions to habitual aversive stimuli.

neuroscience↗

bioRxiv2023

Cellular traits regulate fluorescence-based bio-optical phenotypes of coral photosymbionts living in-hospite

AbstractDiversity across algal family Symbiodiniaceae contributes to the environmental resilience of certain coral species. Chlorophyll-a fluorescence measurements are frequently used to determine symbiont health and resilience, but more work is needed to refine these tools and establish how they relate to underlying cellular traits. We examined trait diversity in symbionts from the generas Cladocopium and Durusdinium, collected from 12 aquacultured coral species. Photophysiological metrics ({Phi}PSII, {sigma}PSII, {rho}, {tau}1, {tau}2, ABQ, NPQ, and qP) were assessed using a prototype multi-spectral fluorometer over a variable light protocol which yielded a total of 1360 individual metrics. Photophysiological metrics were then used to establish four unique phenotypic variants. Corals harboring C15 were predominantly found within a single phenotype which clustered separately from all other coral fragments. The majority of Durusdinium dominated colonies also formed a separate phenotype which it shared with a few C1 dominated corals. C15 and D1 symbionts appear to differ in which mechanisms they employ to dissipate excess light energy. Spectrally dependent variability is also observed across phenotypes that may relate to differences in photopigment utilization. Cell physiology (atomic C:N:P, cell size, chlorophyll-a, neutral lipid content) was also assessed within each sample and differ across phenotypes, linking photophysiological metrics with underlying primary cellular traits. Strong correlations between first- and second-order traits, such as Quantum Yield and cellular N:P content, or light dissipation pathways (qP and NPQ) and C:P underline differences across symbiont types and may also provide a means for using fluorescence-based metrics as biomarkers for certain primary-cellular traits.

physiology↗

bioRxiv2023

Clustering Protein Binding Pockets and Identifying Potential Drug Interactions: A Novel Ligand-based Featurization Method

Protein-ligand interactions are essential to drug discovery and drug development efforts. Desirable on-target or multi-target interactions are a first step in finding an effective therapeutic; undesirable off-target interactions are a first step in assessing safety. In this work, we introduce a novel ligand-based featurization and mapping of human protein pockets to identify closely related protein targets, and to project novel drugs into a hybrid protein-ligand feature space to identify their likely protein interactions. Using structure-based template matches from PDB, protein pockets are featurized by the ligands which bind to their best co-complex template matches. The simplicity and interpretability of this approach provides a granular characterization of the human proteome at the protein pocket level instead of the traditional protein-level characterization by family, function, or pathway. We demonstrate the power of this featurization method by clustering a subset of the human proteome and evaluating the predicted cluster associations of over 7,000 compounds.

synthetic biology↗

bioRxiv2023

Human airway ex vivo models: new tools to study the airway epithelial cell response to SARS-CoV-2 infection

Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models) infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using publicly available and our own bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes (IFI44, IFIT1, IFIT3, IFI35, IRF9, MX1, OAS1, OAS3 and ISG15) and inflammatory genes (NFKBIA, CSF1, FOSL1, IL32 and CXCL10) at day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes (ITGB6, ITGB1 and GJA1) also were altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins also was deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to understand the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.

cell biology↗

bioRxiv2023

Differentiating shame- and guilt-proneness with heart rate variability in Chinese young adults

A high proneness to experience shame and guilt has been associated with psychopathology. Despite their similarity, shame- and guilt-proneness have different psychological and neurobiological correlates. The present study aims to compare the physiological correlates between shame- and guilt-proneness. Resting heart rate variability (HRV), a peripheral biomarker of emotion dysregulation and psychopathology, was measured in a sample of 60 Chinese young adults with two sessions of electrocardiogram recording. Proneness to shame and guilt were measured by the Test of Self-Conscious Affect 3. Hierarchical linear modeling indicated that guilt-proneness was positively associated with HRV while shame-proneness was not. Our findings implied that shame- and guilt-proneness have different relations with HRV. The distinct physiological relations are discussed with respect to the adaptive/maladaptive nature of shame- and guilt-proneness.

neuroscience↗

bioRxiv2022

Magnetic resonance imaging datasets with anatomical fiducials for quality control and registration

Tools available for reproducible, quantitative assessment of brain correspondence have been limited. We previously validated the anatomical fiducial (AFID) placement protocol for point-based assessment of image registration with millimetric (mm) accuracy. In this data descriptor, we release curated AFID placements for some of the most commonly used structural magnetic resonance imaging templates and datasets. The release of our accurate placements allows for rapid quality control of image registration, teaching neuroanatomy, and clinical applications such as disease diagnosis and surgical targeting. We release placements on individual subjects from four datasets (n = 132 subjects for a total of 15,232 fiducials) and more than 10 brain templates (4,288 fiducials), compiling over 300 human rater hours of annotation. We also validate human rater accuracy of released placements to be within 1-2 mm (using a total of 50,336 Euclidean distances), consistent with prior studies. Our data is compliant with the Brain Imaging Data Structure (BIDS) allowing for facile incorporation into modern neuroimaging analysis pipelines. Data is accessible on GitHub (https://github.com/afids/afids-data).

neuroscience↗

bioRxiv2022

A phenomic modeling approach for using chlorophyll-a fluorescence-based measurements on coral photosymbionts: a step towards bio-optical bleaching prediction

We test a newly developed instrument prototype which utilizes time-resolved chlorophyll-a fluorescence techniques and fluctuating light to characterize Symbiodiniaceae functional traits across seven different coral species under cultivation as part of ongoing restoration efforts in the Florida Keys. While traditional chlorophyll-a fluorescence techniques only provide a handful of algal biometrics, the system and protocol we have developed generates > 1000 dynamic measurements in a short ([~]11 min) time frame. Resulting high-content algal biometric data revealed distinct phenotypes, which broadly corresponded to clade-level Symbiodiniaceae designations determined using quantitative PCR. Next, algal biometric data from Acropora cervicornis (10 genotypes) and A. palmata (5 genotypes) coral fragments was correlated with bleaching response metrics collected after a two month-long exposure to high temperature. A network analysis identified 1973 correlations (Spearman R > 0.5) between algal biometrics and various bleaching response metrics. These identified biomarkers of thermal stress were then utilized to train a predictive model, and when tested against the same A. cervicornis and A. palmata coral fragments, yielded high correlation (R = 0.92) with measured thermal response (reductions in absorbance by chlorophyll-a). When applied to all seven coral species, the model ranked fragments dominated by Cladocopium or Breviolum symbionts as more bleaching susceptible than corals harboring thermally tolerant symbionts (Durusdinium). While direct testing of bleaching predictions on novel genotypes is still needed, our device and modeling pipeline may help broaden the scalability of existing approaches for determining thermal tolerance in reef corals. Our instrument prototype and analytical pipeline aligns with recent coral restoration assessments that call for the development of novel tools for improving scalability of coral restoration programs.

ecology↗

bioRxiv2022

Targeting Epsins to Inhibit FGF Signaling while Potentiating TGF-β Signaling Constrains Endothelial-to-Mesenchymal-Transition in Atherosclerosis

BACKGROUNDEpsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease. METHODSUsing single cell RNA sequencing (scRNA-seq), combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knock-out mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTSScRNA-seq and lineage tracing revealed that epsins 1 and 2 promote EndoMT, and the loss of endothelial epsins inhibits EndoMT marker expression as well as transforming growth factor-beta signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 (FGFR1) expression that inhibits TGF-{beta} signaling and EndoMT. Epsins directly bind ubiquitinated FGFR1 through their ubiquitin-interacting motif (UIM), which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic UIM-containing peptide API significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONSWe conclude that epsins potentiate EndoMT during atherogenesis by increasing TGF-{beta} signaling through FGFR1 internalization and degradation. Inhibition of EndoMT by reducing epsin-FGFR1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

cell biology↗

bioRxiv2022

Targeting Epsins by nanotherapy regulates lipid metabolism and promotes 1 ABCG1-mediated cholesterol efflux to fortify atheroma regression

BACKGROUNDExcess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically-target macrophage Epsins with specially-designed S2P-conjugated lipid nanoparticles (NPs), which encapsulate small interfering RNAs to suppress Epsins. METHODSWe used single cell RNA sequencing (scRNA-seq) with our newly developed algorithm MEBOCOST to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (LysM-DKO-ABCG1fl/+). The NPs targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. RESULTSWe revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific NP delivery of Epsin siRNAs, we showed that silencing of macrophage Epsins markedly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1. CONCLUSIONSOur findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux. Novelty and SignificanceO_ST_ABSWHAT IS KNOWN?C_ST_ABSO_LIEpsin endocytic adaptor proteins are upregulated in human and mouse atherosclerotic lesions C_LIO_LILesional macrophages internalize lipids primarily through scavenger receptor-mediated endocytosis such as CD36 and SR-A C_LIO_LIMacrophage-mediated cholesterol efflux and reverse cholesterol transport is crucial to atheroma resolution C_LI WHAT NEW INFORMATION DOES THIS ARTICLE CONTRIBUTE?O_LIScRNA-seq combined with the newly-developed algorithm MEBOCOST reveals that Epsins are involved in macrophage-mediated lipid metabolic regulation C_LIO_LIMacrophage epsins promote lipid uptake by targeting CD36 endocytosis and membrane recycling via the Epsin ENTH domain C_LIO_LIEpsins bind ubiquitinated ABCG1--resulting in the endocytosis and lysosomal degradation of this cholesterol transporter, which reduces cholesterol efflux. C_LIO_LIMacrophage-specific, nanoparticle-mediated RNAi delivery exhibits a therapeutic benefit for the treatment of atherosclerosis C_LIO_LIAtherosclerotic plaque regression using this nanoparticle delivery platform represents a clinically-relevant approach for the treatment of advance atherosclerosis C_LI BRIEF SUMMARYDespite the development of new cholesterol-lowering therapies, including the recently approved PCSK9 small interfering RNA (siRNA) antagonists, patients still face a tremendous risk of developing major acute cardiovascular events resulting from chronic inflammation in the plaque. We employed a novel nanomedicine platform containing a stabilin-2 targeting peptide (S2P) to deliver Epsin-specific siRNAs to lesional macrophages. We discovered that inhibition of these adaptor proteins in lesional macrophages significantly diminished plaque size and necrotic core area, increased fibrous cap thickness, and promoted plaque regression.

cell biology↗