Connecting the tips – a study on sprout fusion in angiogenesis

Angiogenesis is characterized by the sprouting of new vessels from pre-existing ones, with sprouts headed by a specialized endothelial cell (EC) known as the tip cell. The trailing ECs are referred to as stalk cells and help form the vessel proper and lumenize. Newly formed sprouts must come into contact with adjacent tip cells via filopodia to fuse and lumenize to form an additional functional vessel loop able to support blood flow. Here I describe the process of anastomosis in detailed spatial and temporal resolution. Interestingly tip cells do no necessarily form a new connection at their first contact but tend to have a “negotiation” phase whilst tip cells are in contact. This time delay is between 1 hour and 2.5 hours suggesting a possible genetic regulation of anastomosis. Interestingly cessation of filopodia activity is observed upon lumenization of the sprouts and not on the establishment of cell junctions. A gene important in fusion cells in Drosophila tracheal morphogenesis (the equivalent of EC tip cells) was investigated for its role in angiogenesis and anastomosis. Knockdown of heca, the homologue of the drosophila headcase gene, leads to precocious and ectopic connections between the Dorsal Lateral Anastomotic Vessels (DLAVs) over the neural tube in zebrafish embryos. Heca’s function does not appear dependent on the TGF/BMP pathway as in the trachea. Gain of function experiments at the whole organism and cell-autonomous level show that Heca overexpression leads to complete lumenization of some of the migrating intersegmental vessels by 26hpf. This suggests that heca’s possible involvement in instructing ECs to form a lumen or to sense the level of oxygenation/requirement to decrease activity may explain the opposing phenotypes observed. Taken together with observations about lumen formation with initiation of flow and the termination of filopodia activity, the data here presented could mean that lumenization is involved dampening EC activation

Imaging Transient Blood Vessel Fusion Events by Correlative Volume Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 - August 5, 201

Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy

The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism

The effect of simulated microgravity on the osteo-articular system: an in vitro study of long-term culture of cartilage and bone tissue explants in the RCCSTM bioreactor

Long-term spaceflight affects almost all physiological systems in humans and considerable amount of data revealed its serious impact on skeletal homeostasis. While a microgravity environment has been proved to induce significant mineral loss and bone fragility (affecting, specifically, cancellous weight-bearing bones), its effect on articular cartilage (AC) is poorly known. AC is an avascular tissue, composed of relatively few mechanosensitive cells (chondrocytes), that synthesize a mechanically functional extracellular matrix (ECM), composed of collagen, proteoglycans and other proteins. In response to physical factors (e.g. pressure and deformation) chondrocytes regulate AC histomorphology and function, and may affect bone tissue homeostasis. It is then likely that the absence of gravitational load should alter chondrocytes\u2019 activity. In the present study we investigated, in vitro, the effect of long-term exposure to a simulated microgravity condition (vector-averaged gravity) on whole explants of cancellous bone (rat tibial proximal epiphyses) and AC tissue (newborn rabbit knee\u2019s joint). Tissue explants were kept in culture for up to 4 weeks by the use of the Rotary Cell Culture System (RCCSTM) bioreactor, the unique device, operating on the Earth\u2019s surface, capable of successfully simulate a microgravity environment. The analysis of the structural/mechanical properties of cancellous bone explants was performed by a numerical model based on the Cell Method, applied to the 3D reconstruction of micro-computed tomography scans of the bone samples. With regard to AC tissue, functional and structural properties were studied by comparative cell viability, histochemical and molecular analyses performed on either the cellular component or on their ECM. The results obtained demonstrate that, while our RCCSTM-based culture system is able to preserve native tissue architecture and cells\u2019 viability all during the experimental procedure, long-term exposure to a microgravity environment may effectively alter bone cells\u2019 and articular chondrocytes\u2019 physiology