Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 m was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.Many cells in developing organs and tissues have the capacity to detect extracellular chemical gradients and to respond to these gradients by directed positive or negative migration, a process called chemotaxis. In addition, some factors may also regulate chemokinesis which refers to nondirectional cell migration. Directed cell migration is at the heart of embryonic blood vessel formation, where the growing vessels navigate by a combination of secreted chemoattractants and repellents. The leading front of the embryonic vascular sprout holds a tip cell with numerous filopodia that express receptors for sensing secreted and cell-bound guidance cues provided by surrounding cells (1). One of the most well studied factors that control blood vessel formation and function is vascular endothelial growth factor A (VEGFA) 2 (2-5). The effects of VEGFA on endothelial cells have been intensely studied for many years in an array of different model systems (4, 6 -9). However, the ability to generate and maintain stable gradients of soluble factors compatible with cell culture conditions was only recently made possible by the invention of a microfluidic chemotaxis chamber (MCC). Chemotaxis of several cell types, including neutrophils and cancer cells, have been successfully studied in MCCs, but the method has so far not been used to systematically study endothelial cell migration in gradients of chemotactic factors (10, 11).VEGF receptor 2 (VEGFR2) i...

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of singlemolecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods. Counting and cell-to-cell heterogeneitySingle-molecule sensitive methods are needed to determine the number of a particular molecular species and how this number varies over time, in space or between cells. The use of single-molecule counting falls into a few different types of experiments. Inference of kinetics from steady-state distributionsAn obvious strength of single-molecule counting in cells is the possibility to characterize the cell-to-cell variation in molecule numbers and determine the steadystate distribution from snapshots over different cells. These distributions arise from stochasticity in the underlying processes, such as gene expression, fluorophore maturation, and protein partitioning in cell division, and depend on the mechanisms and kinetic rates specific for each process(13). By assuming a dynamic model, it might be possible to fit the model parameters based on the steady-state distribution(14-18) and thus estimate the underlying kinetic rates (Fig. 1A). When using such methods, it is important to keep in mind that different stochastic models can give rise to very similar steady-state distributions (19) and that deterministic differences between individual cells can also be mistaken for stochastic effects(20, 21). Following actual low copy dynamicsFollowing individual cells over time makes it possible to study dynamic correlations that cannot be seen in steady-state distributions. Some classical examples are the early single-molecule studies of bursts in protein (22,23) or RNA(24) expression from the lac operon. Looking at numbers of molecules in single cells over time, it is also possible to study how low copy number dynamics give rise to phenotypic copy number transitions. For example, Choi et al.(25) monitored fluctuations in the expression of fluorescently labeled lacY permeases to deduce how many LacY that are needed to switch the bistability observed for the Lac operon in the presence of Methyl-β-D-thiogalactoside (TMG). Similar in nature is the work by Uphoff et al., which shows that the DNA methylation repair by Ada is turned on by a single expression event which then activates further expression of the protein by positive feedback(26).

BackgroundA function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets.MethodsBioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber.ResultsmiR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro.ConclusionsmiR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature.

Background:The tetraspanin CD63 is known to regulate protein trafficking, leukocyte recruitment, and adhesion processes. Results: Silencing of CD63 disrupts complex formation between ␤1 integrin and VEGFR2, resulting in impaired downstream signaling. Conclusion: CD63 supports VEGFR2 activation and signaling in vitro and in vivo. Significance: A novel role for the tetraspanin CD63 in the convergence between integrin and growth factor signaling in angiogenesis.

Many signals that induce angiogenesis have been identified; however, it is still not clear how these signals interact to shape the vascular system. We have developed a fluidic device for generation of molecular gradients in 3-dimensional cultures of complex tissues and organs in order to create an assay for precise induction and guidance of growing blood vessels. The device features a centrally placed culture chamber, flanked by channels attached to a perfusion system used to generate gradients. A separate network of vacuum channels permits reversible attachment of the device to a flat surface. We show that the fluidic device can be used to create growth factor gradients that induce directional angiogenesis in embryonic mouse kidneys and in clusters of differentiating stem cells. These results demonstrate that the device can be used to accurately manipulate complex morphogenetic processes with a high degree of experimental control.