Fei Yu

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.

Shear Stress–Activated Wnt-Angiopoietin-2 Signaling Recapitulates Vascular Repair in Zebrafish Embryos

Objective— Fluid shear stress intimately regulates vasculogenesis and endothelial homeostasis. The canonical Wnt/&bgr;-catenin signaling pathways play an important role in differentiation and proliferation. In this study, we investigated whether shear stress activated angiopoietin-2 (Ang-2) via the canonical Wnt signaling pathway with an implication in vascular endothelial repair. Approach and Results— Oscillatory shear stress upregulated both TOPflash Wnt reporter activities and the expression of Ang-2 mRNA and protein in human aortic endothelial cells accompanied by an increase in nuclear &bgr;-catenin intensity. Oscillatory shear stress–induced Ang-2 and Axin-2 mRNA expression was downregulated in the presence of a Wnt inhibitor, IWR-1, but was upregulated in the presence of a Wnt agonist, LiCl. Ang-2 expression was further downregulated in response to a Wnt signaling inhibitor, DKK-1, but was upregulated by Wnt agonist Wnt3a. Both DKK-1 and Ang-2 siRNA inhibited endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2. Both Ang-2 and Axin-2 mRNA downregulation was recapitulated in the heat-shock–inducible transgenic Tg(hsp70l:dkk1-GFP) zebrafish embryos at 72 hours post fertilization. Ang-2 morpholino injection of Tg (kdrl:GFP) fish impaired subintestinal vessel formation at 72 hours post fertilization, which was rescued by zebrafish Ang-2 mRNA coinjection. Inhibition of Wnt signaling with IWR-1 also downregulated Ang-2 and Axin-2 expression and impaired vascular repair after tail amputation, which was rescued by zebrafish Ang-2 mRNA injection. Conclusions— Shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and Wnt-Ang-2 signaling is recapitulated in zebrafish embryos with a translational implication in vascular development and repair.

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.

Hemodynamics and ventricular function in a zebrafish model of injury and repair.

Myocardial infarction results in scar tissue and irreversible loss of ventricular function. Unlike humans, zebrafish has the capacity to remove scar tissue after injury. To assess ventricular function during repair, we synchronized microelectrocardiogram (μECG) signals with a high-frequency ultrasound pulsed-wave (PW) Doppler to interrogate cardiac hemodynamics. μECG signals allowed for identification of PW Doppler signals for passive (early [E]-wave velocity) and active ventricular filling (atrial [A]-wave velocity) during diastole. The A wave (9.0±1.2 cm·s(-1)) is greater than the E wave (1.1±0.4 cm·s(-1)), resulting in an E/A ratio <1 (0.12±0.05, n=6). In response to cryocauterization to the ventricular epicardium, the E-wave velocity increased, accompanied by a rise in the E/A ratio at 3 days postcryocauterization (dpc) (0.55±0.13, n=6, p<0.001 vs. sham). The E waves normalize toward the baseline, along with a reduction in the E/A ratio at 35 dpc (0.36±0.06, n=6, p<0.001 vs. sham) and 65 dpc (0.2±0.16, n=6, p<0.001 vs. sham). In zebrafish, E/A<1 at baseline is observed, suggesting the distinct two-chamber system in which the pressure gradient across the atrioventricular valve is higher compared with the ventriculobulbar valve. The initial rise and subsequent normalization of E/A ratios support recovery in the ventricular diastolic function.

Evolving Cardiac Conduction Phenotypes in Developing Zebrafish Larvae: Implications to Drug Sensitivity

Cardiac arrhythmias include problems with impulse formation and/or conduction abnormalities. Zebrafish (Danio rerio) is an emerging model system for studying the cardiac conduction system. However, real-time recording of the electrocardiogram remains a challenge. In the present study, we assessed the feasibility of recording electrical cardiogram (ECG) signals from the zebrafish larvae using the micropipette electrodes, and demonstrated the dynamic changes in ECG signals and their sensitivity to Amiodarone during the developmental stages. We observed that ECG signals revealed P waves and QRS complexes at 7 days postfertilization (dpf). T waves started to develop at 14 dpf. Distinct P waves, QRS complexes, and T waves were similar to those of adult zebrafish at 35 dpf, accompanied by a statistically significant decrease in QRS intervals (from 256 ± 16 ms at 7 dpf to 54 ± 6 ms, p < 0.01, n = 5). In response to Amiodarone, ECG signals showed QRS prolongation from 7 to 35 dpf (p < 0.05, n = 5). Hence, micropipette electrodes can be applied to detect evolving ECG signals from the developing zebrafish larvae, thus providing a noninvasive and nonparalyzing approach to investigate cardiac conduction phenotypes in response to genetic, epigenetic, or pharmacologic perturbation.

Electrochemical impedance spectroscopy to characterize inflammatory atherosclerotic plaques

Despite advances in diagnosis and therapy, atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in the Western world. Predicting metabolically active atherosclerotic lesions has remained an unmet clinical need. We hereby developed an electrochemical strategy to characterize the inflammatory states of high-risk atherosclerotic plaques. Using the concentric bipolar microelectrodes, we sought to demonstrate distinct Electrochemical Impedance Spectroscopic (EIS) measurements for unstable atherosclerotic plaques that harbored active lipids and inflammatory cells. Using equivalent circuits to simulate vessel impedance at the electrode-endoluminal tissue interface, we demonstrated specific electric elements to model working and counter electrode interfaces as well as the tissue impedance. Using explants of human coronary, carotid, and femoral arteries at various Stary stages of atherosclerotic lesions (n=15), we performed endoluminal EIS measurements (n=147) and validated with histology and immunohistochemistry. We computed the vascular tissue resistance using the equivalent circuit model and normalized the resistance to the lesion-free regions. Tissue resistance was significantly elevated in the oxLDL-rich thin-cap atheromas (1.57±0.40, n=14, p<0.001) and fatty streaks (1.36±0.28, n=33, p<0.001) as compared with lesion-free region (1.00±0.18, n=82) or oxLDL-absent fibrous atheromas (0.86±0.30, n=12). Tissue resistance was also elevated in the calcified core of fibrous atheroma (2.37±0.60, n=6, p<0.001). Despite presence of fibrous structures, tissue resistance between ox-LDL-absent fibroatheroma and the lesion-free regions was statistically insignificant (0.86±0.30, n=12, p>0.05). Hence, we demonstrate that the application of EIS strategy was sensitive to detect fibrous cap oxLDL-rich lesions and specific to distinguish oxLDL-absent fibroatheroma.

Stretchable electrochemical impedance sensors for intravascular detection of lipid-rich lesions in New Zealand White rabbits

Flexible electronics have enabled catheter-based intravascular sensing. However, real-time interrogation of unstable plaque remains an unmet clinical challenge. Here, we demonstrate the feasibility of stretchable electrochemical impedance spectroscopy (EIS) sensors for endoluminal investigations in New Zealand White (NZW) rabbits on diet-induced hyperlipidemia. A parylene C (PAC)-based EIS sensor mounted on the surface of an inflatable silicone balloon affixed to the tip of an interrogating catheter was deployed (1) on the explants of NZW rabbit aorta for detection of lipid-rich atherosclerotic lesions, and (2) on live animals for demonstration of balloon inflation and EIS measurements. An input peak-to-peak AC voltage of 10 mV and sweeping-frequency from 300 kHz to 100 Hz were delivered to the endoluminal sites. Balloon inflation allowed EIS sensors to be in contact with endoluminal surface. In the oxidized low-density-lipoprotein (oxLDL)-rich lesions from explants of fat-fed rabbits, impedance magnitude increased significantly by 1.5-fold across the entire frequency band, and phase shifted ~5° at frequencies below 10 kHz. In the lesion-free sites of the normal diet-fed rabbits, impedance magnitude increased by 1.2-fold and phase shifted ~5° at frequencies above 30 kHz. Thus, we demonstrate the feasibility of stretchable intravascular EIS sensors for identification of lipid rich lesions, with a translational implication for detecting unstable lesions.

Elevated electrochemical impedance in the endoluminal regions with high shear stress: Implication for assessing lipid-rich atherosclerotic lesions

BACKGROUND Identifying metabolically active atherosclerotic lesions remains an unmet clinical challenge during coronary intervention. Electrochemical impedance (EIS) increased in response to oxidized low density lipoprotein (oxLDL)-laden lesions. We hereby assessed whether integrating EIS with intravascular ultrasound (IVUS) and shear stress (ISS) provided a new strategy to assess oxLDL-laden lesions in the fat-fed New Zealand White (NZW) rabbits. METHODS AND RESULTS A micro-heat transfer sensor was deployed to acquire the ISS profiles at baseline and post high-fat diet (HD) in the NZW rabbits (n=8). After 9 weeks of HD, serum oxLDL levels (mg/dL) increased by 140 fold, accompanied by a 1.5-fold increase in kinematic viscosity (cP) in the HD group. Time-averaged ISS (ISSave) in the thoracic aorta also increased in the HD group (baseline: 17.61±0.24 vs. 9 weeks: 25.22±0.95dyne/cm(2), n=4), but remained unchanged in the normal diet group (baseline: 22.85±0.53dyn/cm(2) vs. 9 weeks: 22.37±0.57dyne/cm(2), n=4). High-frequency intravascular ultrasound (IVUS) revealed atherosclerotic lesions in the regions with augmented ISSave, and concentric bipolar microelectrodes demonstrated elevated EIS signals, which were correlated with prominent anti-oxLDL immuno-staining (oxLDL-free regions: 497±55Ω, n=8 vs. oxLDL-rich lesions: 679±125Ω, n=12, P<0.05). The equivalent circuit model for tissue resistance between the lesion-free and ox-LDL-rich lesions further validated the experimental EIS signals. CONCLUSIONS By applying electrochemical impedance in conjunction with shear stress and high-frequency ultrasound sensors, we provided a new strategy to identify oxLDL-laden lesions. The study demonstrated the feasibility of integrating EIS, ISS, and IVUS for a catheter-based approach to assess mechanically unstable plaque.

Assessing Mitochondrial Redox Status by Flow Cytometric Methods: Vascular Response to Fluid Shear Stress

Mitochondria are an important source of superoxide production contributing to physiological and pathological responses, including vascular oxidative stress that is relevant to cardiovascular diseases. Vascular oxidative stress is intimately linked with pro‐inflammatory states and atherosclerosis. Oxidized low‐density lipoprotein (OxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. Hemodynamic, specifically, fluid shear stress imparts both biomechanical and metabolic effects on vasculature. Mitochondria are an important source of superoxide production contributing to vascular oxidative stress with relevance to cardiovascular diseases. We hereby present biophysical and biochemical approaches, including fluorescence‐activated cell sorting, to assess the dynamics of vascular redox status. Curr. Protoc. Cytom. 58:9.37.1‐9.37.14.

Flexible intravascular thermal sensors to assess atherosclerosis-mediated changes in hemodynamics

In this study, we introduced an in vivo convective heat transfer-based micro-electromechanical system (MEMS) sensor that measured real-time experimental vascular shear stress values in the aortas of New Zealand White rabbits (n=4). Experiments were performed to measure flow profile changes in response to hyper-cholesterol diet and an irregular tachycardia condition. We have shown that these conditions induce shear stress changes indicative of pre-atherosclerosis regions by the MEMS thermal sensor.