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Insana Lab: Ultrasonic Imaging - The University of Illinois at Urbana-Champaign

Project 4: Atherosclerosis and the role of cell adhesion and blood flow dynamics
Jean K. Tsou [jktsou.ucdavis.edu]

 Fig 1. Cardiovascular disease from atherosclerosis is a lead cause of mortality in the United States. Nascent atheromas commonly form in regions of arterial branching and curvature, such as the infra-renal abdominal aorta, carotid sinus, coronary, iliac, and superficial femoral arteries (Fig. 1a)(G Helmlinger 1995). Atherogenesis preferentially occurs in vessel walls near bifurcations and points of blood flow recirculation and stasis, where the magnitude of wall shear stress is significantly lower and exhibits directional changes (Fig. 1b)(AM Malek 1999). Atherogenesis involves dysfunction of the vascular endothelium, the monolayer of cells lining the inner surfaces of all blood vessels. It is initiated when white blood cells pass through the endothelial cell (EC) lining to enter the intimal space and transform into macrophages. With the accumulation of macrophages and lipid in the intima, the plaque grows into lumen perturbing and eventually reducing blood flow. Also small blood clots forming near the flow disturbance may be shed to cause a stroke or pulmonary embolism. (Fig. 1c)

 Fig 2a illustrates the process of atherogenesis in the cellular level. Abnormal wall shear stress patterns and the presence of cytokines and other factors act to stimulate the ECs to produce an inflammatory response that includes adhesion molecules (E-selectin, intravascular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1)). The ligands and integrins on the monocytes bind to the adhesion molecules expressed on the endothelial cell surface and transmigrate into the subendothelial space. The monocytes then transform into macrophages and bind to highly-oxidized low-density lipoprotein (LDL) to form foam cells. The death of foam cells leaves behind a growing mass of extracellular lipids and other cell debris along with the accumulation of smooth muscle cells, which later form fibrous plaques. Vulnerable plaques with thin fibrous caps have high risk to be ruptured and form thrombus inside the lumen.(Lusis 2000)

 Fig 2b. The range of normal shear stress in an artery is between 10-70 dyne/cm2, and the time averaged shear stress for a healthy artery is around 12-15 dyne/cm2 (AM Malek 1999). In contrast, the magnitude of the shear stress in the atherosclerosis-prone regions is significantly lower (4 dyne/cm2). Detailed studies have been performed to investigate the influence of the magnitude of laminar shear stress on endothelial cells, but only at particular shear values that are considered as high (12 or 20 dyne/cm2) and low (2 or 4 dyne/cm2) shear stress. The difference in magnitude usually results in opposite changes in endothelial cell functions, for example low shear stress (2 dyne/cm2) upregulates VCAM-1 (S Mohan 1999) expression while high stress (20 dyne/cm2) attenuates it (JJ Chiu 2004). However, the change in EC functions in the intermediate shear stress range remains unclear.

 Fig 3. The flow chamber master (Fig. 3a) consists of two major components: the linear shear stress flow chamber and the vacuum channel network. The spider web-like pattern is a network of vacuum channels that helps sealing the flow chamber onto the cover slips surface, where cells were seeded on. The final flow chamber is transparent to visible light and can be placed under a microscope to observe cell behavior during the shear flow experiments. The flow chamber pattern in Fig. 3a was first created using computer software (Freehand, Macromedia) and printed on a transparency that served as a photo-mask (Fig. 3b) in UV-photolithography to generate the master mold. In this procedure, a thin layer of photoresist was spin-coated onto a silicon wafer and exposed to UV light through the photomask. Then a developing reagent was applied to dissolve the unexposed regions. The resulted bas-relief structure functioned as a master for fabricating poly(dimethylsiloxame) (PDMS) molds. The surface of silicon/photoresist master was treated with fluorinated ailanes to prevent irreversible bonding to PDMS. A mixture of PDMS prepolymer and curing agent was poured onto the silicon master and cured. The PDMS is then peeled off the master and produces the replica of the flow chamber design.

 Fig 4. The design of the linear shear flow chamber was adapted from Usami's work (S Usami 1993) based on Hele-Shaw flow theory. By setting the side walls of the flow chamber to be coincident with the streamlines of a two dimensional stagnation flow and making the end of the channel shaped to match the iso-potential lines, this flow allows the wall shear stress to decreases linearly along the center line of the channel (Fig. 4a). The shear stress at the wall  decreases linearly across the flow chamber. Along the center of the flow chamber the shear stress is a function of viscosity  , volumetric flow rate (Q), channel height (h = 100  ), entrance width (w1 = 2 mm), channel length (L) and position (x).

Fig. 4b shows the setup of the in vitro flow experiments on human aortic endothelial cells (HAECs). The cell cover slip is temporarily bound to the PDMS flow chamber by vacuum. Flow medium was infused into the chamber by a syringe pump to create a known, spatially varying shear field.


 Fig 5. After applying a shearing stress for four hours to the HAECs, the PDMS flow chamber was carefully removed from the cover slip without damage to the cell monolayer. Then fluorescently-labeled antibodies were applied and a series of fluorescence images were acquired at different regions of the flow channel that correspond to different shear stress levels (Fig. 5 c-e). The mean pixel intensity for each region of interest was calculated from 8-10 images. The fluorescence intensity in the images is positively correlated the level of protein expression on the endothelial cell surface. The result of VCAM-1 expression represented in fluorescence pixel intensity is plotted with respect to shear stress in Fig. 5b. With increasing shear magnitude, the level of VCAM-1 expression gradually decreases (Fig 5c), and the change is more significantly when shear stress is less than 6 dyne/cm2. Compared to the static control, dramatic increases in VCAM-1 are found when the shear stress is lower than 5 dyne/cm2. Similar results for E-selectin and ICAM-1 expression are found in Figs 5d,e, respectively.

 Fig 6. Leukocyte recruitment is mediated by the expression of adhesion molecules on the surface of the endothelium. Recruitment can be separated into three steps: rolling, arresting (or adhesion) and transmigration (Fig.6b). Leukocyte rolling along the endothelial surface is mediated by selectins which bind to carbohydrate ligands on the leukocytes. Firm adhesion of monocytes to the endothelium can be mediated by the integrin VLA-4 that interacts with VCAM-1 on the ECs. Monocytes isolated from human blood were defused over the endothelial cells at 2 dyne/cm2 after four hours of shearing. A series of image sequences were recorded at different locations on the linear shear flow chamber (Fig. 6a). The number of monocytes that were rolling, adhering and transmigrating were counted offline. Fig. 6c shows the number of monocytes interacting with endothelial cells when the monolayers were preconditioned at different level of shear stress (low shear: 2 dyne/cm2; med shear: 6 dyne/cm2 and high shear: 12 dyne/cm2) for four hours. The variation in the number of monocytes that were rolling and adhering to the EC surface was positively correlated with the alteration in expression level of E-selectin and VCAM-1 with respect to shear stress.

References:

[1] AM Malek, S. A., S Izumo (1999). "Hemodyamic shear stress and its role in atherosclerosis." JAMA 282: 2035-2042.
[2] G Helmlinger, B. B. a. R. N. (1995). "Calcium reponses of endothelial cell monolayers subjected to pulsatile and steady laminar flow differ." Am J Physiol 269: C367-C375.
[3] JJ Chiu, P. L., CN Chen, CI Lee, SF Chang, LJ Chen, SC Lien, YC Kao, S Usami, S Chien (2004). "Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-a in endothelial cells." Arterioscler Thromb Vasc Biol 24: 73-79.
[4] Lusis, A. (2000). "Atherosclerosis." Nature 407: 233-241.
[5] S Mohan, N. M., AJ Valente, EA Sprague (1999). "Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion." Am J Physiol 276(C1100-C1107).
[6] S Usami, H. C., Y Zhao, S chien, R Skalak (1993). "Design and construction of a linear shear stress flow chamber." Annals of Biomedical Engineering 21: 77-83.

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