Hypoxia suppresses glucose-induced increases in collective cell migration in vascular endothelial cell monolayers.

Blood glucose levels fluctuate during daily life, and the oxygen concentration is low compared to the atmosphere. Vascular endothelial cells (ECs) maintain vascular homeostasis by sensing changes in glucose and oxygen concentrations, resulting in collective migration. However, the behaviors of ECs in response to high-glucose and hypoxic environments and the underlying mechanisms remain unclear. In this study, we investigated the collective migration of ECs simultaneously stimulated by changes in glucose and oxygen concentrations. Cell migration in EC monolayer formed inside the media channels of microfluidic devices was observed while varying the glucose and oxygen concentrations. The cell migration increased with increasing glucose concentration under normoxic condition but decreased under hypoxic condition, even in the presence of high glucose levels. In addition, inhibition of mitochondrial function reduced the cell migration regardless of glucose and oxygen concentrations. Thus, oxygen had a greater impact on cell migration than glucose, and aerobic energy production in mitochondria plays an important mechanistic role. These results provide new insights regarding vascular homeostasis relative to glucose and oxygen concentration changes.

Behavior of breast cancer cells under oxygen concentration gradients in a microfluidic device.

The tumor microenvironment (TME) is known as a chronic hypoxic environment, with spatiotemporal variation in oxygen concentration depending on the distance from blood vessels and the blood supply. In our previous studies, cancer cell behavior was observed under hypoxic conditions with spatial variation of oxygen concentration (oxygen concentration gradients); however, that under oxygen concentration gradients at low oxygen levels found in the TME has not been studied. In this study, we investigated the behavior of breast cancer cells at various oxygen concentration gradients, generated using a microfluidic device with oxygen concentration controllability. The results showed that cell distribution was altered in response to oxygen concentration, and tended to increase in a specific region at around 5% O2. Evaluation of changes in cell numbers due to proliferation, migration, and cell death indicated that proliferation strongly affected cell distribution.

The aerotaxis of Dictyostelium discoideum is independent of mitochondria, nitric oxide and oxidative stress.

Spatial and temporal variations of oxygen environments affect the behaviors of various cells and are involved in physiological and pathological events. Our previous studies with Dictyostelium discoideum as a model of cell motility have demonstrated that aerotaxis toward an oxygen-rich region occurs below 2% O2. However, while the aerotaxis of Dictyostelium seems to be an effective strategy to search for what is essential for survival, the mechanism underlying this phenomenon is still largely unclear. One hypothesis is that an oxygen concentration gradient generates a secondary oxidative stress gradient that would direct cell migration towards higher oxygen concentration. Such mechanism was inferred but not fully demonstrated to explain the aerotaxis of human tumor cells. Here, we investigated the role on aerotaxis of flavohemoglobins, proteins that can both act as potential oxygen sensors and modulators of nitric oxide and oxidative stress. The migratory behaviors of Dictyostelium cells were observed under both self-generated and imposed oxygen gradients. Furthermore, their changes by chemicals generating or preventing oxidative stress were tested. The trajectories of the cells were then analyzed through time-lapse phase-contrast microscopic images. The results indicate that both oxidative and nitrosative stresses are not involved in the aerotaxis of Dictyostelium but cause cytotoxic effects that are enhanced upon hypoxia.

Interstitial-fluid shear stresses induced by vertically oscillating head motion lower blood pressure in hypertensive rats and humans.

The mechanisms by which physical exercise benefits brain functions are not fully understood. Here, we show that vertically oscillating head motions mimicking mechanical accelerations experienced during fast walking, light jogging or treadmill running at a moderate velocity reduce the blood pressure of rats and human adults with hypertension. In hypertensive rats, shear stresses of less than 1 Pa resulting from interstitial-fluid flow induced by such passive head motions reduced the expression of the angiotensin II type-1 receptor in astrocytes in the rostral ventrolateral medulla, and the resulting antihypertensive effects were abrogated by hydrogel introduction that inhibited interstitial-fluid movement in the medulla. Our findings suggest that oscillatory mechanical interventions could be used to elicit antihypertensive effects.

Microfluidic platform for the reproduction of hypoxic vascular microenvironments.

Vascular endothelial cells (ECs) respond to mechanical stimuli caused by blood flow to maintain vascular homeostasis. Although the oxygen level in vascular microenvironment is lower than the atmospheric one, the cellular dynamics of ECs under hypoxic and flow exposure are not fully understood. Here, we describe a microfluidic platform for the reproduction hypoxic vascular microenvironments. Simultaneous application of hypoxic stress and fluid shear stress to the cultured cells was achieved by integrating a microfluidic device and a flow channel that adjusted the initial oxygen concentration in a cell culture medium. An EC monolayer was then formed on the media channel in the device, and the ECs were observed after exposure to hypoxic and flow conditions. The migration velocity of the ECs immediately increased after flow exposure, especially in the direction opposite to the flow direction, and gradually decreased, resulting in the lowest value under the hypoxic and flow exposure condition. The ECs after 6-h simultaneous exposure to hypoxic stress and fluid shear stress were generally aligned and elongated in the flow direction, with enhanced VE-cadherin expression and actin filament assembly. Thus, the developed microfluidic platform is useful for investigating the dynamics of ECs in vascular microenvironments.

Development of Ultrasound Phantom Made of Transparent Material: Feasibility of Optical Particle Image Velocimetry.

OBJECTIVE: The need for ultrasound flow phantoms to validate ultrasound systems requires the development of materials that can clearly visualize the flow inside for measurement purposes. METHODS: A transparent ultrasound flow phantom material composed of poly(vinyl alcohol) hydrogel (PVA-H) with dimethyl sulfoxide (DMSO) and water solution manufactured using the freezing method and mixed with quartz glass powder to exhibit scattering effects is proposed. To achieve transparency of the hydrogel phantom, the refractive index (RI) was changed to match that of the glass by modifying the PVA concentration and the ratio of DMSO to water in the solvent. The feasibility of optical particle image velocimetry (PIV) was verified by comparing an acrylic rectangular cross-section channel with a rigid wall. After the feasibility tests, an ultrasound flow phantom was fabricated to conduct ultrasound B-mode visualization and Doppler-PIV comparison. DISCUSSION: The results revealed that the PIV measured through PVA-H material exhibited 0.8% error in the measured maximum velocity compared with PIV through the acrylic material. B-mode images are similar to real tissue visualization with a limitation of a higher sound velocity, when compared with human tissue, of 1792 m/s. Doppler measurement of the phantom revealed approximately 120% and 19% overestimation of maximum and mean velocities, respectively, compared with those from PIV. CONCLUSION: The proposed material possesses the advantage of the single-phantom ability to improve the ultrasound flow phantom for validation of flow.

Region-based SVD processing of high-frequency ultrafast ultrasound to visualize cutaneous vascular networks.

Observing alterations in cutaneous vasculature in response to any disease or pathology is considered as a potential diagnostic marker in the progression and cure of a disease. To observe skin morphologies and tissue conditions, high-frequency ultrasound (HFUS) has been used in dermatology, although its ability to selectively visualize micro-vessels is limited due to insufficient Doppler sensitivity to peripheral slow-speed blood flow. In recent studies, this issue has been improved by increasing the sensitivity of Doppler imaging to slow flow, leveraging advanced cutter filtering approaches based on singular value decomposition (SVD) techniques that aid to effectively extract flow signals overlapped with tissue echo signals. Nevertheless, in skin imaging, variations in flow speed, diameter, and depth of the blood vessels at different skin layers can make clutter filtering challenging because these variations are problematic in selecting the optimal cut-off value for the SVD filtering. In this study, we aimed to devise a novel region-based SVD filtering approach for ultrafast HFUS data to visualize cutaneous vascular networks. The proposed method divides the acquired high-framerate data into two regions based on B-mode cutaneous morphological identification (dermis layer and subcutaneous tissue). Singular value decomposition processing was performed on each region to effectively extract the desired flow signal, and the processed regions were merged to generate a single power Doppler image, thereby highlighting the appearance of a complete cutaneous vascular network. Finally, top-hat transform was applied to the power Doppler image to further suppress the background noises and enhances the visibility of the micro-vessels. Experimental observations of the human cutaneous circulation showed that the image quality (contrast-to-noise ratio) through the region-based SVD filtering was measured to be 4.1 dB (before top-hat filtering) and 5.2 dB (after top-hat filtering), which were improved from 3.4 dB and 4.0 dB obtained using the global SVD approach with and without top-hat filtering, respectively. We envisioned that this approach would provide diverse applications in the diagnosis of cutaneous disorders.

Stiffness of primordial germ cells is required for their extravasation in avian embryos.

Unlike mammals, primordial germ cells (PGCs) in avian early embryos exploit blood circulation to translocate to the somatic gonadal primordium, but how circulating PGCs undergo extravasation remains elusive. We demonstrate with single-cell level live-imaging analyses that the PGCs are arrested at a specific site in the capillary plexus, which is predominantly governed by occlusion at a narrow path in the vasculature. The occlusion is enabled by a heightened stiffness of the PGCs mediated by actin polymerization. Following the occlusion, PGCs reset their stiffness to soften in order to squeeze through the endothelial lining as they transmigrate. Our discovery also provides a model for the understanding of metastasizing cancer extravasation occurring mainly by occlusion.

A Review of Functional Analysis of Endothelial Cells in Flow Chambers.

The vascular endothelial cells constitute the innermost layer. The cells are exposed to mechanical stress by the flow, causing them to express their functions. To elucidate the functions, methods involving seeding endothelial cells as a layer in a chamber were studied. The chambers are known as parallel plate, T-chamber, step, cone plate, and stretch. The stimulated functions or signals from endothelial cells by flows are extensively connected to other outer layers of arteries or organs. The coculture layer was developed in a chamber to investigate the interaction between smooth muscle cells in the middle layer of the blood vessel wall in vascular physiology and pathology. Additionally, the microfabrication technology used to create a chamber for a microfluidic device involves both mechanical and chemical stimulation of cells to show their dynamics in in vivo microenvironments. The purpose of this study is to summarize the blood flow (flow inducing) for the functions connecting to endothelial cells and blood vessels, and to find directions for future chamber and device developments for further understanding and application of vascular functions. The relationship between chamber design flow, cell layers, and microfluidics was studied.

Model-based estimation of QT intervals of mouse fetal electrocardiogram.

BACKGROUND: Abnormal prolongation in the QT interval or long QT syndrome (LQTS) is associated with several cardiac complications such as sudden infant death syndrome (SIDS). LQTS is believed to be linked to genetic mutations which can be understood by using animal models, such as mice models. Nevertheless, the research related to fetal QT interval in mice is still limited because of challenges associated with T wave measurements in fetal electrocardiogram (fECG). Reliable measurement of T waves is essential for estimating their end timings for QT interval assessment. RESULTS: A mathematical model was used to estimate QT intervals. Estimated QT intervals were validated with Q-aortic closure (Q-Ac) intervals of Doppler ultrasound (DUS) and comparison between both showed good agreement with a correlation coefficient higher than 0.88 (r > 0.88, P < 0.05). CONCLUSION: Model-based estimation of QT intervals can help in better understanding of QT intervals in fetal mice.

P21-activated kinase regulates oxygen-dependent migration of vascular endothelial cells in monolayers.

The collective migration of vascular endothelial cells plays important roles in homeostasis and angiogenesis. Oxygen concentration in vivo, which is lower than in the atmosphere and changes due to diseases, is a key factor affecting the cellular dynamics of vascular endothelial cells. We previously reported that hypoxic conditions promote the internalization of vascular endothelial (VE)-cadherin, a specific cell-cell adhesion molecule, and increase the velocity of the collective migration of vascular endothelial cells. However, the mechanism through which cells regulate collective migration as affected by oxygen tension is not fully understood. Here, we investigated oxygen-dependent collective migration, focusing on intracellular protein p21-activated kinase (PAK) and hypoxia-inducing factor (HIF)-1α. A monolayer of human umbilical vein vascular endothelial cells (HUVECs) was formed in a microfluidic device with controllability of oxygen tension. The HUVECs were then exposed to various oxygen conditions in a range from 0.8% to 21% O2, with or without PAK inhibition or chemical stabilization of HIF-1α. Collective cell migration was measured by particle image velocimetry with time-lapse phase-contrast microscopic images. Localizations of VE-cadherin and HIF-1α were quantified by immunofluorescent staining. The collective migration of HUVECs varied in an oxygen-dependent fashion; the migration speed was increased by hypoxic exposure down to 1% O2, while it decreased under an extremely low oxygen tension of less than 1% O2. PAK inhibition suppressed the hypoxia-induced increase of the migration speed by preventing VE-cadherin internalization into HUVECs. A decrease in the migration speed was also obtained by chemical stabilization of HIF-1α, suggesting that excessive accumulation of HIF-1α diminishes collective cell migration. These results indicate that the oxygen-dependent variation of the migration speed of vascular endothelial cells is mediated by the regulation of VE-cadherin through the PAK pathway, as well as other mechanisms via HIF-1α, especially under extreme hypoxic conditions.

Hypoxia triggers collective aerotactic migration in Dictyostelium discoideum.

Using a self-generated hypoxic assay, we show that the amoeba Dictyostelium discoideum displays a remarkable collective aerotactic behavior. When a cell colony is covered, cells quickly consume the available oxygen (O2) and form a dense ring moving outwards at constant speed and density. To decipher this collective process, we combined two technological developments: porphyrin-based O2 -sensing films and microfluidic O2 gradient generators. We showed that Dictyostelium cells exhibit aerotactic and aerokinetic response in a low range of O2 concentration indicative of a very efficient detection mechanism. Cell behaviors under self-generated or imposed O2 gradients were modeled using an in silico cellular Potts model built on experimental observations. This computational model was complemented with a parsimonious 'Go or Grow' partial differential equation (PDE) model. In both models, we found that the collective migration of a dense ring can be explained by the interplay between cell division and the modulation of aerotaxis.

Cancer cell migration and cancer drug screening in oxygen tension gradient chip.

Cancer metastasis, which is prevalent in malignant tumors, is present in a variety of cases depending on the primary tumor and metastatic site. The cancer metastasis is affected by various factors that surround and constitute a tumor microenvironment. One of the several factors, oxygen tension, can affect cancer cells and induce changes in many ways, including motility, directionality, and viability. In particular, the oxygen tension gradient is formed within a tumor cluster and oxygen is lower toward the center of the cluster from the perivascular area. The simple and efficient designing of the tumor microenvironment using microfluidic devices enables the simplified and robust platform of the complex in vivo microenvironment while observing a clear cause-and-effect between the properties of cancer cells under oxygen tension. Here, a microfluidic device with five channels including a gel channel, media channels, and gas channels is designed. MDA-MB-231cells are seeded in the microfluidic device with hydrogel to simulate their three-dimensional movement in the body. The motility and directionality of the cancer cells under the normoxic and oxygen tension gradient conditions are compared. Also, the viability of the cancer cells is analyzed for each condition when anticancer drugs are applied. Unlike the normoxic condition, under the oxygen tension gradient, cancer cells showed directionality toward higher oxygen tension and decreased viability against the certain anticancer drug. The simplified design of the tumor microenvironment through microfluidic devices enables comprehension of the response of cancer cells to varying oxygen tensions and cancer drugs in the hypoxic tumor microenvironment.

Hydrostatic pressure promotes endothelial tube formation through aquaporin 1 and Ras-ERK signaling.

Vascular tubulogenesis is tightly linked with physiological and pathological events in the living body. Endothelial cells (ECs), which are constantly exposed to hemodynamic forces, play a key role in tubulogenesis. Hydrostatic pressure in particular has been shown to elicit biophysical and biochemical responses leading to EC-mediated tubulogenesis. However, the relationship between tubulogenesis and hydrostatic pressure remains to be elucidated. Here, we propose a specific mechanism through which hydrostatic pressure promotes tubulogenesis. We show that pressure exposure transiently activates the Ras/extracellular signal-regulated kinase (ERK) pathway in ECs, inducing endothelial tubulogenic responses. Water efflux through aquaporin 1 and activation of protein kinase C via specific G protein-coupled receptors are essential to the pressure-induced transient activation of the Ras/ERK pathway. Our approach could provide a basis for elucidating the mechanopathology of tubulogenesis-related diseases and the development of mechanotherapies for improving human health.

Microfluidic platform for three-dimensional cell culture under spatiotemporal heterogeneity of oxygen tension.

Cells in a tumor microenvironment are exposed to spatial and temporal variations in oxygen tension due to hyperproliferation and immature vascularization. Such spatiotemporal oxygen heterogeneity affects the behavior of cancer cells, leading to cancer growth and metastasis, and thus, it is essential to clarify the cellular responses of cancer cells to oxygen tension. Herein, we describe a new double-layer microfluidic device allowing the control of oxygen tension and the behavior of cancer cells under spatiotemporal oxygen heterogeneity. Two parallel gas channels were located above the media and gel channels to enhance gas exchange, and a gas-impermeable polycarbonate film was embedded in the device to prevent the diffusion of atmospheric oxygen. Variations in oxygen tension in the device with the experimental parameters and design variables were investigated computationally and validated by using oxygen-sensitive nanoparticles. The present device can generate a uniform hypoxic condition at oxygen levels down to 0.3% O2, as well as a linear oxygen gradient from 3% O2 to 17% O2 across the gel channel within 15 min. Moreover, human breast cancer cells suspended in type I collagen gel were introduced in the gel channel to observe their response under controlled oxygen tension. Hypoxic exposure activated the proliferation and motility of the cells, which showed a local maximum increase at 5% O2. Under the oxygen gradient condition, the increase in the cell number was relatively high in the central mild hypoxia region. These findings demonstrate the utility of the present device to study cellular responses in an oxygen-controlled microenvironment.

Migration of vascular endothelial cells in monolayers under hypoxic exposure.

The hypoxic microenvironment existing in vivo is known to significantly affect cell morphology and dynamics, and cell group behaviour. Collective migration of vascular endothelial cells is essential for vasculogenesis and angiogenesis, and for maintenance of monolayer integrity. Although hypoxic stress increases vascular endothelial permeability, the changes in collective migration and intracellular junction morphology of vascular endothelial cells remain poorly understood. This study reveals the migration of confluent vascular endothelial cells and changes in their adherens junction, as reflected by changes in the vascular endothelial (VE)-cadherin distribution, under hypoxic exposure. Vascular endothelial monolayers of human umbilical vein endothelial cells (HUVECs) were formed in microfluidic devices with controllability of oxygen tension. The oxygen tension was set to either normoxia (21% O2) or hypoxia (<3% O2) by supplying gas mixtures into separate gas channels. The migration velocity of HUVECs was measured using particle image velocimetry with a time series of phase-contrast microscopic images of the vascular endothelial monolayers. Hypoxia inducible factor-1α (HIF-1α) and VE-cadherin in HUVECs were observed after exposure to normoxic or hypoxic conditions using immunofluorescence staining and quantitative confocal image analysis. Changes in the migration speed of HUVECs were observed in as little as one hour after exposure to hypoxic condition, showing that the migration speed was increased 1.4-fold under hypoxia compared to that under normoxia. Nuclear translocation of HIF-1α peaked after the hypoxic gas mixture was supplied for 2 h. VE-cadherin expression was also found to be reduced. When ethanol was added to the cell culture medium, cell migration increased. By contrast, by strengthening VE-cadherin junctions with forskolin, cell migration decreased gradually in spite the effect of ethanol to stimulate migration. These results indicate that the increase of cell migration by hypoxic exposure was attributable to loosening of intercellular junction resulting from the decrease of VE-cadherin expression.

Viscosity Estimation of a Suspension with Rigid Spheres in Circular Microchannels Using Particle Tracking Velocimetry.

Suspension flows are ubiquitous in industry and nature. Therefore, it is important to understand the rheological properties of a suspension. The key to understanding the mechanism of suspension rheology is considering changes in its microstructure. It is difficult to evaluate the influence of change in the microstructure on the rheological properties affected by the macroscopic flow field for non-colloidal particles. In this study, we propose a new method to evaluate the changes in both the microstructure and rheological properties of a suspension using particle tracking velocimetry (PTV) and a power-law fluid model. Dilute suspension (0.38%) flows with fluorescent particles in a microchannel with a circular cross section were measured under low Reynolds number conditions (Re ≈ 10-4). Furthermore, the distribution of suspended particles in the radial direction was obtained from the measured images. Based on the power-law index and dependence of relative viscosity on the shear rate, we observed that the non-Newtonian properties of the suspension showed shear-thinning. This method will be useful in revealing the relationship between microstructural changes in a suspension and its rheology.

Ultrasound Imaging of Mouse Fetal Intracranial Hemorrhage Due to Ischemia/Reperfusion.

Despite vast improvement in perinatal care during the 30 years, the incidence rate of neonatal encephalopathy remains unchanged without any further Progress towards preventive strategies for the clinical impasse. Antenatal brain injury including fetal intracranial hemorrhage caused by ischemia/reperfusion is known as one of the primary triggers of neonatal injury. However, the mechanisms of antenatal brain injury are poorly understood unless better predictive models of the disease are developed. Here we show a mouse model for fetal intracranial hemorrhage in vivo developed to investigate the actual timing of hypoxia-ischemic events and their related mechanisms of injury. Intrauterine growth restriction mouse fetuses were exposed to ischemia/reperfusion cycles by occluding and opening the uterine and ovarian arteries in the mother. The presence and timing of fetal intracranial hemorrhage caused by the ischemia/reperfusion were measured with histological observation and ultrasound imaging. Protein-restricted diet increased the risk of fetal intracranial hemorrhage. The monitoring of fetal brains by ultrasound B-mode imaging clarified that cerebral hemorrhage in the fetal brain occurred after the second ischemic period. Three-dimensional ultrasound power Doppler imaging visualized the disappearance of main blood flows in the fetal brain. These indicate a breakdown of cerebrovascular autoregulation which causes the fetal intracranial hemorrhage. This study supports the fact that the ischemia/reperfusion triggers cerebral hemorrhage in the fetal brain. The present method enables us to noninvasively create the cerebral hemorrhage in a fetus without directly touching the body but with repeated occlusion and opening of the uterine and ovarian arteries in the mother.

Endothelial monolayer permeability under controlled oxygen tension.

Endothelial permeability has been extensively investigated in the context of pathologies such as cancer and also in studies of drug delivery from the circulation. Hypoxia is a critical regulator of endothelial cell (EC) behavior and affects the barrier function of endothelial linings, yet its role has been little studied. This paper reveals the effect of hypoxia on the permeability of an EC monolayer by cellular experiments using a microfluidic device and a conventional cell culture dish. Human umbilical vein endothelial cells (HUVECs) were seeded into one microfluidic channel, creating an EC monolayer on each vertical surface of a collagen gel confined to a central chamber. Oxygen tension was regulated to produce normoxic (21% O2) or hypoxic (3% O2) conditions by the supply of gas mixtures of oxygen, carbon dioxide, and nitrogen at predefined ratios into channels fabricated into the device. Permeability of the EC monolayer quantified by analyzing diffusion of fluorescence-labelled dextrans into the collagen gel increases with barrier function loss by 6 hour hypoxic exposure, showing 11-fold and 4-fold increases for 70 kDa and 10 kDa dextrans, respectively, on average. Consistent with this, subsequent immunofluorescent staining and separate western blot analysis of HUVECs on a culture dish demonstrate loose cell-cell adhesion resulting from internalization of VE-cadherin under hypoxia. Thus, hypoxic stress increases endothelial permeability by altering cell-cell junction integrity.