The History of Antithrombotic Therapy: The Discovery of Heparin, the Vitamin K Antagonists, and the Utility of Aspirin.

The administration of intravenous heparin to postoperative patients by Barritt and Jordan reduced the incidence of fatal and nonfatal pulmonary embolism and established heparin as the standard for parenteral anticoagulation. The coumarin family of vitamin K antagonists quickly became the standard for long-term oral anticoagulation. Aspirin became a widely used antithrombotic agent after the discovery that chronic oral administration reduced the incidence of secondary strokes and myocardial infarction. This article gives a brief history of antithrombotic therapy, including the discovery of heparin, the vitamin k antagonists, and the utility of aspirin.

A clinically relevant in vivo zebrafish model of human multiple myeloma to study preclinical therapeutic efficacy.

Patient-derived multiple myeloma (MM) cells are difficult to establish in culture or propagate in vivo in murine model. Here, we describe a zebrafish xenograft model that permits rapid, reliable growth of human MM cells injected into the perivitelline space of albino zebrafish (Casper) embryos 48 hours postfertilization. MM1S and MM1R MM cell lines and primary CD138(+) MM cells were stained with CM-Dil red fluorescent dye and suspended in Matrigel prior to their injection. The cells grew at the site of injection and disseminated throughout the developing embryos and larvae. Tumor size was quantified by fluorescent microscopy, and cell fate was followed for 4 days. All of the cell line xenografts showed responses similar to those previously observed with in vitro assays. CD138(+) plasma cell xenografts derived from MM patients also grew and were inhibited by the same drugs patients had responded to clinically. Using this technique, we can assess drug sensitivity or resistance with a small number of MM cells in a short period. This raises the possibility that one might be able to assess drug sensitivity in real time with readily obtainable clinical samples.

Cancer Cell Dissemination and Homing to the Bone Marrow in a Zebrafish Model.

Advancement of many solid tumors and hematologic malignancies is frequently characterized by dissemination and homing of cancer cells to the bone marrow (BM). Methods to quantitatively characterize these key steps of the metastatic cascade in mammalian models are currently limited and do not offer opportunities to perform rapid, large-scale genomic, or drug screening. Because of their optical clarity, we used zebrafish to develop an in vivo model of cancer cell dissemination and homing to the BM. We performed intracardiac injection of multiple myeloma (MM) cells derived from human BM or cell lines and monitored their migration to the caudal hematopoietic tissue (CHT), the region where hematopoiesis occurs in the zebrafish embryo, which recapitulates a BM-like niche. Transcriptomic analyses confirmed that MM cells homing to the CHT displayed gene-expression differences compared with MM cells outside of the CHT, including significant enrichment for genes known to regulate interleukin-6 (IL6) signaling, cell adhesion, and angiogenesis. Collectively, our findings point to the zebrafish as a valuable model in which to study cancer cell homing to the hematopoietic niche and to establish a screening platform for the identification of factors and mechanisms contributing to the early steps of bone metastasis.

The identification and characterization of zebrafish hematopoietic stem cells.

HSCs are defined by their ability to self-renew and maintain hematopoiesis throughout the lifespan of an organism. The optical clarity of their embryos and the ease of genetic manipulation make the zebrafish (Danio rerio) an excellent model for studying hematopoiesis. Using flow cytometry, we identified 2 populations of CD41-GFP(+) cells (GFP(hi) and GFP(lo)) in the whole kidney marrow of Tg(CD41:GFP) zebrafish. Past studies in humans and mice have shown that CD41 is transiently expressed in the earliest hematopoietic progenitors and is then silenced, reappearing in the platelet/thrombocyte lineage. We have transplanted flow-sorted GFP(hi) and GFP(lo) cells into irradiated adult zebrafish and assessed long-term hematopoietic engraftment. Transplantation of GFP(hi) cells did not reconstitute hematopoiesis. In contrast, we observed multilineage hematopoiesis up to 68 weeks after primary and secondary transplantation of GFP(lo) cells. We detected the CD41-GFP transgene in all major hematopoietic lineages and CD41-GFP(+) cells in histologic sections of kidneys from transplant recipients. These studies show that CD41-GFP(lo) cells fulfill generally accepted criteria for HSCs. The identification of fluorescent zebrafish HSCs, coupled with our ability to transplant them into irradiated adult recipients, provide a valuable new tool to track HSC homing, proliferation, and differentiation into hematopoietic cells.

Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.

Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10-30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.

PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts.

A zebrafish heart can fully regenerate after amputation of up to 20% of its ventricle. During this process, newly formed coronary blood vessels revascularize the regenerating tissue. The formation of coronary blood vessels during zebrafish heart regeneration likely recapitulates embryonic coronary vessel development, which involves the activation and proliferation of the epicardium, followed by an epithelial-to-mesenchymal transition. The molecular and cellular mechanisms underlying these processes are not well understood. We examined the role of PDGF signaling in explant-derived primary cultured epicardial cells in vitro and in regenerating zebrafish hearts in vivo. We observed that mural and mesenchymal cell markers, including pdgfrß, are up-regulated in the regenerating hearts. Using a primary culture of epicardial cells derived from heart explants, we found that PDGF signaling is essential for epicardial cell proliferation. PDGF also induces stress fibers and loss of cell-cell contacts of epicardial cells in explant culture. This effect is mediated by Rho-associated protein kinase. Inhibition of PDGF signaling in vivo impairs epicardial cell proliferation, expression of mesenchymal and mural cell markers, and coronary blood vessel formation. Our data suggest that PDGF signaling plays important roles in epicardial function and coronary vessel formation during heart regeneration in zebrafish.

The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish.

The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.

The zebrafish vitronectin receptor: characterization of integrin alphaV and beta3 expression patterns in early vertebrate development.

alphaVbeta3 is a receptor for vitronectin and other extracellular matrix ligands, and it has been implicated in angiogenesis and osteoclast function in mammals. We have cloned full-length cDNAs of zebrafish integrin alphaV (itgalphaV), and two paralogous zebrafish beta3 integrins (itgbeta3.1 and itgbeta3.2). Whole-mount in situ hybridization analysis revealed that alphaV and beta3.1 share overlapping expression domains in apical ectodermal ridge, ventricular myocardium, hypothalamus, posterior tuberculum, medial tectal proliferation zone, and in the odontogenic field of the bilateral pharyngeal dentitions. In contrast to beta3.1, beta3.2 is transiently expressed throughout the developing embryo. In situ hybridization profiles and heterologous expression of proteins in tissue culture cells suggest that beta3.1 is the major beta3 paralog that associates with alphaV in zebrafish. Furthermore, when beta3.1 expression profiles are compared to those of other potential alphaV partners (beta1, beta5, and beta8), pharyngeal dentitions appear to represent a unique expression field for alphaV and beta3.1.

Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development.

Although the ontogeny of hematopoietic stem cells (HSCs) in vertebrates has been studied intensely, a lineage relationship between the HSCs found in the developmentally successive hematopoietic organs remains to be shown. By using an in situ photoactivatable cell tracer in the transparent zebrafish embryo, we demonstrated that definitive blood precursors appeared between the dorsal aorta and axial vein, validating the homology of this tissue with the AGM (aorta-gonad-mesonephros) of amniotes. These cells first migrated through the blood to a previously undescribed caudal hematopoietic tissue (CHT), where they differentiated, expanded, and further migrated to seed the definitive hematopoietic organs, the thymus and kidney. Immigrants on the way to the thymus expressed c-myb and ikaros but not rag1; they were probably no longer HSCs, however, because they lacked scl and runx1 expression, unlike immigrants to the kidney. The CHT thus has a hematopoietic function similar to that of the mammalian fetal liver.

Inherited platelet disorders.

The inherited platelet disorders are a heterogeneous collection of rare diseases that are infrequently encountered in clinical practice. They are, however, fascinating abnormalities, which have taught us a great deal about normal platelet biochemistry and physiology. In this section of the presentation we will review disorders of the platelet membrane, platelet granule packaging disorders, the hereditary macrothrombocytopenias, platelet signaling disorders and disorders of platelet coagulant function. The molecular basis of the disorders, the cardinal features of their clinical presentation and best methods to make their diagnosis and the latest information regarding therapy will be presented.

Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.

Thrombocytes are the nucleated equivalent of platelets in nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alpha(IIb), glycoprotein IIb [GPIIb]) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP)-tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP(high) subset, which had the morphologic appearance of mature thrombocytes, and a GFP(low) subset with an immature appearance, suggesting that they may be thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP+ cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP+ cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify thrombocytes, thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.

An initiative in mentoring to promote residents' and faculty members' careers.

Internal medicine trainees and faculty recognize the value of effective mentoring to help meet the personal and professional needs of residents. However, the paradigm of the mentor-trainee relationship is seriously threatened by increased clinical, research, and administrative demands on both faculty and housestaff. Moreover, the current criteria for promotion in most teaching hospitals emphasize scholarship, rather than citizenship, so activities such as mentoring devolve to a lower priority. In 2000, the Department of Medicine at Brigham and Women's/Faulkner Hospital initiated a program to improve the effectiveness of housestaff mentoring and recognize faculty contributions to resident career development. The authors report the feedback received from a survey of the 2002-03 medical housestaff (74% response rate) and describe their experiences with the initiation of this program. Over 90% of the housestaff respondents thought it important that the Department assigns an individual faculty mentor. In practice, time-consuming professional responsibilities made meetings difficult, but most pairs supplemented their interactions with e-mail. Discussions primarily focused on career advice and support. Housestaff thought mentors were helpful and available when needed. The department has established new metrics for recognizing faculty mentoring and now publicly rewards mentoring excellence. Of note, unassigned mentoring has increased since the initiation of this program. The authors conclude that the formal mentoring program has ensured that all trainees are provided with a mentor, which has facilitated faculty-housestaff interactions and increased recognition of faculty contributions to mentoring.