| dc.description.abstract | Vascular diseases, such as stroke and heart attacks, account for more than 50% of
abnormal death worldwide. The cause of these diseases is linked to malfunctions of
vascular endothelial cells, in particular the endothelial glycocalyx. This study
investigates the location and stability of the endothelial glycocalyx under different
flow conditions in vitro.
AFM (Atomic Force Microscopy) micro indentation is carried out on endothelial
cell membrane to determine its Young’s modulus. The Young’s modulus of the
glycocalyx layer is then deduced from measurements on cell membranes with, and
those without, the glycocalyx layer. Heparan sulphate (HS) is an important
component of the glycocalyx and can be removed by the enzyme heparinase-III
(Hep-III). Our results show the glycocalyx on cultured Human Umbilical Vein
Endothelial Cells (HUVECs) has a Young’s modulus of ~0.64Kpa. We further
observe how the Young’s modulus of the endothelial cell membrane decreases with
time, as the glycocalyx layer redevelops, following its removal by Hep-III.
Steady and oscillatory shear stimulations are used in flow chamber experiments.
Under 24 hours’ steady shear stimulation (12.6 dyn/cm2), cells are seen to elongate
and reorient parallel to the flow direction. The glycocalyx is seen to shift to the
peripheral region of the cell surface. With actin depolymerisation treatment,
significant shedding of the glycocalyx from the luminal surface of the cell is
observed. This occurs together with the loss of focal adhesions on the basal
membrane. When endothelial cells are subjected to 24 hours’ oscillating shear stress,
the size of the cell increases as the oscillatory reversal time (time between changes
in oscillatory flow direction) increases. Measurements are taken with oscillatory
flow reversal programmed at 5s, 10s and 15s. The angle (between the long axis of
the cell and the flow direction) and the aspect ratio (long axis vs short axis) change
from 41.57° and 1.72 : 1 (static) to 40.18° and 3.26 : 1 (5s), 36.71° and 4.17 : 1
(10s), 26.5° and 4.39 : 1 (15s). Both the height and the area of the cell increase.
The Young’s modulus of the endothelial cell membrane is measured
under oscillatory flows with different reversal time and compared to that under
static flow conditions. An increase in the Young’s modulus is observable under
oscillatory flows, with the most significant change occurring at the edge (i.e.
periphery) of the cell membrane area. As the oscillatory reversal time increases
from 5s to 15s, the Young's modulus of the cell membrane increases. In the apical
areas of the cell membrane, the increase is less significant. These results indicate
that the thickness of the glycocalyx decreases as cells are exposed to oscillatory
flows, and the loss is most significant in the peripheral region of the cell membrane.
As the oscillatory reversal time increases from 5s to 15s, so the loss in the
glycocalyx increases. | en_US |
