**3.2.2 Regional inflammation and adhesion molecule expression**

WSS magnitude and duration was investigated to determine the effect these factors have on inflammatory response. Straight, cylindrical models were perfused under 4.5, 9 and 18 dyne/cm2 inlet WSS conditions for various time periods (up to 24 hours). VCAM-1 and ICAM-1 mRNA expression decreased with increasing WSS magnitude and time, indicative of an atheroprotective phenotype. An increase in WSS magnitude resulted in a decrease in E-selectin mRNA expression; however, E-selectin expression increased from 0-12 hours, and sharply fell by 24 hours.

Inflammation was considered in the stenosis model by observing regional endothelial CAM expression using immunostaining and confocal microscopy. Perfusions with neutrophils were run at 1.25 dyne/cm2, revealing an increase in CAM expression at the peak of the stenosis, Figure 8 (Rouleau et al., 2010a). Furthermore, perfusions were run at a higher inlet WSS of 6.25 dyne/cm2, with no noticeable difference in regional CAM expression. These WSS values are consistent with the conditions used for neutrophil adhesion experiments.

TNF-α Stimulated 24 hrs – Static – Regional Analysis

Fig. 8. Regional ICAM and VCAM expression. Copyright Springer, Annals of Biomedical Engineering, 38, 2010, pp. 2797, Neutrophil Adhesion on Endothelial Cells in a Novel Asymmetric Stenosis Model: Effect of Wall Shear Stress Gradients. Rouleau, L.; Copland, I; Tardif, J-C.; Mongrain, R. & Leask, R., Figure 6 with kind permission from Springer Science+Business Media B.V.

Using inlet WSS values of 4.5, 9 and 18 dyne/cm2, more physiologically relevant hemodynamics were present. During these perfusions, ICAM-1, VCAM-1 and E-selectin levels were quantified and NF-κB translocation was observed to provide a robust picture of the regional inflammation around the stenosis. Similar to the experiments at 1.25 and 6.25 dyne/cm2, there was an upregulation of ICAM-1 and VCAM-1 at the stenosis peak and in the proximal and recirculation zones. Similarly, E-selectin and NF-κB were also upregulated in these areas. It can then be concluded that there is a higher likelihood of increased neutrophil adhesion around the stenosis of the model.

### **3.2.3 Regional neutrophil adhesion**

202 Biomedical Science, Engineering and Technology

WSS magnitude and duration was investigated to determine the effect these factors have on inflammatory response. Straight, cylindrical models were perfused under 4.5, 9 and 18 dyne/cm2 inlet WSS conditions for various time periods (up to 24 hours). VCAM-1 and ICAM-1 mRNA expression decreased with increasing WSS magnitude and time, indicative of an atheroprotective phenotype. An increase in WSS magnitude resulted in a decrease in E-selectin mRNA expression; however, E-selectin expression increased from 0-12 hours, and

Inflammation was considered in the stenosis model by observing regional endothelial CAM expression using immunostaining and confocal microscopy. Perfusions with neutrophils were run at 1.25 dyne/cm2, revealing an increase in CAM expression at the peak of the stenosis, Figure 8 (Rouleau et al., 2010a). Furthermore, perfusions were run at a higher inlet WSS of 6.25 dyne/cm2, with no noticeable difference in regional CAM expression. These WSS values are consistent with the conditions used for neutrophil adhesion experiments.

Inlet Proximal Peak Recirculation Distal

Fig. 8. Regional ICAM and VCAM expression. Copyright Springer, Annals of Biomedical Engineering, 38, 2010, pp. 2797, Neutrophil Adhesion on Endothelial Cells in a Novel Asymmetric Stenosis Model: Effect of Wall Shear Stress Gradients. Rouleau, L.; Copland, I;

Tardif, J-C.; Mongrain, R. & Leask, R., Figure 6 with kind permission from Springer

**3.2.2 Regional inflammation and adhesion molecule expression** 

TNF-α Stimulated 24 hrs – Static – Regional Analysis

sharply fell by 24 hours.

Science+Business Media B.V.

50um

VCAM-1

ICAM-1

In perfusion experiments, it was shown that both WSS magnitude and perfusion time significantly affected the adhesion of a leukocyte cell line (NB4 cells). The trends showed that flow conditioned cells resulted in reduced adhesion of the NB4 cells for both the TNF-α stimulated and non-stimulated ECs. The two WSS magnitudes investigated, 1.25 dynes/cm2 and 6.25 dynes/cm2, resulted in a 3 fold and 15 fold decrease in adhesion, when compared to cells that were kept static prior to the adhesion experiments, respectively. Furthermore, experiments were run under even higher WSS conditions (12.5 dynes/cm2) and it was found that very few cells were able to adhere to the ECs. This data demonstrates the influence of hemodynamic and attractive (ligand-receptor) forces acting on the neutrophils. The higher hemodynamic forces push the neutrophils off of the binding sites, overcoming the attractive forces which form during the adhesion of the neutrophils. For both the low (1.25 dynes/cm2) and high (6.25 dynes/cm2) shear stress conditions it was found that an increase in perfusion time from 1 to 6 hours resulted in an increase in adhesion, with a more noticeable increase occurring in non-stimulated ECs, potentially showing that at the shorter time point in TNF-α stimulated cells, a maximum *in vitro* adhesion is reached.

### *Regional neutrophil adhesion*

The stenosis model presents a unique 3D environment which allowed for the investigation of the spatial differences in adhesion on and around a stenosis. Videos of the adhesion assays showed that there was a region of flow recirculation downstream of the stenosis. Immediately downstream of the separation point there was a distinct line of NB4 cell adhesion. It is postulated that this focal neutrophil adhesion was facilitated by low WSS and minimal fluid momentum caused by backflow in the recirculation zone.

For the rest of the analysis, the average adhesion was evaluated for each region of the model, Figure 9. TNF-α stimulation increased adhesion in all regions save for the stenosis peak. It was found that both the WSS magnitude and perfusion duration affected the incidence of adhesion. For example, it was found that at low inlet WSS (1.25 dyne/cm2), ECs in the recirculation and distal regions showed a significant increase in adhesion from 1 to 6 hours.

It was found that in general, the recirculation zone tended to have the highest cell adhesion. It is hypothesized that the recirculation of NB4 cells results in a higher concentration of cells flowing along the endothelium (Rouleau et al., 2010a). Furthermore, the leukocytes have a lower momentum in the recirculation zone due to the decreased shear. This would allow for an increase in adhesion in this region. The endothelium in this location is also exposed to reduced WSS, leading to an increased inflammatory response.

The proximal and distal regions have lower incidence of adhesion than the recirculation region but more than at the stenosis peak. Interestingly, there seemed to be greater adhesion in the proximal region than the inlet of the model which may be due to a positive wall shear gradient. As the fluid reaches the stenosis, the projected surface area decreases resulting in an increase in WSS. Hinds et al. found a similar result in their studies using monocytes (Hinds et al., 2001). Comparing these two results, it can be seen that there is increased adhesion of leukocytes to ECs in the presence of complex wall shear stress gradients.

Fig. 9. Regional neutrophil (NB4) adhesion in the asymmetric tissue culture model. Copyright Springer, Annals of Biomedical Engineering, 38, 2010, pp. 2798, Neutrophil Adhesion on Endothelial Cells in a Novel Asymmetric Stenosis Model: Effect of Wall Shear Stress Gradients. Rouleau, L.; Copland, I; Tardif, J-C.; Mongrain, R. & Leask, R., Figure 7 (c) & (d) with kind permission from Springer Science+Business Media B.V.

In all instances, the stenosis peak had relatively low adhesion, which is a result of the high shear forces. By the peak of the stenosis, WSS values were appreciably larger than those found in the inlet (Figure 7). These hemodynamic forces exceed the adhesive force needed for the neutrophils to adhere. Extending static adhesion past 1 hour resulted in little or no additional adhesion. This was likely due to a lack of adhesion sites for the NB4s to bind to. Ultimately, under any conditions, there will be a point where the endothelium becomes saturated with bound NB4s and simply cannot facilitate further adhesion, *in vitro*. Although this leaves a certain limitation on the results of the aforementioned experiments, it also demonstrates the high levels of focal adhesion which can occur as a result of endothelial inflammation.
