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Distinguishing endothelial responses to impingement force, wall shear stress, and wall shear stress gradient
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Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
6792 We, 08:45-09:00 (P28) Flow-activated chloride channels in aortic endothelium: sensitivity and functional implications M. Gautam 1, '~ Shen 1, T.L. Thirkill 2, G.C. Douglas2, A.I. Barakat1. 1Department of Mechanical and Aeronautical Engineering, 2Department of Ceil Biology and Human Anatomy, University of California, Davis, USA Activation of ion channels is among the most rapid flow responses in vascular endothelial cells (ECs); therefore, these channels are considered as candidate flow sensors. Previously, we had reported that flow activates an outwardrectifying CI- current in bovine aortic ECs (BAECs). In the present study, we used whole-cell patch clamping to characterize the sensitivity of the CIcurrent to the magnitude and type of applied shear stress. We also studied the desensitization dynamics of the current and the functional implications of its activation. Application of shear stress caused rapid development of a CI- current that was effectively blocked by the CI- channel antagonist NPPB (100 ~tmol/L). The current initiated at a shear stress as low as 0.3dyne/cm 2 and saturated above 3.5 dyne/cm 2. The peak flow-induced current was -2.5 times that of the baseline current present in BAECs under no flow conditions, and this peak was attained -2 minutes after the onset of flow for all shear stresses studied (0.3-14 dyne/cm2). In the presence of sustained flow, the CI- current exhibited complete desensitization in -50% of the cells, while the remaining cells showed limited desensitization. Oscillatory flow with a physiological frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the CI- current, while lower oscillation frequencies led to partial recovery of the current. Non-reversing pulsatile flow, generally considered athero-protective, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the CI- current is responsive to shear stress rather than shear rate, suggesting that the effect of flow on CI- channels is direct. Blocking the flow-activated CI- current inhibited the phosphorylation by flow of the serine/threonine kinase Akt. Given the importance of Akt for EC survival and function, our results point to an important functional role for flow-sensitive CI- channels. 6607 We, 09:00-09:15 (P28) Unique endothelial gene expression sensitivities to shear stress magnitude and spatial gradient J.A. LaMack, M.H. Friedman. Department ef Biomedical Engineering, Duke University, Durham, NC, USA The apparent tendency of atherosclerotic lesions to form in complex blood flow environments has led to many theories regarding the importance of hemodynamic forces in endothelium-mediated atherosusceptibility. Following up on our recent finding that a composite function of spatial shear stress gradient and magnitude predicts local transendothelial macromolecular permeability in the porcine iliac artery in vive, here we study the effects of the two hemodynamic variables on the expression of several shear responsive genes in vitro. This study is the first in which the effect of spatial gradient has been isolated from that of shear stress magnitude and in which interactions between the two have been studied. Converging-width flow channels were designed to create spatial shear stress gradients over cultured porcine aortic endothelial cells. Cells were collected from locations within the channels selected so that the effect of shear stress gradient could be studied at various fixed levels of shear stress magnitude, and vice versa. Quantitative real-time PCR was used to measure the relative mRNA expression of eNOS, c-jun, MCP-1, ICAM-1, and VCAM-1 in each sample. VCAM-1 expression was insensitive to either variable, and the remaining four genes each had unique sensitivities, eNOS showed a strong positive dependence upon magnitude only. The expression of c-jun was weakly correlated with magnitude and gradient, but not on their interaction. MCP-1 expression correlated inversely with gradient and also depended on the interaction of gradient with magnitude. ICAM-1 expression also depended on the interaction term, and it increased with magnitude. The results support the notion that cells are able to sense each hemodynamic force independently and that signaling pathways evoked by them are partially shared. 6315 We, 09:15-09:30 (P28) Distinguishing endothelial responses to impingement force, wall shear stress, and wall shear stress gradient E. Metaxa1,2, J. Kolega4, M.P. Szymanski 1,2, Z.J. Wang 1,2, D.D. Swartz2,3, H. Meng 1,2,5. 1Department of Mechanical and Aerospace Engineering, State University of New York at Buffalo, Buffalo, USA, 2 Toshiba Stroke Research Center, State University of New York at Buffalo, Buffalo, USA, 3Department of Pediatrics, State University of New York at Buffalo, Buffalo, USA, 4Department of Anatomy and Ceil Biology, State University of New York at Buffalo, Buffalo, USA, 5Department of Neurosurgery, State University of New York at Buffalo, Buffalo, USA Cerebral Aneurysms (CAs) are a major cause of subarachnoid hemorrhage, which is characterized by high rates of morbidity and mortality. However, the
Oral Presentations mechanisms underlying the initiation and development of Cerebral Aneurysms are not known. Vascular remodeling processes have been shown to be highly dependent on the endothelial cells (ECs) and to be strongly influenced by the local hemodynamic environment. CAs are associated with hypertension and develop at curvatures and apices of bifurcations, where hemodynamics is complex, characterized by overlapping regions of impinging flow, high Wall Shear Stress (WSS) and high WSS gradient. In order to understand how ECs respond to the various flow dynamic factors that are involved in this complex hemodynamic environment, two in vitro systems were designed. In the first, ECs are exposed to flow in a tapered cell chamber, in which WSS ranges from 10-100 dynes/cm 2 and pressure either normal (100mmHg) or high (150mmHg). In the second, a flow jet impinges on a monolayer of ECs creating areas of quantifiable flow stagnation, high WSS gradient and a combination of high WSS gradient and high WSS. In both systems, ECs maintained cell-substrate and cell-cell attachments and displayed orientation that was highly dependent on their specific flow environment. In addition, EC proliferation depended on the flow dynamic conditions, with EC proliferation increasing at higher WSS. These experiments elucidate the response of ECs to specific, controlled flow dynamic factors that are involved in the complex hemodynamic environment of arterial bifurcations, sites prone to aneurysm development. 5168 We, 09:30-09:45 (P28) Endothelial cell morphology and response to shear stress in an asymmetric stenosis model L. Rouleau 1, M. Farcas 1, J.-C. Tardif3, E. Thorin 3, R. Mongrain2,3, R.L. Leask 1,3. 1Department of Chemical Engineering, McGill University, Montreal, Canada, 2Department of Mechanical Engineering, McGill University, Montreal, Canada, 3Mentreal Heart Institute, Montreal, Canada Coronary artery disease (CAD) is the leading cause of death and hospitalization in the World. Hemodynamic forces are known to play a key role in the development of CAD and have been linked to endothelial cell (EC) dysfunction. Flow in stenotic vessels creates a complex pattern of hemodynamics in that it includes both high and low shear stress regions. In vitro studies examining the effect of shear stress on endothelial cells have used unrealistic and simplified models, which cannot reproduce physiological conditions. In this study, a transparent and compliant 50% occlusion idealized asymmetric vascular stenosis model was designed and the response of endothelial cells to shear stress was characterized. Human abdominal aortic endothelial cells were seeded repeatedly onto fibronectin coated models and grown for 24 hours. The models were exposed for different times to a steady flow of 100mL/min resulting in physiological fluid parameters. The models were fixed using paraformaldehyde and stained using a crystal violet solution. EC in the static and disturbed flow conditions randomly aligned and had a cobblestone shape whereas when they were subjected to laminar flow they aligned with the flow and had a spindle-like shape. A morphometric analysis verified that there is a significant difference in terms of shape index (One-way Anova, P <0.05) and angle of orientation (F-test, P <0.05), the shape index value and the angle of orientation variance being similar in the disturbed regions and in the static models and significantly different than in the laminar flow regions. This innovative technique provides a more realistic model to study endothelial cell response to shear stress. A confluent monolayer of EC was grown within the model and morphology was analyzed. In the future, this model will provide a better understanding of the mechanisms involved in atherosclerosis and of the influence of hemodynamics on CAD. 7885 We, 11:00-11:30 (P32) Harmonic environment and frequency response of vascular cells. Toward a definition of disturbed flow M.H. Friedman. Department of Biomedical Engineering, Duke University, Durham NC, USA Atherosclerosis is often said to localize preferentially in regions of "disturbed flow", but the term in this usage does not have the precision it possesses in fluid mechanics. We hypothesize that the atherogenicity of "disturbed flow" can be related to the harmonic content of the fluid dynamic shear at the arterial wall. Computations of vascular shear distributions in real arterial geometries reveal that, at sites where the time-average shear stress is low, the harmonic content of the shear waveform is abnormally high. Cellular mechanotransduction necessarily involves mechanical elements whose response might be expected to be frequency dependent. Gene expression profiles of cultured vascular endothelial cells under pulsatile flow are strongly frequency dependent, lending support to this hypothesis at the molecular level. Research supported by NIH Grant HL050442.
Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
6792 We, 08:45-09:00 (P28) Flow-activated chloride channels in aortic endothelium: sensitivity and functional implications M. Gautam 1, '~ Shen 1, T.L. Thirkill 2, G.C. Douglas2, A.I. Barakat1. 1Department of Mechanical and Aeronautical Engineering, 2Department of Ceil Biology and Human Anatomy, University of California, Davis, USA Activation of ion channels is among the most rapid flow responses in vascular endothelial cells (ECs); therefore, these channels are considered as candidate flow sensors. Previously, we had reported that flow activates an outwardrectifying CI- current in bovine aortic ECs (BAECs). In the present study, we used whole-cell patch clamping to characterize the sensitivity of the CIcurrent to the magnitude and type of applied shear stress. We also studied the desensitization dynamics of the current and the functional implications of its activation. Application of shear stress caused rapid development of a CI- current that was effectively blocked by the CI- channel antagonist NPPB (100 ~tmol/L). The current initiated at a shear stress as low as 0.3dyne/cm 2 and saturated above 3.5 dyne/cm 2. The peak flow-induced current was -2.5 times that of the baseline current present in BAECs under no flow conditions, and this peak was attained -2 minutes after the onset of flow for all shear stresses studied (0.3-14 dyne/cm2). In the presence of sustained flow, the CI- current exhibited complete desensitization in -50% of the cells, while the remaining cells showed limited desensitization. Oscillatory flow with a physiological frequency of 1 Hz, as occurs in disturbed flow zones prone to atherosclerosis, failed to elicit the CI- current, while lower oscillation frequencies led to partial recovery of the current. Non-reversing pulsatile flow, generally considered athero-protective, was as effective in eliciting the current as steady flow. Measurements using fluids of different viscosities indicated that the CI- current is responsive to shear stress rather than shear rate, suggesting that the effect of flow on CI- channels is direct. Blocking the flow-activated CI- current inhibited the phosphorylation by flow of the serine/threonine kinase Akt. Given the importance of Akt for EC survival and function, our results point to an important functional role for flow-sensitive CI- channels. 6607 We, 09:00-09:15 (P28) Unique endothelial gene expression sensitivities to shear stress magnitude and spatial gradient J.A. LaMack, M.H. Friedman. Department ef Biomedical Engineering, Duke University, Durham, NC, USA The apparent tendency of atherosclerotic lesions to form in complex blood flow environments has led to many theories regarding the importance of hemodynamic forces in endothelium-mediated atherosusceptibility. Following up on our recent finding that a composite function of spatial shear stress gradient and magnitude predicts local transendothelial macromolecular permeability in the porcine iliac artery in vive, here we study the effects of the two hemodynamic variables on the expression of several shear responsive genes in vitro. This study is the first in which the effect of spatial gradient has been isolated from that of shear stress magnitude and in which interactions between the two have been studied. Converging-width flow channels were designed to create spatial shear stress gradients over cultured porcine aortic endothelial cells. Cells were collected from locations within the channels selected so that the effect of shear stress gradient could be studied at various fixed levels of shear stress magnitude, and vice versa. Quantitative real-time PCR was used to measure the relative mRNA expression of eNOS, c-jun, MCP-1, ICAM-1, and VCAM-1 in each sample. VCAM-1 expression was insensitive to either variable, and the remaining four genes each had unique sensitivities, eNOS showed a strong positive dependence upon magnitude only. The expression of c-jun was weakly correlated with magnitude and gradient, but not on their interaction. MCP-1 expression correlated inversely with gradient and also depended on the interaction of gradient with magnitude. ICAM-1 expression also depended on the interaction term, and it increased with magnitude. The results support the notion that cells are able to sense each hemodynamic force independently and that signaling pathways evoked by them are partially shared. 6315 We, 09:15-09:30 (P28) Distinguishing endothelial responses to impingement force, wall shear stress, and wall shear stress gradient E. Metaxa1,2, J. Kolega4, M.P. Szymanski 1,2, Z.J. Wang 1,2, D.D. Swartz2,3, H. Meng 1,2,5. 1Department of Mechanical and Aerospace Engineering, State University of New York at Buffalo, Buffalo, USA, 2 Toshiba Stroke Research Center, State University of New York at Buffalo, Buffalo, USA, 3Department of Pediatrics, State University of New York at Buffalo, Buffalo, USA, 4Department of Anatomy and Ceil Biology, State University of New York at Buffalo, Buffalo, USA, 5Department of Neurosurgery, State University of New York at Buffalo, Buffalo, USA Cerebral Aneurysms (CAs) are a major cause of subarachnoid hemorrhage, which is characterized by high rates of morbidity and mortality. However, the
Oral Presentations mechanisms underlying the initiation and development of Cerebral Aneurysms are not known. Vascular remodeling processes have been shown to be highly dependent on the endothelial cells (ECs) and to be strongly influenced by the local hemodynamic environment. CAs are associated with hypertension and develop at curvatures and apices of bifurcations, where hemodynamics is complex, characterized by overlapping regions of impinging flow, high Wall Shear Stress (WSS) and high WSS gradient. In order to understand how ECs respond to the various flow dynamic factors that are involved in this complex hemodynamic environment, two in vitro systems were designed. In the first, ECs are exposed to flow in a tapered cell chamber, in which WSS ranges from 10-100 dynes/cm 2 and pressure either normal (100mmHg) or high (150mmHg). In the second, a flow jet impinges on a monolayer of ECs creating areas of quantifiable flow stagnation, high WSS gradient and a combination of high WSS gradient and high WSS. In both systems, ECs maintained cell-substrate and cell-cell attachments and displayed orientation that was highly dependent on their specific flow environment. In addition, EC proliferation depended on the flow dynamic conditions, with EC proliferation increasing at higher WSS. These experiments elucidate the response of ECs to specific, controlled flow dynamic factors that are involved in the complex hemodynamic environment of arterial bifurcations, sites prone to aneurysm development. 5168 We, 09:30-09:45 (P28) Endothelial cell morphology and response to shear stress in an asymmetric stenosis model L. Rouleau 1, M. Farcas 1, J.-C. Tardif3, E. Thorin 3, R. Mongrain2,3, R.L. Leask 1,3. 1Department of Chemical Engineering, McGill University, Montreal, Canada, 2Department of Mechanical Engineering, McGill University, Montreal, Canada, 3Mentreal Heart Institute, Montreal, Canada Coronary artery disease (CAD) is the leading cause of death and hospitalization in the World. Hemodynamic forces are known to play a key role in the development of CAD and have been linked to endothelial cell (EC) dysfunction. Flow in stenotic vessels creates a complex pattern of hemodynamics in that it includes both high and low shear stress regions. In vitro studies examining the effect of shear stress on endothelial cells have used unrealistic and simplified models, which cannot reproduce physiological conditions. In this study, a transparent and compliant 50% occlusion idealized asymmetric vascular stenosis model was designed and the response of endothelial cells to shear stress was characterized. Human abdominal aortic endothelial cells were seeded repeatedly onto fibronectin coated models and grown for 24 hours. The models were exposed for different times to a steady flow of 100mL/min resulting in physiological fluid parameters. The models were fixed using paraformaldehyde and stained using a crystal violet solution. EC in the static and disturbed flow conditions randomly aligned and had a cobblestone shape whereas when they were subjected to laminar flow they aligned with the flow and had a spindle-like shape. A morphometric analysis verified that there is a significant difference in terms of shape index (One-way Anova, P <0.05) and angle of orientation (F-test, P <0.05), the shape index value and the angle of orientation variance being similar in the disturbed regions and in the static models and significantly different than in the laminar flow regions. This innovative technique provides a more realistic model to study endothelial cell response to shear stress. A confluent monolayer of EC was grown within the model and morphology was analyzed. In the future, this model will provide a better understanding of the mechanisms involved in atherosclerosis and of the influence of hemodynamics on CAD. 7885 We, 11:00-11:30 (P32) Harmonic environment and frequency response of vascular cells. Toward a definition of disturbed flow M.H. Friedman. Department of Biomedical Engineering, Duke University, Durham NC, USA Atherosclerosis is often said to localize preferentially in regions of "disturbed flow", but the term in this usage does not have the precision it possesses in fluid mechanics. We hypothesize that the atherogenicity of "disturbed flow" can be related to the harmonic content of the fluid dynamic shear at the arterial wall. Computations of vascular shear distributions in real arterial geometries reveal that, at sites where the time-average shear stress is low, the harmonic content of the shear waveform is abnormally high. Cellular mechanotransduction necessarily involves mechanical elements whose response might be expected to be frequency dependent. Gene expression profiles of cultured vascular endothelial cells under pulsatile flow are strongly frequency dependent, lending support to this hypothesis at the molecular level. Research supported by NIH Grant HL050442.