Exposure of particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultra fine particles (UFP) from diesel vehicle engines have been shown to be pro-atherogenic in apoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induced vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intra-cellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O2·-) production in human aortic endothelial cells (HAEC). Flow cytometry (FACS) showed that UFP increased MitoSOX Red intensity specific for mitochondrial superoxide. Protein carbonyl content is increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated hemeoxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pre-treatment with antioxidant, N-acetyl cysteine (NAC), significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP stimulated O2·- production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation play an important role in UFP-induced oxidative stress and stress response gene expression.

The zebrafish (Danio rerio) is an emerging model for cardiovascular research. The zebrafish heart regenerates after 20% ventricular amputation. However, assessment of the physiological responses during heart regeneration has been hampered by the small size of the heart and the necessity of conducting experiments in an aqueous environment. We developed a methodology to monitor a real-time surface electrocardiogram (ECG) by the use of micro-electrodes, signal amplification, and a low pass-filter at a sampling rate of 1 kHz. Wavelet transform was used to further remove ambient noises. Rather than paralyzing the fish, we performed mild sedation by placing the fish in a water bath mixed with MS-222 (tricane methanesulfonate). We recorded distinct P waves for atrial contraction, QRS complexes for ventricular depolarization, and QT intervals for ventricular repolarization prior to, and 2 and 4 days post-amputation (dpa). Sedation reduced the mean fish heart rate from 149 +/- 18 to 90 +/- 17 beats/min. The PR and QRS intervals remained unchanged in response to ventricular apical amputation (n = 6, p > 0.05). Corrected QT intervals (QTc) were shortened 4 dpa (n = 6, p < 0.05). In a parallel study, histology revealed that apical thrombi were replaced with fibrin clots and collagen fibers. Atrial arrhythmia was noted in response to prolonged sedation. Unlike the human counterpart, ventricular tachycardia or fibrillation was not observed in response to ventricular amputation 2 and 4 dpa. Taken together, we demonstrated a minimally invasive methodology to monitor zebrafish heart function, electrical activities, and regeneration in real-time.

Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO 2 À ) production via NADPH oxidase and c-Jun NH 2 -terminal kinase ( JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (AE3 dyn=cm 2 ) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO 2 À 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 O 2 À and mtO 2 À production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 ( JNK inhibitor) significantly attenuated OSS-mediated mtO 2 À production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO 2 À production via NADPH oxidase and JNK activation relevant for vascular oxidative stress. Antioxid. Redox Signal. 15, 1379-1388.

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.

Fluid shear stress modulates vascular production of endothelial superoxide anion (O2 ⅐ Ϫ ) and nitric oxide ( ⅐ 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 (ave) of 23 dyn/cm 2 upregulated Mn-SOD mRNA expression at a higher level than did OSS at ave ϭ 0.02 dyn/cm 2 Ϯ 3.0 dyn ⅐ cm Ϫ2 ⅐ s Ϫ1 and at 1 Hz (PSS by 11.3 Ϯ 0.4-fold vs. OSS by 5.0 Ϯ 0.5-fold vs. static condition; P Ͻ 0.05, n ϭ 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 Ͻ 0.05, n ϭ 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P Ͻ 0.5, n ϭ 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. superoxide dismutase; superoxide anion; nitric oxide; nitrotyrosine; low-density lipoprotein

Vascular inflammatory responses are intimately linked with oxidative stress, favoring the development of pre-atherosclerotic lesions. We proposed that oxidized low density lipoprotein (oxLDL) and foam cell infiltrates in the subendothelial layer engendered distinct electrochemical properties that could be measured in terms of the electrochemical impedance spectroscopy (EIS). Concentric bipolar microelectrodes were applied to interrogate EIS of aortas isolated from fat-fed New Zealand White (NZW) rabbits and explants of human aortas. Frequency-dependent EIS measurements were assessed between 10 kHz and 100 kHz, and were significantly elevated in the pre-atherosclerotic lesions in which oxLDL and macrophage infiltrates were prevalent (At 100 kHz: aortic arch lesion = 26.7 ± 2.7 kΩ vs. control = 15.8 ± 2.4 kΩ; at 10 kHz: lesions = 49.2 ± 7.3 kΩ vs. control = 27.6 ± 2.7 kΩ, n = 10, p<0.001). Similarly, EIS measurements were significantly elevated in the human descending aorta where pre-atherosclerotic lesions or fatty streaks were prominent. EIS measurements remained unchanged in spite of various depths of electrode submersion or orientation of the specimens. Hence, the concentric bipolar microelectrodes provided a reliable means to measure endoluminal electrochemical modifications in regions of pro-inflammatory with high spatial resolution and reproducibility albeit uneven lesion topography and non-uniform current distribution.

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