Visualization of Endothelial Actin Cytoskeleton in the Mouse Retina

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation

N-CAM Exhibits a Regulatory Function in Pathological Angiogenesis in Oxygen Induced Retinopathy

Background: Diabetic retinopathy and retinopathy of prematurity are diseases caused by pathological angiogenesis in the retina as a consequence of local hypoxia. The underlying mechanism for epiretinal neovascularization (tuft formation), which contributes to blindness, has yet to be identified. Neural cell adhesion molecule (N-CAM) is expressed by Muller cells and astrocytes, which are in close contact with the retinal vasculature, during normal developmental angiogenesis. Methodology/Principal Findings: Notably, during oxygen induced retinopathy (OIR) N-CAM accumulated on astrocytes surrounding the epiretinal tufts. Here, we show that N-CAM ablation results in reduced vascular tuft formation due to reduced endothelial cell proliferation despite an elevation in VEGFA mRNA expression, whereas retinal developmental angiogenesis was unaffected. Conclusion/Significance: We conclude that N-CAM exhibits a regulatory function in pathological angiogenesis in OIR. This is a novel finding that can be of clinical relevance in diseases associated with proliferative vasculopathy

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

Copyright: © 2021 Edgar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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.L.T.E, C.A.F, H.G., and M.O.B. would like to graciously acknowledge our funding as part of a Foundation Leducq Transatlantic Network of Excellence (17 CVD 03, https://www.mdc-berlin.de/leducq-attract). M.O.B is supported by grants from EPSRC (EP/R029598/1, EP/R021600/1). C.A.F was supported by European Research Council starting grant (679368), the Fundação para a Ciência e a Tecnologia funding (grants: PTDC/MED-PAT/31639/2017; PTDC/BIA-CEL/32180/2017; CEECIND/04251/2017). C.A.F. and M.O.B are supported by a grant from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 801423. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Remodeling of an in vitro microvessel exposed to cyclic mechanical stretch.

In the lung, vascular endothelial cells experience cyclic mechanical strain resulting from rhythmic breathing motions and intraluminal blood pressure. Mechanical stress creates evident physiological, morphological, biochemical, and gene expression changes in vascular endothelial cells. However, the exact mechanisms of the mechanical signal transduction into biological response remain to be clarified. Besides, the level of mechanical stress is difficult to determine due to the complexity of the local distension patterns in the lung and thus assumed to be the same as the one acting on the alveolar epithelium. Existing in vitro models used to investigate the effect of mechanical stretch on endothelial cells are usually limited to two-dimensional (2D) cell culture platforms, which poorly mimic the typical three-dimensional structure of the vessels. Therefore, the development of an advanced in vitro vasculature model that closely mimics the dynamic of the human lung vasculatures is highly needed. Here, we present the first study that investigates the interplay of the three-dimensional (3D) mechanical cyclic stretch and its magnitude with vascular endothelial growth factor (VEGF) stimulation on a 3D perfusable vasculature in vitro. We studied the effects of the cyclic strain on a perfusable 3D vasculature, either made of human lung microvascular endothelial cells or human umbilical vein endothelial cells embedded in a gel layer. The in vitro 3D vessels underwent both in-vivo-like longitudinal and circumferential deformations, simultaneously. Our results showed that the responses of the human lung microvascular endothelial cells and human umbilical vein endothelial cells to cyclic stretch were in good agreement. Although our 3D model was in agreement with 2D model in predicting a cytoskeletal remodeling in response to different magnitudes of cyclic stretch, however we observed several phenomena in 3D model that 2D model was unable to predict to. Angiogenic sprouting induced by VEGF decreased significantly in presence of cyclic stretch. Similarly, while treatment with VEGF increased vascular permeability, the cyclic stretch restored vascular barrier tightness and significantly decreased vascular permeability. One of the major findings of this study was that a 3D microvasculature can be exposed to a much higher mechanical cyclic stress level than reported in the literature without any dysfunction of its barrier. For higher magnitudes of the cyclic stretch, the applied longitudinal strain level was 14% and the associated circumferential strain reached the equivalent of 63%. In sharp contrast with our findings, such strain typically leads to the disruption of the endothelial barrier in a 2D stretching assay and is considered pathological. This highlights the importance of 3D modeling to investigate mechanobiology effects rather than using a simple endothelial monolayer which truly recapitulates the in vivo situation

Does self-control depletion affect risk attitudes?

A core prediction of recent “dual-self” models is that risk attitudes depend on self-control. While these models have received a lot of attention, empirical evidence regarding their predictions is lacking. We derive hypotheses from three prominent models for choices between risky monetary payoffs under regular and reduced self-control. We test the hypotheses in a lab experiment, using a well-established ego depletion task to reduce self-control, and measuring risk attitudes via finely graduated choice lists. Manipulation checks document the effectiveness of the depletion task. We find no systematic evidence in favor of the theoretical predictions. In particular, depletion does not increase risk aversion

Long-Lasting Enhancement of Visual Perception with Repetitive Noninvasive Transcranial Direct Current Stimulation

Understanding processes performed by an intact visual cortex as the basis for developing methods that enhance or restore visual perception is of great interest to both researchers and medical practitioners. Here, we explore whether contrast sensitivity, a main function of the primary visual cortex (V1), can be improved in healthy subjects by repetitive, noninvasive anodal transcranial direct current stimulation (tDCS). Contrast perception was measured via threshold perimetry directly before and after intervention (tDCS or sham stimulation) on each day over 5 consecutive days (24 subjects, double- blind study). tDCS improved contrast sensitivity from the second day onwards, with significant effects lasting 24 h. After the last stimulation on day 5, the anodal group showed a significantly greater improvement in contrast perception than the sham group (23 vs. 5%). We found significant long-term effects in only the central 2–4° of the visual field 4 weeks after the last stimulation. We suspect a combination of two factors contributes to these lasting effects. First, the V1 area that represents the central retina was located closer to the polarization electrode, resulting in higher current density. Second, the central visual field is represented by a larger cortical area relative to the peripheral visual field (cortical magnification). This is the first study showing that tDCS over V1 enhances contrast perception in healthy subjects for several weeks. This study contributes to the investigation of the causal relationship between the external modulation of neuronal membrane potential and behavior (in our case, visual perception). Because the vast majority of human studies only show temporary effects after single tDCS sessions targeting the visual system, our study underpins the potential for lasting effects of repetitive tDCS-induced modulation of neuronal excitability

Music-evoked incidental happiness modulates probability weighting during risky lottery choices

We often make decisions with uncertain consequences. The outcomes of the choices we make are usually not perfectly predictable but probabilistic, and the probabilities can be known or unknown. Probability judgments, i.e., the assessment of unknown probabilities, can be influenced by evoked emotional states. This suggests that also the weighting of known probabilities in decision making under risk might be influenced by incidental emotions, i.e., emotions unrelated to the judgments and decisions at issue. Probability weighting describes the transformation of probabilities into subjective decision weights for outcomes and is one of the central components of cumulative prospect theory (CPT) that determine risk attitudes. We hypothesized that music-evoked emotions would modulate risk attitudes in the gain domain and in particular probability weighting. Our experiment featured a within-subject design consisting of four conditions in separate sessions. In each condition, the 41 participants listened to a different kind of music-happy, sad, or no music, or sequences of random tones-and performed a repeated pairwise lottery choice task. We found that participants chose the riskier lotteries significantly more often in the "happy" than in the "sad" and "random tones" conditions. Via structural regressions based on CPT, we found that the observed changes in participants' choices can be attributed to changes in the elevation parameter of the probability weighting function: in the "happy" condition, participants showed significantly higher decision weights associated with the larger payoffs than in the "sad" and "random tones" conditions. Moreover, elevation correlated positively with self-reported music-evoked happiness. Thus, our experimental results provide evidence in favor of a causal effect of incidental happiness on risk attitudes that can be explained by changes in probability weighting

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

The Genetic Basis of Moyamoya Disease

Moyamoya disease (MMD) is a rare cerebrovascular disease characterized by progressive spontaneous bilateral occlusion of the intracranial internal cerebral arteries (ICA) and their major branches with compensatory capillary collaterals resembling a "puff of smoke" (Japanese: Moyamoya) on cerebral angiography. These pathological alterations of the vessels are called Moyamoya arteriopathy or vasculopathy and a further distinction is made between primary and secondary MMD. Clinical presentation depends on age and population, with hemorrhage and ischemic infarcts in particular leading to severe neurological dysfunction or even death. Although the diagnostic suspicion can be posed by MRA or CTA, cerebral angiography is mandatory for diagnostic confirmation. Since no therapy to limit the stenotic lesions or the development of a collateral network is available, the only treatment established so far is surgical revascularization. The pathophysiology still remains unknown. Due to the early age of onset, familial cases and the variable incidence rate between different ethnic groups, the focus was put on genetic aspects early on. Several genetic risk loci as well as individual risk genes have been reported; however, few of them could be replicated in independent series. Linkage studies revealed linkage to the 17q25 locus. Multiple studies on the association of SNPs and MMD have been conducted, mainly focussing on the endothelium, smooth muscle cells, cytokines and growth factors. A variant of the RNF213 gene was shown to be strongly associated with MMD with a founder effect in the East Asian population. Although it is unknown how mutations in the RNF213 gene, encoding for a ubiquitously expressed 591 kDa cytosolic protein, lead to clinical features of MMD, RNF213 has been confirmed as a susceptibility gene in several studies with a gene dosage-dependent clinical phenotype, allowing preventive screening and possibly the development of new therapeutic approaches. This review focuses on the genetic basis of primary MMD only