Tzung K. Hsiai

Pulsatile Versus Oscillatory Shear Stress Regulates NADPH Oxidase Subunit Expression. Implication for Native LDL Oxidation

Abstract— Shear stress regulates endothelial nitric oxide and superoxide (O2−·) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 &mgr;g/mL of native LDL were exposed to (1) pulsatile flow with a mean shear stress (&tgr;ave) of 25 dyne/cm2 and (2) oscillating flow at &tgr;ave of 0. After 4 hours, aliquots of culture medium were collected for high-performance liquid chromatography analyses of electronegative LDL species, described as LDL− and LDL2−. In response to oscillatory shear stress, gp91phox mRNA expression was upregulated by 2.9±0.3-fold, and its homologue, Nox4, by 3.9±0.9-fold (P <0.05, n=4), with a corresponding increase in O2−· production rate. The proportion of LDL− and LDL2− relative to static conditions increased by 67±17% and 30±7%, respectively, with the concomitant upregulation of monocyte chemoattractant protein-1 expression and increase in monocyte/BAEC binding (P <0.05, n=5). In contrast, pulsatile flow downregulated both gp91phox and Nox4 mRNA expression (by 1.8±0.2-fold and 3.0±0.12-fold, respectively), with an accompanying reduction in O2−· production, reduction in the extent of LDL modification (51±12% for LDL− and 30±7% for LDL2−), and monocyte/BAEC binding. The flow-dependent LDL oxidation is determined in part by the NADPH oxidase activity. The formation of modified LDL via O2−· production may also affect the regulation of monocyte chemoattractant protein-1 expression and monocyte/BAEC binding.

Monocyte recruitment to endothelial cells in response to oscillatory shear stress

Leukocyte recruitment to endothelial cells is a critical event in inflammatory responses. The spatial, temporal gradients of shear stress, topology, and outcome of cellular interactions that underlie these responses have so far been inferred from static imaging of tissue sections or studies of statically cultured cells. In this report, we developed micro‐electromechanical systems (MEMS) sensors, comparable to a single endothelial cell (EC) in size, to link real‐time shear stress with monocyte/EC binding kinetics in a complex flow environment, simulating the moving and unsteady separation point at the arterial bifurcation with high spatial and temporal resolution. In response to oscillatory shear stress (τ) at ± 2.6 dyn/cm2 at a time‐averaged shear stress (τave) = 0 and 0.5 Hz, individual monocytes displayed unique to‐and‐fro trajectories undergoing rolling, binding, and dissociation with other monocyte, followed by solid adhesion on EC. Our study quantified individual monocyte/EC binding kinetics in terms of displacement and velocity profiles. Oscillatory flow induces up‐regulation of adhesion molecules and cytokines to mediate monocyte/EC interactions over a dynamic range of shear stress ± 2.6 dyn/cm2 (P=0.50. n=10).—Hsiai, T. K., Cho, S. K., Wong, P. K., Ing, M., Salazar, A., Sevanian, A., Navab, M., Demer, L. L., Ho, C.‐M. Monocyte recruitment to endothelial cells in response to oscillatory shear stress. FASEB J. 17, 1648–1657 (2003)

Ultrafine particles from diesel vehicle emissions at different driving cycles induce differential vascular pro-inflammatory responses: Implication of chemical components and NF-κB signaling

BackgroundEpidemiological evidence supports the association between exposure to ambient particulate matter (PM) and cardiovascular diseases. Chronic exposure to ultrafine particles (UFP; Dp <100 nm) is reported to promote atherosclerosis in ApoE knockout mice. Atherogenesis-prone factors induce endothelial dysfunction that contributes to the initiation and progression of atherosclerosis. We previously demonstrated that UFP induced oxidative stress via c-Jun N-terminal Kinases (JNK) activation in endothelial cells. In this study, we investigated pro-inflammatory responses of human aortic endothelial cells (HAEC) exposed to UFP emitted from a diesel truck under an idling mode (UFP1) and an urban dynamometer driving schedule (UFP2), respectively. We hypothesize that UFP1 and UFP2 with distinct chemical compositions induce differential pro-inflammatory responses in endothelial cells.ResultsUFP2 contained a higher level of redox active organic compounds and metals on a per PM mass basis than UFP1. While both UFP1 and UFP2 induced superoxide production and up-regulated stress response genes such as heme oxygenease-1 (HO-1), OKL38, and tissue factor (TF), only UFP2 induced the expression of pro-inflammatory genes such as IL-8 (2.8 ± 0.3-fold), MCP-1 (3.9 ± 0.4-fold), and VCAM (6.5 ± 1.1-fold) (n = 3, P < 0.05). UFP2-exposed HAEC also bound to a higher number of monocytes than UFP1-exposed HAEC (Control = 70 ± 7.5, UFP1 = 106.7 ± 12.5, UFP2 = 137.0 ± 8.0, n = 3, P < 0.05). Adenovirus NF-κB Luciferase reporter assays revealed that UFP2, but not UFP1, significantly induced NF-κB activities. NF-κB inhibitor, CAY10512, significantly abrogated UFP2-induced pro-inflammatory gene expression and monocyte binding.ConclusionWhile UFP1 induced higher level of oxidative stress and stress response gene expression, only UFP2, with higher levels of redox active organic compounds and metals, induced pro-inflammatory responses via NF-κB signaling. Thus, UFP with distinct chemical compositions caused differential response patterns in endothelial cells.

Ultrafine particles from diesel engines induce vascular oxidative stress via JNK activation

Exposure to particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultrafine particles (UFP) from diesel vehicle engines have been shown to be proatherogenic in ApoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induce vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intracellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O(2)(-)) production in human aortic endothelial cells (HAEC). Flow cytometry showed that UFP increased MitoSOX red intensity specific for mitochondrial superoxide. Protein carbonyl content was increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated heme oxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pretreatment with the antioxidant N-acetylcysteine significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with the JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP-stimulated O(2)(-) production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation plays an important role in UFP-induced oxidative stress and stress response gene expression.

Endothelial cell dynamics under pulsating flows: significance of high versus low shear stress slew rates (d(tau)/dt).

AbstractShear stress modulates endothelial cell (EC) remodeling via realignment and elongation. We provide the first evidence that the upstroke slopes of pulsatile flow, defined as shear stress slew rates (positive ∂τ/∂τ affect significantly the rates at which ECs remodel. We designed a novel flow system to isolate various shear stress slew rates by precisely controlling the frequency, amplitude, and time-averaged shear stress τave of pulsatile flow. Bovine aortic endothelial cell (BAEC) monolayers were exposed to three conditions: (1) pulsatile flow (1 Hz) at high slew rate (293 dyn/cm2 s), (2) pulsatile flow (1 Hz) at low slew rate (71 dyn/cm2 s), and (3) steady laminar flow at ∂τ/∂t=0. All of the three conditions were operated at τave=50{dyn/cm}2. BAEC elongation and alignment were measured over 17 h. We were able to demonstrate the effects of shear stress slew rates ∂τ/∂t on EC remodeling at a fixed spatial shear stress gradient (∂τ/∂x). We found that pulsatile flow significantly increased the rates at which EC elongated and realigned, compared to steady flow at ∂τ/∂t=0. Furthermore, EC remodeling was faster in response to high than to low slew rates at a given tau τave

A complex flow pattern of low shear stress and flow reversal promotes monocyte binding to endothelial cells.

Predilection sites for atherosclerosis within the vasculature are characterized by low shear stress and flow reversal. In this study, endothelial cells were exposed to a complex flow pattern that was characterized by particle velocity determination. Bovine aortic endothelial cells exposed to low shear stress and flow reversal demonstrated higher levels of monocyte binding compared to endothelial cells exposed to one-directional flow. In addition, endothelial cells exposed to low shear stress and flow reversal responded to inflammatory stimuli with substantial increases in monocyte binding, similar to that seen in cells exposed to one-directional flow. These findings suggest a mechanism by which areas of low shear stress and flow reversal are predisposed to the development of atherosclerotic lesions.

Chemical gating of In2O3 nanowires by organic and biomolecules

In2O3 nanowire transistors were used to investigate the chemical gating effect of organic molecules and biomolecules with amino or nitro groups. The nanowire conductance changed dramatically after adsorption of these molecules. Specifically, amino groups in organic molecules such as butylamine, donated electrons to In2O3 nanowires and thus led to enhanced carrier concentrations and conductance, whereas molecules with nitro groups such as butyl nitrite made In2O3 nanowires less conductive by withdrawing electrons. In addition, intrananowire junctions created by partial exposure of the nanowire device to butyl nitrite were investigated, and pronounced rectifying current–voltage characteristics were obtained. Furthermore, chemical gating by low-density lipoprotein cholesterol, the offending agent in coronary heart diseases, was also observed and attributed to the amino groups carried by the bio species.

Oxidized Low-Density Lipoprotein-Activated c-Jun NH2-Terminal Kinase Regulates Manganese Superoxide Dismutase Ubiquitination: Implication for Mitochondrial Redox Status and Apoptosis

Objective—Oxidized low-density lipoprotein (oxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. However, the signal pathways and molecular mechanism remain unknown. In this study, we investigated the role of manganese superoxide dismutase (Mn-SOD) on oxLDL-induced apoptosis via c-Jun NH2-terminal kinase (JNK)-mediated ubiquitin/proteasome pathway. Methods and Results—OxLDL induced JNK phosphorylation that peaked at 30 minutes in human aortic endothelial cells. Fluorescence-activated cell sorting analysis revealed that oxLDL increased mitochondrial superoxide production by 1.88±0.19-fold and mitochondrial membrane potential by 18%. JNK small interference RNA (siJNK) reduced oxLDL-induced mitochondrial superoxide production by 88.4% and mitochondrial membrane potential by 61.7%. OxLDL did not affect Mn-SOD mRNA expression, but it significantly reduced Mn-SOD protein level, which was restored by siJNK. Immunoprecipitation by ubiquitin antibody revealed that oxLDL increased ubiquitination of Mn-SOD, which was inhibited by siJNK. OxLDL-induced caspase-3 activities were also attenuated by siJNK but were enhanced by Mn-SOD small interfering RNA. Furthermore, overexpression of Mn-SOD abrogated oxLDL-induced caspase-3 activities. Conclusion—OxLDL-induced JNK activation regulates mitochondrial redox status and Mn-SOD protein degradation via JNK-dependent ubiquitination, leading to endothelial cell apoptosis.

Flexible Polymer Sensors for In Vivo Intravascular Shear Stress Analysis

Hemodynamic forces, specifically fluid shear stress, play an important role in the focal nature of arterial plaque formation known as atherosclerosis. We hereby developed biocompatible and flexible intravascular microelectromechanical systems sensor to measure real-time shear stress in the aortas of New Zealand white (NZW) rabbits. Titanium (Ti) and platinum (Pt) were deposited on silicon wafers and patterned to form the sensing elements. The polymer, parylene C, provided insulation to the electrode leads and flexibility to the sensors. Based on heat transfer principle, the heat dissipation from the sensors to the blood flow altered the resistance of the sensing elements, from which shear stress was calibrated. The resistance of the sensing element was measured at approximately 1.0 kOmega , and the temperature coefficient of resistance was at approximately 0.16%/degC. The individual sensors were packaged to the catheter for intravascular deployment in the aortas of NZW rabbits (n = 5) . The sensor was capable of resolving spatial- and time-varying components of shear stress in the abdominal aorta. Computational fluid dynamic code based on non-Newtonian fluid properties showed comparable results within an acceptable range of experimental errors ( plusmn9%) for the maximal and minimal values in shear stress during one cardiac cycle. Therefore, we demonstrated the capability of biocompatible sensors for real-time shear stress measurement in vivo with a potential to advance the understanding between the blood flow and vascular disease.

Ambient Ultrafine Particles Alter Lipid Metabolism and HDL Anti-Oxidant Capacity in LDLR-null Mice

Exposure to ambient particulate matter (PM) is a risk factor for cardiovascular diseases. The redox-active ultrafine particles (UFPs) promote vascular oxidative stress and inflammatory responses. We hypothesized that UFPs modulated lipid metabolism and anti-oxidant capacity of high density lipoprotein (HDL) with an implication in atherosclerotic lesion size. Fat-fed low density lipoprotein receptor-null (LDLR−/−) mice were exposed to filtered air (FA) or UFPs for 10 weeks with or without administering an apolipoprotein A-I mimetic peptide made of D-amino acids, D-4F. LDLR−/− mice exposed to UFPs developed a reduced plasma HDL level (P < 0.01), paraoxonase activity (P < 0.01), and HDL anti-oxidant capacity (P < 0.05); but increased LDL oxidation, free oxidized fatty acids, triglycerides, serum amyloid A (P < 0.05), and tumor necrosis factor α (P < 0.05), accompanied by a 62% increase in the atherosclerotic lesion ratio of the en face aortic staining and a 220% increase in the cross-sectional lesion area of the aortic sinus (P < 0.001). D-4F administration significantly attenuated these changes. UFP exposure promoted pro-atherogenic lipid metabolism and reduced HDL anti-oxidant capacity in fat-fed LDLR−/− mice, associated with a greater atherosclerotic lesion size compared with FA-exposed animals. D-4F attenuated UFP-mediated pro-atherogenic effects, suggesting the role of lipid oxidation underlying UFP-mediated atherosclerosis.