Human brain microvascular endothelial cells resist elongation due to shear stress

Abstract

Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical cord endothelial vein cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm−2. From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood–brain barrier.

Real-time quantification of endothelial response to shear stress and vascular modulators

“…This transition from cobblestone to spindle-like morphology is well known for HUVECs and bovine aortic endothelial cells. 19–22 In our experiments, this transition is observed to occur between 17 and 24 hours at full shear stress. This is slower than some reports, 23 but within 24 hours for a transition from cobblestone to spindle-like morphology reported elsewhere.…”

“…13, 20 Although our time-averaged shear stress is similar to previous studies (4 – 16 dyne cm −2 ), 13, 20 the unique pulsatile waveform generated by our peristaltic pump may be a source of variation in biomechanical stress applied on cells; we have robustly characterized our pulsatile flow profile previously. 19 Quantitative analysis of cell morphology shows that the inverse aspect ratio, a measure of cell shape and elongation (Figure 1D), slightly increases to about 0.65 independent of shear stress and then decreases to between 0.45 to 0.55 at longer times (Figure 3A). The cobblestone morphology is clearly visible from 7 – 17 hours following exposure to shear stress and corresponds to a maximum in the inverse aspect ratio.…”

“…24, 25 The average cell area decreases to a steady state value, approximately 1500 μm 2 , for all shear stresses after 10 hours (Figure 3C). 19 This initial decrease in cell area, while maintaining confluence over a fixed area, implies a net increase in the number of cells. When the rate of proliferation is quantified, as discussed below, and plotted versus cell area (Figure 3D), it shows that the cell area decreases when the proliferation rate increases and halts when the proliferation rate reaches a minimum.…”

“…19 Images of cell monolayers from time-lapse movies were imported into ImageJ and the cell borders were delineated automatically using a custom macro. 19 At each time point, images from the three locations within respective channels were analyzed and averaged. Experiments with different media conditions were performed in triplicate.…”

“…19 Briefly, image sequences of cell monolayers from time-lapse movies were imported into OpenPIV and analyzed using particle image velocimetry (PIV). 19 The cell speed obtained from the PIV algorithm was about 50% lower than that obtained from manually tracking individual cells (see Supplmentary Material). We quantified RMS displacement and directional bias by tracking endothelial cell position within the monolayer (Supplemental Figure 2).…”