Biology subjects

Li, A.

Publications and source records attributed to Li, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Glucokinase links metabolism and lineage plasticity in neuroendocrine prostate cancer via interaction with AKT1

Cancer cells frequently rewire their metabolism to sustain growth and survival under stress. Despite the critical role of metabolic adaptation in tumorigenesis, how specific metabolic enzymes regulate lineage plasticity remains unclear. Here, through FDG-PET imaging and transcriptomic analyses, we reveal markedly elevated glucose uptake in neuroendocrine prostate cancer (NEPC) and identify glucokinase (GCK) as a MYCN-induced metabolic enzyme. Beyond its metabolic role, GCK is indispensable for maintaining the neuroendocrine lineage of prostate cancer cells by establishing a functional circuit with AKT1 through reciprocal regulation--AKT1 binds and phosphorylates GCK at S373, whereas GCK phosphorylates AKT1 at S473. Pharmacological disruption of this AKT1-GCK axis suppresses tumor growth in NEPC mouse models and patient-derived xenografts. Altogether, our findings uncover both the metabolic and noncanonical kinase functions of GCK and establish the AKT1-GCK axis as a key link between metabolic reprogramming and neuroendocrine lineage transition in prostate cancer.

cancer biology↗

bioRxiv2025

Evolutionarily Conserved T-tubule-Mitochondria Contacts in Striated Muscle

The intricate architecture of skeletal muscle is essential for its contractile function, with the spatial organization of organelles such as T-tubules and mitochondria playing a crucial role in maintaining muscle performance and health1,2. T-tubules propagate electrical signals deep into muscle fibers to ensure synchronous contraction, while mitochondria provide the necessary energy for muscle contraction3-5. Previous studies have extensively documented the interactions between T-tubules and the sarcoplasmic reticulum (SR), which are essential for excitation-contraction coupling6,7. However, the detailed spatial relationship between T-tubules and mitochondria has remained relatively unexplored. In this study, we utilized zebrafish and mice as model organisms to investigate the contacts between T-tubules and mitochondria. Using array tomography, deep learning-based analysis, and advanced microscopy techniques, we discovered extensive membrane contact sites, termed T-tubule-Mitochondria (TTM) contacts. These contacts are evolutionarily conserved across vertebrates, including skeletal and cardiac muscles. Our findings reveal a previously unrecognized, intimate spatial coupling between T-tubules and mitochondria, with significant implications for muscle physiology and pathology. Understanding these TTM contacts could provide new insights into the mechanisms underlying muscle function and the development of muscle-related diseases.

cell biology↗

bioRxiv2025

Primate-specific adaptation of Ku protects transcriptomic integrity by suppressing Alu-mediated alternative splicing

Accurate pre-mRNA splicing is essential for the transfer of genetic information but faces unique challenge in higher primates due to the massive expansion of intronic Alu elements1-3. While studying Ku, the Ku70/Ku80 heterodimer best known for initiating non-homologous end-joining (NHEJ) by encircling DNA ends4, we discovered that Ku expression increased markedly during primate evolution in parallel with Alu expansion5. Ku binds double-stranded RNA (dsRNA) stem-loops, including those at the antisense Alu (asAlu) elements within introns5. Here, we show that Ku-depletion in human cells has a broad impact on splicing largely independent of cell-cycle states, NHEJ, or innate immune signaling, significantly affecting ~8-10% of quantifiable alternative splicing events. Mechanistically, Ku directly binds exonic asAlu to prevent their aberrant inclusion and binds asAlu within inverted-repeat Alu (irAlu) pairs flanking canonical exons to prevent exon skipping6. Among human tissues, Ku expression in the brain is consistently ~50% lower, correlating with more permissive expression of Alu-derived splice variants, particularly those encoding mitochondrial proteins and RNA-binding factors. Correspondingly, heterozygous Ku loss in patient causes developmental delay, neurological dysfunction, and acidosis. Together our findings identified Ku as a critical suppressor of Alu-associated alternative splicing co-opted during evolution with implications for primate brain function and human disease.

molecular biology↗

bioRxiv2025

Synaptopodin KO rat for assessing the dendritic spine apparatus and axonal cisternal organelle in synaptic plasticity, development, and behavior

The actin-binding protein synaptopodin (Synpo) regulates the cytoskeleton and organization of endoplasmic reticulum, thereby amplifying intracellular Ca2+ signaling. Knockout (KO) mouse models have been used to study the role of Synpo in kidney and brain functions, where it supports stress fiber formation, as well as long-term potentiation (LTP) and learning, respectively. Here, we generated Synpo KO rats using CRISPR-Cas9, and show they are viable but have reduced body weight after postnatal days 35-45, along with shorter limb bone length. Their basal kidney function is normal into early adulthood. Serial section electron microscopy from Synpo KO rat hippocampus reveals the absence of the spine apparatus in dendrites and cisternal organelle in the axon initial segment (AIS), two Synpo-dependent specializations of smooth endoplasmic reticulum. The AIS in KO was still innervated by inhibitory synapses despite the total loss of the cisternal organelle. Synpo KO rats also showed reduced LTP. Previously unknown KO effect of Synpo on body stature could have an inadvertent impact on behavioral outcomes. Furthermore, rats have a well-defined developmental onset of LTP, compared to the variable onset of LTP in mice. This, combined with known species differences in behavior, makes our KO rat model a valuable resource for assessing the role of Synpo in development, learning, synaptic plasticity, and a wide range of other biological functions.

neuroscience↗

bioRxiv2025

Repurposed endogenous virus-like vesicles mediate dendritic cell long-range antigen presentation and T cell activation for enhanced cancer vaccination

Dendritic cells (DCs) release extracellular vesicles (DEVs) that amplify antigen presentation while incorporating patient-derived, rapidly evolving antigens. By enriching peptide-MHC and co-stimulatory ligands orders-of-magnitude above donor-cell levels, DEVs emerge as potent vesicle-vaccines, although efficacy remains limited by unclear mechanisms. We show that DCs repurpose viral components to generate endogenous virus-like vesicles (VLVs) that preferentially carry peptide-MHC and high-density co-stimulatory ligands, intensifying and extending antigen presentation. Upon antigen exposure, Arc partners with endogenous envelope proteins to assemble VLVs that directly engage T cells and trigger intrinsic adjuvanticity via viral mimicry. Arc-/- DEVs failed to prime antigen-specific T cell responses, whereas Arc overexpression with its 5'-UTR stem-loop shifted DEVs toward VLVs that trafficked to lymphoid organs, drove rapid CD4-assisted priming and durable CD8-biased memory, suppressed melanoma, and prolonged survival. These reveal a viral-mimicry mechanism enabling long-range immune activation and support Arc+ VLVs as an antigen-agnostic vaccine for cancer immunotherapy. HighlightsO_LIArc+ VLVs intensify and extend DC antigen presentation in vivo C_LIO_LIArc+ VLVs directly engage T cells and trigger viral-mimic adjuvanticity C_LIO_LIArc-/- DEVs fail to prime antigen-specific T cell responses C_LIO_LIEngineered Arc+ VLVs drive durable CD8-biased memory and tumor control C_LIO_LIArc+ VLV vaccination provides long-range and cross-tumor protection in vivo C_LI

bioengineering↗

bioRxiv2025

Dissociating the neural codes for multiple pitch perception in humans

Pitch is crucial for speech and music perception, yet its neural code remains contested. The classic rate-place theory suggests that pitch is computed from spatial patterns of average neural firing rates along the cochlear tonotopic axis. These cues are thought to be robust for isolated harmonic complex tones (HCTs) but degraded for spectrally dense mixtures of simultaneous HCTs (e.g., musical chords) due to auditory filtering. In many cases, pitch perception remains feasible for HCT mixtures, but it is unclear whether listeners utilize residual rate-place cues or alternative neural codes. To adjudicate between these possibilities, we generated rate-place metamers (META), which are synthetic stimuli with simulated auditory-nerve average-rate responses that are nearly identical to those of original pitch-evoking stimuli (ORIG). Using these stimuli, listeners completed four psychophysical experiments that spanned a wide range of acoustic and cognitive complexity, from single-HCT pitch discrimination to harmonic-expectation judgments. Combining the behavioral data with predictive models of listener performance, we found that listener performance on single pitch discrimination tasks could be adequately explained by a rate-place model, with or without simultaneous pitch maskers (Experiments 1 and 2). More complex tasks involving attention to all pitches in a three-pitch mixture (Experiments 3 and 4) showed a pattern of results more difficult to account for with a rate-place model, perhaps suggesting a role for integration of temporal cues. Significance StatementEveryday listening typically involves parsing or integrating several simultaneous pitches, for example, while listening to music or conversations in noisy environments, rather than hearing a single, isolated pitch. Despite their ubiquity, the neural mechanisms for encoding multiple pitches are not well understood, which partly explains the limitations of modern assistive listening devices in noisy settings. To address this, we developed model-based synthetic stimuli to measure how distinct pitch cues contribute to pitch perception. This approach advances our understanding of the neural basis of music perception, with a goal of informing the design of more effective hearing prostheses.

neuroscience↗

bioRxiv2025

"Awe-scillations": EEG spectral and complexity representations of awe

Awe is a positive emotion often accompanied by sensations of vastness and unity, with known benefits for well-being and social behavior. However, its neural underpinnings remain poorly understood. We recorded electroencephalography (EEG) and autonomic physiology in 23 healthy older participants while they watched a nature-based audiovisual film and subjectively rated awe events. Awe was the predominant emotion reported - though other positive emotions (e.g., joy) were also highly rated. Awe events were associated with decreased skin conductance level (SCL), and decreased EEG alpha and theta spectral power - physiological changes associated with low arousal and positively valenced emotional states. Interestingly, awe events exhibited increased Lempel Ziv Complexity (LZC) - indicating heightened neural signal entropy and increased richness of the conscious experience. LZC was also positively associated with the intensity of the awe ratings and negatively associated with SCL. Three additional datasets with separate independent induction methods (video clips and pharmacological induction via N,N-dimethyltryptamine) also showed positive occipital LZC associations with awe - suggesting some generalizability of LZC as a neurophysiological marker of awe. These results suggest that awe evokes neurophysiological states linked to the subjective affective experience. Impact StatementAwe, an emotion of increasing interest, has been studied with fMRI and peripheral physiology - but few studies have used electroencephalography (EEG). In this EEG study, we utilize a movie-watching paradigm to explore potential physiological correlates of awe. The results present EEG-based complexity increases as a potential correlate of awe, though showing limited generalization to independent datasets and limited uniqueness compared to joy, another positive emotion.

neuroscience↗

bioRxiv2025

Cascade-Targeting Nanoparticles for Reversing Chemoresistance in Osteosarcoma

Osteosarcoma (OS) remains the most prevalent primary malignant bone tumor in adolescents, with chemotherapy resistance significantly limiting the efficacy of current treatments. To address the challenges of chemoresistance driven by DNA repair mechanisms and drug efflux, we developed a biomimetic cascade-targeting drug delivery system, TAT-mPDO@cRGD-M. This system combines a polydopamine (mPDA) core with strong photothermal and drug-loading capacities, enabling the co-delivery of cisplatin (CDDP) and olaparib (OLA). Surface functionalization with the nuclear localization peptide TAT and cloaking with osteosarcoma cell membranes modified with cRGD peptides allows for selective tumor recognition, nuclear targeting, and stimuli-responsive drug release. Upon near-infrared (NIR) irradiation, the platform enhances DNA damage, suppresses the PI3K-AKT signaling pathway, and promotes apoptosis while inhibiting epithelial-mesenchymal transition (EMT). In vitro and in vivo studies demonstrated that TAT-mPDO@cRGD-M exhibits potent antitumor activity against cisplatin-resistant osteosarcoma, maintaining strong therapeutic efficacy under low-power NIR irradiation without causing significant damage to normal tissues. This study highlights a highly specific and biocompatible therapeutic approach that offers promising potential for overcoming chemotherapy resistance in osteosarcoma and advancing clinical treatment strategies.

cancer biology↗

bioRxiv2025

CRISPR-engineered deletion of POGZ alters transcription factor binding at promoters of genes involved in synaptic signaling

One of the seminal discoveries from genetic studies of autism spectrum disorder and related neurodevelopmental disorders (NDD) has been that loss-of-function (LoF) mutations in many genes that impact chromatin and transcriptional regulation confer substantial liability to NDD. Haploinsufficiency of the epigenetic regulator POGZ represents one of the strongest such associations; however, little is known about the direct or indirect regulatory targets of POGZ, or the mechanisms by which loss of this chromatin modifier alters early neuronal development and synaptic functions. Here, we created an allelic series of CRISPR-engineered human induced pluripotent stem cell (hiPSC) clones harboring mono- and biallelic POGZ deletions. In hiPSC-derived neural stem cells (NSC) and Neurogenin 2-induced neurons (iN), POGZ LoF altered the expression of genes associated with critical cellular processes and neuronal functions, including synaptic and intracellular signaling and extracellular matrix organization. Our multiomics profiling also showed altered footprinting of critical transcription factors (e.g., activator protein 1 complexes) that were enriched at promoters of differentially expressed genes associated with synaptic function. To further interrogate the shared molecular changes in neuronal development associated with NDD and POGZ regulation, we compared our results to deletions of the transcription factor MEF2C and the sodium channel gene SCN2A that we generated in these same isogenic iN. These analyses revealed strong enrichment of extracellular matrix and intracellular signaling disruption associated with POGZ and MEF2C deletion, whereas POGZ and SCN2A haploinsufficiency exhibited shared transcriptional effects on gene modules enriched for NDD-associated genes with opposing regulatory effects. Notably, we also observed alterations to synaptic firing rate and neurite extension with biallelic deletions, but not heterozygous lines, suggesting subtle effects in neuronal development associated with haploinsufficiency. Overall, these shared molecular consequences suggest key points of convergence that connect epigenetic regulation to neuronal function in the etiology of neurodevelopmental pathologies.

genomics↗

bioRxiv2025

Igniting full-length isoform analysis in single-cell and spatial RNA-seq data with FLAMESv2

Long-read single-cell RNA-sequencing enables the profiling of RNA isoform expression and alternative splicing at single cell resolution. However, diverse single-cell technologies and sparse isoform data demand flexible and accurate analysis tools. We introduce FLAMESv2, a highly modular and protocol-agnostic R/Bioconductor package for long-read single-cell RNA-seq data analysis. FLAMESv2 supports a wide range of single-cell and spatial protocols, is highly configurable, scales to allow multi-sample analysis and provides versatile visualisation and analysis outputs. We demonstrate its compatibility with both droplet-based and combinatorial barcoding single-cell methods, as well as spatial transcriptomics workflows. Benchmarking confirms FLAMESv2 achieves field-leading performance across key analysis tasks. Applying FLAMESv2 to in vitro differentiation of stem cells into neurons, we identify cell-types, differentiation trajectories, expression of annotated and novel isoforms and isoform expression diversity and heterogeneity within individual cells. FLAMESv2 provides a comprehensive, flexible approach to analysing long-read single-cell RNA-sequencing, unlocking this powerful methodology for RNA isoform characterisation.

bioinformatics↗

bioRxiv2025

A simple, open-source restraint system for magnetic resonance imaging in awake rats

Magnetic resonance imaging (MRI) is a critical tool for translational neuroscience, offering cross-species insights into brain structure and function; however, its application in preclinical research is constrained by routine anesthesia use or sedation, which alters neural activity and limits comparisons to awake human imaging. Awake rodent functional MRI (fMRI) provides a powerful platform for investigating brain function under physiologically relevant conditions, but implementation is limited by technical challenges, particularly head motion and stress during scanning. Most restraint systems employ initial anesthesia, compromising translatability of findings, and highlighting the need for improved designs. We developed a novel restraint system optimized for awake rat fMRI. The system consists of modular 3D-printed components and can be assembled in under five minutes. It is accompanied by a protocol that includes head-post implantation followed by an 11-day habituation period post-surgical recovery. The system eliminates the need for isoflurane anesthesia, ear bars, and bite bars, reducing stress and improving animal comfort. It supports integration with behavioral paradigms such as pupil tracking and licking responses. High-resolution T2-weighted anatomical images and functional scans obtained using the system showed excellent spatial clarity and minimal motion artifacts. Quality control metrics, including head motion parameters and temporal signal-to-noise ratio, confirmed the systems stability and suitability for awake imaging. Functional connectivity analysis revealed robust positive correlations between functionally relevant regions. This system offers a scalable, reproducible, and animal-friendly solution for awake rat fMRI. While the current design limits direct cranial access for multimodal recordings, it enables high-quality, behaviorally enriched imaging without anesthesia. Significance Statement: Most rodent fMRI studies, including awake studies, rely on anesthesia, which profoundly alters brain activity and limits the interpretation of the data. This study presents a novel restraint system that enables high-quality fMRI in fully awake rats, eliminating the need for anesthesia, ear bars, and bite bars. By reducing stress and motion, this simple restraint system allows for investigation of neural activity and connectivity without confounds from sedation or anesthesia. Its open-source, modular design supports behavioral tasks and broad accessibility, making it a valuable tool for neuroscience research seeking to bridge the gap between preclinical imaging and real-world brain function.

neuroscience↗

bioRxiv2025

Structured Chemical Reaction Modeling with Multitask Graph Neural Networks

Modeling chemical reactions requires connecting fine-grained atom-bond edits with broader semantic categories. Yet, most machine learning approaches model these aspects in isolation: atom mapping, reaction center identification, and reaction classification are treated as separate problems. This separation limits accuracy, interpretability, and generalization. In this work, we argue that multitask learning provides a natural and powerful framework for reaction modeling. By jointly predicting mappings, centers, and classes within a single graph neural network, models can leverage structural dependencies between tasks. We proposed MARCC (Mapping-Assisted Reaction Center and Classification), a multitask architecture that achieves state-of-the-art performance on the USPTO-50K benchmark. MARCC demonstrates that multitask supervision not only improves accuracy across all tasks but also provides a structured representation of reaction mechanisms.

bioinformatics↗

bioRxiv2025

Modtector: Ultra-Fast Modification Signal Mining on Mapped Sequencing Reads

SummaryCurrent tools for RNA epitranscriptomic modification and structural feature analysis are often fragmented, focusing on single signal types and struggling with processing efficiency, especially as sequencing data volumes increase and single-cell technologies advance. To address this challenge, we developed Modtector, an efficient and versatile tool for unified extraction of modification signals from aligned sequencing data. By integrating dual-signal recognition within a single framework, Modtector employs a "count-then-correct" approach to handle both mutation and stop signals, significantly reducing computational complexity. This results in a multi-fold performance improvement compared to existing tools, particularly when processing large-genome, high-coverage datasets--such as completing the analysis of HEK293 22G data in 5 minutes-- demonstrating its potential for large-scale data analysis. Availability and implementationThe manual is available at Readthedocs (https://modtector.readthedocs.io/), and source code is available at GitHub (https://github.com/TongZhou2017/modtector) and Crate.io (https://crates.io/crates/modtector). Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/681300v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1535c98org.highwire.dtl.DTLVardef@1e40f39org.highwire.dtl.DTLVardef@1a37476org.highwire.dtl.DTLVardef@3315c5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Maternal Influenza A Virus Infection Induces Antiviral and Immune Dysregulation in the Placenta and Fetus Without Vertical Transmission

Influenza A virus (IAV) infection during pregnancy is associated with stillbirth and preterm birth, but the degree to which IAV alters placental and fetal immunity is poorly understood. The objective of our study was to determine the immunologic impact of maternal IAV infection on the placenta and fetus in a pigtail macaque (Macaca nemestrina) model. Pregnant pigtail macaques were inoculated with 107 plaque forming units (PFU) of IAV [A/California/07/2009 (H1N1)] and underwent necropsy 5 days post-infection (N=11). Results were compared to uninfected historical controls (N=16). IAV inoculation induced maternal pneumonia in all cases. Stillbirth occurred in 18% (2/11) of IAV-infected pregnancies, but not in controls. While vertical transmission was not observed, low-level IAV viral RNA was detected in two placentas. In the placenta, maternal IAV infection was associated with increased IL-1{beta}, IL-18, and IFN-{beta} levels, and an upregulated type I interferon (IFN) transcriptional response. IAV infection was also associated with significantly higher frequencies of intermediate and non-classical monocytes, plasmacytoid dendritic cells, CD4 T cells, and NKT cells in the fetus (lung, lymph node, blood). Although placental immune and transcriptional perturbations were rarely correlated with maternal IAV disease indicators (e.g., maternal lung viral load/IFN-/IFN-{beta}/IL-6), there were consistent and significant correlations between these metrics and perturbed immune cell populations in the fetus (CD4+ and CD8+ T cells, plasmacytoid dendritic cells, monocyte sub-populations). Maternal IAV infection disrupted both placental and fetal immune environments, but only fetal immune alterations correlated with maternal lung disease severity. One Sentence SummaryMaternal influenza A virus infection in pregnant pigtail macaques dysregulates placental and fetal immunity, with disease severity correlating strongly with fetal, but not placental, immune perturbations.

systems biology↗

bioRxiv2025

Multi-immersion Oblique Plane Microscope (miOPM): A reconfigurable platform for high-resolution Light-Sheet Fluorescence Microscopy.

Light-Sheet Fluorescence Microscopy (LSFM) enables gentle, rapid, and efficient volumetric imaging of biological specimens. Despite its potential, LSFM has fragmented into multiple variants, each narrowly optimized for specific samples or imaging regimes, limiting its broad applicability. To overcome this barrier, we introduce the multi-immersion oblique plane microscope (miOPM), an adaptable LSFM platform for high-resolution imaging ranging from subcellular dynamics to whole organisms and cleared tissues. miOPM uniquely supports seamless interchangeability of oil, water, and air objectives, and maintains diffraction limited performance across a refractive index range of 1.33 - 1.51. It is compatible with standard sample mounting, current clearing protocols and high-throughput 3D imaging. We leverage miOPM to image diverse biological specimens at multiple spatial scales. By greatly expanding the adaptability and ease of use of LSFM, miOPM is poised to democratize advanced three-dimensional imaging.

bioengineering↗

bioRxiv2025

PI-FC: Pre-training Individual-specific Functional Connectome through State-invariant Contrastive Learning

Functional MRI enables non-invasive mapping of brain connectivity, yet its clinical translation remains hindered by uncontrolled state-dependent variability that obscures individual-specific signatures during routine scanning. Here we introduce PI-FC -- a deep learning framework leveraging state-invariant contrastive learning to extract stable individual brain signatures across diverse arousal levels, cognitive states, and temporal scales spanning tens of seconds to hours. PI-FC achieves equivalent phenotypic prediction accuracy using substantially reduced scanning time, and eliminates state-dependent effects varying task demands and brain states. Trained on 36,119 subjects across 8 independent datasets, our model demonstrates superior cross-site generalization and outperforms traditional functional connectome (FC) in predicting neuropsychiatric conditions including schizophrenia, autism, depression, and anxiety. Furthermore, PI-FC enables zero-shot inference of brain age, biological sex, and cognitive ability without site-specific retraining. Overall, PI-FC represents a robust, clinically scalable framework that overcomes fundamental barriers to real-world deployment of precision functional neuroimaging.

neuroscience↗

bioRxiv2025

Von Willebrand Factor Deficiency Impairs Vascular Morphogenesis via Angiopoietin-2: Relevance for Gut Angiodysplasia

Management of recurrent gastrointestinal (GI) bleeding is a clinical unmet need for patients with Von Willebrand disease (VWD) and is linked to the presence of gut vascular malformations (angiodysplasia). We previously demonstrated that von Willebrand factor (VWF) regulates angiogenesis and vascular integrity, the likely mechanism underlying angiodysplasia. VWF controls the storage of the angiogenesis regulator Angiopoietin-2 (Angpt-2) in endothelial cells (EC), suggesting a candidate for the genesis of angiodysplasia; however, no direct evidence of the role of Angpt-2 in VWF-dependent angiogenesis is available. Here we use VWF-deficient HUVEC, and endothelial colony forming cells (ECFCs) from severe VWD patients and find that loss of VWF in EC results in increased Angpt-2 expression through a positive feedback loop via the Angpt-2-TIE2-AKT-FOXO1 pathway. We also show an imbalance of the Angpt/Tie2 pathway in vivo. In the gut of VWF-deficient mice, Angpt-2 expression is increased whilst Angpt-1 expression is decreased; this correlates with reduced expression of the pericyte marker NG2. These data suggest that VWF regulates the Angpt/Tie2 balance in the gut. To investigate the functional defects caused by loss of VWF, we use a fibrin bead assay and show that VWF-deficient HUVEC present increased sprouting. We develop a microfluidic model of 3D vasculogenesis/angiogenesis and find that ECFCs from VWD patients exhibit defective remodeling and abnormal lumen formation compared to healthy controls. Importantly, inhibition of Angpt-2 reduces sprouting in VWF-deficient HUVEC and normalises vascular networks in ECFCs from severe VWD, suggesting Angpt-2 inhibitors may be effective in VWD patients with GI bleeding and angiodysplasia. Key pointsO_LIEndothelial VWF regulates multiple steps of angiogenesis, including sprouting and lumen formation. C_LIO_LIVWF regulates Angpt-2 storage and expression, and Angpt-2 blockade normalises defective angiogenesis in VWD ECFCs. C_LI Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/675051v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@150eb15org.highwire.dtl.DTLVardef@179e6beorg.highwire.dtl.DTLVardef@1bcadborg.highwire.dtl.DTLVardef@a30fb8_HPS_FORMAT_FIGEXP M_FIG VWF deficiency in HUVECs results in increased Angpt-2 release and expression via the Tie2-Akt-FOXO1 pathway. A. Model pathway for the regulation of Angpt-2 levels by VWF. Loss of VWF results in increased Angpt-2 release from endothelial cells; Angpt-2 binds to and inhibits the Tie2 receptor, decreasing its phosphorylation as well as the downstream phosphorylation of Akt and FOXO1; this leads to FOXO1 activation and an increase expression of Angpt-2, generating a feedback loop. B. Model for the pathogenesis of angiodysplasia in VWD. In the absence of VWF, the increase in Angpt-2 disrupts vascular morphogenesis through multiple mechanisms: increased sprouting, impaired vascular remodelling and an imbalance between Angpt-1 and Angpt-2 in the gut. Figure generated with Biorender. C_FIG

cell biology↗

bioRxiv2025

Tissue Kallikrein 1 cleaves complement factor C3 and activates the alternative complement pathway

Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA), characterized by microvascular endothelial damage and severe renal injury, negatively affects HSCT survivorship with high mortality and long-term renal morbidity. After HSCT, innate immunity and inflammation are often dysregulated. The alternative complement pathway (AP) of the innate immune system is overactivated in HSCT-TMA, but the mechanisms of its initiation are poorly described. Complement component C3 of the AP can be cleaved by proteases outside of the AP. Because mRNA expression of tissue kallikrein 1 (KLK1), an inflammatory serine protease that produces kinins, has been found to be markedly elevated in the renal endothelium of inflamed mice, we hypothesized that increased KLK1 activity during inflammation contributes to AP overactivation and endothelial injury in HSCT-TMA. We assessed AP activation, KLK1 activity, endothelial injury, and renal function in HSCT-TMA experimental models and disease settings and investigated C3 cleavage and AP activation by KLK1. We found that patients with HSCT-TMA had significantly increased AP activation and decreased KLK1 inactivation at TMA diagnosis compared to pre-HSCT. Mice challenged with HSCT-TMA triggers cyclosporine A (CsA) and lipopolysaccharide (LPS) exhibited increased AP activation, renal endothelial injury, and impaired renal function in the setting of decreased KLK1 inactivation. We further demonstrated that KLK1 cleaved AP component C3 to C3b that functionally activated the AP. Our data indicate a noncanonical mechanism for AP activation by KLK1 in settings of HSCT-TMA. Key PointsO_LITissue kallikrein (KLK1) functionally cleaves complement factor C3. C_LIO_LIReduced KLK1 inhibition is associated with activation of the alternative pathway of complement. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/674260v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@8f260aorg.highwire.dtl.DTLVardef@a6511org.highwire.dtl.DTLVardef@7160f6org.highwire.dtl.DTLVardef@194d36d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO HSCT-TMA triggers (e.g. inflammation, immunosuppression) upregulate expression of the inflammatory serine protease KLK1, which cleaves C3 to initiate AP activation and amplification, leading to renal endothelial injury. Created with BioRender.com under its Academic License Terms with Baylor College of Medicine. C_FIG

molecular biology↗