The means by which extracellular matrix density regulates three-dimensional capillary morphogenesis is unclear. To study this phenomenon, we utilized a fibrin-based in vitro assay in which a fibroblast monolayer is plated atop a fibrin gel approximately 2.5 mm away from endothelial cell-coated beads within the matrix. Increasing fibrin density from 2.5 to 10 mg/ml resulted in a threefold reduction in capillary network formation. However, distributing fibroblasts throughout the matrix completely eliminated this inhibitory effect, resulting in robustly vascularized matrices suitable for in vivo applications, as functional anastomoses formed between the implanted tissues and host vasculature when implanted into immune-compromised mice. Dense matrices did not stimulate fibroblast-mediated matrix remodeling: differentiation into myofibroblasts, matrix production, and protease secretion were not enhanced by the dense condition. Instead, quantifying diffusivity of FITC-dextran (molecular mass 10, 40, 70, and 150 kDa) through fibrin revealed a two- to threefold decrease within the 10 mg/ml matrices. Thus, distributing a proangiogenic source (fibroblasts) throughout the matrix stimulates capillary network formation by overcoming this diffusion restriction due to significantly reduced diffusion distances. Although roles for matrix stiffness and ligand binding density have previously been identified, our results emphasize the importance of diffusion restrictions in limiting capillary morphogenesis.

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We synthesize a large gene expression data set using dbEST and UniGene. We use guilt-by-association (GBA) to analyze this data set and identify coexpressed genes. One module, or group of genes, was found to be coexpressed mainly in tissue extracted from breast and ovarian cancers, but also found in tissue from lung cancers, brain cancers, and bone marrow. This module contains at least six members that are believed to be involved in either transcritional regulation (PDEF, H2AFO, NUCKS) or the ubiquitin proteasome pathway (PSMD7, SQSTM1, FLJ10111). We confirm these observations of coexpression by real-time RT–PCR analysis of mRNA extracted from four model breast epithelial cell lines

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Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for >3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.