Prof Wen Wang
PhD, CEng, FIMechE, FHEA, FAIMBE, FREng
My work focuses on vascular bioengineering and biomaterial mechanics, including studies on the endothelial glycocalyx, vascular stem cells and progenator cells, transmembrane and transcapillary exchange. Working at the interface between biomedical engineering, biomaterials and cell mechanics, I have led a number of multidisciplinary projects in the UK, as well as international projects between the UK, US, China and Japan. Due to limitation of the space, I will only present our research on the vascular endothelial glycocalyx. We have a number of other projects, including microcapsules as vehicles for drug delivery and vascular stem cells in collaboration with colleagues in Medical Schools at Queen Mary and King's College London. Details of these studies can be found in our publication lists.
Studies on the glycocalyx
a) its spatial distribution and temporal development on the endothelial cell membrane;
b) mechanical properties of the glycocalyx;
c) effects of fluid shear stress on its distribution on the cell membrane; and
d) its stability and interplay with the cytoskeletal actin nextwork.
The endothelial glycocalyx is a carbohydrate–protein layer that lines the luminal surface of the endothelium. It anchors to the cell membrane via its core proteins that share extended link to the actin cytoskeleton. Those protein domains and the attached carbohydrates are susceptible to pathological changes, contributing to vascular diseases.
a) Spatio-temporal development of the glycocalyx
We observe the spatial distribution of the endothelial glycocalyx layer using laser-scanning confocal microscopy. Cells after different days in culture are studied to evaluate the temporal development of the glycocalyx layer. Comparisons are made between the controls and enzyme-treated groups.
Spatial distribution of the glycocalyx layer on live HUVECs in vitro and its temporal development from day 1 to day 21. The main panel shows the enface image at a given z-depth. The bottom and side panels show the x–z and y–z cross-sectional images, respectively. Scale bar, 10 μm, in the main panel. (a) Control, (b) after neuraminidase treatment.
Our in vitro results using confocal microscopy show the development of the endothelial glycocalyx over time. The growth appears to initiate from the edge of cells and takes up to two weeks to cover the apical region of the cell membrane (i.e. region above the nucleus). The mechanism underlining this spatial distribution and temporal change needs further investigation, but it seems functionally beneficial as the glycocalyx layer covers the intercellular gaps for the endothelium to fulfil its role as a selective semi-permeable membrane for the circulating blood. Our three-dimensional reconstructed images, although limited by the confocal resolution in the z-direction, indicate that the thickness of the glycocalyx layer is approximately 1 μm. This finding agrees reasonably well with previous studies.
b) Mechanical property of the glycocalyx
we have investigated mechanical properties of the glycocalyx layer using AFM nano-indentation. The Young's modulus of the glycocalyx is calculated by analysing testing results on the HUVEC cell membrane with and without the glycocalyx layer.
(a) Schematics of AFM probing of HUVECs in vitro, (b) rectangular cantilever, pyramidal-shaped tip (enlarged panel), the end of the tip is semi-spherical (further enlarged panel), (c) height image of HUVECs, (d) phase image of HUVECs. (Online version in colour.)
The Young's modulus of the HUVEC membrane in vitro. HUVECs cultured for 3, 7, 14 and 21 days are tested using AFM indentation. Comparisons are made between different locations (i.e. apical, middle and edge) on the cell membrane, as well as between the control groups (Con) and neuraminidase-treated groups (Nase) at different days. Black bars, apical; grey bars, middle;
Indentation of the cell membrane of the initial 200 nm (after bending of the cantilever is deducted) enables us to estimate the Young's modulus of the glycocalyx layer by comparing the change in the cell membrane Young's modulus with and without the glycocalyx. We can simplify the flexible glycocalyx layer on a deformable cell membrane to a two springs in series system. Our results suggest that the cell membrane Young's modulus without the glycocalyx layer is approximately 2.13 kPa (average of day 14 and 21), and with the glycocalyx layer, the overall Young's modulus is approximately 0.34 kPa (average of day 14 and 21 control groups). This leads to the Young's modulus of the glycocalyx layer alone to be approx. 0.39 kPa.
c) Shear stress-induced redistribution of the glycocalyx
We study the effects of shear stress on the spatial distribution of the glycocalyx on endothelial cell membranes, using a flow chamber. Two parameters are used to characterize the spatial distribution of the glycocalyx: (a) the percentage area of the cell membrane covered by the glycocalyx layer and (b) the fluorescence intensity ratio between the apical and edge regions of endothelial cells. They give quantitative measures of the observed changes in the distribution of the glycocalyx following shear exposure. Comparisons are made at different time points after shear stimulation to study the redistribution of the glycocalyx on the cell membrane. Further studies are carried out to analyze the recovery of the glycocalyx layer following its enzyme degradation in either static or shear flow conditions.
a Schematic drawing of the steady flow bioreactor; b Schematic drawing of the rectangular parallel plate flow chamber; c Stack images of the glycocalyx (top), cytoplasm (middle) and nucleus (bottom) of day 14 HUVECs after 24 h of shear stimulation. Bar =10μ m ; d Corresponding images in black and white for the percentage area analysis
Recovery of the glycocalyx on 14 days old HUVECs after neuraminidase treatment. Main panels are the x−y cross-section images, bar=10μ m . Bottom and side panels are the x−z and y−z cross-section images, respectively. Arrow in c indicates the flow direction. a HUVECs treated with neuraminidase; b after 24 h recovery in a static medium; c after 24 h recovery in shear flow of 12 dyn / cm 2
The percentage area of cell membrane covered by the glycocalyx after neuraminidase treatment (control) and 24 h recovery in either a static medium (red bars) or in a flow chamber (blue bars). 4D, 7D, 10D, 14D and 21D groups are HUVECs cultured for different days in medium (for development of the glycocalyx) before experiments
Ratio of WGA intensity between the apical and edge regions of cell membrane. Controls are results immediately after neuraminidase treatment. They are compared to results after 24 h recovery in either a static medium (red bars) or under 12 dyn / cm 2 shear stress (blue bars). Results for 4D, 7D, 10D, 14D and 21D HUVECs groups are presented
d) Stabiity of the glycocalyx
We investigate the contribution of the actin cytoskeleton to the structural stability of the glycocalyx in vitro. Specifically, we establish two states of actin depolymerisation with the use of cytochalasin D (CD). One is rapid depolymerisation which quickly disrupts the actin using a high concentration of CD, and the other is prolonged actin disruption by employing a low CD concentration to persistently depolymerise the actin cytoskeleton.
Endothelial glycocalyx is preserved during rapid actin depolymerisation and subsequent repolymerisation. Confluent HUVECs were incubated in medium supplemented with 1000 nM cytochalasin D (CD) for 10 min. CD+Rec10: CD plus 10 min recovery, CD+Rec60: CD plus 60 min recovery. a Immunofluorescence images show that the actin cytoskeleton is depolymerised with CD treatment, followed by partial recovery at 10 min and fully repolymerisation at 60 min in fresh CD-free medium. b WGA staining is maintained over the cell surface during the rapid actin depolymerisation and the subsequent repolymerisation. In particular, under rapid depolymerisation, cells retract severely, leading to significant folding of the membrane protrusion and concentrated WGA at this region (as indicated by triangle). b′ x−z cross-sectional images correspond to the dash lines drawn in the x−y planes. The WGA layer on top of the apical cell surface remains continuous. Scale bar=50 μm. The actin cytoskeleton and WGA are quantified over a whole cell as outlined in yellow. c Changes in the number of actin filaments per cell. **P<0.01 by ANOVA with Dunnett’s T3. d Changes in the projected cell area during the rapid actin depolymerisation and the subsequent repolymerisation. **P<0.01 by ANOVA with Dunnett’s T3. e The mean fluorescence intensity (MFI) of the WGA on the cell surface (junctional regions excluded) remains largely unchanged, which is consistent with results in b