Human endothelial cells in high glucose: New clues from culture in 3D microfluidic chips.

Several studies have demonstrated the role of high glucose in promoting endothelial dysfunction utilizing traditional two-dimensional (2D) culture systems, which, however, do not replicate the complex organization of the endothelium within a vessel constantly exposed to flow. Here we describe the response to high glucose of micro- and macro-vascular human endothelial cells (EC) cultured in biomimetic microchannels fabricated through soft lithography and perfused to generate shear stress. In 3D macrovascular EC exposed to a shear stress of 0.4 Pa respond to high glucose with cytoskeletal remodeling and alterations in cell shape. Under the same experimental conditions, these effects are more pronounced in microvascular cells that show massive cytoskeletal disassembly and apoptosis after culture in high glucose. However, when exposed to a shear stress of 4 Pa, which is physiological in the microvasculature, human dermal microvascular endothelial cells (HDMEC) show alterations of the cytoskeleton but no apoptosis. This result emphasizes the sensitivity of HDMEC to different regimens of flow. No significant variations in the thickness of glycocalyx were detected in both human endothelial cells from the umbilical vein and HDMEC exposed to high glucose in 3D, whereas clear differences emerge between cells cultured in static 2D versus microfluidic channels. We conclude that culture in microfluidic microchannels unveils unique insights into endothelial dysfunction by high glucose.

The effect of shear stress reduction on endothelial cells: A microfluidic study of the actin cytoskeleton.

Reduced blood flow, as occurring in ischemia or resulting from exposure to microgravity such as encountered in space flights, induces a decrease in the level of shear stress sensed by endothelial cells forming the inner part of blood vessels. In the present study, we use a microvasculature-on-a-chip device in order to investigate in vitro the effect of such a reduction in shear stress on shear-adapted endothelial cells. We find that, within 1 h of exposition to reduced wall shear stress, human umbilical vein endothelial cells undergo reorganization of their actin skeleton with a decrease in the number of stress fibers and actin being recruited into the cells' peripheral band, indicating a fairly fast change in the cells' phenotype due to altered flow.

Vascular bifurcation mapping with photoacoustic microscopy.

The early detection of microvascular changes in cancer diagnosis is needed in the clinic. A change in the vascular bifurcation density is a biomarker for the sprouting activity. Here, Optical-Resolution PhotoAcoustic Microscopy is used for quantitative vascular bifurcation mapping in 2D after the creation of Virtual Tubes out of Bifurcations. In stacks of OR-PAM images of the hemoglobin distribution, bifurcations become tubes and are selected by the 3D tubeness filter. These fast analyses will be compared to a classical approach and are easier to implement for functional analysis of the vascular bifurcation density in healthy and diseased tissues.

Paroxysmal Permeability Disorders: Development of a Microfluidic Device to Assess Endothelial Barrier Function.

Background: Paroxysmal Permeability Disorders (PPDs) are pathological conditions caused by periodic short lasting increase of endothelial permeability, in the absence of inflammatory, degenerative, ischemic vascular injury. PPDs include primary angioedema, idiopathic systemic capillary leak syndrome and some rare forms of localized retroperitoneal-mediastinal edema. Aim: to validate a microfluidic device to study endothelial permeability in flow conditions. Materials and Methods: we designed a microchannel network (the smallest channel is 30µm square section). Human Umbilical Vein Endothelial Cells (HUVECs) were cultured under constant shear stress in the networks. Endothelial permeability assessment was based on interaction of biotinylated fibronectin used as a matrix for HUVECs and FITC-conjugated avidin. The increase in endothelial permeability was identified as changes in fluorescence intensity detected by confocal fluorescent microscopy. Results: The microchannels were constantly perfused with a steady flow of culture medium, ensuring a physiologically relevant level of shear stress at the wall of ~0.2 Pa. Our preliminary results demonstrated that circulation of culture medium or plasma from healthy volunteers was associated with low fluorescence of fibronectin matrix. When bradykinin diluted in culture medium was perfused, an increase in average fluorescence was detected. Conclusion: Our microvasculature model is suitable to study endothelial functions in physiological flow conditions and in the presence of factors like bradykinin known as mediator of several PPDs. Therefore, it can be a promising tool to better understand the mechanisms underlying disorders of endothelial permeability.

Multi-wavelength photo-acoustic microscopy in the frequency domain for simultaneous excitation and detection of dyes.

An optical-resolution photoacoustic microscope with modulated CW laser diodes allowing multi-channel imaging is presented that can be used for both imaging biological tissues and for targeted photo-dynamic therapy (PDT) varying the optical power and exposure time. The effects of this therapy are immediately monitored in order to optimize the time of irradiation. After the description of the experimental setup, in vitro and in vivo applications are presented on a synthetic sample and on the mouse ear using hemoglobin as endogenous and methylene blue as exogenous dye for imaging and PDT, respectively.

Nonlinear laser dynamics induced by frequency shifted optical feedback: application to vibration measurements.

In this article, we study the nonlinear dynamics of a laser subjected to frequency shifted optical reinjection coming back from a vibrating target. More specifically, we study the nonlinear dynamical coupling between the carrier and the vibration signal. The present work shows how the nonlinear amplification of the vibration spectrum is related to the strength of the carrier and how it must be compensated to obtain accurate (i.e., without bias) vibration measurements. The theoretical predictions, confirmed by numerical simulations, are in good agreement with the experimental data. The main motivation of this study is the understanding of the nonlinear response of a laser optical feedback imaging sensor for quantitative phase measurements of small vibrations in the case of strong optical feedback.