Blood flow modulation of vascular dynamics

Purpose of review: Blood flow is intimately linked with cardiovascular development, repair and dysfunction. The current review will build on the fluid mechanical principle underlying haemodynamic shear forces, mechanotransduction and metabolic effects. Recent findings: Pulsatile flow produces both time (∂τ/∂t) and spatial-varying shear stress (∂τ/∂x) to modulate vascular oxidative stress and inflammatory response with pathophysiological significance to atherosclerosis. The characteristics of haemodynamic shear forces, namely, steady laminar (∂τ/∂t = 0), pulsatile shear stress (PSS: unidirectional forward flow) and oscillatory shear stress (bidirectional with a near net 0 forward flow), modulate mechano-signal transduction to influence metabolic effects on vascular endothelial function. Atheroprotective PSS promotes antioxidant, anti-inflammatory and antithrombotic responses, whereas atherogenic oscillatory shear stress induces nicotinamide adenine dinucleotide phosphate oxidase–JNK signalling to increase mitochondrial superoxide production, protein degradation of manganese superoxide dismutase and post-translational protein modifications of LDL particles in the disturbed flow-exposed regions of vasculature. In the era of tissue regeneration, shear stress has been implicated in reactivation of developmental genes, namely, Wnt and Notch signalling, for vascular development and repair. Summary: Blood flow imparts a dynamic continuum from vascular development to repair. Augmentation of PSS confers atheroprotection and reactivation of developmental signalling pathways for regeneration

Moving Domain Computational Fluid Dynamics to Interface with an Embryonic Model of Cardiac Morphogenesis

Peristaltic contraction of the embryonic heart tube produces time- and spatial-varying wall shear stress (WSS) and pressure gradients (∇P) across the atrioventricular (AV) canal. Zebrafish (Danio rerio) are a genetically tractable system to investigate cardiac morphogenesis. The use of Tg(fli1a:EGFP)y1 transgenic embryos allowed for delineation and two-dimensional reconstruction of the endocardium. This time-varying wall motion was then prescribed in a two-dimensional moving domain computational fluid dynamics (CFD) model, providing new insights into spatial and temporal variations in WSS and ∇P during cardiac development. The CFD simulations were validated with particle image velocimetry (PIV) across the atrioventricular (AV) canal, revealing an increase in both velocities and heart rates, but a decrease in the duration of atrial systole from early to later stages. At 20-30 hours post fertilization (hpf), simulation results revealed bidirectional WSS across the AV canal in the heart tube in response to peristaltic motion of the wall. At 40-50 hpf, the tube structure undergoes cardiac looping, accompanied by a nearly 3-fold increase in WSS magnitude. At 110-120 hpf, distinct AV valve, atrium, ventricle, and bulbus arteriosus form, accompanied by incremental increases in both WSS magnitude and ∇P, but a decrease in bi-directional flow. Laminar flow develops across the AV canal at 20-30 hpf, and persists at 110-120 hpf. Reynolds numbers at the AV canal increase from 0.07±0.03 at 20-30 hpf to 0.23±0.07 at 110-120 hpf (p< 0.05, n=6), whereas Womersley numbers remain relatively unchanged from 0.11 to 0.13. Our moving domain simulations highlights hemodynamic changes in relation to cardiac morphogenesis; thereby, providing a 2-D quantitative approach to complement imaging analysis. © 2013 Lee et al

Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping.

Tissue clearing methods promise to provide exquisite three-dimensional imaging information; however, there is a need for simplified methods for lower resource settings and for non-fluorescence based phenotyping to enable light microscopic imaging modalities. Here we describe the simplified CLARITY method (SCM) for tissue clearing that preserves epitopes of interest. We imaged the resulting tissues using light sheet microscopy to generate rapid 3D reconstructions of entire tissues and organs. In addition, to enable clearing and 3D tissue imaging with light microscopy methods, we developed a colorimetric, non-fluorescent method for specifically labeling cleared tissues based on horseradish peroxidase conversion of diaminobenzidine to a colored insoluble product. The methods we describe here are portable and can be accomplished at low cost, and can allow light microscopic imaging of cleared tissues, thus enabling tissue clearing and imaging in a wide variety of settings

Flow-Responsive Vascular Endothelial Growth Factor Receptor-Protein Kinase C Isoform Epsilon Signaling Mediates Glycolytic Metabolites for Vascular Repair

Aims: Hemodynamic shear stress participates in maintaining vascular redox status. Elucidating flow-mediated endothelial metabolites enables us to discover metabolic biomarkers and therapeutic targets. We posited that flow-responsive vascular endothelial growth factor receptor (VEGFR)-protein kinase C isoform epsilon (PKCɛ)-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) signaling modulates glycolytic metabolites for vascular repair. Results: Bidirectional oscillatory flow (oscillatory shear stress [OSS]: 0.1 ± 3 dyne·cm^(−2) at 1 Hz) upregulated VEGFR-dependent PKCɛ expression to a greater degree than did unidirectional pulsatile flow (pulsatile shear stress [PSS]: 23 ± 8 dyne·cm^(−2) at 1 Hz) in human aortic endothelial cells (p < 0.05, n = 3). PSS and OSS further upregulated PKCɛ-dependent PFKFB3 expression for glycolysis (p < 0.05, n = 4). Constitutively active PKCɛ increased, whereas dominant-negative PKCɛ reduced both basal and maximal extracellular acidification rates for glycolytic flux (p < 0.01, n = 4). Metabolomic analysis demonstrated an increase in PKCɛ-dependent glycolytic metabolite, dihydroxyacetone (DHA), but a decrease in gluconeogenic metabolite, aspartic acid (p < 0.05 vs. control, n = 6). In a New Zealand White rabbit model, both PKCɛ and PFKFB3 immunostaining was prominent in the PSS- and OSS-exposed aortic arch and descending aorta. In a transgenic Tg(flk-1:EGFP) zebrafish model, GATA-1a morpholino oligonucleotide injection (to reduce viscosity-dependent shear stress) impaired vascular regeneration after tail amputation (p < 0.01, n = 20), which was restored with PKCɛ messenger RNA (mRNA) rescue (p < 0.05, n = 5). As a corollary, siPKCɛ inhibited tube formation and vascular repair, which were restored by DHA treatment in our Matrigel and zebrafish models. Innovation and Conclusion: Flow-sensitive VEGFR-PKCɛ-PFKFB3 signaling increases the glycolytic metabolite, dihydroxyacetone, to promote vascular repair

3-D Electrochemical Impedance Spectroscopy Mapping of Arteries to Detect Metabolically Active but Angiographically Invisible Atherosclerotic Lesions

We designed a novel 6-point electrochemical impedance spectroscopy (EIS) sensor with 15 combinations of permutations for the 3-D mapping and detection of metabolically active atherosclerotic lesions. Two rows of 3 stretchable electrodes circumferentially separated by 120° were mounted on an inflatable balloon for intravascular deployment and endoluminal interrogation. The configuration and 15 permutations of 2-point EIS electrodes allowed for deep arterial penetration via alternating current (AC) to detect varying degrees of lipid burden with distinct impedance profiles (Ω). By virtue of the distinctive impedimetric signature of metabolically active atherosclerotic lesions, a detailed impedance map was acquired, with the 15 EIS permutations uncovering early stages of disease characterized by fatty streak lipid accumulation in the New Zealand White rabbit model of atherosclerosis. Both the equivalent circuit and statistical analyses corroborated the 3-D EIS permutations to detect small, angiographically invisible, lipid-rich lesions, with translational implications for early atherosclerotic disease detection and prevention of acute coronary syndromes or strokes

Light-Sheet Imaging to Elucidate Cardiovascular Injury and Repair

Purpose of Review: Real-time 3-dimensional (3-D) imaging of cardiovascular injury and regeneration remains challenging. We introduced a multi-scale imaging strategy that uses light-sheet illumination to enable applications of cardiovascular injury and repair in models ranging from zebrafish to rodent hearts. Recent Findings: Light-sheet imaging enables rapid data acquisition with high spatiotemporal resolution and with minimal photo-bleaching or photo-toxicity. We demonstrated the capacity of this novel light-sheet approach for scanning a region of interest with specific fluorescence contrast, thereby providing axial and temporal resolution at the cellular level without stitching image columns or pivoting illumination beams during one-time imaging. This cutting-edge imaging technique allows for elucidating the differentiation of stem cells in cardiac regeneration, providing an entry point to discover novel micro-circulation phenomenon with clinical significance for injury and repair. Summary: These findings demonstrate the multi-scale applications of this novel light-sheet imaging strategy to advance research in cardiovascular development and regeneration

Advanced microscopy to elucidate cardiovascular injury and regeneration: 4D light-sheet imaging

The advent of 4-dimensional (4D) light-sheet fluorescence microscopy (LSFM) has provided an entry point for rapid image acquisition to uncover real-time cardiovascular structure and function with high axial resolution and minimal photo-bleaching/-toxicity. We hereby review the fundamental principles of our LSFM system to investigate cardiovascular morphogenesis and regeneration after injury. LSFM enables us to reveal the micro-circulation of blood cells in the zebrafish embryo and assess cardiac ventricular remodeling in response to chemotherapy-induced injury using an automated segmentation approach. Next, we review two distinct mechanisms underlying zebrafish vascular regeneration following tail amputation. We elucidate the role of endothelial Notch signaling to restore vascular regeneration after exposure to the redox active ultrafine particles (UFP) in air pollutants. By manipulating the blood viscosity and subsequently, endothelial wall shear stress, we demonstrate the mechanism whereby hemodynamic shear forces impart both mechanical and metabolic effects to modulate vascular regeneration. Overall, the implementation of 4D LSFM allows for the elucidation of mechanisms governing cardiovascular injury and regeneration with high spatiotemporal resolution

Wearable multi-channel microelectrode membranes for elucidating electrophysiological phenotypes of injured myocardium

Understanding the regenerative capacity of small vertebrate models has provided new insights into the plasticity of injured myocardium. Here, we demonstrate the application of flexible microelectrode arrays (MEAs) in elucidating electrophysiological phenotypes of zebrafish and neonatal mouse models of heart regeneration. The 4-electrode MEA membranes were designed to detect electrical signals in the aquatic environment. They were micro-fabricated to adhere to the non-planar body surface of zebrafish and neonatal mice. The acquired signals were processed to display an electrocardiogram (ECG) with high signal-to-noise-ratios, and were validated via the use of conventional micro-needle electrodes. The 4-channel MEA provided signal stability and spatial resolution, revealing the site-specific electrical injury currents such as ST-depression in response to ventricular cryo-injury. Thus, our polymer-based and wearable MEA membranes provided electrophysiological insights into long-term conduction phenotypes for small vertebral models of heart injury and regeneration with a translational implication for monitoring cardiac patients