Biology subjects

Nguyen, V.

Publications and source records attributed to Nguyen, V..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

CD14 plays a critical role in pain and inflammation across multiple models of post-traumatic osteoarthritis

Inflammation is a primary driver of osteoarthritis (OA), and no therapies exist to halt or delay disease progression or substantially ameliorate the chronic pain, inflammation and disability that are characteristic of disease. Soluble CD14 (sCD14), a co-factor that enhances inflammatory toll-like receptor signaling, is present in synovial fluid in patients with OA and positively associates with joint space narrowing, synovial macrophage content, and pain. In this study, we show that increased sCD14 within human synovial fluid correlates with joint effusion volume and increased knee hyperalgesia. Next, we evaluated CD14 as a potential therapeutic target in three pre-clinical models of post traumatic OA (PTOA), using both genetic deficiency and pharmacologic blockade to modulate its activity. We demonstrate that deficiency or blockade of CD14 results in significant protection from increased evoked pain behaviors and from OA driven mobility impairments (i.e. decreased cage activity) across models that differ in severity, and across male and female cohorts. Using flow cytometry, single cell transcriptomics, and spatial proteomics, we further show that CD14-deficiency drastically influences the local synovial inflammatory landscape post-injury, reducing monocyte and macrophage populations and modulating local fibroblast populations. Targeting CD14 via genetic deficiency or therapeutic blockade revealed no substantial protection, but no worsening, of cartilage degeneration. Ultimately, our results provide strong support that targeting synovial inflammation through blockade of CD14 can safely ameliorate OA pain and disability after a pre-disposing injury. One Sentence SummaryThe study demonstrates the key role of CD14 in pain, mobility loss, and inflammation in PTOA, and demonstrates the therapeutic potential of CD14 blockade for OA pain relief.

immunology↗

bioRxiv2025

The Establishment of Prostate-specific, SKP2 Humanized Mice by CRISPR Knock-in Method Reveals Neoplastic Initiation and Microenvironmental Reprogramming

A recent study has shown that SKP2 inactivation can prevent cancer initiation by extension of total cell cycle duration without perturbing normal division, which suggests a new strategy for cancer prevention. However, direct in vivo evidence for human SKP2 on cancer initiation and prostatic microenvironment is still lacking and a prostate-specific SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting human SKP2. We therefore have established a prostate-specific human SKP2 (hSKP2) knock-in mouse model by a CRISPR knock-in approach. Overexpression of hSKP2, which is driven by an endogenous mouse probasin promoter, induces prostatic lesions including hyperplasia, mouse prostate intraepithelial neoplasia (mPIN), and low-grade carcinoma and increases prostate weights. Transcriptional profiling by RNA-sequencing analysis revealed significant gene expression alterations in epithelial to mesenchymal transition (EMT), extracellular matrix, and interferon signaling in the prostate of hSKP2 knock-in mice compared to wild-type mice. Single cell deconvolution showed an increase of fibroblasts population and a decrease of CD8+ T cell and B cell populations in the prostate of hSKP2-knock-in mice. Consistently with these results from the SKP2 humanized mouse, overexpression of hSKP2 in human prostate cancer PC3 cells markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways, including FMOD, THY1, PFKP, USP18, IL15, etc. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decrease the viability and alter the morphologies of organoids of hSKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific hSKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.

cancer biology↗

bioRxiv2025

Degraded mapping of disparity tuning in visual cortex explains deficits in binocular depth perception

Sensory cortex is highly organized, but the function of this organization is contentious. We show that closing one eye during the developmental critical period in juvenile mice (monocular deprivation) caused lasting impairments in binocular depth perception and disrupted cortical maps of binocular disparity tuning. In normal mice, disparity tuning was concentrated in the central visual field, particularly with respect to azimuth and in higher visual areas (HVAs). Additionally, neurons transitioned from encoding nearer to farther stereoscopic depths from the center toward the peripheral visual field, especially along elevation and within HVAs. Monocular deprivation did not reduce overall disparity selectivity across the neuronal population, but rather weakened the retinotopic and hierarchical organization of disparity tuning and reduced trial-to-trial reliability of neuronal responses. Analysis of single-trial responses from neurons tuned to near disparities showed that this reduced response reliability is sufficient to explain the impaired depth discrimination observed in deprived mice.

neuroscience↗

bioRxiv2024

BRAF V600 and ErbB inhibitors directly activate GCN2 in an off-target manner to limit cancer cell proliferation

Targeted kinase inhibitors are well known for their promiscuity and off-target effects. Herein, we define an off-target effect in which several clinical BRAFV600 inhibitors, including the widely used dabrafenib and encorafenib, interact directly with GCN2 to activate the Integrated Stress Response and ATF4. Blocking this off-target effect by co-drugging with a GCN2 inhibitor in A375 melanoma cells causes enhancement rather than suppression of cancer cell outgrowth, suggesting that the off-target activation of GCN2 is detrimental to these cells. This result is mirrored in PC9 lung cancer cells treated with erlotinib, an EGFR inhibitor, that shares the same off-target activation of GCN2. Using an in silico kinase inhibitor screen, we identified dozens of FDA-approved drugs that appear to share this off-target activation of GCN2 and ATF4. Thus, GCN2 activation may modulate the therapeutic efficacy of some kinase inhibitors, depending on the cancer context.

cancer biology↗

bioRxiv2024

Failure to reproduce the effect of procedural memory interference on wakeful consolidation of episodic memory in younger and older adults

Brown and Robertson (2007) revealed that skill learning interferes with the wakeful consolidation of episodic memories in young adults. This finding is commonly used as evidence that episodic and procedural memories should not be learned in close temporal proximity but has not been reproduced by an independent laboratory. Additionally, older adults experience episodic memory deficits, but it is unknown whether this group is also vulnerable to this type of interference. We aimed to reproduce Brown and Robertsons (2007) finding in younger adults, while also comparing the magnitude of interference between younger and older adults. Forty younger (18-40 years; n =20) and older adults ([≥]55 years; n = 20) visited the laboratory in the morning and acquired episodic memories (a list of words) immediately before a procedural finger-tapping (procedural) task. Half of all participants were exposed to a learnable sequential structure. In the afternoon of the same day, participants were asked to recall the episodic memories from the morning session. We found weak evidence of interference for both age groups and no statistical difference in interference between groups. Our results suggest that the interfering effects of these memory types may be negligible or overestimated, and that these memory types can be acquired together without interference.

neuroscience↗

bioRxiv2024

Deciphering regulation of FOXP3 expression in human conventional T cells

FOXP3 is a lineage-defining transcription factor (TF) for immune-suppressive regulatory T cells (Tregs). While mice exclusively express FOXP3 in Tregs, humans also transiently express FOXP3 in stimulated conventional CD4+ T cells (Tconvs). Mechanisms governing these distinct expression patterns remain unknown. Here, we performed CRISPR screens tiling the FOXP3 locus and targeting TFs in human Tregs and Tconvs to discover cis-regulatory elements (CREs) and trans-regulators of FOXP3. Tconv FOXP3 expression depended on a subset of Treg CREs and Tconv-selective positive (TcNS+) and negative (TcNS-) CREs. The CREs are occupied and regulated by TFs we identified as critical regulators of FOXP3. Finally, mutagenesis of murine TcNS- revealed that it is critical for restriction of FOXP3 expression to Tregs. We discover CRE and TF circuitry controlling FOXP3 expression and reveal evolution of mechanisms regulating a gene indispensable to immune homeostasis. HighlightsO_LIComprehensive CRISPR maps of CREs and TFs controlling FOXP3 in human Tregs and Tconvs C_LIO_LIKey TFs that control FOXP3 directly occupy and regulate CREs forming TF-CRE circuits C_LIO_LIA previously unknown negative CRE stringently restricts FOXP3 to Tregs in mice C_LI

immunology↗

bioRxiv2024

epiTCR-KDA: Knowledge Distillation model on Dihedral Angles for TCR-peptide prediction

MotivationAntigen recognition by T-cell receptors (TCRs) triggers cascades of immune responses. Successful predictions of the TCR and antigen (as peptide) bindings therefore signify the advancements in immunotherapy. However, most of current TCR-peptide interaction predictors fail to predict unseen data. This limitation may be derived from the conventional usage of TCR and/or peptide sequences as input, which may not adequately reflect their structural characteristics. Therefore, incorporating the TCR and peptide structural information into the prediction model to improve the generalizability is necessary. ResultsWe presented epiTCR-KDA as a new predictor of TCR-peptide binding that utilises structural information, specifically the dihedral angles between the residues of both the peptide and the TCR. This structural descriptor was integrated into a model constructed using knowledge distillation to enhance its generalizability. The epiTCR-KDA demonstrated competitive prediction performance, with an AUC of 0.99 for seen data and AUC of 0.86 for unseen data. Across multiple public datasets, epiTCR-KDA consistently outperformed other predictors, such as epiTCR, NetTCR, BERTrand, TEIM-Seq, TEINet, and ImRex, maintaining a median AUC of 0.9 (ranging from 0.82 to 0.91). Further analysis of epiTCR-KDA performance indicated that the cosine similarity of the dihedral angle vectors between the unseen testing data and training data is crucial for its stable performance. In conclusion, our epiTCR-KDA model, with its capacity to predict for unseen data, has brought us one step closer toward the development of a highly effective pipeline for affordable antigen-based immunotherapy. Availability and implementationepiTCR-KDA is available on GitHub (https://github.com/ddiem-ri-4D/epiTCR-KDA)

bioinformatics↗

bioRxiv2024

A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice

BackgroundSmall artery remodeling and endothelial dysfunction are hallmarks of hypertension. Growing evidence supports a likely causal association between cardiovascular diseases and the presence of endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire additional mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. MethodsUsing a partial reprogramming of ECs, via overexpression of Oct-3/4, Sox-2, and Klf-4 (OSK) transcription factors, we aimed to bring ECs back to a youthful phenotype in hypertensive mice. Primary ECs were infected with lentiviral vectors (LV) containing the specific EC marker cadherin 5 (Cdh5) and the fluorescent reporter enhanced green fluorescence protein (EGFP) with empty vector (LVCO) or with OSK (LV-OSK). Confocal microscopy and western blotting analysis were used to confirm the OSK overexpression. Cellular migration, senescence, and apoptosis were evaluated. Human aortic ECs (HAoECs) from male and female normotensive and hypertensive patients were analyzed after OSK or control treatments for their endothelial nitric oxide synthase (eNOS) levels, nitric oxide (NO), and genetic profile. Male and female normotensive (BPN/3J) and hypertensive (BPH/2J) mice were treated with an intravenous (i.v.) injection of LVCO or LV-OSK and evaluated 10 days post-infection. The blood pressure, cardiac function, vascular reactivity of small arteries, in vivo EGFP signal and EndMT inhibition were analyzed. ResultsOSK overexpression induced partial EC reprogramming in vitro, and these cells showed endothelial progenitor cell (EPC)-like features with lower migratory capability. OSK treatment of hypertensive BPH/2J mice normalized blood pressure and resistance arteries hypercontractility, via the attenuation of EndMT and elastin breaks. EGFP signal was detected in vivo in the prefrontal cortex of both BPN/3J and BPH/2J-treated mice, but OSK induced angiogenesis only in male BPN/3J mice. OSK-treated human ECs from hypertensive patients showed high eNOS activation and NO production, with low ROS formation. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in human ECs from hypertensive patients. ConclusionOverall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

physiology↗

bioRxiv2024

An shRNA screen in primary human beta cells identifies the serotonin 1F receptor as a negative regulator of survival during transplant

Islet transplantation can cure type 1 diabetes, but peri-transplant beta cell death limits this procedure to those with low insulin requirements. Improving human beta cell survival or proliferation may make islet transplantation a possibility for more type 1 patients. To identify novel regulators of beta cell survival and proliferation, we conducted a pooled small hairpin RNA (shRNA) screen in primary human beta cells transplanted into immunocompromised mice. shRNAs targeting several cyclin dependent kinase inhibitors were enriched after transplant. Here, we focused on the Gi/o-coupled GPCR, serotonin 1F receptor (HTR1F, 5-HT1F) which our screen identified as a negative regulator of beta cell numbers after transplant. In vitro, 5-HT1F knockdown induced human beta cell proliferation but only when combined with harmine and exendin-4. In vivo, knockdown of 5-HT1F reduced beta cell death during transplant. To demonstrate the feasibility of targeting 5-HT1F in islet transplant, we identified and validated a small molecule 5-HT1F antagonist. This antagonist increased glucose stimulated insulin secretion from primary human islets and cAMP accumulation in primary human beta cells. Finally, the 5-HT1F antagonist improved glycemia in marginal mass, human islet transplants into immunocompromised mice. We identify 5-HT1F as a novel druggable target to improve human beta cell survival in the setting of islet transplantation. One Sentence SummarySerotonin 1F receptor (5-HT1F) negatively regulates insulin secretion and beta cell survival during transplant.

cell biology↗

bioRxiv2024

Rapid Motor Adaptation via Population-level Modulation of Cerebellar Error Signals

A core principle of cerebellar learning theories is that climbing fibers from the inferior olive convey error signals about movement execution to Purkinje cells in the cerebellar cortex. These inputs trigger synaptic changes which are purported to drive progressive adjustment of future movements. Individually, binary complex spike signals lack information about the sign and magnitude of errors which presents a problem for cerebellar learning paradigms exhibiting fast adaptation. Using a newly developed behavioral paradigm in mice, we introduced sensorimotor perturbations into a simple joystick pulling behavior and found parasagittal bands of Purkinje cells with reciprocal modulation of complex spike activity. Whereas complex spiking showed little modulation in the unperturbed condition, alternating bands were activated or inhibited when the perturbation was introduced, and this modulation encoded the sign and magnitude of the resulting sensorimotor mismatch. These findings provide an important piece of information for the understanding of cerebellar learning that helps to explain how the cerebellum could use supervised learning to quickly adapt motor behavior in response to perturbations. One-Sentence SummaryPopulations of Purkinje cells in the cerebellum facilitate trial-by-trial movement refinement by converting binary signals from movement errors into graded signals that encode the errors direction and magnitude.

neuroscience↗

bioRxiv2023

Design of parallel ????-sheet nanofibrils using Monte-Carlo search, coarse-grained simulations, and experimental testing

Peptide self-assembly into amyloid fibrils provides numerous applications in drug delivery and biomedical engineering applications. We augment our previously-established computational screening technique along with experimental biophysical characterization to discover 7-mer peptides that self-assemble into "parallel {beta}-sheets", i.e., {beta}-sheets with N-terminus-to-C-terminus {beta}-strand vectors oriented in parallel. To accomplish the desired {beta}-strand organization, we applied the PepAD amino acid sequence design software to the Class-1 cross-{beta} spine defined by Sawaya et al. This molecular configuration includes two layers of parallel {beta}-sheets stacked such that N-terminus-to-C-terminus vectors are oriented antiparallel for molecules on adjacent {beta}-sheets. The first cohort of PepAD identified peptides were examined for their fibrillation behavior in DMD/PRIME20 simulations, and the top performing sequence was selected as a prototype for a subsequent round of sequence refinement. The two rounds of design resulted in a library of eight 7-mer peptides. In DMD/PRIME20 simulations, five of these peptides spontaneously formed fibril-like structures with a predominantly parallel {beta}-sheet arrangement, two formed fibril-like structure with <50% in parallel {beta}-sheet arrangement and one remained a random coil. Among the eight candidate peptides produced by PepAD and DMD/PRIME20, five were synthesized and purified. All five assembled into amyloid fibrils composed of parallel {beta}-sheets based on Fourier Transform Infrared Spectroscopy, Circular Dichroism, Electron Microscopy, and Thioflavin-T fluorescence spectroscopy measurements.

bioengineering↗

bioRxiv2023

Dissecting Multiparametric Cerebral Hemodynamics using Integrated Ultrafast Ultrasound and Multispectral Photoacoustic Imaging

Understanding brain-wide hemodynamic responses to different stimuli at high spatiotemporal resolutions can help study neuro-disorders and brain functions. However, the existing brain imaging technologies have limited resolution, sensitivity, imaging depth and provide information about only one or two hemodynamic parameters. To address this, we propose a multimodal functional ultrasound and photoacoustic (fUSPA) imaging platform, which integrates ultrafast ultrasound and multispectral photoacoustic imaging methods in a compact head-mountable device, to quantitatively map cerebral blood volume (CBV), cerebral blood flow (CBF), oxygen saturation (SO2) dynamics as well as contrast agent enhanced brain imaging with high spatiotemporal resolutions. After systematic characterization, the fUSPA system was applied to quantitatively study the changes in brain hemodynamics and vascular reactivity at single vessel resolution in response to hypercapnia stimulation. Our results show an overall increase in brain-wide CBV, CBF, and SO2, but regional differences in singular cortical veins and arteries and a reproducible anti-correlation pattern between venous and cortical hemodynamics, demonstrating the capabilities of the fUSPA system for providing multiparametric cerebrovascular information at high-resolution and sensitivity, that can bring insights into the complex mechanisms of neurodiseases.

bioengineering↗

bioRxiv2023

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains

A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of Ppar- and Cdx1 in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis.

genomics↗

bioRxiv2023

NUCLEOPORIN1 mediates proteasome-based degradation of ABI5 to regulate Arabidopsis seed germination

NUCLEOPORIN1 (NUP1), a member of the Nuclear Pore Complex (NPC), is located on the inner side of the nuclear membrane. It is highly expressed in seeds; however, its role in seeds including germination has not been explored yet. Here, we identified an abscisic acid (ABA) hypersensitive phenotype of nup1 during germination. ABA treatment drastically changes the expression pattern of thousands of genes in nup1, including the major transcription factors (TFs) involved in germination, ABI3, ABI4, and ABI5. Double mutant analysis of NUP1 and these ABA-related genes showed that mutations in ABI5 can rescue the phenotype of nup1, suggesting that NUP1 acts upstream of ABI5 to regulate seed germination. ABI5, a key negative regulator of germination, is abundant in dry seeds and rapidly degrades during germination. However, its spatiotemporal regulation and interaction with other molecular players during degradation remained to be fully elucidated. We found that NUP1 is physically associated with ABI5 and the 26S proteasome. Mutation in NUP1 delayed ABI5 degradation through its post-translational retention in nucleolus under abiotic stress. Taken together, our findings suggest that NUP1 anchors the proteasome to NPC and modulates seed germination through proteasome-mediated degradation of ABI5 in the vicinity of NPC in the nucleoplasm.

plant biology↗

bioRxiv2023

A conserved graft formation process in Picea abies and Arabidopsis thaliana identifies the PAT gene family as central regulators of wound healing

The widespread use of plant grafting has enabled eudicots and gymnosperms to join with closely related species and grow as one. Gymnosperms have dominated forests for over 200 million years and despite their economic and ecological relevance, we know little about how they graft. Here, we developed a micrografting method in conifers using young tissues that allowed efficient grafting between closely related species and distantly related genera. Conifer graft junctions rapidly connected vasculature and differentially expressed thousands of genes including auxin and cell wall-related genes. By comparing these genes to those induced during Arabidopsis thaliana graft formation, we found a common activation of cambium, cell division, phloem and xylem-related genes. A gene regulatory network analysis in Picea abies (Norway spruce) predicted that PHYTOCHROME A SIGNAL TRANSDUCTION 1 (PAT1) acted as a core regulator of graft healing. This gene was strongly upregulation during both P. abies and Arabidopsis grafting, and Arabidopsis mutants lacking PAT-genes failed to attach tissues or successfully graft. Complementing Arabidopsis PAT mutants with the P. abies PAT1 homolog rescued tissue attachment and enhance callus formation. Together, our data demonstrate an ability for young tissues to facilitate grafting with distantly related species and identifies the PAT gene family as conserved regulators of graft healing and tissue regeneration.

plant biology↗

bioRxiv2023

Perturbations of glutathione and sphingosine metabolites in Port Wine Birthmark patient-derived induced pluripotent stem cells

Port Wine Birthmark (PWB) is a congenital vascular malformation in the skin, occurring in 1-3 per 1,000 live births. We recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, which we aimed to explore in this study. Metabolites were separated by ultra-performance liquid chromatography and were screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant analysis, multivariate and univariate analysis were used to identify differential metabolites (DMs). KEGG analysis was used for the enrichment of metabolic pathways. A total of 339 metabolites were identified. There were 22 DMs confirmed with 9 downregulated DMs including sphingosine and 13 upregulated DMs including glutathione in PWB iPSCs as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key factors associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1 (HIF1), glutathione S-transferase Pi 1 (GSTP1), {gamma}-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skins. Our data demonstrate that there are perturbations in sphingolipid and cellular redox homeostasis in the PWB vasculature, which may facilitate cell survival and pathological progression. Our data imply that upregulation of glutathione may contribute to laser-resistant phenotypes in the PWB vasculature.

cell biology↗

bioRxiv2023

Hippo and Wnt pathways are impaired in Port Wine Birthmark-derived induced pluripotent stem cells and endothelial cells

AbstractO_ST_ABSBackgroundC_ST_ABSPort wine birthmark (PWB) is a congenital vascular malformation resulting from developmentally defective endothelial cells (ECs). Developing clinically relevant disease models for PWB studies is currently an unmet need. ObjectiveOur study aims to generate PWB-derived induced pluripotent stem cells (iPSCs) and iPSC-derived ECs that preserve disease-related phenotypes. MethodsPWB iPSCs were generated by reprogramming lesional dermal fibroblasts and differentiated into ECs. RNA-seq was performed to identify differentially expressed genes (DEGs) and enriched pathways. The functional phenotypes of iPSC-derived ECs were characterized by capillary-like structure (CLS) formation in vitro and Geltrex plug-in assay in vivo. ResultsHuman PWB and control iPSC lines were generated through reprogramming of dermal fibroblasts by introducing the "Yamanaka factors" (Oct3/4, Sox2, Klf4, c-Myc) into them; the iPSCs were successfully differentiated into ECs. These iPSCs and their derived ECs were validated by expression of a series of stem cell and EC biomarkers, respectively. PWB iPSC-derived ECs showed impaired CLS in vitro with larger perimeters and thicker branches as compared to control iPSC-derived ECs. In the plug-in assay, perfused human vasculature formed by PWB iPSC- derived ECs showed bigger perimeters and greater densities than those formed by control iPSC- derived ECs in severe combined immune deficient (SCID) mice. The transcriptome analysis showed that dysregulated pathways of stem cell differentiation, Hippo, Wnt, and focal adhesion persisted through differentiation of PWB iPSCs to ECs. Functional enrichment analysis showed that Hippo and Wnt pathway-related PWB DEGs are enriched for vasculature development, tube morphology, endothelium development, and EC differentiation. Further, members of the zinc finger (ZNF) gene family were overrepresented among the DEGs in PWB iPSCs. ZNF DEGs confer significant functions in transcriptional regulation, chromatin remodeling, protein ubiquitination, and retinoic acid receptor signaling. Furthermore, NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were dysregulated in PWB ECs as readouts of impaired differentiation. ConclusionsPWB iPSC-derived ECs render a novel and clinically-relevant disease model by retaining pathological phenotypes. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated, which may contribute to the development of differentiation-defective ECs in PWB. Bulleted statementsO_ST_ABSWhat is already known about this topic?C_ST_ABSO_LIPort Wine Birthmark (PWB) is a congenital vascular malformation with an incidence rate of 0.1 - 0.3 % per live births. C_LIO_LIPWB results from developmental defects in the dermal vasculature; PWB endothelial cells (ECs) have differentiational impairments. C_LIO_LIPulse dye laser (PDL) is currently the preferred treatment for PWB; unfortunately, the efficacy of PDL treatment of PWB has not improved over the past three decades. C_LI What does this study add?O_LIInduced pluripotent stem cells (iPSCs) were generated from PWB skin fibroblasts and differentiated into ECs. C_LIO_LIPWB ECs recapitulated their pathological phenotypes such as forming enlarged blood vessels in vitro and in vivo. C_LIO_LIHippo and Wnt pathways were dysregulated in PWB iPSCs and ECs. C_LIO_LIZinc-finger family genes were overrepresented among the differentially expressed genes in PWB iPSCs. C_LIO_LIDysregulated NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were enriched in PWB ECs. C_LI What is the translational message?O_LITargeting Hippo and Wnt pathways and Zinc-finger family genes could restore the physiological differentiation of ECs. C_LIO_LITargeting NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways could mitigate the pathological progression of PWB. C_LIO_LIThese mechanisms may lead to the development of paradigm-shifting therapeutic interventions for PWB. C_LI

cell biology↗

bioRxiv2023

Non-coding sequence variation reveals fragility within interleukin 2 feedback circuitry and shapes autoimmune disease risk

Genetic variants associated with human autoimmune diseases commonly map to non-coding control regions, particularly enhancers that function selectively in immune cells and fine-tune gene expression within a relatively narrow range of values. How such modest, cell-type-selective changes can meaningfully shape organismal disease risk remains unclear. To explore this issue, we experimentally manipulated species-conserved enhancers within the disease-associated IL2RA locus and studied accompanying changes in the progression of autoimmunity. Perturbing distinct enhancers with restricted activity in conventional T cells (Tconvs) or regulatory T cells (Tregs)--two functionally antagonistic T cell subsets--caused only modest, cell-type-selective decreases in IL2ra expression parameters. However, these same perturbations had striking and opposing effects in vivo, completely preventing or severely accelerating disease in a murine model of type 1 diabetes. Quantitative tissue imaging and computational modelling revealed that each enhancer manipulation impinged on distinct IL-2-dependent feedback circuits. These imbalances altered the intracellular signaling and intercellular communication dynamics of activated Tregs and Tconvs, producing opposing spatial domains that amplified or constrained ongoing autoimmune responses. These findings demonstrate how subtle changes in gene regulation stemming from non-coding variation can propagate across biological scales due to non-linearities in intra- and intercellular feedback circuitry, dramatically shaping disease risk at the organismal level.

immunology↗