Human brain microvascular endothelial cells resist elongation due to shear stress

Highlights

• Human brain microvascular endothelial cells (HBMECs) do not elongate and align in response to shear stress.

• Actin filaments in HBMECs are not aligned in the direction of flow.

• HBMECs may be programmed to respond differently to physical stimuli compared to endothelial cells from larger vessels.

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 vein endothelial 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.

Introduction

Blood flow results in a frictional drag, or shear stress, on the endothelial lining of vessel walls parallel to the direction of flow. These stresses play an important role in regulating endothelial cell morphology and function, and in mediating a wide range of signaling and transport processes between the vascular system and surrounding tissue (Aird, 2007a, Aird, 2007b, Chien, 2007, Davies, 1995, Johnson et al., 2011).

Endothelial cells in straight sections of large resected vessels and away from branch points exhibit an elongated, spindle-like morphology (Davies, 1995, Dolan et al., 2013, Kibria et al., 1980, Levesque et al., 1986, Nerem et al., 1981, Reidy and Langille, 1980, Silkworth and Stehbens, 1975, Zand et al., 1988). When subjected to a physiological shear stress in 2D cell culture, confluent monolayers of bovine aortic endothelial cells (BAEs), human umbilical vein endothelial cells (HUVECs), and primary baboon artery endothelial cells (BAECs) undergo a transition from a cobblestone morphology to an elongated spindle-like morphology and align in the direction of flow (Blackman, 2002, Chien, 2007, Chiu et al., 1998, Davies, 1995, Ensley et al., 2012, Eskin et al., 1984, Levesque and Nerem, 1985, Levesque and Nerem, 1989, Malek and Izumo, 1996, Simmers et al., 2007). A similar morphological response has been reported for human abdominal aortic endothelial cells seeded onto the inner surface of a polydimethyl siloxane tube (Farcas et al., 2009, Rouleau et al., 2010). The response of BAEs and HUVECs to shear stress results in a morphology similar to that of endothelial cells in resected vessels, which provides evidence that mechano-transduction modulates cellular function and is important in maintaining vascular homeostasis (Chien, 2007, Johnson et al., 2011).

Morphological parameters associated with endothelial cells in confluent monolayers in response to shear stress and resected vessels are summarized in Table 1. Endothelial cells in straight sections of the aorta across several animal species are characterized by an inverse aspect ratio (IAR, cell width/cell length) of about 0.20, a circularity of about 0.3, and an average orientation angle with respect to the flow direction (θ) of 5–15°. Similar morphological parameters have been reported for 2D confluent monolayers of BAEs and HUVECs in cell culture under shear stress. The somewhat larger variability in morphological parameters seen in cell culture is due in part to the different experimental conditions and the fact that the morphology is often characterized at a single time point.

In previous work we have reported on the influence of curvature on the morphology of endothelial cells. By seeding confluent monolayers of endothelial cells on collagen-coated glass rods of different diameters, we studied the influence of curvature on endothelial cell morphology (Ye et al., 2014). To minimize the effects of curvature, HUVEC cells elongate and align in the axial direction with decreasing diameter. In contrast, human brain microvascular endothelial cells (HBMECs) do not elongate or align in the axial direction but wrap around in the radial direction with little change in morphology as the diameter decreases (Ye et al., 2014). The endothelial cells in the brain microvasculature are highly specialized, with an array of transporters, efflux pumps, and tight junctions that are an important component of the blood–brain barrier, regulating transport into and out of the brain (Wong et al., 2013). These results suggest that HBMECs may also display a unique morphological phenotype.

Elongation and alignment in response to shear stress is thought to be a universal phenotype of endothelial cells. However, our previous work suggests that brain microvascular endothelial cells may be programmed to respond differently to physical stimuli, such as curvature, compared to endothelial cells from larger vessels. Therefore, here we compare the morphological response of HBMECs, representative of brain capillaries, and HUVECs, representative of large vessels, to shear stress. We show that HBMECs do not elongate and align in response to physiological shear stress. In addition, we show that actin fibers are randomly oriented within HBMECs and do not align with flow. These results suggest that HBMECs are programmed to resist elongation and alignment in response to shear stress. This phenotype may be associated with the unique properties of the blood–brain barrier.