Nelson Jen

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.

A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids

Previous studies from our group have demonstrated that oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) activates over 1000 genes in human aortic endothelial cells (HAECs). Prominent among these are genes regulating inflammation, cholesterol homeostasis, antioxidant enzymes, and the unfolded protein response. Previous studies from our lab and others suggested that transcriptional regulation by Ox-PAPC may be controlled, at least in part, by reactive oxygen species. We now present evidence that Ox-PAPC activation of NADPH oxidase 4 (NOX4) is responsible for the regulation of two of these important groups of genes: those controlling inflammation and those involved in sterol regulation. Our data demonstrate that Ox-PAPC increases reactive oxygen species formation in HAECs as seen by DCF fluorescence. NOX4 is the major molecule responsible for this increase because downregulation of NOX4 and its components (p22(phox) and rac1) blocked the Ox-PAPC effect. Our data show that Ox-PAPC did not change NOX4 transcription levels but did induce recruitment of rac1 to the membrane for NOX4 activation. We present evidence that vascular endothelial growth factor receptor 2 (VEGFR2) activation is responsible for rac1 recruitment to the membrane. Finally, we demonstrate that knockdown of NOX4 and its components rac1 and p22(phox) decreases Ox-PAPC induction of inflammatory and sterol regulatory genes, but does not affect Ox-PAPC transcriptional regulation of other genes for antioxidants and the unfolded protein response. In summary, we have identified a VEGFR2/NOX4 regulatory pathway by which Ox-PAPC controls important endothelial functions.

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.

Effect of exposure to atmospheric ultrafine particles on production of free fatty acids and lipid metabolites in the mouse small intestine.

Background: Exposure to ambient ultrafine particulate matter (UFP) is a well-recognized risk factor for cardiovascular and respiratory diseases. However, little is known about the effects of air pollution on gastrointestinal disorders. Objective: We sought to assess whether exposure to ambient UFP (diameter < 180 nm) increased free fatty acids and lipid metabolites in the mouse small intestine. Methods: Ldlr-null mice were exposed to filtered air (FA) or UFP collected at an urban Los Angeles, California, site that was heavily affected by vehicular emissions; the exposure was carried out for 10 weeks in the presence or absence of D-4F, an apolipoprotein A-I mimetic peptide with antioxidant and anti-inflammation properties on a high-fat or normal chow diet. Results: Compared with FA, exposure to UFP significantly increased intestinal hydroxyeicosatetraenoic acids (HETEs), including 15-HETE, 12-HETE, 5-HETE, as well as hydroxyoctadecadienoic acids (HODEs), including 13-HODE and 9-HODE. Arachidonic acid (AA) and prostaglandin D2 (PGD2) as well as some of the lysophosphatidic acids (LPA) in the small intestine were also increased in response to UFP exposure. Administration of D-4F significantly reduced UFP-mediated increase in HETEs, HODEs, AA, PGD2, and LPA. Although exposure to UFP further led to shortened villus length accompanied by prominent macrophage and neutrophil infiltration into the intestinal villi, administration of D-4F mitigated macrophage infiltration. Conclusions: Exposure to UFP promotes lipid metabolism, villus shortening, and inflammatory responses in mouse small intestine, whereas administration of D-4F attenuated these effects. Our findings provide a basis to further assess the mechanisms underlying UFP-mediated lipid metabolism in the digestive system with clinical relevance to gut homeostasis and diseases. Citation: Li R, Navab K, Hough G, Daher N, Zhang M, Mittelstein D, Lee K, Pakbin P, Saffari A, Bhetraratana M, Sulaiman D, Beebe T, Wu L, Jen N, Wine E, Tseng CH, Araujo JA, Fogelman A, Sioutas C, Navab M, Hsiai TK. 2015. Effect of exposure to atmospheric ultrafine particles on production of free fatty acids and lipid metabolites in the mouse small intestine. Environ Health Perspect 123:34–41; http://dx.doi.org/10.1289/ehp.1307036

4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.

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.

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.

Compact plane illumination plugin device to enable light sheet fluorescence imaging of multi-cellular organisms on an inverted wide-field microscope

We developed a compact plane illumination plugin (PIP) device which enabled plane illumination and light sheet fluorescence imaging on a conventional inverted microscope. The PIP device allowed the integration of microscope with tunable laser sheet profile, fast image acquisition, and 3-D scanning. The device is both compact, measuring approximately 15 by 5 by 5 cm, and cost-effective, since we employed consumer electronics and an inexpensive device molding method. We demonstrated that PIP provided significant contrast and resolution enhancement to conventional microscopy through imaging different multi-cellular fluorescent structures, including 3-D branched cells in vitro and live zebrafish embryos. Imaging with the integration of PIP greatly reduced out-of-focus contamination and generated sharper contrast in acquired 2-D plane images when compared with the stand-alone inverted microscope. As a result, the dynamic fluid domain of the beating zebrafish heart was clearly segmented and the functional monitoring of the heart was achieved. Furthermore, the enhanced axial resolution established by thin plane illumination of PIP enabled the 3-D reconstruction of the branched cellular structures, which leads to the improvement on the functionality of the wide field microscopy.

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.