Biology subjects

Oppenheim, O.

Publications and source records attributed to Oppenheim, O..

4 recordsLinked to original sources

bioRxiv2025

Disrupted endothelial cell-cell dynamics in ALK1 and SMAD4 deficiency drive arteriovenous malformations

Arteriovenous malformations (AVMs) are a hallmark of hereditary haemorrhagic telangiectasia (HHT) and arise from abnormal vascular remodelling. Although AVM formation has been associated with disruptions in the BMP9/10 signalling pathway, the distinct contributions of its components remain unclear. Here, we combine in vitro mosaic endothelial cell (EC) cultures with agent-based modelling (ABM) to investigate how knockdown of the BMP9/10 pathway components ALK1 and SMAD4 alters cell-cell interactions and collective vascular organisation. Using cell tracking data, we inferred the mechanical interactions between neighbouring ECs in 2D monolayers by applying Approximate Bayesian Computation to an ABM of cell migration. SMAD4 knockdown increased the motile forces generated by ECs, while ALK1 knockdown weakened the ability of cells to push apart and rearrange with their neighbours, both resulting in greater mixing and fluidity within the cell layer. When these altered interaction profiles were incorporated into an ABM of collective EC dynamics during vascular remodelling, they gave rise to distinct AVM-prone phenotypes. SMAD4-deficient populations exhibited local blockages as highly polarised cells migrated excessively, whereas ALK1-deficient populations disrupted vascular patterning by impairing coordinated polarity and neighbour separation, favouring flow reversal. Our findings suggest that canonical BMP9/10 pathway defects destabilise the biomechanical balance of endothelial interactions, leading to emergent collective behaviours that predispose vessels to AVM formation. Significance StatementArteriovenous malformations (AVMs) are abnormal connections between arteries and veins that disrupt blood flow and can cause stroke or life-threatening bleeding. They are a hallmark of hereditary haemorrhagic telangiectasia (HHT), a genetic vascular disorder, but the cellular events that initiate AVMs remain unclear. Using a combination of endothelial cell cultures and computational modelling, we show that loss of two key HHT-linked genes, ALK1 and SMAD4, disrupts the coordinated movement and interactions of endothelial cells. Although both deficiencies lead to AVM-like behaviours, they do so through distinct mechanisms. SMAD4 loss drives excessive collective migration, whereas ALK1 loss impairs polarity and neighbour separation. These findings provide a mechanistic framework for how genetic defects could contribute to cell-cell dynamics observed in vascular malformations.

cell biology↗

bioRxiv2025

Divergent endothelial mechanisms drive arteriovenous malformations in Alk1 and Smad4 loss-of-function

BackgroundHereditary hemorrhagic telangiectasia is a genetic disorder caused by loss-of-function mutations in components of the bone morphogenetic protein signaling pathway, leading to arteriovenous malformations. Most prior work has treated BMP-component mutations as mechanistically interchangeable, yet whether distinct genes converge on a shared mechanism remains unclear. We aimed to understand the molecular relationship between BMP signaling and endothelial flow response that leads to AVM formation. MethodsWe expose human endothelial monolayers treated with siRNA against SMAD4 or ALK1 to laminar flow and analyze flow-responsive transcriptomics, flow-responsive BMP signaling activation dynamics, cell polarity and morphology. We analyze the cell-autonomous and non-cell-autonomous migration dynamics of ECs treated with siSMAD4 or siALK1. Using the postnatal mouse retina model, we study EC distribution changes over time in mosaic settings, and assess the remodelling capabilities of Smad4iECKO or Alk1iECKO, relative to littermate controls. ResultsThis study shows that mutations in SMAD4 or ALK1 lead to fundamentally distinct mechanisms of malformation formation. SMAD4 deficiency enhances endothelial responses to blood flow, including transcriptional activation and migration against flow, causing excessive capillary pruning and the development of single large shunts. In contrast, ALK1 deficiency disrupts flow sensing, impairs cell polarization and migration, and promotes a persistent angiogenic state, resulting in dense, hypervascularized networks. RNA sequencing reveals that these transcriptional changes precede flow onset, suggesting early defects in endothelial fate specification. Mosaic in vitro models show that mutant cells co-opt neighboring wild-type cells, while in vivo tracking confirms mutation-specific migration behavior. ConclusionsThese findings reveal divergent cellular programs driving arteriovenous malformations and underscore the need for gene-specific diagnostic and therapeutic strategies. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIThis study identifies gene-specific mechanisms of AVM formation in endothelial cells with SMAD4 or ALK1 loss-of-function, two major mutations underlying Hereditary Hemorrhagic Telangiectasia. C_LIO_LIUsing multiple side-by-side analyses, the study reveals that SMAD4 deficiency enhances flow-mediated remodeling, while ALK1 deficiency impairs flow sensing and promotes angiogenic persistence. C_LIO_LIRNA sequencing showed that several transcriptional differences precede the onset of flow, indicating early divergence in endothelial cell fate specification and mechanosensory behavior. C_LIO_LIMosaic culture and retinal models demonstrate that mutant cells exert distinct non-cell-autonomous effects on wild-type endothelial cells. C_LI What Are the Clinical Implications?O_LIThis work challenges the prevailing assumption of a unified pathogenic pathway in HHT and supports a model of mutation-specific AVM pathogenesis, with distinct cellular and molecular drivers. C_LIO_LIThe findings provide a mechanistic basis for the variable clinical presentation seen in patients with different HHT mutations. C_LIO_LIIdentification of early transcriptional signatures, such as persistent ANGPT2 or DPP4 upregulation, may offer new biomarkers or therapeutic targets for early intervention before AVMs become clinically symptomatic. C_LIO_LIPersonalized therapeutic strategies could reduce reliance on invasive treatments and shift toward mechanism-based, precision medicine approaches in HHT and related vascular disorders. C_LI

developmental biology↗

bioRxiv2024

Polarity-JaM: An image analysis toolbox for cell polarity, junction and morphology quantification

Cell polarity involves the asymmetric distribution of cellular components such as signaling molecules and organelles within a cell, asymmetries of a cell"s shape as well as contacts with neighbouring cells. Gradients and mechanical forces often act as global cues that bias cell polarity and orientation, and polarity is coordinated by communication between adjacent cells. Advances in fluorescence microscopy combined with deep learning algorithms for image segmentation open up a wealth of possibilities to understand cell polarity behaviour in health and disease. We have therefore developed the open-source package Polarity-JaM, which offers versatile methods for performing reproducible exploratory image analysis. Multi-channel single cell segmentation is performed using a flexible and userfriendly interface to state-of-the-art deep learning algorithms. Interpretable single-cell features are automatically extracted, including cell and organelle orientation, cell-cell contact morphology, signaling molecule gradients, as well as collective orientation, tissue-wide size and shape variation. Circular statistics of cell polarity, including polarity indices, confidence intervals, and circular correlation analysis, can be computed using our web application. We have developed data graphs for comprehensive visualisation of key statistical measures and suggest the use of an adapted polarity index when the expected polarisation direction or the direction of a global cue is known a priori. The focus of our analysis is on fluorescence image data from endothelial cells (ECs) and their polarisation behaviour. ECs line the inside of blood vessels and are essential for vessel formation and repair, as well as for various cardiovascular diseases, cancer, and inflammation. However, the general architecture of the software will allow it to be applied to other cell types and image modalities. The package is built in in Python, allowing researchers to seamlessly integrate Polarity-JaM into their image and data analysis workflows, see https://polarityjam. readthedocs.io. In addition, a web application for statistical analysis, available at www.polarityjam.com, and a Napari plugin are available, each with a graphical user interface to facilitate exploratory analysis.

bioinformatics↗

bioRxiv2023

Reconstructing stochastic cell population trajectories reveals regulators and heterogeneity of endothelial flow-migration coupling driving vascular remodelling

Emerging concepts of developmental vascular remodelling recently identified that selectively labelled sprouting tip cells and/or venous endothelial cells (ECs) accumulate in developing arteries, suggesting directional migration of specific ECs drives artery formation. However, a general population analysis and detailed quantitative investigation of migratory mechanisms is so far lacking. Here, we developed a universally applicable quantitative approach and a computational model allowing to track and simulate stochastically labelled EC populations irrespective of labelling density and origin. Dynamic mapping of EC distributions in a bespoke coordinate system revealed how ECs move during the most active remodelling phases in the mouse retina. Simulation and parameter sensitivity analysis illustrated that the population shift from veins to arteries cannot be explained by random walk, but best fits to a tuneable dual force-field between shear-force induced directionality against blood flow, and hypoxia mediated VEGFA-gradients. High migration rates require only weak flow-migration coupling, whereas low migration rates require strong coupling to the flow direction. Functional analysis identified Cdc42 as the critical mediator of overall population movement from veins to arteries, yet with surprising heterogeneity suggesting the existence of distinct cell populations. This new quantitative understanding will enable future tailored intervention and tuning of the remodelling process.

developmental biology↗