Hemodynamic Forces Regulate Embryonic Stem Cell Commitment to Vascular Progenitors

Pluripotent embryonic stem can (ES) cells can differentiate into all cell lineages. During the process of embryonic development, ES cells are exposed to fluid flow or blood flow generated by the contracting heart. Absence of fluid flow results in the formation of abnormal cardiac chambers and valve formation. Thus, hemodynamic forces and ES cell differentiation to vascular progenitor cells (VPCs) are of emerging interests for restoring endothelial dysfunction, inducing angiogenesis, and forming blood vessel networks. Hemodynamic forces such as fluid shear stress increase the percentage of cells in the S and G2-M phases, and induce decondensation of chromatin for gene transcription. Fluid shear stress further accelerates ES commitment to CD31+ VPC vascular progenitor cells. These ES-derived CD31+ cells express endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). They are also capable of LDL uptake and tubular network formation. In this context, understanding hemodynamic forces and ES cell kinetics of differentiation towards endothelial lineage has potential therapeutic applications for repairing vascular damage and engineering vascular graft. Multidisciplinary team approach will likely garner momentum and synergize expertise to address the current road blocks in basic stem cell research for engraftable, restorative, low immunogenic, and non-tumorigenic endothelial progenitors in high purity and stability

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

Flexible and waterproof micro-sensors to uncover zebrafish circadian rhythms: The next generation of cardiac monitoring for drug screening

Flexible electronics are the next generation of sensors for mobile health and implantation. Zebrafish (Danio rerio) is an emergent strategy for pre-clinical drug development and toxicity testing. To address the confounding effects from sedation of fish and removal from the aquatic habitat for micro-electrocardiogram (µECG) measurements, we developed waterproof and wearable sensors to uncover the circadian variation in heart rate (HR) and heart rate variability (HRV) ( Massin et al., 2000). The parylene-C based ECG sensor consisted of an ultra-soft silicone integrated jacket designed to wrap around the fish during swimming. The Young’s modulus of this silicone jacket matched with the fish surface, and an extended parylene cable connected the underwater chest electrodes with the out-of water electronics. In addition, embedded micro-glass spheres in the silicone effectively reduced the effective density of the jacket to ~1 g cm^(−3). These innovations enabled physiological ECG telemetry in the fish’s natural habitat without the need for sedation. Furthermore, a set of non-linear signal processing techniques filtered out the breathing and electromagnetic artifacts from the recorded signals. We observed a reduction in mean HR and an increase in HRV over 24 h at 10 dpa, accompanied by QT prolongation as well as diurnal variations, followed by normalization in mean HR and QT intervals at 26 days post ventricular amputation (dpa). We revealed Amiodarone-mediated QTc prolongation, HR reduction and HRV increase otherwise masked by sedation. The novel features of the flexible silicon jacket for µECG telemetry unraveled the biological clock and normalization of QT intervals at 26 dpa, providing the first evidence of new physiological phenomena during cardiac injury and repair as well as cardiac drug-mediated aberrant rhythms. Thus, the light weight and waterproof design holds promise to advance the next generation of mobile health and drug discovery

Flexible microelectrode arrays to interface epicardial electrical signals with intracardial calcium transients in zebrafish hearts

The zebrafish (Danio rerio) is an emerging genetic model for regenerative medicine. In humans, myocardial infarction results in the irreversible loss of cardiomyocytes. However, zebrafish hearts fully regenerate after a 20% ventricular resection, without either scarring or arrhythmias. To study this cardiac regeneration, we developed implantable flexible multi-microelectrode membrane arrays that measure the epicardial electrocardiogram signals of zebrafish in real-time. The microelectrode electrical signals allowed for a high level of both temporal and spatial resolution (~20 μm), and the signal to noise ratio of the epicardial ECG was comparable to that of surface electrode ECG (7.1 dB vs. 7.4 dB, respectively). Processing and analysis of the signals from the microelectrode array demonstrated distinct ECG signals: namely, atrial conduction (P waves), ventricular contraction (QRS), and ventricular repolarization (QT interval). The electrical signals were in synchrony with optically measured Calcium concentration gradients in terms of d[Ca^(2+)]/dt at both whole heart and tissue levels. These microelectrodes therefore provide a real-time analytical tool for monitoring conduction phenotypes of small vertebral animals with a high temporal and spatial resolution

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

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

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

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