Cytoplasmic accumulation and plasma membrane association of anillin and Ect2 promote confined migration and invasion.

Cells migrating in confinement experience mechanical challenges whose consequences on cell migration machinery remain only partially understood. Here, we demonstrate that a pool of the cytokinesis regulatory protein anillin is retained during interphase in the cytoplasm of different cell types. Confinement induces recruitment of cytoplasmic anillin to plasma membrane at the poles of migrating cells, which is further enhanced upon nuclear envelope (NE) rupture(s). Rupture events also enable the cytoplasmic egress of predominantly nuclear RhoGEF Ect2. Anillin and Ect2 redistributions scale with microenvironmental stiffness and confinement, and are observed in confined cells in vitro and in invading tumor cells in vivo. Anillin, which binds actomyosin at the cell poles, and Ect2, which activates RhoA, cooperate additively to promote myosin II contractility, and promote efficient invasion and extravasation. Overall, our work provides a mechanistic understanding of how cytokinesis regulators mediate RhoA/ROCK/myosin II-dependent mechanoadaptation during confined migration and invasive cancer progression.

The influence of physiological and pathological perturbations on blood-brain barrier function.

The blood-brain barrier (BBB) is located at the interface between the vascular system and the brain parenchyma, and is responsible for communication with systemic circulation and peripheral tissues. During life, the BBB can be subjected to a wide range of perturbations or stresses that may be endogenous or exogenous, pathological or therapeutic, or intended or unintended. The risk factors for many diseases of the brain are multifactorial and involve perturbations that may occur simultaneously (e.g., two-hit model for Alzheimer's disease) and result in different outcomes. Therefore, it is important to understand the influence of individual perturbations on BBB function in isolation. Here we review the effects of eight perturbations: mechanical forces, temperature, electromagnetic radiation, hypoxia, endogenous factors, exogenous factors, chemical factors, and pathogens. While some perturbations may result in acute or chronic BBB disruption, many are also exploited for diagnostic or therapeutic purposes. The resultant outcome on BBB function depends on the dose (or magnitude) and duration of the perturbation. Homeostasis may be restored by self-repair, for example, via processes such as proliferation of affected cells or angiogenesis to create new vasculature. Transient or sustained BBB dysfunction may result in acute or pathological symptoms, for example, microhemorrhages or hypoperfusion. In more extreme cases, perturbations may lead to cytotoxicity and cell death, for example, through exposure to cytotoxic plaques.

Modeling angiogenesis in the human brain in a tissue-engineered post-capillary venule.

Angiogenesis plays an essential role in embryonic development, organ remodeling, wound healing, and is also associated with many human diseases. The process of angiogenesis in the brain during development is well characterized in animal models, but little is known about the process in the mature brain. Here, we use a tissue-engineered post-capillary venule (PCV) model incorporating stem cell derived induced brain microvascular endothelial-like cells (iBMECs) and pericyte-like cells (iPCs) to visualize the dynamics of angiogenesis. We compare angiogenesis under two conditions: in response to perfusion of growth factors and in the presence of an external concentration gradient. We show that both iBMECs and iPCs can serve as tip cells leading angiogenic sprouts. More importantly, the growth rate for iPC-led sprouts is about twofold higher than for iBMEC-led sprouts. Under a concentration gradient, angiogenic sprouts show a small directional bias toward the high growth factor concentration. Overall, pericytes exhibited a broad range of behavior, including maintaining quiescence, co-migrating with endothelial cells in sprouts, or leading sprout growth as tip cells.

Corrigendum: The influence of physiological and pathological perturbations on blood-brain barrier function.

[This corrects the article DOI: 10.3389/fnins.2023.1289894.].

Three-dimensional microenvironment regulates gene expression, function, and tight junction dynamics of iPSC-derived blood-brain barrier microvessels.

The blood-brain barrier (BBB) plays a pivotal role in brain health and disease. In the BBB, brain microvascular endothelial cells (BMECs) are connected by tight junctions which regulate paracellular transport, and express specialized transporter systems which regulate transcellular transport. However, existing in vitro models of the BBB display variable accuracy across a wide range of characteristics including gene/protein expression and barrier function. Here, we use an isogenic family of fluorescently-labeled iPSC-derived BMEC-like cells (iBMECs) and brain pericyte-like cells (iPCs) within two-dimensional confluent monolayers (2D) and three-dimensional (3D) tissue-engineered microvessels to explore how 3D microenvironment regulates gene expression and function of the in vitro BBB. We show that 3D microenvironment (shear stress, cell-ECM interactions, and cylindrical geometry) increases BBB phenotype and endothelial identity, and alters angiogenic and cytokine responses in synergy with pericyte co-culture. Tissue-engineered microvessels incorporating junction-labeled iBMECs enable study of the real-time dynamics of tight junctions during homeostasis and in response to physical and chemical perturbations.

Hypoxia-induced blood-brain barrier dysfunction is prevented by pericyte-conditioned media via attenuated actomyosin contractility and claudin-5 stabilization.

The blood-brain barrier (BBB) regulates molecular and cellular entry from the cerebrovasculature into the surrounding brain parenchyma. Many diseases of the brain are associated with dysfunction of the BBB, where hypoxia is a common stressor. However, the contribution of hypoxia to BBB dysfunction is challenging to study due to the complexity of the brain microenvironment. In this study, we used a BBB model with brain microvascular endothelial cells and pericytes differentiated from iPSCs to investigate the effect of hypoxia on barrier function. We found that hypoxia-induced barrier dysfunction is dependent upon increased actomyosin contractility and is associated with increased fibronectin fibrillogenesis. We propose a role for actomyosin contractility in mediating hypoxia-induced barrier dysfunction through modulation of junctional claudin-5. Our findings suggest pericytes may protect brain microvascular endothelial cells from hypoxic stresses and that pericyte-derived factors could be candidates for treatment of pathological barrier-forming tissues.

Role of iPSC-derived pericytes on barrier function of iPSC-derived brain microvascular endothelial cells in 2D and 3D.

BACKGROUND: Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell-cell contact. METHODS: We differentiated RFP-pericytes and GFP-BMECs from hiPSCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. RESULTS: We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm2). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. CONCLUSIONS: We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS.

Chipping Away at Blood-Brain-Barrier Modeling.

In this issue, Vatine et al. (2019) present a fully human blood-brain barrier chip that accurately predicts drug permeability and can be perfused with whole blood. Utilizing patient-derived tissue, they recapitulate disease-specific defects and establish a platform to advance drug screening and disease modeling.

Benchmarking in vitro tissue-engineered blood-brain barrier models.

The blood-brain barrier (BBB) plays a key role in regulating transport into and out of the brain. With increasing interest in the role of the BBB in health and disease, there have been significant advances in the development of in vitro models. The value of these models to the research community is critically dependent on recapitulating characteristics of the BBB in humans or animal models. However, benchmarking in vitro models is surprisingly difficult since much of our knowledge of the structure and function of the BBB comes from in vitro studies. Here we describe a set of parameters that we consider a starting point for benchmarking and validation. These parameters are associated with structure (ultrastructure, wall shear stress, geometry), microenvironment (basement membrane and extracellular matrix), barrier function (transendothelial electrical resistance, permeability, efflux transport), cell function (expression of BBB markers, turnover), and co-culture with other cell types (astrocytes and pericytes). In suggesting benchmarks, we rely primarily on imaging or direct measurements in humans and animal models.

Engineering the human blood-brain barrier in vitro.

The blood-brain barrier (BBB) is the interface between the vasculature and the brain, regulating molecular and cellular transport into the brain. Endothelial cells (ECs) that form the capillary walls constitute the physical barrier but are dependent on interactions with other cell types. In vitro models are widely used in BBB research for mechanistic studies and drug screening. Current models have both biological and technical limitations. Here we review recent advances in stem cell engineering that have been utilized to create innovative platforms to replicate key features of the BBB. The development of human in vitro models is envisioned to enable new mechanistic investigations of BBB transport in central nervous system diseases.

Using surface plasmon resonance imaging to study bacterial biofilms.

This paper describes the use of Surface Plasmon Resonance imaging (SPRi) as an emerging technique to study bacterial physiology in real-time without labels. The overwhelming majority of bacteria on earth exist in large multicellular communities known as biofilms. Biofilms are especially problematic because they facilitate the survival of pathogens, leading to chronic and recurring infections as well as costly industrial complications. Monitoring biofilm accumulation and removal is therefore critical in these and other applications. SPRi uniquely provides label-free, high-resolution images of biomass coverage on large channel surfaces up to 1 cm(2) in real time, which allow quantitative assessment of biofilm dynamics. The rapid imaging capabilities of this technique are particularly relevant for multicellular bacterial studies, as these cells can swim several body lengths per second and divide multiple times per hour. We present here the first application of SPRi to image Escherichia coli and Pseudomonas aeruginosa cells moving, attaching, and forming biofilms across a large surface. This is also the first time that biofilm removal has been visualized with SPRi, which has important implications for monitoring the biofouling and regeneration of fluidic systems. Initial images of the removal process show that the biofilm releases from the surface as a wave along the direction of the fluid flow.