light-sheet-fluorescence-microscopy-to-capture-4-dimensional-images

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Shenzhen Technology University

A total synthesis of the naturally occurring hydroxy ketone PEIPC 1, a compound that plays a role in endothelial activation in atherosclerosis, has been completed via a triply convergent preparation of a protected EI derivative 13 from 3,5-diacetoxycyclopentene 7, pentane-1,5-diol, and vinyllithium, using Sharpless epoxidation and enzymatic resolution as key steps. Final coupling with lyso-PC 16 and silyl group deprotection gave PECPC 2 and PEIPC 1, which showed the same activity as natural PECPC and PEIPC. [reaction: see text]

Total synthesis of the epoxy isoprostane phospholipids PEIPC and PECPC.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) is present in oxidative modified LDL and accumulates in lesions of many chronic inflammatory diseases, such as atherosclerosis. In a microarray study, OxPAPC has been demonstrated to modulate the expression of >700 genes in human aortic endothelial cells. We found that the levels of mRNA for OKL38 [also named Bone marrow Derived Growth Factor (BDGI)], a tumor growth inhibitor, were strongly increased by OxPAPC. Here, we report that OKL38 is regulated by an oxidative signal induced by OxPAPC and its component lipid 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine. The stimulation of OKL38 by OxPAPC depends on superoxide production, because the NADPH oxidase (Nox) inhibitor apocynin and the superoxide scavenger N-acetyl cysteine block this stimulation. Oxidative stress by tert-butylhydroquinone treatment also induced the expression of OKL38. The stimulation of OKL38 expression by OxPAPC is mediated via transcription factor nuclear factor E2-related factor (Nrf2), a common factor involved in the regulation of oxidative stress-stimulated genes. Activation of Nrf2 induces the expression of OKL38, whereas small interfering RNA knockdown of Nrf2 blocks the stimulation of OKL38 by OxPAPC. Our results suggest that OKL38 is regulated via the Nox/Nrf2 pathway in response to oxidative stress stimuli.

OKL38 is an oxidative stress response gene stimulated 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.

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

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.

Oxidized low-density lipoprotein-activated c-Jun NH2-terminal kinase regulates manganese superoxide dismutase ubiquitination: implication for mitochondrial redox status and apoptosis.

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.

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

Fluid shear stress intimately regulates vasculogenesis and endothelial homeostasis. The canonical Wnt/β-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.

Shear stress-activated Wnt-angiopoietin-2 signaling recapitulates vascular repair in zebrafish embryos.

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.

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

Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Autophagy is tightly regulated in intracellular bulk degradation/recycling system for maintaining cellular homeostasis. We postulated that disturbed flow modulates autophagy with an implication in mitochondrial superoxide (mtO2(•-)) production.

Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis.

Four-point electrode systems are commonly used for electric impedance measurements of biomaterials and tissues. We introduce a 2-point system to reduce electrode polarization for heterogeneous measurements of vascular wall. Presence of endoluminal oxidized low density lipoprotein (oxLDL) and lipids alters the electrochemical impedance that can be measured by electrochemical impedance spectroscopy (EIS). We developed a catheter-based 2-point micro-electrode configuration for intravascular deployment in New Zealand White rabbits. An array of 2 flexible round electrodes, 240 µm in diameter and separated by 400 µm was microfabricated and mounted on an inflatable balloon catheter for EIS measurement of the oxLDL-rich lesions developed as a result of high-fat diet-induced hyperlipidemia. Upon balloon inflation, the 2-point electrode array conformed to the arterial wall to allow deep intraplaque penetration via alternating current (AC). The frequency sweep from 10 to 300 kHz generated an increase in capacitance, providing distinct changes in both impedance (Ω) and phase (ϕ) in relation to varying degrees of intraplaque lipid burden in the aorta. Aortic endoluminal EIS measurements were compared with epicardial fat tissue and validated by intravascular ultrasound and immunohistochemistry for plaque lipids and foam cells. Thus, we demonstrate a new approach to quantify endoluminal EIS via a 2-point stretchable electrode strategy.

Two-Point Stretchable Electrode Array for Endoluminal Electrochemical Impedance Spectroscopy Measurements of Lipid-Laden Atherosclerotic Plaques.

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.

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

 Obesity is associated with an increased risk of nonalcoholic fatty liver disease (NAFLD). While Magnetic Resonance Imaging (MRI) is a non-invasive gold standard to detect fatty liver, we demonstrate a low-cost and portable electrical impedance tomography (EIT) approach with circumferential abdominal electrodes for liver conductivity measurements.  A finite element model (FEM) was established to simulate decremental liver conductivity in response to incremental liver lipid content. To validate the FEM simulation, we performed EIT imaging on an  porcine liver in a non-conductive tank with 32 circumferentially-embedded electrodes, demonstrating a high-resolution output given  information on location and geometry. To further examine EIT capacity in fatty liver detection, we performed EIT measurements in age- and gender-matched New Zealand White rabbits (3 on normal, 3 on high-fat diets). Liver conductivity values were significantly distinct following the high-fat diet ( = 0.003 vs. normal diet, n=3), accompanied by histopathological evidence of hepatic fat accumulation. We further assessed EIT imaging in human subjects with MRI quantification for fat volume fraction based on Dixon procedures, demonstrating average liver conductivity of 0.331 S/m for subjects with low Body-Mass Index (BMI < 25 kg/m²) and 0.286 S/m for high BMI (> 25 kg/m²). : We provide both the theoretical and experimental framework for a multi-scale EIT strategy to detect liver lipid content. Our preliminary studies pave the way to enhance the spatial resolution of EIT as a marker for fatty liver disease and metabolic syndrome.

Non-Invasive Electrical Impedance Tomography for Multi-Scale Detection of Liver Fat Content.

Hemodynamic shear force has been implicated as modulating Notch signaling-mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function.

Article	Year
Total synthesis of the epoxy isoprostane phospholipids PEIPC and PECPC. Organic letters\| PubMed ID: 16119935	2005
OKL38 is an oxidative stress response gene stimulated by oxidized phospholipids. Journal of lipid research\| PubMed ID: 17192422	2007
A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids. Free radical biology & medicine\| PubMed ID: 19375500	2009
Oxidized low-density lipoprotein-activated c-Jun NH2-terminal kinase regulates manganese superoxide dismutase ubiquitination: implication for mitochondrial redox status and apoptosis. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 20139358	2010
Stretchable electrochemical impedance sensors for intravascular detection of lipid-rich lesions in New Zealand White rabbits. Biosensors & bioelectronics\| PubMed ID: 24333932	2014
Shear stress-activated Wnt-angiopoietin-2 signaling recapitulates vascular repair in zebrafish embryos. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 25147335	2014
Effect of exposure to atmospheric ultrafine particles on production of free fatty acids and lipid metabolites in the mouse small intestine. Environmental health perspectives\| PubMed ID: 25170928	2015
Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis. Antioxidants & redox signaling\| PubMed ID: 26120766	2015
Two-Point Stretchable Electrode Array for Endoluminal Electrochemical Impedance Spectroscopy Measurements of Lipid-Laden Atherosclerotic Plaques. Annals of biomedical engineering\| PubMed ID: 26857007	2016
4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation. The Journal of clinical investigation\| PubMed ID: 27018592	2016
Non-Invasive Electrical Impedance Tomography for Multi-Scale Detection of Liver Fat Content. Theranostics\| PubMed ID: 29556346	2018
Spatial and temporal variations in hemodynamic forces initiate cardiac trabeculation. JCI insight\| PubMed ID: 29997298	2018

Rongsong Li

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