Towards definition of an ECM parts list: An advance on GO categories

Those of us interested in the extracellular matrix (ECM) are faced with significant challenges of definition. ECM proteins are large, complex and assembled into crosslinked insoluble matrices. This has meant that defining the biochemical composition of ECMs has been difficult. Nonetheless, protein chemistry and molecular biology have defined many familiar ECM proteins — collagens, proteoglycans, laminins, thrombospondins, tenascins, fibronectins, etc. With the completion of many genomes it should now be possible to develop complete “parts lists” for the ECM. Such lists are needed for analyzing data from “omic” approaches such as expression arrays, latest-generation sequencing and proteomics. These approaches generate long lists and it is typically necessary to extract from those lists the genes/proteins of interest. Anyone who attempts to do this using the commonly used gene ontology (GO) categories soon discovers that they are largely useless for defining ECM proteins. Many ECM proteins are unannotated and those which are, are sorted, with little evidence of logic or consistency, into diverse categories such “extracellular matrix,” “basement membrane,” “cell surface” and many others. The human and mouse orthologs are often found in different categories and attempts to use GO categories to extract a complete list of ECM genes or proteins from a data set are unsatisfactory at best

Alternative Splicing of Endothelial Fibronectin Is Induced by Disturbed Hemodynamics and Protects Against Hemorrhage of the Vessel Wall

Objective—Abnormally low-flow conditions, sensed by the arterial endothelium, promote aneurysm rupture. Fibronectin (FN) is among the most abundant extracellular matrix proteins and is strongly upregulated in human aneurysms, suggesting a possible role in disease progression. Altered FN splicing can result in the inclusion of EIIIA and EIIIB exons, generally not expressed in adult tissues. We sought to explore the regulation of FN and its splicing and their possible roles in the vascular response to disturbed flow. Approach and Results—We induced low and reversing flow in mice by partial carotid ligation and assayed FN splicing in an endothelium-enriched intimal preparation. Inclusion of EIIIA and EIIIB was increased as early as 48 hours, with negligible increases in total FN expression. To test the function of EIIIA and EIIIB inclusion, we induced disturbed flow in EIIIAB[superscript −/−] mice unable to include these exons and found that they developed focal lesions with hemorrhage and hypertrophy of the vessel wall. Acute deletion of floxed FN caused similar defects in response to disturbed flow, consistent with a requirement for the upregulation of the spliced isoforms, rather than a developmental defect. Recruited macrophages promote FN splicing because their depletion by clodronate liposomes blocked the increase in endothelial EIIIA and EIIIB inclusion in the carotid model. Conclusions—These results uncover a protective mechanism in the inflamed intima that develops under disturbed flow, by showing that splicing of FN mRNA in the endothelium, induced by macrophages, inhibits hemorrhage of the vessel wall.National Institutes of Health (U.S.) (Grant 5F32HL110484)National Institutes of Health (U.S.) (Grant PO1-HL66105)Howard Hughes Medical InstituteNational Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

Intravital imaging of metastasis in adult Zebrafish

Background Metastasis is a major clinical problem whose biology is not yet fully understood. This lack of understanding is especially true for the events at the metastatic site, which include arrest, extravasation, and growth into macrometastases. Intravital imaging is a powerful technique that has shown great promise in increasing our understanding of these events. To date, most intravital imaging studies have been performed in mice, which has limited its adoption. Zebrafish are also a common system for the intravital imaging of metastasis. However, as imaging in embryos is technically simpler, relatively few studies have used adult zebrafish to study metastasis and none have followed individual cells at the metastatic site over time. The aim of this study was to demonstrate that adult casper zebrafish offer a convenient model system for performing intravital imaging of the metastatic site over time with single-cell resolution. Methods ZMEL1 zebrafish melanoma cells were injected into 6 to 10-week-old casper fish using an intravenous injection protocol. Because casper fish are transparent even as adults, they could be imaged without surgical intervention. Individual cells were followed over the course of 2 weeks as they arrested, extravasated, and formed macroscopic metastases. Results Our injection method reliably delivered cells into circulation and led to the formation of tumors in multiple organs. Cells in the skin and sub-dermal muscle could be imaged at high resolution over 2 weeks using confocal microscopy. Arrest was visualized and determined to be primarily due to size restriction. Following arrest, extravasation was seen to occur between 1 and 6 days post-injection. Once outside of the vasculature, cells were observed migrating as well as forming protrusions. Conclusions Casper fish are a useful model for studying the events at the metastatic site using intravital imaging. The protocols described in this study are relatively simple. Combined with the reasonably low cost of zebrafish, they offer to increase access to intravital imaging.National Cancer Institute (U.S.) (Grant P30-CA14051)National Institutes of Health (U.S.) (Grant 5-T32-GM007287

Tumor Angiogenesis in the Absence of Fibronectin or Its Cognate Integrin Receptors

Binding of α5β1 and αvβ3/β5 integrin receptors on the endothelium to their fibronectin substrate in the extracellular matrix has been targeted as a possible means of blocking tumor angiogenesis and tumor growth. However, clinical trials of blocking antibodies and peptides have been disappointing despite promising preclinical results, leading to questions about the mechanism of the inhibitors and the reasons for their failure. Here, using tissue-specific and inducible genetics to delete the α5 and αv receptors in the endothelium or their fibronectin substrate, either in the endothelium or globally, we show that both are dispensable for tumor growth, in transplanted tumors as well as spontaneous and angiogenesis-dependent RIP-Tag-driven pancreatic adenocarcinomas. In the nearly complete absence of fibronectin, no differences in vascular density or the deposition of basement membrane laminins, ColIV, Nid1, Nid2, or the TGFβ binding matrix proteins, fibrillin-1 and -2, could be observed. Our results reveal that fibronectin and the endothelial fibronectin receptor subunits, α5 and αv, are dispensable for tumor angiogenesis, suggesting that the inhibition of angiogenesis induced by antibodies or small molecules may occur through a dominant negative effect, rather than a simple functional block.National Institutes of Health (U.S.) (Grant 5F32HL110484)National Institutes of Health (U.S.) (Grant PO1-HL66105)Howard Hughes Medical InstituteNational Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

Endothelium-derived fibronectin regulates neonatal vascular morphogenesis in an autocrine fashion

Fibronectin containing alternatively spliced EIIIA and EIIIB domains is largely absent from mature quiescent vessels in adults, but is highly expressed around blood vessels during developmental and pathological angiogenesis. The precise functions of fibronectin and its splice variants during developmental angiogenesis however remain unclear due to the presence of cardiac, somitic, mesodermal and neural defects in existing global fibronectin KO mouse models. Using a rare family of surviving EIIIA EIIIB double KO mice, as well as inducible endothelial-specific fibronectin-deficient mutant mice, we show that vascular development in the neonatal retina is regulated in an autocrine manner by endothelium-derived fibronectin, and requires both EIIIA and EIIIB domains and the RGD-binding α5 and αv integrins for its function. Exogenous sources of fibronectin do not fully substitute for the autocrine function of endothelial fibronectin, demonstrating that fibronectins from different sources contribute differentially to specific aspects of angiogenesis

Requirement for the NF-kappa B family member Re1A in the development of secondary lymphoid organs

The transcription factor nuclear factor (NF)-kappaB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-kappaB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-kappaB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the trice that are born lack lymph nodes, foyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell-dependent antigen responses. Analyses of TNFR1/1RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs

Extracellular Matrix Proteins in Hemostasis and Thrombosis

The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets