Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions

Topography of Extracellular Matrix Mediates Vascular Morphogenesis and Migration Speeds in Angiogenesis

The extracellular matrix plays a critical role in orchestrating the events necessary for wound healing, muscle repair, morphogenesis, new blood vessel growth, and cancer invasion. In this study, we investigate the influence of extracellular matrix topography on the coordination of multi-cellular interactions in the context of angiogenesis. To do this, we validate our spatio-temporal mathematical model of angiogenesis against empirical data, and within this framework, we vary the density of the matrix fibers to simulate different tissue environments and to explore the possibility of manipulating the extracellular matrix to achieve pro- and anti-angiogenic effects. The model predicts specific ranges of matrix fiber densities that maximize sprout extension speed, induce branching, or interrupt normal angiogenesis, which are independently confirmed by experiment. We then explore matrix fiber alignment as a key factor contributing to peak sprout velocities and in mediating cell shape and orientation. We also quantify the effects of proteolytic matrix degradation by the tip cell on sprout velocity and demonstrate that degradation promotes sprout growth at high matrix densities, but has an inhibitory effect at lower densities. Our results are discussed in the context of ECM targeted pro- and anti-angiogenic therapies that can be tested empirically

Preconditioning-induced ischemic tolerance: a window into endogenous gearing for cerebroprotection

Ischemic tolerance defines transient resistance to lethal ischemia gained by a prior sublethal noxious stimulus (i.e., preconditioning). This adaptive response is thought to be an evolutionarily conserved defense mechanism, observed in a wide variety of species. Preconditioning confers ischemic tolerance if not in all, in most organ systems, including the heart, kidney, liver, and small intestine. Since the first landmark experimental demonstration of ischemic tolerance in the gerbil brain in early 1990's, basic scientific knowledge on the mechanisms of cerebral ischemic tolerance increased substantially. Various noxious stimuli can precondition the brain, presumably through a common mechanism, genomic reprogramming. Ischemic tolerance occurs in two temporally distinct windows. Early tolerance can be achieved within minutes, but wanes also rapidly, within hours. Delayed tolerance develops in hours and lasts for days. The main mechanism involved in early tolerance is adaptation of membrane receptors, whereas gene activation with subsequent de novo protein synthesis dominates delayed tolerance. Ischemic preconditioning is associated with robust cerebroprotection in animals. In humans, transient ischemic attacks may be the clinical correlate of preconditioning leading to ischemic tolerance. Mimicking the mechanisms of this unique endogenous protection process is therefore a potential strategy for stroke prevention. Perhaps new remedies for stroke are very close, right in our cells

European Heart Rhythm Association (EHRA)/Heart Rhythm Society (HRS)/Asia Pacific Heart Rhythm Society (APHRS)/Latin American Heart Rhythm Society (LAHRS) Expert Consensus Statement on the state of genetic testing for cardiac diseases.

Genetic testing has advanced significantly since the publication of the 2011 HRS/EHRA Expert Consensus Statement on the State of Genetic Testing for the Channelopathies and Cardiomyopathies.1 In addition to single-gene testing, there is now the ability to perform whole-exome sequencing (WES) and whole-genome sequencing (WGS). There is growing appreciation of oligogenic disorders,2,3 the role of modifier genes,2 and the use of genetic testing for risk stratification, even in common cardiac diseases such as coronary artery disease or atrial fibrillation (AFib), including a proposal for a score awaiting validation.4 This document reviews the state of genetic testing at the present time, and addresses the questions of what tests to perform and when to perform them. It should be noted that, as articulated in a 1999 Task Force Document by the European Society of Cardiology (ESC) on the legal value of medical guidelines,5 ‘The guidelines from an international organization, such as the ESC, have no specific legal territory and have no legally enforcing character. Nonetheless, in so far as they represent the state-of-the-art, they may be used as indicating deviation from evidence-based medicine in cases of questioned liability’. In the case of potentially lethal and treatable conditions such as catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS), it is the responsibility of the physician, preferably in conjunction with an expert genetics team, to communicate to the patient/family the critical importance of family screening, whether this be facilitated by cascade genetic testing or by broader clinical family screenin

Microfluidics: reframing biological enquiry

The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science