Cancer cells traveling to distant tissues during metastasis must survive passing through the circulation. However, the influence of this fluid microenvironment on these cells is poorly understood. It was previously viewed that exposure to the hemodynamic shear forces within circulation was inhospitable to cancer cells, causing the cells to be destroyed. Recent evidence indicates that transformed cells are markedly more resistant to fluid shear stress when compared to non-transformed epithelial cells. Furthermore, these cells selectively adapt following exposure to fluid shear stresses and become more resistant to subsequent exposures to shear stress. The mechanisms behind this difference in phenotype and induced resistance are investigated. The elastic modulus, a measure of stiffness, may play a role in resistance and is shown to be altered upon exposure to fluid shear forces. Additionally, plasma membrane repair is a critical process in the resistance phenotype as cells sustain damage but are able to maintain viability. Cytoskeletal dynamics are also shown to play a role in resistance to fluid shear forces.

There is increasing interest in assessing the role of hemodynamics in aneurysm growth and rupture mechanism. Identification of the indicators of rupture risk can prove very valuable in the clinical management of patients. If rupture risk of aneurysms can be predicted, immediate preemptive treatments can be done for the high risk patients whereas others can avoid the risky intervention. Retrospective studies have been performed in the past to filter out indices that differentiate ruptured aneurysms from unruptured aneurysms. However, these differences may not necessarily translate to differences between aneurysms that present unruptured but fork towards growth/rupture and unruptured aneurysms that are invariably stable. The hypothesis of the present study is that hemodynamic indices of unruptured aneurysms when they first presented can be used to predict their longitudinal outcome. A prospective longitudinal cohort study was designed to test this hypothesis. Four clinical centers participated in this study and a total of 198 aneurysms were recruited. These aneurysms were chosen by the physicians to be kept under watchful waiting. Three-dimensional models of aneurysms and their contiguous vasculature generated using the initial scans of patients were used for computational fluid dynamic (CFD) simulations. Both pulsatile and steady flow analyses were performed for each patient. By collating all the prominent hemodynamic indices available in aneurysm literature and developing a few new indices, 25 hemodynamic indices were estimated for each subject. For statistical analysis, it was hypothesized a priori that low wall shear area is different between stable and unstable aneurysms. All other indices were tested in a post-hoc manner. The longitudinal outcome information of these patients was recorded at the clinical centers and the author was blinded until all analyses were complete. Aneurysms that grew during the follow up period were labeled as "grown" and otherwise they were called "stable" by the radiologists. After the hemodynamic analysis was complete, a non-parametric Mann Whitney U test was performed to determine if any index can statistically differentiate the two groups ("grown" versus "stable"). It was found that none of the indices distinguished the two groups with statistical significance. Comparison of the steady and pulsatile flow analysis suggested that the patient population is stratified in the same order by an index, irrespective of whether the index is computed using a steady or pulsatile flow simulation. Pearson correlation coefficient was obtained between basic geometric indices and hemodynamic indices of this population. No strong correlation was found in between morphology and hemodynamics, suggesting uniqueness of the hemodynamic indices. The hypothesis motivating the present study is that aneurysm blood flow based indices can be used as prognostic indicators of growth and/or rupture risk. This study is the first to analyze intracranial aneurysm hemodynamics of a large cohort in a longitudinal prospective manner. Results of the present study indicate that quantitative hemodynamics cannot be used to predict the longitudinal outcome of an aneurysm. Further studies are needed to gain additional clinical insights.

Ph.D. / Biomedical Engineering / The cardiovascular disease atherosclerosis is directly linked to the functions of the endothelium, the monolayer of endothelial cells (ECs) that line the lumen of all blood vessels. EC functions are affected by fluid shear stress (FSS), the tangential force exerted by flowing blood. In vivo FSS is determined by vascular geometry with relatively straight vessels producing high, unidirectional FSS and vessel branch points and curvatures producing low, oscillatory FSS. While these distinct FSS conditions differentially regulate EC functions, they also dramatically affect EC shape and cytoskeletal structure. High and unidirectional FSS induces EC elongation and cytoskeletal alignment, while concurrently promoting EC functions that are atheroprotective. In contrast, low and oscillatory FSS induces cobblestone-shaped ECs with randomly oriented cytoskeletal features, while simultaneously promoting EC functions that create an athero-prone vascular environment. Whether these distinct EC shapes and cytoskeletal structures influence EC functions, independent of FSS, is largely unknown. The overall hypothesis of this study is that cell shape and cytoskeletal structure regulate EC functions through mechanisms that are independent of FSS. Due to advances in surface engineering in the field of micropatterning, EC shape can be controlled independent of external forces by creating spatially localized surface cues. In this research, lanes of protein were micropatterned on glass surfaces to induce EC elongated shape in the absence of FSS. In Aim 1, micropattern-elongated EC (MPEC) shape and cytoskeletal structure were fully characterized and determined to be comparable to FSS-elongated ECs. Thus, inducing EC elongation on micropatterned lanes provides a platform for studying the functional consequences of EC shape, independent of FSS. Using this model, the following important markers of EC functions related to atherosclerosis were evaluated to determine the influence of EC shape and cytoskeletal alignment: extracellular matrix deposition (Aim 2), inflammatory function(Aim 3), and thrombotic potential (Aim 4). The results indicate that EC-elongated shape and cytoskeletal alignment participate in promoting selected EC functions that are protective against atherosclerosis, independent of FSS. Since EC shape is governed by the cytoskeleton, this data suggests that the cytoskeleton plays an active role in the regulation of EC functions that promote cardiovascular health.

Collecting microlymphatics play a vital role in promoting lymph flow from the initial lymphatics in the interstitial spaces to the large transport lymph ducts. In most tissues, the primary mechanism for producing this flow is the spontaneous contractions of the lymphatic wall. Individual units, known as lymphangion, are separated by valves that help prevent backflow when the vessel contracts, thus promoting flow through the lymphatic network. Lymphatic contractile activity is inhibited by flow in isolated lymphatics, however there are virtually no in situ measurements of lymph flow in these vessels. Initially, a high speed imaging system was set up to image in situ preparations at 500 fps. These images were then manually processed to extract information regarding lymphocyte velocity (-4 to 10 mm/sec), vessel diameter (25 to 165 um), and particle location. Fluid modeling was performed to obtain reasonable estimates of wall shear stress (-8 to 17 dynes/cm2). One of the difficulties encountered was the time consuming methods of manual particle tracking. Using previously captured images, an image correlation method was developed to automate lymphatic flow measurements and to track wall movements as the vessel contracts. Using this method the standard error of prediction for velocity measurements was 0.4 mm/sec and for diameter measurements it was 7.0 µm. It was found that the actual physical quantity being measured through this approach is somewhere between the spatially averaged velocity and the maximum velocity of a Poiseuille flow model.

This research was conducted to investigate the connective tissue contribution to toughness of cow beef and to find means to decrease it. Intra muscular connective tissue (IMCT) content of meat from cows (~6 years) and heifers (~16 months) varied significantly among muscles (P<0.0001) and maturity groups (P<0.05). Amount of total collagen in IMCT was a constant (37.3-46.3 %) among muscles and between maturity groups. Shear force of <i>biceps femoris</i>, <i>semimembranosus</i> and <i>longissimus</i> muscles had increased significantly with animal maturity (P<0.0001). Shear stress of <i>gluteus medius</i> was similar between maturity groups. Collagen solubility decreased with animal maturity, except for <i>biceps femoris</i>. <p> The impact of the temperature of aqueous heating (55 to 95 ºC) and time on thermolabile proteins, amorphous proteins, Ehrlich chromogen, pyridinoline, thickness change, shrinkage, weight gain, shear force, amide bands and morphology of epimysium was studied. Collagen contributed to 90% (w/w) of epimysial proteins. At 55 ºC, epimysial properties were changed only after exposure to long heating times. Shear stress values of raw cow (39.6 N/mm2) and heifer (30.8 N/mm2) epimysium decreased significantly to 11.6 and 2.1 N/mm2, respectively, at 70 ºC. Amount of epimysial amorphous collagen (14-16% w/w) detected after heating at 70 ºC and above was not related to shear stress decrease. Before and after heating, cow epimysium contained more pyridinoline cross-links than heifer epimysium.<p> The effects of strong and weak acids and alkalis on epimysial properties were studied following heating at 55 and 70 ºC for 15 min. As the concentration of HCl (0.1-0.5 M) and pre-equilibration time were increased at 70 ºC, shear stress decreased to <2 N/mm2. Increasing concentration of CH3COOH (0.1-0.5 M) and pre-equilibration times had decreased shear stress to ~5 N/mm2. At 55 ºC, HCl was not superior to CH3COOH in its ability to decrease epimysial shear stress. Increasing concentration of NaOH (0.01-0.05 M) and high temperature decreased shear stress to ~3 N/mm2. Lack of a shear stress decrease at 55 ºC and increased thermal denaturation temperature (66 ºC compared to 63 ºC in water), indicated that NH4OH had an epimysial stabilization effect, which was not eliminated at 55 ºC.

During inflammation, leukocytes modulate α4β1(VLA-4) integrin avidity in order to rapidly stabilize nascent adhesive contacts to VCAM-1-expressing endothelial cells and resist detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization under conditions of fluid flow remains poorly understood. We modeled leukocyte (U937 cell, mouse lymphocyte and human monocyte) arrest and adhesion stabilization through the use of a parallel plate flow chamber and visualized cells by phase contrast or fluorescent confocal microscopy. Live cell imaging with Lifeact-transfected U937 cells revealed that mechanical forces imparted by fluid flow induced formation of upstream tension-bearing anchors attached to the VCAM-1-coated surface. Scanning electron microscopy confirmed that flow-induced mechanical force culminates in the formation of structures that anchor monocyte adhesion. These structures are critical for adhesion stabilization, since disruption of actin polymerization dramatically inhibited VLA-4-dependent resistance to detachment, but did not affect VLA-4 expression, affinity modulation, and clustering or constitutive linkage to F-actin. Transfection of dominant-negative constructs and inhibition of kinase function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. Rap1 was shown to be critical for resistance to flow-induced detachment and accumulated in its GTP form at the sites of anchor formation. A key mediator of force-induced Rac activation and actin polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension-bearing anchors and blockade of PI3K or deficiency of PI3Kγ isoform reproduced the adhesion defect produced by inhibition of actin polymerization. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces; these included activation of Rap1, phosphoinositide 3-kinase γ-isoform and Rac, but not Cdc42.

The lymphatic system plays an important role in fluid and protein balance within the interstitial spaces. Its dysfunction could result in a number of debilitating diseases, namely lymphedema. Lymphatic vessels utilize both intrinsic and extrinsic mechanisms to pump lymph. Intrinsic pumping involves the active contraction of vessels, a phenomenon that is regulated in part by nitric oxide (NO) produced by lymphatic endothelial cells (LECs). NO production by arterial endothelial cells has been shown to be sensitive to both shear stress and stretch. Therefore, because of the unique mechanical environment of the LECs, we hypothesize that mechanical forces play an important role in regulation of the lymphatic pumping. Parallel-plate flow chambers and indenter-based cyclic stretch devices were constructed and used to apply mechanical loads to LECs. In addition, high-throughput micro-scale channels were developed and tested for shear experiments to address the need to increase the productivity and high- resolution imaging. Twenty-four hours treatment of LECs with different shear stress conditions showed a shear-dependent elevation in NO production. Moreover, 2.5 folds increase in cumulative NO was observed for stretched cells compared to the unstretched cells over six hours period. In conclusion, the upregulation observed in NO production under mechanical stimuli suggest new regulatory mechanisms that can be pharmaceutically targeted. These results provide an unprecedented insight into lymphatic pumping mechanism.

When cultivated properly in bioreactors, microalgae have been found to produce vast amounts of biomass. In the case of diatom cultivation where the organisms will fall out of suspension quite easily, paddle wheels or pumps are the primary means to maintain the necessary velocity in the raceway. This study will focus on the potentially harmful shear stress these devices may impart onto the organisms. The system used to impart shear stress to a diatom culture was a cone and plate viscometer. Cells were counted using a fluorescein diacetate staining method with a fluorescent and brightfield microscope. Under the white light all cells were visible while only the healthy cells were visible under fluorescent light. The sample was exposed to shear stress with the cone and plate viscometer at 6 Pascals for 10 minutes and compared against a non-sheared sample. For each sample, 5 pairs of white and fluorescent light images were captured, counted, and averaged. A non-sheared sample was paired with a sheared sample to calculate the decrease in cell viability. The slope was calculated from the plot of shear stress and cell viability for 9 strains. In each case shear stress resulted in a significant decrease in cell viability; however, there was no statistical difference between strains. While effective, this method would be impractical for a commercial algae cultivation facility as the viscometer in this study costs approximately $100,000. Therefore, tests were performed to determine if a rotary mixer could be substituted for the viscometer. The hypothesis was that the cell damage was a product of shear stress and exposure time. For the viscometer test, the shear exposure was 3600 Pa s. Two rotational mixer tests were performed, one at 1250 RPM for 7 hours and one at 313 RPM for 28 hours, providing the same 3600 Pa s shear exposure. After staining, cell viability decreased 35.62% and 11.07% in the 1250 RPM and 313 RPM test, respectively. This difference was significant compared to the 6.04% decrease in the viscometer test. The increased cell damage was attributed to turbulence in the mixer tests and the basis for further study.

Flow-mediated dilation (FMD) refers to the relaxation of vascular smooth muscle and the subsequent dilation of the vessel in response to increases in shear stress on the endothelial lining accompanying increases in blood flow. The phenomenon has been shown to be endothelium dependent and as such is used clinically and experimentally as an index of endothelial health. FMD can be assessed by imaging a conduit artery with ultrasound during a period of reactive hyperaemia, typically following a period of prior blood flow occlusion achieved by the inflation of a pneumatic cuff around the limb distal to the imaging site. Previous studies have shown that the health of the endothelium is predictive of the health of the cardiovascular system as a whole. This thesis set out to scrutinize the FMD test as a marker for endothelial health by testing the following five hypotheses: 1. A short burst of high shear is not adequate to elicit the FMD response. 2. Brachial artery dilation following 15 minutes of occlusion is a clearer indicator of endothelium dependent FMD than 5 minutes of occlusion with exercise. 3. Oscillating the post occlusion shear stress will decrease FMD compared to unidirectional shear). 4. Heavy dynamic hand grip exercise 6 minutes before an occlusion-only FMD protocol will result in an enhanced FMD response. 5. Long term bed-rest inactivity will attenuate the FMD response and an exercise program will preserve endothelial function. The experiments documented in Chapter 2 found that a 20-s shear stress stimulus following 15 min of forearm circulatory occlusion was not adequate to induce an FMD response compared to longer durations of shear and there was a progressive reduction in FMD when the magnitude of the initial peak shear was reduced by limiting the duration of prior occlusion. Also, the FMD response was correlated with the total shear to time of peak diameter for all shear durations and peaks that were studied while the same was not true of peak shear. In Chapter 3 it was revealed that an uncoupling of the shear-to-dilation ratio occurred when dynamic exercise was added to the FMD test as both 15 min of occlusion (15OC) and 5 min of occlusion with 1 min of exercise (1EXin5OC) yielded similar FMD responses, even though the shear stimulus was increased with the addition of exercise. Increased plasma nitrite during hyperaemia was observed only in the 15OC protocol, suggesting that the exercise in the 1EXin5OC protocol initiates dilatory mechanisms that are not as heavily reliant on the shear sensitive nitric oxide pathway . In Chapter 4 it was shown that 5 min of intense dynamic hand grip exercise (5EX) produced a greater dilation than either continuous (15OC) or intermittent (IO) shear following 15OC. Total shear to the time of peak diameter (AUCshear) and peak shear were both correlated to %dilation following 15OC; however this relationship was lost during 5EX and IO. The results of this study echoed the suggestion in Chapter 3 that there was an uncoupling of the intensity of the shear stimulus and the magnitude of vasodilatation when exercise was introduced, and adds that it may be in part due to the oscillatory nature of the shear profile during exercise. The acute effects of local exercise on the FMD response following 15OC were examined in Chapter 5. FMD in the brachial artery was blunted following dynamic hand grip exercise, even though the shear stimulus was greater during PostEX. Nitrite was significantly elevated in CON at 15s while PostEX nitrite was significantly elevated at 30s post cuff release but not different from CON at 15s. The results of this study suggested that prior exercise had a negative effect on FMD which may be related to exercise blunting post occlusion endothelial N ̇O production. Chapter 6 examined the effect of 56 days of head-down tilt bed rest (HDBR) and an exercise countermeasure on conduit artery FMD following release of distal limb ischemia and NMD following sublingual administration of 0.3 mg of nitroglycerin. HDBR without EX decreased the resting diameter of the popliteal artery while EX increased the diameter. HDBR had no effect on the resting diameter of the brachial artery. FMD was elevated in all groups for the brachial but only in the non-exercisers for the popliteal. When change in resting diameter was taken into account the preserved FMD in EX was removed. NMD was not altered by HDBR in any group. There was enhanced endothelial function relative to intrinsic dilatory capacity in both the brachial and popliteal arteries post HDBR. The results from Chapter 2 support hypothesis 1, showing that a 20 second burst of high shear stimulus was not adequate to elicit the FMD response during reactive hyperaemia. It is not clear whether hypothesis 2 was supported or not given that the results from Chapter 3 showed on the one hand that the %FMD did not change with the addition of exercise in the occlusion but on the other hand the shear to dilation ratio was altered. The finding, in Chapter 4, that FMD was not reduced when the hyperaemia was intermittent does not support hypothesis 3. In opposition to hypothesis 4, Chapter 5 showed that %FMD was reduced following bouts of heavy hand grip exercise; however the absolute magnitude of vessel diameter was similar in both post exercise and control tests. Finally, hypothesis 5 was also contradicted, with Chapter 6 showing that long term bed-rest enhanced rather than attenuated the FMD response in both arm and leg arteries, while an exercise countermeasure preserved pre-bed-rest FMD in the legs only. In addition to the specific hypotheses tested, there was evidence that acute exercise evoked dilatory mechanisms in the conduit arteries that were not shear/endothelium dependent given that the shear to dilation relationship was uncoupled during, following, and in occlusion protocols that include exercise. The precise mechanisms by which this is achieved are still unknown, but it may be partially due to the oscillatory nature of the elevated blood flow during exercise. I conclude that inference of cardiovascular health from endothelial function by the evaluation of %FMD should be approached with caution, especially in the event that physical activity is involved.

Flow-mediated dilation (FMD) refers to the relaxation of vascular smooth muscle and the subsequent dilation of the vessel in response to increases in shear stress on the endothelial lining accompanying increases in blood flow. The phenomenon has been shown to be endothelium dependent and as such is used clinically and experimentally as an index of endothelial health. FMD can be assessed by imaging a conduit artery with ultrasound during a period of reactive hyperaemia, typically following a period of prior blood flow occlusion achieved by the inflation of a pneumatic cuff around the limb distal to the imaging site. Previous studies have shown that the health of the endothelium is predictive of the health of the cardiovascular system as a whole. This thesis set out to scrutinize the FMD test as a marker for endothelial health by testing the following five hypotheses: 1. A short burst of high shear is not adequate to elicit the FMD response. 2. Brachial artery dilation following 15 minutes of occlusion is a clearer indicator of endothelium dependent FMD than 5 minutes of occlusion with exercise. 3. Oscillating the post occlusion shear stress will decrease FMD compared to unidirectional shear). 4. Heavy dynamic hand grip exercise 6 minutes before an occlusion-only FMD protocol will result in an enhanced FMD response. 5. Long term bed-rest inactivity will attenuate the FMD response and an exercise program will preserve endothelial function. The experiments documented in Chapter 2 found that a 20-s shear stress stimulus following 15 min of forearm circulatory occlusion was not adequate to induce an FMD response compared to longer durations of shear and there was a progressive reduction in FMD when the magnitude of the initial peak shear was reduced by limiting the duration of prior occlusion. Also, the FMD response was correlated with the total shear to time of peak diameter for all shear durations and peaks that were studied while the same was not true of peak shear. In Chapter 3 it was revealed that an uncoupling of the shear-to-dilation ratio occurred when dynamic exercise was added to the FMD test as both 15 min of occlusion (15OC) and 5 min of occlusion with 1 min of exercise (1EXin5OC) yielded similar FMD responses, even though the shear stimulus was increased with the addition of exercise. Increased plasma nitrite during hyperaemia was observed only in the 15OC protocol, suggesting that the exercise in the 1EXin5OC protocol initiates dilatory mechanisms that are not as heavily reliant on the shear sensitive nitric oxide pathway . In Chapter 4 it was shown that 5 min of intense dynamic hand grip exercise (5EX) produced a greater dilation than either continuous (15OC) or intermittent (IO) shear following 15OC. Total shear to the time of peak diameter (AUCshear) and peak shear were both correlated to %dilation following 15OC; however this relationship was lost during 5EX and IO. The results of this study echoed the suggestion in Chapter 3 that there was an uncoupling of the intensity of the shear stimulus and the magnitude of vasodilatation when exercise was introduced, and adds that it may be in part due to the oscillatory nature of the shear profile during exercise. The acute effects of local exercise on the FMD response following 15OC were examined in Chapter 5. FMD in the brachial artery was blunted following dynamic hand grip exercise, even though the shear stimulus was greater during PostEX. Nitrite was significantly elevated in CON at 15s while PostEX nitrite was significantly elevated at 30s post cuff release but not different from CON at 15s. The results of this study suggested that prior exercise had a negative effect on FMD which may be related to exercise blunting post occlusion endothelial N ̇O production. Chapter 6 examined the effect of 56 days of head-down tilt bed rest (HDBR) and an exercise countermeasure on conduit artery FMD following release of distal limb ischemia and NMD following sublingual administration of 0.3 mg of nitroglycerin. HDBR without EX decreased the resting diameter of the popliteal artery while EX increased the diameter. HDBR had no effect on the resting diameter of the brachial artery. FMD was elevated in all groups for the brachial but only in the non-exercisers for the popliteal. When change in resting diameter was taken into account the preserved FMD in EX was removed. NMD was not altered by HDBR in any group. There was enhanced endothelial function relative to intrinsic dilatory capacity in both the brachial and popliteal arteries post HDBR. The results from Chapter 2 support hypothesis 1, showing that a 20 second burst of high shear stimulus was not adequate to elicit the FMD response during reactive hyperaemia. It is not clear whether hypothesis 2 was supported or not given that the results from Chapter 3 showed on the one hand that the %FMD did not change with the addition of exercise in the occlusion but on the other hand the shear to dilation ratio was altered. The finding, in Chapter 4, that FMD was not reduced when the hyperaemia was intermittent does not support hypothesis 3. In opposition to hypothesis 4, Chapter 5 showed that %FMD was reduced following bouts of heavy hand grip exercise; however the absolute magnitude of vessel diameter was similar in both post exercise and control tests. Finally, hypothesis 5 was also contradicted, with Chapter 6 showing that long term bed-rest enhanced rather than attenuated the FMD response in both arm and leg arteries, while an exercise countermeasure preserved pre-bed-rest FMD in the legs only. In addition to the specific hypotheses tested, there was evidence that acute exercise evoked dilatory mechanisms in the conduit arteries that were not shear/endothelium dependent given that the shear to dilation relationship was uncoupled during, following, and in occlusion protocols that include exercise. The precise mechanisms by which this is achieved are still unknown, but it may be partially due to the oscillatory nature of the elevated blood flow during exercise. I conclude that inference of cardiovascular health from endothelial function by the evaluation of %FMD should be approached with caution, especially in the event that physical activity is involved.