3DVascNet: An Automated Software for Segmentation and Quantification of Mouse Vascular Networks in 3D.

BACKGROUND: Analysis of vascular networks is an essential step to unravel the mechanisms regulating the physiological and pathological organization of blood vessels. So far, most of the analyses are performed using 2-dimensional projections of 3-dimensional (3D) networks, a strategy that has several obvious shortcomings. For instance, it does not capture the true geometry of the vasculature and generates artifacts on vessel connectivity. These limitations are accepted in the field because manual analysis of 3D vascular networks is a laborious and complex process that is often prohibitive for large volumes. METHODS: To overcome these issues, we developed 3DVascNet, a deep learning-based software for automated segmentation and quantification of 3D retinal vascular networks. 3DVascNet performs segmentation based on a deep learning model, and it quantifies vascular morphometric parameters such as vessel density, branch length, vessel radius, and branching point density. We tested the performance of 3DVascNet using a large data set of 3D microscopy images of mouse retinal blood vessels. RESULTS: We demonstrated that 3DVascNet efficiently segments vascular networks in 3D and that vascular morphometric parameters capture phenotypes detected by using manual segmentation and quantification in 2 dimension. In addition, we showed that, despite being trained on retinal images, 3DVascNet has high generalization capability and successfully segments images originating from other data sets and organs. CONCLUSIONS: Overall, we present 3DVascNet, a freely available software that includes a user-friendly graphical interface for researchers with no programming experience, which will greatly facilitate the ability to study vascular networks in 3D in health and disease. Moreover, the source code of 3DVascNet is publicly available, thus it can be easily extended for the analysis of other 3D vascular networks by other users.

Endothelial cell invasion is controlled by dactylopodia.

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

Defective Flow-Migration Coupling Causes Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. METHODS: Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. RESULTS: ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. CONCLUSIONS: Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.

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.

FOXO1 couples metabolic activity and growth state in the vascular endothelium.

Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.

Methods to quantify endothelial cell front-rear polarity in vivo and in vitro.

PURPOSE OF REVIEW: Endothelial cell (EC) front-rear (axial) polarization in response to chemokines and shear stress is fundamental for angiogenesis. This review provides an overview of the in vitro and in vivo methods that are currently available to quantify EC axial polarity. RECENT FINDINGS: Innovative methodologies and new animal models have been developed to evaluate EC axial polarity. Micropatterning, wound healing and microfluidic assays allow interrogation of signalling mechanisms in vitro. Mouse and zebrafish transgenic lines, in combination with advances in imaging techniques and computational tools, enable interrogation of physiological functions of EC axial polarity in vascular biology during development and in pathology in vivo. SUMMARY: We present a literature-based review of the methods available to study EC polarity. Further refinement of quantitative methods to analyse EC axial polarity using deep learning-based computational tools will generate new understanding on the aetiology of vascular malformations.

Phosphoinositide 3-Kinase-Regulated Pericyte Maturation Governs Vascular Remodeling.

BACKGROUND: Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. METHODS: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreERT2 mice into RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kß isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. RESULTS: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kß, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kß inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. CONCLUSIONS: Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kß activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kß activity.

Alterations in Skin Blood Flow at the Fingertip Are Related to Mortality in Patients With Circulatory Shock.

OBJECTIVES: Skin blood flow is rapidly altered during circulatory shock and may remain altered despite apparent systemic hemodynamic stabilization. We evaluated whether changes in skin blood flow during circulatory shock were related to survival. DESIGN: Prospective study. SETTING: Thirty-five-bed medical-surgical university hospital department of intensive care. SUBJECTS: Twenty healthy volunteers and 70 patients with circulatory shock (< 12 hr duration), defined as the need for vasopressors to maintain mean arterial pressure greater than or equal to 65 mm Hg and signs of altered tissue perfusion. INTERVENTIONS: We assessed skin blood flow using skin laser Doppler on the fingertip for 3 minutes at basal temperature (SBFBT) and at 37°C (SBF37) (thermal challenge test) once in volunteers and at the time of inclusion and after 6, 24, 48, 72, and 96 hours in patients with shock. Capillary refill time and peripheral perfusion index were measured at the same time points on the contralateral hand. MEASUREMENTS AND MAIN RESULTS: The thermal challenge response (ΔSBF/ΔT) was calculated using the following formula: (SBF37-SBFBT)/(37-basal temperature). Area under the receiver operating characteristic curves were calculated to evaluate variables predictive of ICU mortality. At inclusion, skin blood flow and ΔSBF/ΔT were lower in patients than in volunteers. Baseline skin blood flow (31 [17-113] vs 16 [9-32] arbitrary perfusion units; p = 0.01) and ΔSBF/ΔT (4.3 [1.7-10.9] vs 0.9 [0.4-2.9] arbitrary perfusion unit/s) were greater in survivors than in nonsurvivors. Capillary refill time was shorter in survivors than in nonsurvivors; peripheral perfusion index was similar in the two groups. ΔSBF/ΔT (area under the receiver operating characteristic curve 0.94 [0.88-0.99]) and SBFBT (area under the receiver operating characteristic curve 0.83 [0.73-0.93]) had the best predictive value for ICU mortality with cutoff values less than or equal to 1.25 arbitrary perfusion unit/°C (sensitivity 88%, specificity 89%) and less than or equal to 21 arbitrary perfusion unit (sensitivity 84%, specificity 81%), respectively. CONCLUSIONS: Alterations in fingertip skin blood flow can be evaluated using a laser Doppler thermal challenge technique in patients with circulatory shock and are directly related to outcome. These novel monitoring techniques could potentially be used to guide resuscitation.

PAR-3 controls endothelial planar polarity and vascular inflammation under laminar flow.

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3ß activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3ß complexes controls local GSK3ß activity and thereby regulates endothelial planar polarity. The spatial information for GSK3ß activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced anti-inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.

GNrep mouse: A reporter mouse for front-rear cell polarity.

Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front-rear polarity axis for directional movement. Although front-rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live-imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front-rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus-to-Golgi axis to each cell, which functions as a readout for cell front-rear polarity. As a proof-of-principle, we validated the efficiency of the GNrep line using an endothelial-specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large-scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front-rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.

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.].

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase.

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.

SRF selectively controls tip cell invasive behavior in angiogenesis.

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Laminin-binding integrins induce Dll4 expression and Notch signaling in endothelial cells.

RATIONALE: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. OBJECTIVE: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. METHODS AND RESULTS: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2ß1 and α6ß1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2ß1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. CONCLUSIONS: We show that, in endothelial cells, ligation of α2ß1 and α6ß1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo.

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin ß1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditions.

The ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress.

Cell painting transfer increases screening hit rate.

Drug discovery uses high throughput screening to identify compounds that interact with a molecular target or that alter a phenotype favorably. The cautious selection of molecules used for such a screening is instrumental and is tightly related to the hit rate. In this work, we wondered if cell painting, a general-purpose image-based assay, could be used as an efficient proxy for compound selection, thus increasing the success rate of a specific assay. To this end, we considered cell painting images with 30,000 molecules treatments, and selected compounds that produced a visual effect close to the positive control of an assay, by using the Frechet Inception Distance. We then compared the hit rates of such a preselection with what was actually obtained in real screening campaigns. As a result, cell painting would have permitted a significant increase in the success rate and, even for one of the assays, would have allowed to reach 80% of the hits with 10 times fewer compounds to test. We conclude that images of a cell painting assay can be directly used for compound selection prior to screening, and we provide a simple quantitative approach in order to do so.