WASp controls oriented migration of endothelial cells to achieve functional vascular patterning.

Endothelial cell migration and proliferation are essential for the establishment of a hierarchical organization of blood vessels and optimal distribution of blood. However, how these cellular processes are quantitatively coordinated to drive vascular network morphogenesis remains unknown. Here, using the zebrafish vasculature as a model system, we demonstrate that the balanced distribution of endothelial cells, as well as the resulting regularity of vessel calibre, is a result of cell migration from veins towards arteries and cell proliferation in veins. We identify the Wiskott-Aldrich Syndrome protein (WASp) as an important molecular regulator of this process and show that loss of coordinated migration from veins to arteries upon wasb depletion results in aberrant vessel morphology and the formation of persistent arteriovenous shunts. We demonstrate that WASp achieves its function through the coordination of junctional actin assembly and PECAM1 recruitment and provide evidence that this is conserved in humans. Overall, we demonstrate that functional vascular patterning in the zebrafish trunk is established through differential cell migration regulated by junctional actin, and that interruption of differential migration may represent a pathomechanism in vascular malformations.

Mathematical models of developmental vascular remodelling: A review.

Over the past 40 years, there has been a strong focus on the development of mathematical models of angiogenesis, while developmental remodelling has received little such attention from the mathematical community. Sprouting angiogenesis can be seen as a very crude way of laying out a primitive vessel network (the raw material), while remodelling (understood as pruning of redundant vessels, diameter control, and the establishment of vessel identity and hierarchy) is the key to turning that primitive network into a functional network. This multiscale problem is of prime importance in the development of a functional vasculature. In addition, defective remodelling (either during developmental remodelling or due to a reactivation of the remodelling programme caused by an injury) is associated with a significant number of diseases. In this review, we discuss existing mathematical models of developmental remodelling and explore the important contributions that these models have made to the field of vascular development. These mathematical models are effectively used to investigate and predict vascular development and are able to reproduce experimentally observable results. Moreover, these models provide a useful means of hypothesis generation and can explain the underlying mechanisms driving the observed structural and functional network development. However, developmental vascular remodelling is still a relatively new area in mathematical biology, and many biological questions remain unanswered. In this review, we present the existing modelling paradigms and define the key challenges for the field.

Novel linkage approach to join community-acquired and national data.

BACKGROUND: Community optometrists in Scotland have performed regular free-at-point-of-care eye examinations for all, for over 15 years. Eye examinations include retinal imaging but image storage is fragmented and they are not used for research. The Scottish Collaborative Optometry-Ophthalmology Network e-research project aimed to collect these images and create a repository linked to routinely collected healthcare data, supporting the development of pre-symptomatic diagnostic tools. METHODS: As the image record was usually separate from the patient record and contained minimal patient information, we developed an efficient matching algorithm using a combination of deterministic and probabilistic steps which minimised the risk of false positives, to facilitate national health record linkage. We visited two practices and assessed the data contained in their image device and Practice Management Systems. Practice activities were explored to understand the context of data collection processes. Iteratively, we tested a series of matching rules which captured a high proportion of true positive records compared to manual matches. The approach was validated by testing manual matching against automated steps in three further practices. RESULTS: A sequence of deterministic rules successfully matched 95% of records in the three test practices compared to manual matching. Adding two probabilistic rules to the algorithm successfully matched 99% of records. CONCLUSIONS: The potential value of community-acquired retinal images can be harnessed only if they are linked to centrally-held healthcare care data. Despite the lack of interoperability between systems within optometry practices and inconsistent use of unique identifiers, data linkage is possible using robust, almost entirely automated processes.

Corneal biomechanics are not exclusively compromised in high myopia.

INTRODUCTION: Research assuming linearity has concluded that corneal biomechanics are compromised in high myopia. We investigated whether this assumption was appropriate and re-examined these associations across different levels of myopia. METHODS: Myopic (spherical equivalent refraction, SER ≤ -0.50 D) eyes of 10,488 adults aged 40-69 years without any history of systemic and ocular conditions were identified in the UK Biobank. Ordinary least squares (OLS) regression was employed to test the linear association between corneal hysteresis (CH) or corneal resistance factor (CRF), separately, and SER while controlling for age, sex, corneal radius and intraocular pressure. Quantile regression (QR) was used to test the same set of associations across 49 equally spaced conditional quantiles of SER. RESULTS: In OLS regression, each standard deviation (SD) decrease in CH and CRF was associated with 0.08 D (95% CI: 0.04-0.12; p < 0.001) and 0.10 D (95% CI: 0.04-0.15; p < 0.001) higher myopia, respectively. However, residual analysis indicated that the linearity assumption was violated. QR revealed no evidence of a significant association between CH/CRF and SER in low myopia, but a significant (p < 0.05) positive association became evident from -2.78 D (0.06 and 0.08 D higher myopia per SD decrease in CH and CRF). The magnitude of association increased exponentially with increasing myopia: in the -5.03 D quantile, every SD decrease in CH and CRF was associated with 0.17 D (95% CI: 0.08-0.25; p < 0.001) and 0.21 D (95% CI: 0.10-0.31; p < 0.001) higher myopia. In the -8.63 D quantile, this further increased to 0.54 D (95% CI: 0.33-0.76; p < 0.001) and 0.67 D (95% CI: 0.41-0.93; p < 0.001) higher myopia per SD decrease in CH and CRF. CONCLUSIONS: Corneal biomechanics appeared compromised from around -3.00 D. These changes were observed to be exponential with increasing myopia.

Factors associated with pathologic myopia onset and progression: A systematic review and meta-analysis.

PURPOSE: To synthesise evidence across studies on factors associated with pathologic myopia (PM) onset and progression based on the META-analysis for Pathologic Myopia (META-PM) classification framework. METHODS: Findings from six longitudinal studies (5-18 years) were narratively synthesised and meta-analysed, using odds ratio (OR) as the common measure of association. All studies adjusted for baseline myopia, age and sex at a minimum. The quality of evidence was rated using the Grades of Recommendation, Assessment, Development and Evaluation framework. RESULTS: Five out of six studies were conducted in Asia. There was inconclusive evidence of an independent effect (or lack thereof) of ethnicity and sex on PM onset/progression. The odds of PM onset increased with greater axial length (pooled OR: 2.03; 95% CI: 1.71-2.40; p < 0.001), older age (pooled OR: 1.07; 1.05-1.09; p < 0.001) and more negative spherical equivalent refraction, SER (OR: 0.77; 0.68-0.87; p < 0.001), all of which were supported by an acceptable level of evidence. Fundus tessellation was found to independently increase the odds of PM onset in a population-based study (OR: 3.02; 2.58-3.53; p < 0.001), although this was only supported by weak evidence. There was acceptable evidence that greater axial length (pooled OR: 1.23; 1.09-1.39; p < 0.001), more negative SER (pooled OR: 0.87; 0.83-0.92; p < 0.001) and higher education level (pooled OR: 3.17; 1.36-7.35; p < 0.01) increased the odds of PM progression. Other baseline factors found to be associated with PM progression but currently supported by weak evidence included age (pooled OR: 1.01), severity of myopic maculopathy (OR: 3.61), intraocular pressure (OR: 1.62) and hypertension (OR: 0.21). CONCLUSIONS: Most PM risk/prognostic factors are not supported by an adequate evidence base at present (an indication that PM remains understudied). Current factors for which an acceptable level of evidence exists (limited in number) are unmodifiable in adults and lack personalised information. More longitudinal studies focusing on uncovering modifiable factors and imaging biomarkers are warranted.

Compressed vessels bias red blood cell partitioning at bifurcations in a hematocrit-dependent manner: Implications in tumor blood flow.

The tumor microenvironment is abnormal and associated with tumor tissue hypoxia, immunosuppression, and poor response to treatment. One important abnormality present in tumors is vessel compression. Vessel decompression has been shown to increase survival rates in animal models via enhanced and more homogeneous oxygenation. However, our knowledge of the biophysical mechanisms linking tumor decompression to improved tumor oxygenation is limited. In this study, we propose a computational model to investigate the impact of vessel compression on red blood cell (RBC) dynamics in tumor vascular networks. Our results demonstrate that vessel compression can alter RBC partitioning at bifurcations in a hematocrit-dependent and flow rate-independent manner. We identify RBC focusing due to cross-streamline migration as the mechanism responsible and characterize the spatiotemporal recovery dynamics controlling downstream partitioning. Based on this knowledge, we formulate a reduced-order model that will help future research to elucidate how these effects propagate at a whole vascular network level. These findings contribute to the mechanistic understanding of hemodilution in tumor vascular networks and oxygen homogenization following pharmacological solid tumor decompression.

Red blood cell lingering modulates hematocrit distribution in the microcirculation.

The distribution of red blood cells (RBCs) in the microcirculation determines the oxygen delivery and solute transport to tissues. This process relies on the partitioning of RBCs at successive bifurcations throughout the microvascular network, and it has been known since the last century that RBCs partition disproportionately to the fractional blood flow rate, therefore leading to heterogeneity of the hematocrit (i.e., volume fraction of RBCs in blood) in microvessels. Usually, downstream of a microvascular bifurcation, the vessel branch with a higher fraction of blood flow receives an even higher fraction of RBC flux. However, both temporal and time-average deviations from this phase-separation law have been observed in recent studies. Here, we quantify how the microscopic behavior of RBC lingering (i.e., RBCs temporarily residing near the bifurcation apex with diminished velocity) influences their partitioning, through combined in vivo experiments and in silico simulations. We developed an approach to quantify the cell lingering at highly confined capillary-level bifurcations and demonstrate that it correlates with deviations of the phase-separation process from established empirical predictions by Pries et al. Furthermore, we shed light on how the bifurcation geometry and cell membrane rigidity can affect the lingering behavior of RBCs; e.g., rigid cells tend to linger less than softer ones. Taken together, RBC lingering is an important mechanism that should be considered when studying how abnormal RBC rigidity in diseases such as malaria and sickle-cell disease could hinder the microcirculatory blood flow or how the vascular networks are altered under pathological conditions (e.g., thrombosis, tumors, aneurysm).

Abnormal morphology biases hematocrit distribution in tumor vasculature and contributes to heterogeneity in tissue oxygenation.

Oxygen heterogeneity in solid tumors is recognized as a limiting factor for therapeutic efficacy. This heterogeneity arises from the abnormal vascular structure of the tumor, but the precise mechanisms linking abnormal structure and compromised oxygen transport are only partially understood. In this paper, we investigate the role that red blood cell (RBC) transport plays in establishing oxygen heterogeneity in tumor tissue. We focus on heterogeneity driven by network effects, which are challenging to observe experimentally due to the reduced fields of view typically considered. Motivated by our findings of abnormal vascular patterns linked to deviations from current RBC transport theory, we calculated average vessel lengths [Formula: see text] and diameters [Formula: see text] from tumor allografts of three cancer cell lines and observed a substantial reduction in the ratio [Formula: see text] compared to physiological conditions. Mathematical modeling reveals that small values of the ratio λ (i.e., [Formula: see text]) can bias hematocrit distribution in tumor vascular networks and drive heterogeneous oxygenation of tumor tissue. Finally, we show an increase in the value of λ in tumor vascular networks following treatment with the antiangiogenic cancer agent DC101. Based on our findings, we propose λ as an effective way of monitoring the efficacy of antiangiogenic agents and as a proxy measure of perfusion and oxygenation in tumor tissue undergoing antiangiogenic treatment.

Loss of the heterogeneous expression of flippase ATP11B leads to cerebral small vessel disease in a normotensive rat model.

Cerebral small vessel disease (SVD) is the leading cause of vascular dementia, causes a quarter of strokes, and worsens stroke outcomes. The disease is characterised by patchy cerebral small vessel and white matter pathology, but the underlying mechanisms are poorly understood. This microvascular and tissue damage has been classically considered secondary to extrinsic factors, such as hypertension, but this fails to explain the patchy nature of the disease, the link to endothelial cell (EC) dysfunction even when hypertension is absent, and the increasing evidence of high heritability to SVD-related brain damage. We have previously shown the link between deletion of the phospholipase flippase Atp11b and EC dysfunction in an inbred hypertensive rat model with SVD-like pathology and a single nucleotide polymorphism (SNP) in ATP11B associated with human sporadic SVD. Here, we generated a novel normotensive transgenic rat model, where Atp11b is deleted, and show pathological, imaging and behavioural changes typical of those in human SVD, but that occur without hypertension. Atp11bKO rat brain and retinal small vessels show ECs with molecular and morphological changes of dysfunction, with myelin disruption in a patchy pattern around some but not all brain small vessels, similar to the human brain. We show that ATP11B/ATP11B is heterogeneously expressed in ECs in normal rat and human brain even in the same transverse section of the same blood vessel, suggesting variable effects of the loss of ATP11B on each vessel and an explanation for the patchy nature of the disease. This work highlights a link between inherent EC dysfunction and vulnerability to SVD white matter damage with a marked heterogeneity of ECs in vivo which modulates this response, occurring even in the absence of hypertension. These findings refocus our strategies for therapeutics away from antihypertensive (and vascular risk factor) control alone and towards ECs in the effort to provide alternative targets to prevent a major cause of stroke and dementia.

On the preservation of vessel bifurcations during flow-mediated angiogenic remodelling.

During developmental angiogenesis, endothelial cells respond to shear stress by migrating and remodelling the initially hyperbranched plexus, removing certain vessels whilst maintaining others. In this study, we argue that the key regulator of vessel preservation is cell decision behaviour at bifurcations. At flow-convergent bifurcations where migration paths diverge, cells must finely tune migration along both possible paths if the bifurcation is to persist. Experiments have demonstrated that disrupting the cells' ability to sense shear or the junction forces transmitted between cells impacts the preservation of bifurcations during the remodelling process. However, how these migratory cues integrate during cell decision making remains poorly understood. Therefore, we present the first agent-based model of endothelial cell flow-mediated migration suitable for interrogating the mechanisms behind bifurcation stability. The model simulates flow in a bifurcated vessel network composed of agents representing endothelial cells arranged into a lumen which migrate against flow. Upon approaching a bifurcation where more than one migration path exists, agents refer to a stochastic bifurcation rule which models the decision cells make as a combination of flow-based and collective-based migratory cues. With this rule, cells favour branches with relatively larger shear stress or cell number. We found that cells must integrate both cues nearly equally to maximise bifurcation stability. In simulations with stable bifurcations, we found competitive oscillations between flow and collective cues, and simulations that lost the bifurcation were unable to maintain these oscillations. The competition between these two cues is haemodynamic in origin, and demonstrates that a natural defence against bifurcation loss during remodelling exists: as vessel lumens narrow due to cell efflux, resistance to flow and shear stress increases, attracting new cells to enter and rescue the vessel from regression. Our work provides theoretical insight into the role of junction force transmission has in stabilising vasculature during remodelling and as an emergent mechanism to avoid functional shunting.

Emergent cell-free layer asymmetry and biased haematocrit partition in a biomimetic vascular network of successive bifurcations.

Blood is a vital soft matter, and its normal circulation in the human body relies on the distribution of red blood cells (RBCs) at successive bifurcations. Understanding how RBCs are partitioned at bifurcations is key for the optimisation of microfluidic devices as well as for devising novel strategies for diagnosis and treatment of blood-related diseases. We report the dynamics of RBC suspensions flowing through a biomimetic vascular network incorporating three generations of microchannels and two classical types of bifurcations at the arteriole level. Our microfluidic experiments with dilute and semidilute RBC suspensions demonstrate the emergence of excessive heterogeneity of RBC concentration in downstream generations upon altering the network's outflow rates. Through parallel simulations using the immersed-boundary-lattice-Boltzmann method, we reveal that the heterogeneity is attributed to upstream perturbations in the cell-free layer (CFL) and lack of its recovery between consecutive bifurcations owing to suppressed hydrodynamic lift under reduced flow conditions. In the dilute/semidilute regime, this perturbation dominates over the effect of local fractional flow at the bifurcation and can lead to inherently unfavourable child branches that are deprived of RBCs even for equal flow split. Our work highlights the importance of CFL asymmetry cascading down a vascular network, which leads to biased phase separation that deviates from established empirical predictions.

Mechanisms of enhanced drug delivery in brain metastases with focused ultrasound-induced blood-tumor barrier disruption.

Blood-brain/blood-tumor barriers (BBB and BTB) and interstitial transport may constitute major obstacles to the transport of therapeutics in brain tumors. In this study, we examined the impact of focused ultrasound (FUS) in combination with microbubbles on the transport of two relevant chemotherapy-based anticancer agents in breast cancer brain metastases at cellular resolution: doxorubicin, a nontargeted chemotherapeutic, and ado-trastuzumab emtansine (T-DM1), an antibody-drug conjugate. Using an orthotopic xenograft model of HER2-positive breast cancer brain metastasis and quantitative microscopy, we demonstrate significant increases in the extravasation of both agents (sevenfold and twofold for doxorubicin and T-DM1, respectively), and we provide evidence of increased drug penetration (>100 vs. <20 µm and 42 ± 7 vs. 12 ± 4 µm for doxorubicin and T-DM1, respectively) after the application of FUS compared with control (non-FUS). Integration of experimental data with physiologically based pharmacokinetic (PBPK) modeling of drug transport reveals that FUS in combination with microbubbles alleviates vascular barriers and enhances interstitial convective transport via an increase in hydraulic conductivity. Experimental data demonstrate that FUS in combination with microbubbles enhances significantly the endothelial cell uptake of the small chemotherapeutic agent. Quantification with PBPK modeling reveals an increase in transmembrane transport by more than two orders of magnitude. PBPK modeling indicates a selective increase in transvascular transport of doxorubicin through small vessel wall pores with a narrow range of sizes (diameter, 10-50 nm). Our work provides a quantitative framework for the optimization of FUS-drug combinations to maximize intratumoral drug delivery and facilitate the development of strategies to treat brain metastases.

Spatiotemporal Dynamics of Dilute Red Blood Cell Suspensions in Low-Inertia Microchannel Flow.

Microfluidic technologies are commonly used for the manipulation of red blood cell (RBC) suspensions and analyses of flow-mediated biomechanics. To enhance the performance of microfluidic devices, understanding the dynamics of the suspensions processed within is crucial. We report novel, to our knowledge, aspects of the spatiotemporal dynamics of RBC suspensions flowing through a typical microchannel at low Reynolds number. Through experiments with dilute RBC suspensions, we find an off-center two-peak (OCTP) profile of cells contrary to the centralized distribution commonly reported for low-inertia flows. This is reminiscent of the well-known "tubular pinch effect," which arises from inertial effects. However, given the conditions of negligible inertia in our experiments, an alternative explanation is needed for this OCTP profile. Our massively parallel simulations of RBC flow in real-size microfluidic dimensions using the immersed-boundary-lattice-Boltzmann method confirm the experimental findings and elucidate the underlying mechanism for the counterintuitive RBC pattern. By analyzing the RBC migration and cell-free layer development within a high-aspect-ratio channel, we show that such a distribution is co-determined by the spatial decay of hydrodynamic lift and the global deficiency of cell dispersion in dilute suspensions. We find a cell-free layer development length greater than 46 and 28 hydraulic diameters in the experiment and simulation, respectively, exceeding typical lengths of microfluidic designs. Our work highlights the key role of transient cell distribution in dilute suspensions, which may negatively affect the reliability of experimental results if not taken into account.

PolNet: A Tool to Quantify Network-Level Cell Polarity and Blood Flow in Vascular Remodeling.

In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License.

Dynamic endothelial cell rearrangements drive developmental vessel regression.

Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.

Correction: dynamic endothelial cell rearrangements drive developmental vessel regression.

[This corrects the article DOI: 10.1371/journal.pbio.1002125.].

Chaste: an open source C++ library for computational physiology and biology.

Chaste - Cancer, Heart And Soft Tissue Environment - is an open source C++ library for the computational simulation of mathematical models developed for physiology and biology. Code development has been driven by two initial applications: cardiac electrophysiology and cancer development. A large number of cardiac electrophysiology studies have been enabled and performed, including high-performance computational investigations of defibrillation on realistic human cardiac geometries. New models for the initiation and growth of tumours have been developed. In particular, cell-based simulations have provided novel insight into the role of stem cells in the colorectal crypt. Chaste is constantly evolving and is now being applied to a far wider range of problems. The code provides modules for handling common scientific computing components, such as meshes and solvers for ordinary and partial differential equations (ODEs/PDEs). Re-use of these components avoids the need for researchers to 're-invent the wheel' with each new project, accelerating the rate of progress in new applications. Chaste is developed using industrially-derived techniques, in particular test-driven development, to ensure code quality, re-use and reliability. In this article we provide examples that illustrate the types of problems Chaste can be used to solve, which can be run on a desktop computer. We highlight some scientific studies that have used or are using Chaste, and the insights they have provided. The source code, both for specific releases and the development version, is available to download under an open source Berkeley Software Distribution (BSD) licence at http://www.cs.ox.ac.uk/chaste, together with details of a mailing list and links to documentation and tutorials.

Applicability of Oculomics for Individual Risk Prediction: Repeatability and Robustness of Retinal Fractal Dimension Using DART and AutoMorph.

Purpose: To investigate whether fractal dimension (FD)-based oculomics could be used for individual risk prediction by evaluating repeatability and robustness. Methods: We used two datasets: "Caledonia," healthy adults imaged multiple times in quick succession for research (26 subjects, 39 eyes, 377 color fundus images), and GRAPE, glaucoma patients with baseline and follow-up visits (106 subjects, 196 eyes, 392 images). Mean follow-up time was 18.3 months in GRAPE; thus it provides a pessimistic lower bound because vasculature could change. FD was computed with DART and AutoMorph. Image quality was assessed with QuickQual, but no images were initially excluded. Pearson, Spearman, and intraclass correlation (ICC) were used for population-level repeatability. For individual-level repeatability, we introduce measurement noise parameter λ, which is within-eye standard deviation (SD) of FD measurements in units of between-eyes SD. Results: In Caledonia, ICC was 0.8153 for DART and 0.5779 for AutoMorph, Pearson/Spearman correlation (first and last image) 0.7857/0.7824 for DART, and 0.3933/0.6253 for AutoMorph. In GRAPE, Pearson/Spearman correlation (first and next visit) was 0.7479/0.7474 for DART, and 0.7109/0.7208 for AutoMorph (all P < 0.0001). Median λ in Caledonia without exclusions was 3.55% for DART and 12.65% for AutoMorph and improved to up to 1.67% and 6.64% with quality-based exclusions, respectively. Quality exclusions primarily mitigated large outliers. Worst quality in an eye correlated strongly with λ (Pearson 0.5350-0.7550, depending on dataset and method, all P < 0.0001). Conclusions: Repeatability was sufficient for individual-level predictions in heterogeneous populations. DART performed better on all metrics and might be able to detect small, longitudinal changes, highlighting the potential of robust methods.