The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process. blood flow; shear stress; glycocalyx charge; glycocalyx thickness; in vitro model ATHEROSCLEROTIC LESIONS appear in regions of low shear stress in relatively large arteries, such as the carotid bifurcation and the coronary artery (3). Atherosclerosis is initiated by the uptake of low-density lipoprotein (LDL) (24), which is highly associated with hemodynamic stress. Some studies demonstrate that the transport of macromolecules such as albumin across the cell membrane is strongly affected by the shear stress on endothelial cells (17,27). Kudo et al. (11,12) measured the effect of shear stress on albumin uptake into endothelial cells in vitro and reported an increased uptake at lower shear stress and a decreased uptake at higher shear stress. However, the mechanism of this biphasic response of uptake remains unclear.The endothelial cell surface is characterized by various extracellular domains of membrane-bound molecules, the glycocalyx (20), which can sense the shear force of flowing blood (9, 15). Luft (14) visualized the endothelial glycocalyx layer in vitro using ruthenium red staining in an electron microscopic study and found that the glycocalyx layer is about 20 nm thick. Subsequent in vitro electron microscopic observations of the molecules revealed that the glycocalyx thickness is Ͻ100 nm (26). In vivo studies (19, 21) have revealed thicker glycocalyx layers ranging from 0.5 m to over 1.0 m. This difference in thickness is due to the preparation and staining techniques, which cause the collapse of the glycocalyx structures (14,20,32,33).The glycocalyx surface forms a complex three-dimensional array of soluble plasma components, including ...

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

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Control of three-dimensional (3D) microvessel formation is critical for regenerative medicine and tissue engineering because vessels are essential for the formation and maintenance of organ function. In order to function, tissues need an internal network of vessels. To introduce a 3D vessel network deep into the tissue, it is necessary to introduce 3D microvessel control which makes it a critical factor in regenerative medicine and tissue engineering. This study focuses on the effect of the concentration gradient of growth factors used in seeding the endothelial cells (ECs) on the morphology of the network. First, ECs were seeded using two model environments: collagen gel containing bFGF and incubated without bFGF medium (gel-bFGF model), and collagen gel containing no bFGF and incubated with bFGF medium (medium-bFGF model). The networks were observed in 3D with confocal laser scanning microscopy. The migration of ECs on the collagen gel was analyzed to study the effect of the concentration gradient on the network formation process. We found that the ECs of the gel-bFGF model showed significantly longer migration distance and more sprouting points compared with those of the medium-bFGF model. The networks of the gel-bFGF model, expanded mainly in a depth of 20-30 µm, and many reached a depth of 50-60 µm, whereas many networks in the medium-bFGF model expanded in a depth of only 10-20 µm. These results revealed that the initial growth factor distribution affects (a) both EC migration of the network formation process and the number of sprouting points, and (b) network morphology.

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