Lisong Ai

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.

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.

Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration

Fluid shear stress modulates vascular production of endothelial superoxide anion (O2*-) and nitric oxide (*NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress (tau ave) of 23 dyn/cm2 upregulated Mn-SOD mRNA expression at a higher level than did OSS at tau ave = 0.02 dyn/cm2 +/- 3.0 dyn.cm(-2).s(-1) and at 1 Hz (PSS by 11.3 +/- 0.4-fold vs. OSS by 5.0 +/- 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration.

Oscillatory Shear Stress Induces Mitochondrial Superoxide Production: Implication of NADPH Oxidase and c-Jun NH2-Terminal Kinase Signaling

Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•-) production via NADPH oxidase and c-Jun NH(2)-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3 dyn/cm2) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO2•- production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O2•- and mtO2•- production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO2•- production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO2•- production via NADPH oxidase and JNK activation relevant for vascular oxidative stress.

Pulsatile shear stress increased mitochondrial membrane potential: Implication of Mn-SOD

Mitochondrial dysfunction is intimately involved in cardiovascular diseases. Mitochondrial membrane potential (DeltaPsi(m)) is coupled with oxidative phosphorylation to drive ATP synthesis. In this study, we examined the effect of physiological pulsatile shear stress (PSS) on DeltaPsi(m) and the role of Mn-SOD expression on DeltaPsi(m). Confluent human aortic endothelial cells (HAEC) were exposed to PSS, and DeltaPsi(m) was monitored using tetramethylrhodamine methyl ester (TMRM(+)), a mitochondrial membrane potential probe. PSS significantly increased DeltaPsi(m) and the change in DeltaPsi(m) was a dynamic process. DeltaPsi(m) returned to baseline level after PSS for 2h followed by static state for 4h. Mitochondrial Mn-SOD expression and activities were also significantly up-regulated in response to PSS. Silencing Mn-SOD attenuated PSS-mediated DeltaPsi(m) increase while adding Mn-SOD mimetic, MnTMPyP, increased DeltaPsi(m) to the similar extent as induced by PSS. Our findings suggest that PSS-increased mitochondrial DeltaPsi(m), in part, via Mn-SOD up-regulation.

Real-time assessment of flow reversal in an eccentric arterial stenotic model

Plaque rupture is the leading cause of acute coronary syndromes and stroke. Plaque formation, otherwise known as stenosis, preferentially occurs in the regions of arterial bifurcation or curvatures. To date, real-time assessment of stenosis-induced flow reversal remains a clinical challenge. By interfacing microelectromechanical system (MEMS) thermal sensors with the high frequency pulsed wave (PW) Doppler ultrasound, we proposed to assess flow reversal in the presence of an eccentric stenosis. We developed a 3-D stenotic model (inner diameter of 6mm, an eccentric stenosis with a height of 2.75 mm, and width of 21 mm) simulating a superficial arterial vessel. We demonstrated that heat transfer from the sensing element (2 x 80 μm²) to the flow field peaked as a function of flow rates at the throat of the stenosis along the center/midline of arterial model, and dropped downstream from the stenosis, where flow reversal was detected by the high frequency ultrasound device at 45 MHz. Computational fluid dynamics (CFD) codes are in agreement with the ultrasound-acquired flow profiles upstream, downstream, and at the throat of the stenosis. Hence, we characterized regions of eccentric stenosis in terms of changes in heat transfer along the midline of vessel and identified points of flow reversal with high spatial and temporal resolution.

Optimization of intravascular shear stress assessment in vivo

The advent of microelectromechanical systems (MEMS) sensors has enabled real-time wall shear stress (WSS) measurements with high spatial and temporal resolution in a 3-D bifurcation model. To optimize intravascular shear stress assessment, we evaluated the feasibility of catheter/coaxial wire-based MEMS sensors in the abdominal aorta of the New Zealand white (NZW) rabbits. Theoretical and computational fluid dynamics (CFD) analyses were performed. Fluoroscope and angiogram provided the geometry of aorta, and the Doppler ultrasound system provided the pulsatile flow velocity for the boundary conditions. The physical parameters governing the shear stress assessment in NZW rabbits included (1) the position and distance from which the MEMS sensors were mounted to the terminal end of coaxial wire or the entrance length, (L(e)), (2) diameter ratios of aorta to the coaxial wire (D(aorta) /D(coaxial wire)=1.5-9.5), and (3) the range of Reynolds numbers (116-1550). At an aortic diameter of 2.4mm and a maximum Reynolds number of 212 (a mean Reynolds number of 64.2), the time-averaged shear stress (tau(ave)) was computed to be 10.06 dyn cm(-2) with a systolic peak at 33.18 dyn cm(-2). In the presence of a coaxial wire (D(aorta)/D(coaxial wire)=6 and L(e)=1.18 cm), the tau(ave) value increased to 15.54 dyn cm(-2) with a systolic peak at 51.25 dyn cm(-2). Real-time intravascular shear stress assessment by the MEMS sensor revealed an tau(ave) value of 11.92 dyn cm(-2) with a systolic peak at 47.04 dyn cm(-2). The difference between CFD and experimental tau(ave) was 18.5%. These findings provided important insights into packaging the MEMS sensors to optimize in vivo shear stress assessment.

Real-Time Intravascular Shear Stress in the Rabbit Abdominal Aorta

Fluid shear stress is intimately linked with the biological activities of vascular cells. A flexible microelectromechanical system (MEMS) sensor was developed to assess spatial- and temporal-varying components of intravascular shear stress (ISS) in the abdominal aorta of adult New Zealand white (NZW) rabbits. Real-time ISS (ISSreal-time) was analyzed in comparison with computational fluid dynamics (CFD) simulations for wall shear stress (WSS). Three-dimensional abdominal arterial geometry and mesh were created using the GAMBIT software. Simulation of arterial flow profiles was established by FLUENT. The Navier-Stokes equations were solved for non-Newtonian blood flow. The coaxial-wire-based MEMS sensor was deployed into the abdominal arteries of rabbits via a femoral artery cutdown. Based on the CFD analysis, the entrance length of the sensor on the coaxial wire (0.4 mm in diameter) was less than 10 mm. Three-dimensional fluoroscope and contrast dye allowed for visualization of the positions of the sensor and ratios of vessel to coaxial wire diameters. Doppler ultrasound provided the velocity profiles for the CFD boundary conditions. If the coaxial wire were positioned at the center of vessel, the CFD analysis revealed a mean ISS value of 31.1 with a systolic peak at 102.8 dyn ldr cm-2. The mean WSS was computed to be 10.1 dyn ldr cm-2 with a systolic peak at 33.2 dynldrcm-2, and the introduction of coaxial wire increased the mean WSS by 5.4 dyn ldr cm-2 and systolic peak by 18.0 dyn ldr cm-2. Experimentally, the mean ISS was 11.9 dyn ldr cm-2 with a systolic peak at 47.0 dyn ldr cm-2. The waveform of experimental ISS was similar to that of CFD solution with a 30.2% difference in mean and 8.9% in peak systolic shear stress. Despite the difference between CD and experimental results, the flexible coaxial-wire-based MEMS sensors provided a possibility to assess real-time ISS in the abdominal aorta of NZW rabbits.

Monitoring oxidative stress in vascular endothelial cells in response to fluid shear stress: from biochemical analyses to micro- and nanotechnologies.

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherosclerosis. Shear stress induces vascular oxidative stress via the activation of membrane-bound NADPH oxidases present in vascular smooth muscle cells, fibroblasts, and phagocytic mononuclear cells. Shear stress acting on the endothelial cells at arterial bifurcations or branching points regulates both NADPH oxidase and nitric oxide (NO) synthase activities. The former is considered a major source of oxygen-centered radicals (i.e., superoxide anion [O2(.-)]) that give rise to oxidative stress; the latter is a source of nitrogen-centered radicals (i.e., nitric oxide [NO]) that give rise to nitrative/nitrosative stress. In addition to conventional biochemical analyses, the emerging microelectromechanical systems (MEMS) provide spatial and temporal resolutions to investigate the mechanisms whereby the characteristics of shear stress regulate the biological activities of endothelial cells at the complicated arterial geometry. In parallel, the development of MEMS liquid chromatography (LC) provides a new venue to measure circulating oxidized low-density lipoprotein (ox-LDL) particles as a lab-on-a chip platform. Nanowire-based field effect transistors further pave the way for a high throughput approach to analyze the LDL redox state. Integration of MEMS with oxidative biology is synergistic in assessing vascular oxidative stress. The MEMS LC provides an emerging lab-on-a-chip platform for ox-LDL analysis. In this context, this chapter has integrated expertise from the fields of vascular biology and oxidative biology to address the dynamics of inflammatory responses.