Single-Molecule Kinetics in Living Cells.

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 single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

Tetraspanin CD63 promotes vascular endothelial growth factor receptor 2-β1 integrin complex formation, thereby regulating activation and downstream signaling in endothelial cells in vitro and in vivo.

CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of β1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs.

Single molecule methods with applications in living cells.

Our knowledge about dynamic processes in biological cells systems has been obtained roughly on two levels of detail; molecular level experiments with purified components in test tubes and system wide experiments with indirect readouts in living cells. However, with the development of single molecule methods for application in living cells, this partition has started to dissolve. It is now possible to perform detailed biophysical experiments at high temporal resolution and to directly observe processes at the level of molecules in living cells.

A vaccine targeting angiomotin induces an antibody response which alters tumor vessel permeability and hampers the growth of established tumors.

Angiomotin (Amot) is one of several identified angiostatin receptors expressed by the endothelia of angiogenic tissues. We have shown that a DNA vaccine targeting Amot overcome immune tolerance and induce an antibody response that hampers the progression of incipient tumors. Following our observation of increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, we evaluated the effect of anti-Amot vaccination on clinically evident tumors.

Exocyst complex component 3-like 2 (EXOC3L2) associates with the exocyst complex and mediates directional migration of endothelial cells.

The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections.

Discovery of microvascular miRNAs using public gene expression data: miR-145 is expressed in pericytes and is a regulator of Fli1.

Background: A 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.

A fluidic device to study directional angiogenesis in complex tissue and organ culture models.

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.

Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2: effects on chemotaxis and chemokinesis.

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).

Effect of fluid shear stress on endocytosis of heparan sulfate and low-density lipoproteins.

Hemodynamic stress is a critical factor in the onset of atherosclerosis such that reduced rates of shear stress occurring at regions of high curvature are more prone to disease. The level of shear stress has direct influence on the thickness and integrity of the glycocalyx layer. Here we show that heparan sulfate, the main component of the glycocalyx layer, forms an intact layer only on cell surfaces subjected to shear, and not under static conditions.