Leonie Rouleau

Local mechanical and structural properties of healthy and diseased human ascending aorta tissue

OBJECTIVE This study investigates the mechanics and histology of healthy and dilated human ascending aortas (AA). The regional variation in mechanical response and tissue structure were compared. METHODS Rings of human AA from healthy (n=5), dilated tricuspid aortic valve (TAV, n=5), and dilated bicuspid aortic valve (BAV, n=6) patients were mechanically tested. Each aortic ring was sectioned into quadrants-anterior, posterior, medial (inner curvature) and lateral (outer curvature). Low- and high-stress elastic moduli were calculated from the equibiaxial stress strain curve to determine the local mechanical properties. Histological analysis was used to quantify the percent composition of elastin, collagen, and smooth muscle cells. RESULTS BAV tissue was thinnest and contained the largest percent composition of collagen. Both TAV and BAV tissue had significantly less elastin than healthy tissue. At low strain in the circumferential direction, TAV tissue was on average the least stiff. The elastic modulus was dependent on quadrant and tissue type but not direction (isotropic). Generally, the lateral quadrant tissue was the stiffest and the medial quadrant the least stiff. There were no apparent local variations in the tissue histology. CONCLUSIONS Local variations in tissue thickness and mechanical properties were evident in all samples analyzed and may be linked to the type of aortic valve present.

The development of 3-D, in vitro, endothelial culture models for the study of coronary artery disease

The response of the vascular endothelium to wall shear stress plays a central role in the development and progression of atherosclerosis. Current studies have investigated endothelial response using idealized in vitro flow chambers. Such cell culture models are unable to accurately replicate the complex in vivo wall shear stress patterns arising from anatomical geometries. To better understand this implication, we have created both simplified/tubular and anatomically realistic in vitro endothelial flow models of the human right coronary artery. A post-mortem vascular cast of the human left ventricular outflow tract was used to create geometrically accurate silicone elastomer models. Straight, tubular models were created using a custom made mold. Following the culture of human abdominal aortic endothelial cells within the inner lumen, cells were exposed to steady flow (Re = 233) for varying time periods. The resulting cell morphology was analyzed in terms of shape index and angle of orientation relative to the flow direction. In both models a progressive elongation and alignment of the endothelium in the flow direction was observed following 8, 12, and 24 hours. This change, however, was significantly less pronounced in the anatomical model (as observed from morphological variations indicative of localized flow features). Differences were also observed between the inner and outer walls at the disease-prone proximal region. Since morphological adaptation is a visual indication of endothelial shear stress activation, the use of anatomical models in endothelial genetic and biochemical studies may offer better insight into the disease process.

Endothelial Cell Morphologic Response to Asymmetric Stenosis Hemodynamics: Effects of Spatial Wall Shear Stress Gradients

Endothelial cells are known to respond to hemodynamic forces. Their phenotype has been suggested to differ between atheroprone and atheroprotective regions of the vasculature, which are characterized by the local hemodynamic environment. Once an atherosclerotic plaque has formed in a vessel, the obstruction creates complex spatial gradients in wall shear stress. Endothelial cell response to wall shear stress may be linked to the stability of coronary plaques. Unfortunately, in vitro studies of the endothelial cell involvement in plaque stability have been limited by unrealistic and simplified geometries, which cannot reproduce accurately the hemodynamics created by a coronary stenosis. Hence, in an attempt to better replicate the spatial wall shear stress gradient patterns in an atherosclerotic region, a three dimensional asymmetric stenosis model was created. Human abdominal aortic endothelial cells were exposed to steady flow (Re=50, 100, and 200 and tau=4.5 dyn/cm(2), 9 dyn/cm(2), and 18 dyn/cm(2)) in idealized 50% asymmetric stenosis and straight/tubular in vitro models. Local morphological changes that occur due to magnitude, duration, and spatial gradients were quantified to identify differences in cell response. In the one dimensional flow regions, where flow is fully developed and uniform wall shear stress is observed, cells aligned in flow direction and had a spindlelike shape when compared with static controls. Morphological changes were progressive and a function of time and magnitude in these regions. Cells were more randomly oriented and had a more cobblestone shape in regions of spatial wall shear stress gradients. These regions were present, both proximal and distal, at the stenosis and on the wall opposite to the stenosis. The response of endothelial cells to spatial wall shear stress gradients both in regions of acceleration and deceleration and without flow recirculation has not been previously reported. This study shows the dependence of endothelial cell morphology on spatial wall shear stress gradients and demonstrates that care must be taken to account for altered phenotype due to geometric features. These results may help explain plaque stability, as cells in shoulder regions near an atherosclerotic plaque had a cobblestone morphology indicating that they may be more permeable to subendothelial transport and express prothrombotic factors, which would increase the risk of atherothrombosis.

Effect of simvastatin on Kruppel-like factor2, endothelial nitric oxide synthase and thrombomodulin expression in endothelial cells under shear stress

AIMS Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) and fluid wall shear stress have been reported to modulate the expression of genes related to inflammation, blood coagulation, thrombosis, and vascular constriction in cultured endothelial cells. In this study, we investigated the combined effect of laminar shear stress (LSS) and statins on endothelial cell gene expression. MAIN METHODS Kruppel-like factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) mRNA and protein expression were evaluated in human abdominal aortic endothelial cells (HAAEC) treated with simvastatin (0.1, 1 or 10 microM) at various levels of LSS (0, 1.25, 12.5 or 25 dynes/cm(2)). KEY FINDINGS As expected, simvastatin and LSS separately enhanced KLF2, eNOS, and TM mRNA expressions. The combination of simvastatin and LSS resulted in significantly higher mRNA levels of all three genes compared to cells treated with LSS only. The highest KLF2, eNOS, and TM mRNA levels were detected at 10 microM simvastatin and 25 dynes/cm(2). Under these conditions, eNOS and TM protein levels were also elevated. Combining LSS and simvastatin produced an overall additive increase in KLF2, eNOS, and TM mRNA. Treatment of the endothelial cells with 10 microM simvastatin and 200 microM mevalonate completely eliminated the effect of simvastatin. SIGNIFICANCE Our results suggest an additive increase in KLF2, eNOS, and TM expressions when simvastatin and LSS are combined. These results may help to explain the proposed non-lipid lowering benefits of statins observed in the clinic.

Impact of degradation products of sulfamethoxazole on mammalian cultured cells

Sulfamethoxazole (SMX) is a widely used antibiotic which has been detected in surface water samples in the ng/L range and also detected in drinking water samples. To limit the environmental impact, ozonation treatment of waste streams has been proposed. However, the degradation products created by ozonation as well as their toxicity have not been reported. In this study, we investigated the degradation products of SMX formed during ozonation and the effects of these products on mammalian cultured cells. In addition to alcohols and nitrates, sulfanilamide was identified as the larger molecular weight compound of the degradation products detected. Cells exposed to the degradation products of SMX maintained their polyhedral geometry longer than the control cells. Proliferation of the cells exposed to the degradation products was not negatively affected when compared with the control cells. The results of this study show that bioactive degradation products can be formed by ozonation of SMX.

The Response of Human Aortic Endothelial Cells in a Stenotic Hemodynamic Environment: Effect of Duration, Magnitude, and Spatial Gradients in Wall Shear Stress

Inflammation plays a key role in the development and stability of coronary plaques. Endothelial cells alter their expression in response to wall shear stress (WSS). Straight/tubular and asymmetric stenosis models were designed to study the localized expression of atheroprone molecules and inflammatory markers due to the presence of the spatial wall shear stress gradients created by an eccentric plaque. The effects of steady wall shear stress duration (0-24 h) and magnitude (4.5-18 dynes/cm(2)) were analyzed in human abdominal aortic endothelial cells through quantitative real-time polymerase chain reaction (PCR) and immunofluorescence analysis in straight/tubular models. Regional expression was assessed by immunofluorescence and confocal microscopy in stenosis models. Under steady fully developed flow, endothelial cells exhibited a sustained increase in levels of atheroprotective genes with WSS duration and magnitude. The local response in the stenosis model showed that expression of endothelial nitric oxide synthase and Kruppel-like factor 2 is magnitude rather than gradient dependent. A WSS magnitude dependent transient increase in translocation of transcription factor nuclear factor kappaB was observed. Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin exhibited a sustained increase in protein expression with time. The mRNA levels of these molecules were transiently upregulated and this was followed by a decrease in expression to levels lower than static controls. Regionally, increased inflammatory marker expression was observed in regions of WSS gradients both proximal and distal to the stenosis when compared with the uniform flow regions, whereas the atheroprotective markers were expressed to a greater extent in regions of elevated WSS magnitudes. The results from the straight/tubular model cannot explain the regional variation seen in the stenosis models. This may help explain the localization of inflammatory cells at the shoulders of plaques in vivo.

Concentration and Time Effects of Dextran Exposure on Endothelial Cell Viability, Attachment, and Inflammatory Marker Expression In Vitro

Dextran is commonly used to alter growth medium rheological properties for in vitro flow experiments in order to match physiological parameters. Despite its acceptance in literature, few studies have examined dextran effects on cells. In this study, we investigated changes in endothelial cell function due to dextran, under static and flow conditions, in a concentration and time-dependent manner. Dextran increased endothelial cell viability, decreased their ability to attach to culture plates and decreased leukocyte adhesion to endothelial cells. Under static conditions, dextran increased protein and mRNA expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in a concentration and time-dependent manner and caused the nuclear translocation of NF-κB. Steady laminar wall shear stress modulated the effects of dextran on ICAM-1, VCAM-1, and NF-κB expression in straight/tubular in vitro models. When the expression was normalized to their respective time matched static dextran control, it did not affect the ability to detect changes caused by shear on the mRNA expression of ICAM-1 and VCAM-1. This study demonstrates that dextran can alter endothelial cell function and therefore, caution is advised and time matched dextran controls are necessary when using dextran for dynamic cell studies.

Laminar shear stress prevents simvastatin-induced adhesion molecule expression in cytokine activated endothelial cells

In addition to lowering cholesterol, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, have been shown to modulate gene expression in endothelial cells. The effect of statins on cell adhesion molecule expression is unclear and largely unexplored in endothelial cells exposed to shear stress, an important regulator of endothelial cell function. In this study, the effect of simvastatin on vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression was evaluated in human abdominal aortic endothelial cells (HAAEC) conditioned with various levels of laminar wall shear stress with or without tumor necrosis factor alpha (TNFα). As expected, TNFα alone greatly enhanced both VCAM-1 and ICAM-1 mRNA and protein. In static culture, simvastatin potentiated the TNFα-induced increase in VCAM-1 and ICAM-1 mRNA but not total protein at 24 h. Mevalonate, a precursor to cholesterol biosynthesis, eliminated the effect of simvastatin. Exposure of endothelial cells to elevated levels of laminar shear stress during simvastatin treatment prevented the potentiating effect of simvastatin on cell adhesion molecule mRNA. A shear stress of 12.5 dyn/cm² eliminated the increase in VCAM-1 by simvastatin, while 25 dyn/cm² was needed for ICAM-1. We conclude that simvastatin enhances VCAM-1 and ICAM-1 gene expression in TNFα-activated endothelial cells through inhibition of HMG-CoA reductase. High levels of laminar shear stress prevented the upregulation of VCAM-1 and ICAM-1 by simvastatin suggesting that an induction of cell adhesion molecules by statins may not occur in endothelial cells exposed to shear stress from blood flow.

Regional variations in canine descending aortic tissue mechanical properties change with formalin fixation.

BACKGROUND/INTRODUCTION Diseases of the aorta can alter the local mechanical properties of the tissue, leading to aneurysms and plaque instability. Local tissue properties are best evaluated from surgical samples or autopsy tissue using mechanical testing ex vivo. We examined whether formalin-fixed tissues preserve regional and local variations in tissue properties when compared to fresh tissues in order to determine if fixed tissue can be used to infer mechanical changes associated with tissue remodeling. METHODS Equibiaxial mechanical tests were performed on canine descending thoracic aorta to quantify the regional and local tissue stiffness. Samples were taken from four locations along the aorta and from the lateral and medial side at each location. Half of the samples were randomly formalin fixed and used to measure the effect of fixation on local thickness, stiffness, and anisotropy. RESULTS In fresh tissue, regional differences in tissue stiffness and thickness are present. Aortic tissue stiffens and thins along the aorta. Fixation did not alter thickness, significantly increased tissue stiffness, and altered the directional dependency of the mechanical properties (anisotropy) at low strain. Formalin fixation altered local stiffness of the aorta near the aortic arch. CONCLUSION The changes in mechanical properties along the aorta were preserved in formalin-fixed samples. However, our results show that formalin fixation can change the variation in tissue stiffness and significantly affects the anisotropic properties of vascular tissues. Formalin fixation introduces spurious changes in mechanical properties, and we therefore strongly recommend the use of fresh aortic tissues for biomechanical analysis.

Morphological and Functional Flow-Induced Response of Endothelial Cells and Adhesive properties of Leukocytes in 3D Stenotic Models

Endothelial cell dysfunction in response to hemodynamic forces is believed to be a cause of focal atherosclerosis. Flow in stenotic vessels creates complex spatial wall shear stress (WSS) gradients which may alter endothelial cell function and promote adhesion of inflammatory cells. In vitro studies have generally used unrealistic geometries, which cannot reproduce the complexity of physiological hemodynamics. We have developed a three dimensional asymmetric stenosis cell culture model to better study the interaction of endothelial cells with blood components. Human abdominal aortic endothelial cells (ECs) were exposed to steady physiological flows in our models. The adhesive properties of human promyelocytic cells (NB4) following exposure to all-trans-retinoic acid (ATRA) on ECs, were studied. Cells subjected to one dimensional flow aligned in flow direction and had a spindle-like shape when compared to static controls. EC morphology differed in the spatial WSS gradient regions, being randomly oriented and of cobblestone shape. Tumor necrosis factor α stimulation (TNF-α) increased significantly the expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) on ECs as observed by confocal microscopy and western blots. These cell adhesion molecules are known to be involved in inflammation and upregulated under the control of transcription factor nuclear factor κB (NF-κB). Under static conditions, NB4 cells adhered to a greater extent than under flow, with decreased adhesion observed with increasing flowrate. Regionally, cells under flow adhered more in the low wall shear stress recirculation region distal to the stenosis than in the one dimensional flow inlet region. At the proximal shoulder regions, greater adhesion was noticed although this was not significant. This suggests an important shear mediated role of neutrophil-endothelial interactions in the progression of atherosclerosis. Moreover, the regional response to complex hemodynamics may play an important role in plaque stability.