Biology subjects

Vigolo, D.

Publications and source records attributed to Vigolo, D..

12 recordsLinked to original sources

bioRxiv2026

Elevated hydrostatic pressure modulates endothelial junctional mechanotransduction through VE-cadherin remodelling and altered association with YAP1, EPS8: an endothelium-on-chip study

Endothelial dysfunction is a hallmark of numerous vascular pathologies and is strongly influenced by mechanobiological forces within the vascular microenvironment. While the effects of shear stress have been extensively investigated, the mechanisms by which elevated hydrostatic pressure regulates endothelial junctional organisation remain sparsely investigated. Here, we employed a microfluidic platform to investigate the combined effects of low shear stress (1.4 dyne/cm2) and elevated hydrostatic pressure (~3972 Pa) on endothelial junctional dynamics. Elevated hydrostatic pressure induced marked remodelling of VE-cadherin junctions, characterised by formation of serrated, finger-like structures accompanied by increased YAP1 nuclear localisation and reduced YAP1-VE-cadherin cytoplasmic colocalisation compared to shear stress alone conditions. Further, elevated hydrostatic pressure also demonstrated an increase in cytoplasmic accumulation of EPS8, an actin adaptor protein, and increased cytoplasmic EPS8-VE-cadherin colocalisation. These observations were accompanied by functional changes marked by increased endothelial permeability, and enhanced THP-1 monocyte adhesion, thus suggesting activation of mechanosensitive pathways linked to dynamic junctional reorganisation. Inhibition of PI3K at elevated hydrostatic pressure exhibited a thin VE-cadherin patterning and increased cytoplasmic EPS8-VE-cadherin colocalisation, thus demonstrating a prominent role for PI3K signalling in regulating the junction organisation. Interestingly, Piezo-1 activation using Yoda1 produced context-dependent effects. Under shear stress alone, Yoda1 promoted YAP1 nuclear translocation, reduced YAP1-VE-cadherin colocalisation, increased endothelial permeability but strikingly did not impact THP-1 adhesion compared to shear stress alone conditions. In contrast, under elevated hydrostatic pressure conditions, Yoda1 significantly reduced both endothelial permeability and THP-1 adhesion while increasing YAP1-VE-cadherin colocalisation and decreasing YAP1 nuclear accumulation. Collectively, these findings identify a previously underappreciated elevated hydrostatic pressure-Piezo-1-PI3K signalling axis that regulates endothelial barrier integrity and pro-adhesive endothelial activation through coordinated regulation of VE-cadherin, YAP1, and EPS8. These results highlight elevated hydrostatic pressure as a unique mechanobiological stimulus, distinct from that of shear stress alone and provide novel insights into mechanisms underlying microvascular dysfunction.

bioengineering↗

bioRxiv2026

A Reduced Mechanobiological Framework for Platelet Priming: From Hemodynamic Shear to Mechanosensitive Calcium Entry

BackgroundPlatelet activation in flowing blood is a multiscale process in which vessel-scale hemodynamics, red blood cell (RBC) mechanics, adhesive receptor interactions, and intracellular signalling jointly determine thrombotic risk. Individual components are well studied, but a single reduced description that carries each explicitly from vessel-scale flow to mechanosensitive calcium entry, with dimensionally consistent couplings, remains uncommon. ObjectivesWe develop and analyse a reduced, six-module mechanobiological framework for platelet priming spanning the cascade from hemodynamic shear to mechanosensitive calcium entry, and we delineate which elements are supported by existing evidence and which are new, testable hypotheses. MethodsThe framework comprises six coupled modules: (I) hemodynamic forcing from the incompressible Navier-Stokes equations, with an objective principal-strain-rate measure for extensional flow; (II) RBC-mediated platelet margination and near-wall delivery, closed by a near-wall arrival flux; (III) von Willebrand factor (VWF) activation with a bounded kernel and glycoprotein Ib (GPIb) catch-slip capture, resolved through an explicit contact area and a bond-dependent mobility that progressively immobilises wall-interacting platelets; (IV) a single-load membrane-stimulus formulation; (V) mechanosensitive gating and a dimensionally consistent cytosol-store calcium model with extracellular influx; and (VI) a phenomenological mechanical-memory state. We formally derive that the single-platelet stochastic dynamics and the continuum population balance form a Fokker-Planck pair, with the spatially varying diffusivity handled by an explicit drift correction. ResultsThe framework yields a family of mechanochemical dimensionless groups delineating priming regimes. Its central prediction is reformulated as a falsifiable, history-sensitive signature: in a conditioning-test protocol, a low-tension conditioning block charges the memory state, and a fixed sub-threshold test pulse then reports a delay-dependent calcium facilitation that decays on the memory time{tau} m and is distinguishable from no-memory gating, channel adaptation, and residual-calcium priming. We show explicitly that the previously proposed pulsatile-versus-monotone contrast is a nonlinear convexity/thresholding effect of the gating nonlinearity--its difference-in-differences is approximately zero-- and is therefore not a valid test of memory; the conditioning-test signature is. A second prediction links RBC stiffening to reduced near-wall delivery and captured-platelet calcium response, upstream of intrinsic platelet signalling. ConclusionsThe framework provides a dimensionally consistent, mechanistically grounded and hypothesis-generating description linking hemodynamic forcing to mechanosensitive calcium entry. It demonstrates how history-dependent platelet priming may arise from a phenomenological sensitisation state and proposes a conditioning-test protocol for comparison against adhesive, channel and intracellular-store persistence. The framework is calibratable rather than validated, and the quantitative outputs shown use representative uncalibrated parameters.

biophysics↗

bioRxiv2026

Symmetry-breaking flow bifurcation as an under-recognised haemodynamic factor in valve-associated thrombosis

Venous thrombosis commonly develops near venous valve pockets, where disturbed hemodynamics and reduced washout create spatially heterogeneous flow environments. Conventional descriptions of this region emphasize continuously varying quantities such as velocity, shear stress, recirculation, and residence time, but do not address whether post-valve flow can transition between distinct organized states. Here, we combine computational and experimental vein-on-a-chip approaches to resolve Reynolds-number-dependent post-valve flow organization and its downstream transport consequences. In a geometrically symmetric rigid valve, increasing Reynolds number triggered a symmetrybreaking transition from a centered jet to a persistent asymmetric flow state, with repeatable separation between the increasing- and decreasing-Reynolds-number branches under transient forcing. Valve compliance delayed this transition by increasing the effective opening and reducing local inertial forcing, whereas a small bilateral geometric bias promoted earlier branch selection. Fluid-structure interaction further showed that, after symmetry breaking, unequal hydrodynamic loading generated unequal leaflet deformation, providing a mechanism for structural reinforcement of the selected flow state. The Reynolds-number-dependent ordering was reproduced experimentally and remained detectable in whole blood. Downstream transport of RBC-scale particles exhibited a delayed but persistent lateral redistribution relative to the velocity-field response. These results identify post-valve symmetry breaking as a flow-state transition regulated by local inertial forcing, valve mechanics, and geometric bias. Venous-valve hemodynamics can therefore transition between distinct flow states, providing a physical basis for how small mechanical or geometric differences can generate persistent transport heterogeneity relevant to thrombosis.

biophysics↗

bioRxiv2026

Force-Gated Thrombosis (FGT): A Non-Equilibrium Mechanical Theory of Shear-Induced Blood Clot Initiation

Arterial thrombosis is initiated when mechanical forces in flowing blood exceed the activation thresholds of platelets and von Willebrand factor (vWF). Despite extensive experimental characterization of shear-induced platelet aggregation, a unified theoretical framework that maps hemodynamic forcing onto clot nucleation is lacking. Here we present Force-Gated Thrombosis (FGT), a non-equilibrium mechanical theory that treats thrombus formation as a continuous phase transition driven by an effective mechanical forcing {Sigma} ={sigma} + |{nabla}{sigma}| + {beta}{varepsilon}, which combines local wall shear stress{sigma} , shear gradient |{nabla}{sigma}|, and extensional strain rate{varepsilon} . We introduce a dimensionless Thrombosis Number {Theta} = ({Sigma}/{Sigma}c)(P/P0)m(C/C0)n, which incorporates platelet concentration P and coagulation factor concentration C, and governs the transition between stable flow ({Theta} < 1) and active clot growth ({Theta} > 1). The thrombus density is represented by a scalar order parameter{varphi} whose dynamics follow a Ginzburg- Landau free energy functional. For a simplified stenosed artery we derive an analytic closed-form thrombosis onset criterion and a critical flow rate [Formula], where{delta} is stenosis severity. Linear stability analysis shows that perturbations grow at rate{omega} (k) = {Lambda}({Theta}) - D{varphi}k2, becoming unstable when {Theta} > 1. Near threshold the clot volume fraction scales as{varphi} [~] ({Theta} - 1)1/2, a mean-field critical exponent consistent with Ginzburg- Landau theory. Systematic comparison with fifteen published experimental and computational datasets spanning shear rates from 100 to 15,000 s-1 confirms that FGT correctly predicts the existence, location, and approximate severity of pathological thrombus formation across diverse vascular geometries. The theory provides a quantitative bridge between single-molecule mechanobiology and macroscale clinical thrombosis, and yields experimentally testable predictions distinguishing FGT from purely biochemical models.

biophysics↗

bioRxiv2025

Fluorescently Labeled Gradient Hydrogels Reveal Matrix-Dependent Cell Responses to Substrate Stiffness

Microfabricated stiffness gradient hydrogels hold significant value for advancing mechanobiology, tissue engineering, and in vitro tissue models. However, it remains challenging to design these materials given their broad processing parameter space. The continuum of stiffness values also makes it difficult to precisely correlate the local substrate properties and observed biological responses, often relying on cumbersome characterization methods such as atomic force microscopy. To address these bottlenecks, we present a straightforward thermophoresis-based fabrication strategy to pattern stiffness gradients in fluorescein isothiocyanate-labeled hydrogel network, which displays a polymer concentration-dependent fluorescence readout. This approach enables quantitative assessment of the gradient formation process and contactless stiffness mapping via standard microscopy imaging. Using gelatin methacryloyl and Gellan gum as model systems, it is shown that substrate stiffness and extracellular matrix protein composition work together to affect 3T3-L1 fibroblast cell morphology and migration, with the underlying hydrogel type also affecting the outcome. By offering a simple and reliable approach for characterizing stiffness gradient hydrogels, this work advances the thermophoretic fabrication platform, opening avenues for new biomaterial systems for understanding and controlling the cell-material interplay.

bioengineering↗

bioRxiv2025

DNA Partitioning Modulates Liquid-to-Solid Transitions and the Internal Microstructure of FUS Condensates

Protein liquid-liquid phase separation has recently been recognized as an essential process involved in cellular functions, including transcription, translation, and DNA damage repair. However, a further liquid-to-solid transition (LST) of condensates due to mutation or external stimuli can result in aggregation, sometimes pathological. The unique ability of protein condensates to concentrate and sequester biomolecules is at the heart of the regulatory mechanism, controlling the dynamics and function of the condensates. While the recruitment of essential biomolecules, such as RNA has been studied to have the impact to the condensate formation, how DNA partition can affect the phase behaviour and dynamics of protein condensates is not fully elucidated. In this study, we investigate both the short-term and long-term kinetics of double-stranded DNA partitioning into preformed fresh and aged FUS protein condensates. Confocal imaging shows that DNA partition follows the core-shell diffusion pattern within the condensates. LST slows down and reduces FUS condensates ability to recruit DNA but stabilizes the DNA-FUS condensate complex due to the heterogeneous solid network formation. Using the optical technique of Spatial Dynamic Mapping (SDM), we find that DNA partition promotes coalescence and alters the characteristics of the condensates. The partition made the condensates more dynamic in the short term (within minutes) but accelerated LST in long-term incubation (within hours), ultimately leading to an irreversible porous core-shell structure of FUS condensates. Our findings reveal the kinetics of DNA partition during aging and its impact on LST, underlining the modulation of condensate properties by molecule sequestration, shedding light on possible regulation of disease-related LST of biomolecular condensates.

biophysics↗

bioRxiv2025

A Modular Microfluidic System to Generate Gradient Hydrogels with Simple-to-Complex Stiffness Profiles for Mechanobiology

Engineered stiffness gradient hydrogels offer exciting opportunities to probe fundamental mechanobiological processes in vitro. However, the need to spatially manipulate the properties of soft hydrogels at the micron scale poses challenges in developing fabrication platforms that can reliably modulate the gradient gel characteristics according to user needs. This study describes a modular approach leveraging thermophoresis in microfluidics to create high-fidelity stiffness gradients with linear and complex profiles, including periodic and anisotropic gradients. This study describes the platforms design and optimization, demonstrating achievable stiffness ranges and gradient slopes that correlate with many physiologic and diseased tissue types. This platform is also compatible with different hydrogel crosslinking chemistries, providing a versatile tool to engineer microenvironments with increased complexity. Directionally biased fibroblast cell proliferation and migration on fabricated stiffness gradient gelatin methacryloyl (GelMA) hydrogels indicate the effectiveness of this platform in modulating the mechanical microenvironment of cells. The results indicate that both the absolute stiffness range and the pattern of stiffness variation jointly affect cell behaviors. Considering its remarkable flexibility, the fabrication platform can significantly advance the development of biophysical gradient hydrogels that better replicate the intricacies of native tissues and help realize the next breakthroughs in mechanobiology.

bioengineering↗

bioRxiv2025

Elevated hydrostatic pressure acts via piezo-1 to destabilise VE-cadherin junctions: an endothelium-on-chip study

Despite the effects of shear stress on endothelial biology having been extensively researched, the effects of hydrostatic vascular pressure at extremely low shear stresses have been largely ignored. In the current study, we employ a microfluidic organ-on-chip platform to elucidate the time and shear stress dependent effects of elevated hydrostatic pressure on endothelial junctional perturbations. We report that short term (1h) exposure to elevated hydrostatic pressure at high shear stress (1 dyne/cm2) but not low shear stress (0.1 dyne/cm2) caused VE-cadherin to form serrated, finger like projections at the cell-cell junctions and this effect was abrogated upon pharmacologically inhibiting piezo-1 mechanosensory protein. Interestingly, prolonged exposure (24h) to elevated hydrostatic pressure at low (0.1 dyne/cm2) but not high shear stress (1 dyne/cm2) caused VE-cadherin internalisation, thereby increasing the cytoplasmic concentration. Further, we report that this internalisation of VE-cadherin was reversible upon pharmacologically inhibiting piezo-1 in a time-dependent manner wherein after 12h, we observed stable VE-cadherin junctions re-appear at the cell-cell junctions. Overall, we demonstrate that piezo-1 plays a crucial role in the mechanotransduction of elevated hydrostatic pressure by regulating the VE-cadherin dynamics at cell-cell junctions. Targeting piezo-1 may provide a novel therapeutic/diagnostic marker, especially in conditions that involve microvascular dysfunction due to elevated vascular pressures.

bioengineering↗

bioRxiv2024

Neuromorphic Imaging Cytometry on Human Blood Cells

AO_SCPLOWBSTRACTC_SCPLOWImage-enhanced cytometry and sorting are powerful technologies that provide single-cell resolution and, where possible, cell actuation based on spatial and fluorescence characterisation. With the emergence of deep learning (DL), numerous cytometry-related works incorporate DL to assist their research in handling data-intensive and repetitive workloads. The rich spatial information provided by single-cell images has exceptional use with DL models to classify cells, detect rare cell events, disclose irregularity and achieve higher sample purity than a conventional feature-gating strategy. One of the significant challenges in these image-enable technologies is the constrained throughput owing to the data-expensive image acquisition and balancing between speed and resolution. This work introduces a novel paradigm by adopting a bio-inspired neuromorphic photosensor to capture fast-moving cell events. It facilitates a data-efficient, fluorescence-sensitive, fast inference approach to establish a foundation for neuromorphic-enabled cytometry/sorting applications. We have also curated the first neuromorphic-encoded cell dataset, including human blood cells (red blood cells, neutrophils, lymphocytes, thrombocytes), endothelial cells and polystyrene-based microparticles. To evaluate the data quality and potential of DL-based gating, we have directly trained a hybrid classification model based on this dataset, accomplishing a promising performance of 97% accuracy and F1 score with a significant reduction in memory usage and power consumption. Combining neuromorphic imaging and DL holds substantial potential to develop into a next-generation AI-assisted cytometry and sorting application.

cell biology↗

bioRxiv2023

Neuromorphic Cytometry: Implementation on cell counting and size estimation

Flow cytometry is a widespread and high-throughput technology that can measure the features of cells and can be combined with fluorescence analysis for additional phenotypical characterisations but only provide low-dimensional output and spatial resolution. Imaging flow cytometry is another technology that offers rich spatial information, allowing more profound insight into single-cell analysis. However, offering such high-resolution, full-frame feedback can compromise speed and has become a significant trade-off challenge to tackle during development. In addition, the current dynamic range offered by conventional photosensors can only capture limited fluorescence signals, exacerbating the difficulties in elevating performance speed. Neuromorphic photo-sensing architecture focuses on the events of interest via individual-firing pixels to reduce data redundancy and provide low latency in data processing. With the inherent high dynamic range, this architecture has the potential to drastically elevate the performance in throughput by incorporating motion-activated spatial resolution. Herein, we presented an early demonstration of neuromorphic cytometry with the implementation of object counting and size estimation to measure 8 m and 15 m polystyrene-based microparticles and human monocytic cell line (THP-1). In this work, our platform has achieved highly consistent outputs with a widely adopted flow cytometer (CytoFLEX) in detecting the total number and size of the microparticles. Although the current platform cannot deliver multiparametric measurements on cells, future endeavours will include further functionalities and increase the measurement parameters (granularity, cell condition, fluorescence analysis) to enrich cell interpretation.

cell biology↗

bioRxiv2022

Solid/liquid coexistence during aging of FUS condensates

A wide range of macromolecules undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed in a reversible manner, and carry out important functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics and mechanics in time and space, that FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases co-exist simultaneously within the same condensate, resulting in highly inhomogeneous structures. We introduce two new optical techniques, dynamic spatial mapping and reflective confocal dynamic speckle microscopy, and use these to further show that the liquid-to-solid transition is initiated at the interface between the dense phase within condensates and the dilute phase. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a key element in driving pathological protein aggregation.

biophysics↗

bioRxiv2020

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1-4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5-7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquid-liquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.

biophysics↗