Pericytes promote selective vessel regression to regulate vascular patterning.

Blood vessel networks form in a 2-step process of sprouting angiogenesis followed by selective branch regression and stabilization of remaining vessels. Pericytes are known to function in stabilizing blood vessels, but their role in vascular sprouting and selective vessel regression is poorly understood. The endosialin (CD248) receptor is expressed by pericytes associated with newly forming but not stable quiescent vessels. In the present study, we used the Endosialin(-/-) mouse as a means to uncover novel roles for pericytes during the process of vascular network formation. We demonstrate in a postnatal retina model that Endosialin(-/-) mice have normal vascular sprouting but are defective in selective vessel regression, leading to increased vessel density. Examination of the Endosialin(-/-) mouse tumor vasculature revealed an equivalent phenotype, indicating that pericytes perform a hitherto unidentified function to promote vessel destabilization and regression in vivo in both physiologic and pathologic angiogenesis. Mechanistically, Endosialin(-/-) mice have no defect in pericyte recruitment. Rather, endosialin binding to an endothelial associated, but not a pericyte associated, basement membrane component induces endothelial cell apoptosis and detachment. The results of the present study advance our understanding of pericyte biology and pericyte/endothelial cell cooperation during vascular patterning and have implications for the design of both pro- and antiangiogenic therapies.

Membrane type-1 matrix metalloproteinase activity is regulated by the endocytic collagen receptor Endo180.

The molecular interactions leading to organised, controlled extracellular matrix degradation are of central importance during growth, development and tissue repair, and when deregulated contribute to disease processes including cancer cell invasion. There are two major pathways for collagen degradation: one dependent on secreted and membrane-bound collagenases, the other on receptor-mediated collagen internalisation and intracellular processing. Despite the established importance of both pathways, the functional interaction between them is largely unknown. We demonstrate here, that the collagen internalisation receptor Endo180 (also known as CD280, uPARAP, MRC2) is a novel regulator of membrane-bound matrix metalloproteinase (MT1-MMP) activity, MT1-MMP-dependent MMP-2 activation and urokinase plasminogen activator (uPA) activity. We show close correlation between Endo180 expression, collagen accumulation and regulation of MT1-MMP cell-surface localisation and activity. We directly demonstrate, using collagen inhibition studies and non-collagen-binding mutants of Endo180, that the molecular mechanism underlying this regulation is the ability of Endo180 to bind and/or internalise collagens, rather than by acting as an interaction partner for pro-uPA and its receptor uPAR. These studies strongly support a functional interaction between two distinct collagen degradation pathways, define a novel mechanism regulating MT1-MMP activity and might have important implications for organised collagen clearance in the pericellular environment.

Isolation and analysis of large and small vessel endothelial cells.

The in vitro isolation, propagation and study of endothelial cells (EC) is an invaluable means by which the function of the vascular endothelium in physiology and patho-physiology can be explored. In recent years heterogeneity between large and small vessel EC, between arteries and veins, and between microvascular EC derived from different organs has become increasingly apparent. This has led to the development of protocols for the isolation of these different EC. In addition, the data emerging on vascular EC function in gene-targeted mice has highlighted the need for reliable methods of isolation of murine EC. This chapter describes methods for the isolation, characterization and culture of macro- and microvascular EC from a variety of species and introduces simple approaches to investigating their surface antigen expression.

The hyaluronan receptor, CD44.

CD44 is a widely expressed cell surface hyaluronan receptor which plays a key role in mediating cell migration. A number of recent papers demonstrating an interplay between CD44 and matrix metalloproteinases (MMPs) have shed important insights into the molecular mechanisms underlying these events. This has important implication for understanding how mis-regulation of CD44 can contribute to disease pathologies.

An in vivo functional screen identifies ST6GalNAc2 sialyltransferase as a breast cancer metastasis suppressor.

To interrogate the complex mechanisms involved in the later stages of cancer metastasis, we designed a functional in vivo RNA interference (RNAi) screen combined with next-generation sequencing. Using this approach, we identified the sialyltransferase ST6GalNAc2 as a novel breast cancer metastasis suppressor. Mechanistically, ST6GalNAc2 silencing alters the profile of O-glycans on the tumor cell surface, facilitating binding of the soluble lectin galectin-3. This then enhances tumor cell retention and emboli formation at metastatic sites leading to increased metastatic burden, events that can be completely blocked by galectin-3 inhibition. Critically, elevated ST6GALNAC2, but not galectin-3, expression in estrogen receptor-negative breast cancers significantly correlates with reduced frequency of metastatic events and improved survival. These data demonstrate that the prometastatic role of galectin-3 is regulated by its ability to bind to the tumor cell surface and highlight the potential of monitoring ST6GalNAc2 expression to stratify patients with breast cancer for treatment with galectin-3 inhibitors.

Increased surveillance of cells in mitosis by human NK cells suggests a novel strategy for limiting tumor growth and viral replication.

The threat from cancer cells is inherently linked to cell-cycle progression, and viral genomes commonly replicate, for example, within episomes or proviruses, during mitosis. We report here that human natural killer (NK) cells bound cells in mitosis and attacked pathogenic cells in mitosis more effectively than the same cells in other stages of the cell cycle. Thus, cells in mitosis warrant and undergo heightened surveillance, a novel strategy for immunologic assessment of danger. Recognition of cells in mitosis involved ligation of activating NK-cell receptors and binding to target-cell hyaluronan, a component of the pericellular matrix known to be increased during mitosis. Direct interaction between activating NK-cell receptors and hyaluronan is possible, but other mechanisms consistent with our data are also discussed.